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THANKS TO OUR SPONSORS LabEx IBEID TABLE OF CONTENTS Page General information ................................................................................... 6 Social events.............................................................................................. 7 Map of the campus .................................................................................... 8 Scientific program ...................................................................................... 9 Oral presentations.................................................................................... 21 Poster sessions Poster session 1 ................................................................................. 87 Poster session 2 ............................................................................... 201 Poster session 3 ............................................................................... 259 Poster session 4 ............................................................................... 339 Authors and co‐authors index ................................................................ 393 Participant list......................................................................................... 409 Reproduction or exploitation, in any form, of the data included in this document are forbidden. Chairs of the conference Roland Brosch (Chair) and Pedro Alzari, Marco Bellinzoni, Carmen Buchrieser, Laleh Majlessi, Giulia Manina, Roxane Simeone, Ludovic Tailleux (co-chairs) Institut Pasteur, Paris, France Scientific Committee Pedro Alzari, Institut Pasteur, Paris, France Clifton Barry, NIH, Bethesda, USA Priscille Brodin, INSERM and Institut Pasteur de Lille, France Roland Brosch, Institut Pasteur, Paris, France Stewart Cole, EPFL, Lausanne, Switzerland Sabine Ehrt, Weill Cornell Medical College, New York, USA Lalita Ramakrishnan, University of Cambridge, UK Eric Rubin, Harvard TH Chan School of Public Health, Boston, USA Theme of the conference The principle theme of this EMBO conference “Tuberculosis 2016: Interdisciplinary research on tuberculosis and pathogenic mycobacteria”, consists of the latest biological insights and advances on pathogenic mycobacteria that are presently gained by new powerful technologies and sophisticated approaches linked to the domains of genomics, genetics, cell biology, structural biology, medicinal chemistry and/or immunology and how these advances might be used to cope with the enormous problems that pathogenic mycobacteria cause for millions of people today. Pathogenic mycobacteria correspond to a large group of medically important bacteria that cause the major human diseases Tuberculosis, Leprosy, and Buruli ulcer, but are also increasingly important in the context of emerging pathogens in patients with genetic disorders, such as cystic fibrosis patients. These different themes will be presented and discussed by world-renowned scientists from a broad range of different disciplines. This international conference, to be held from September 19-23, 2016, in the Conference centre (CIS) within the campus of the Institut Pasteur in Paris will provide a premium forum for the discussion and exchange of the recent advances in this timely and important topic. Conference Center of the Institut Pasteur - 25-28 rue du Dr Roux - 75724 Paris cedex 15 - France www.tuberculosis2016.org – [email protected] GENERAL INFORMATION W ELCOME DESK OPENING HOURS On the Institut Pasteur campus, the "Plan Vigipirate Attentats" is on, so please make sure to have an official ID or passport on you to enter the campus. If your registration is fully covered, you will receive your complete congress kit including your badge, the certificate of attendance, the conference program and if pre-booked ahead, the voucher for the conference dinner on Wednesday evening. Please wear your badge at all time. If registration was not fully covered, please come directly to the registration desk "on site payment". We accept payment by cash or credit card (Visa or Mastercard). REGISTRATION DESK opens at 2:00 pm on September 19, 2016 in the hall of the CIS Auditorium. Monday Tuesday Wednesday Thursday Friday September 19 September 20 September 21 September 22 September 23 2:00 pm 7:30 pm 8:00 am 7:00 pm 8:00 am 6:00 pm 8:00 am 7:00 pm 8:00 am 5:30 pm ORAL SESSIONS Scientific sessions are taking place in the main auditorium of the "Centre d’information Scientifique", located in the building CIS. POSTER SESSIONS Four poster sessions are scheduled in the hall of the CIS and in the Espace Congrès: Poster session 1: Hall of CIS + Espace Congrès Tuesday September 20, 2016, 1:45 pm - 3:30 pm - [Posters 65-176] Poster session 2: Hall of CIS Wednesday September 21, 2016, 1:45 pm - 3:15 pm - [Posters 177-231] Poster session 3: Hall of CIS Thursday September 22, 2016, 1:30 pm - 3:00 pm - [Posters 232-308] Poster session 4: Hall of CIS Friday September 23, 2016, 1:45 pm - 3:15 pm - [Posters 309-359] Poster numbers are in the programme book. Check the matching number on the board to display your poster in the right place. Magnets are available at the welcome desk to mount your poster. COFFEE BREAKS AND LUNCHES Coffee breaks will be served in the hall of CIS. Lunches buffet will be served in the hall of the CIS and in the Espace Congrès throughout the conference. Access to lunches is limited to participants who registered. 6 Tuberculosis 2016 – September 19-23, 2016 – Paris, France SOCIAL EVENTS You are invited to join us for the Tuberculosis 2016 on • Monday 19 at 7:30 - 9:30 pm: Welcome cocktail (hall of CIS) • Tuesday 20 at 7:00 - 9:00 pm: Wine & Cheese by the posters (hall of CIS) Conference dinner is scheduled on Wednesday 21, at 8.00 pm in the ‘Salon Honnorat-Maison internationale’ at the Cité internationale universitaire de Paris. The Cité Internationale universitaire de Paris was set up as part of the inter-war pacifist movement. Its founders, driven by a humanist ideal, sought to create “a school of human relations to promote peace” with a view to fostering harmony between different nations by furthering friendship between students, researchers and artists from all over the world. Address: 17 boulevard Jourdan - 75014 Paris Access: Metro Line 6 from Pasteur (Nation direction) – stop at Denfert-Rochereau + connection with RER line B (train) from Denfert-Rochereau (Massy Palaiseau direction) – stop at Cité universitaire (one stop). Journey: 15/20 minutes. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 7 M AP OF THE CAMPUS CIS Building Welcome desk Auditorium (plenary sessions) Espace Congrès Coffee breaks, lunches & cocktails Poster sessions 1 - 4 8 Lunches Poster session 1-4 Tuberculosis 2016 – September 19-23, 2016 – Paris, France th Monday, 19 September 2016 2:00 Arrival, Registration and Poster set up 5:40 Welcome addresses by Prof. Christian Bréchot, General Director of the Institut Pasteur and the Organizers 1 Opening Keynote Lecture Session Chair: 6:00 pm 7:20 pm Howard TAKIFF 1 Cellular and molecular insights on the survival of Mycobacterium tuberculosis in 6:00 phagosomes P. Brodin Center for Infection and Immunity, Institut Pasteur de Lille - INSERM U1019, Lille, France 2 Pursuing Edward Jenner’s Revenge: Novel Strategies for Sterilizing 6:40 Mycobacterium tuberculosis Infections W.R. Jacobs Microbiology and Immunilogy, Albert Einstein College of Medicine, New York, United States 7:20 Welcome reception th Tuesday, 20 September 2016 2 Mycobacterial Genomics and Evolution Chair: 8:30 am 10:15 am Philip SUPPLY 3 TB under the sea: re-thinking the origins of Mycobacterium tuberculosis 8:30 T. Stinear Department of Microbiology and Immunology, University of Melbourne, Melbourne (VIC), Australia Tuberculosis 2016 – September 19-23, 2016 – Paris, France 9 4 Comparative genomics and phylogenetics of Uruguayan Mycobacterium bovis 8:55 isolates provide insights into their genetic variability M. Lasserre Unidad de Biología Molecular, Institut Pasteur de Montevideo, Montevideo, Uruguay 5 Generalists and Specialists in Mycobacterium tuberculosis 9:10 S. Gagneux Medical Parasitology & Infection Biology, Swiss Tropical & Public Health Institute, Basel, Switzerland 6 Origin of M. tuberculosis Beijing family and its possible hypervirulent 9:35 determinants Q. Gao Fudan University, Shanghai, China 7 Programmable transcriptional repression in mycobacteria using an orthogonal 10:00 CRISPR interference platform J. Rock Harvard School of Public Health, Boston, United States 10:15 Coffee break 3 Mycobacterial Biology & Physiology I Chair: 10:45 am 12:30 pm Stefan NIEMANN 8 Cell envelope remodeling and evolution of tuberculosis bacilli virulence 10:45 C. Guilhot CNRS, IPBS, Toulouse, France 9 Mycobacterium tuberculosis periplasmic protease MarP activates the 11:10 peptidoglycan hydrolase RipA during acid stress H. Botella Microbiology and Immunology Department, Weill Cornell Medical College, New York, United States 10 The tailoring modifications of Mycobacterium tuberculosis polysaccharides 11:25 M. Jackson Mycobacteria Research Laboratories, Colorado State University, Fort Collins, United States 11 Drug use on a single cell level 11:50 H. Rego Microbial Pathogenesis, Yale, New Haven, United States 10 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 12 Single-cell approaches to unravel mycobacterial individuality and persistence 12:05 G. Manina Junior Group Microbial Individuality and Infection, Institut Pasteur, Paris, France 12:30 Lunch buffet (Hall of CIS + Espace Congrès) 1:45 Poster Session 1 - No. 65 - 176 4 Transport systems & Secretion in Mycobacteria Chair: 3:30 pm 4:50 pm Riccardo MANGANELLI 13 The role of the ESX-5 secretion system, from nutrient acquisition to immune 3:30 modulation W. Bitter VU university medical center, Amsterdam, The Netherlands 14 Intercellular communication links ESX-1 to ESX-4 in mycobacterial conjugation 3:55 K. Derbyshire Department of Biomedical Sciences, University at Albany, United States 15 Structure of the ESX-5 Type VII Secretion System 4:10 K. Beckham EMBL - Hamburg, Germany 16 The Tuberculosis Necrotizing Toxin 4:25 M. Niederweis Department of Microbiology, University of Alabama at Birmingham, Homewood, United States 4:50 Coffee break 5 Mycobacterial Biology & Physiology II Chair: 5:15 pm 7:00 pm Brian ROBERTSON 17 Stress resistance, cell division and in vivo persistence 5:15 S. Ehrt Department of Microbiology & Immunology, Weill Cornell Medicine, New York, United States 18 Inherited cell-surface wave-troughs mark future division sites in mycobacteria 5:40 H.A. Eskandarian Global Health Institute, UPKIN, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Tuberculosis 2016 – September 19-23, 2016 – Paris, France 11 19 The inosine monophosphate dehydrogenase, GuaB2, is a vulnerable new 5:55 bactericidal drug target for tuberculosis V. Mizrahi Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Cape Town, South Africa 20 Functional, regulatory, and evolutionary aspects of the phage-shock-protein 6:20 response of Mycobacterium tuberculosis M. Gennaro PHRI, Rutgers University, Newark, United States 21 Playing nice together - making cell walls in mycobacteria 6:35 E. Rubin Immunology and Infectious Diseases Department, Harvard TH Chan Scholl of Public Health, Boston, United States 7:00 Wine and Cheese st Wednesday, 21 September 2016 6 Biomarkers, Profiling and Beyond Chair: 8:30 am 10:25 am Nathalie WINTER 22 Human CD8+ T-cells recognizing peptides from Mycobacterium tuberculosis 8:30 presented by HLA-E have an unorthodox Th2-like, multifunctional, Mtb-inhibitory phenotype and represent a novel human T-cell subset T. Ottenhoff Leiden University Medical Center, Leiden, The Netherlands 23 Profiling persistent tubercule bacilli from patient sputa during therapy predicts 8:55 early drug efficacy S. Waddell Brighton and Sussex Medical School, University of Sussex, Brighton, United Kingdom 24 Understanding and Intervening in HIV-1 associated Tuberculosis 9:10 R. Wilkinson University of Cape Town, Cape Town, South Africa Imperial College London The Francis Crick Institute, London, United Kingdom 25 Condensation of DNA as a generic stress response in Mycobacterium 9:35 tuberculosis: apoptosis in bacteria? N. Van Der Wel Electron Microscopy Center Amsterdam, Cell Biology and Histology, AMC, Amsterdam, The Netherlands 26 TB Subunit Vaccine Immunity Expressed in The Lung 10:00 P.L. Andersen Statens Serum Institut, Copenhagen, Denmark 10:25 Coffee break 12 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 7 Whole cell vacines and beyond Chair: 10:50 am 12:30 pm Daria BOTTAI 27 VPM1002 as a TB prime vaccine, but also as post-exposure vaccine on the 10:50 horizon L. Grode Business Development, Vakzine Projekt Management GmbH, Honnover, Germany 28 Mycobacterium bovis BCG Δzmp1 – a promising, novel live vaccine candidate 11:15 P. Sander Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland 29 ESX-1 type VII secretion, cytosolic pattern recognition and consequences for 11:40 vaccine design R. Brosch Institut Pasteur, Paris, France 30 MTBVAC: from Discovery to Evaluation in High-Burden Countries 12:05 C. Martin University of Zaragoza, Zaragoza, Spain 12:30 Lunch buffet (Hall of CIS + Espace Congrès) 1:45 Poster Session 2 - No. 177 - 231 8 Biochemistry & Chemical biology Chair: 3:15 pm 4:35 pm Patrick BRENNAN 31 The dynamics of TB lesions revealed by PET/CT scanning using [F-18] trehalose 3:15 C. Barry Tuberculosis Research Section - LCID, NIH, Bethesda, United States 32 Flipping trehalose monomycolate across the inner membrane of mycobacteria 3:40 S.S. Chng National University of Singapore, Singapore 33 Mechanisms of high fidelity DNA replication in Mycobacterium tuberculosis 3:55 M. Lamers Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom 34 Structural basis of phosphatidylinositol mannosides biosynthesis in 4:10 mycobacteria M. Guerin CICbioGUNE, Spain 4:35 Coffee break Tuberculosis 2016 – September 19-23, 2016 – Paris, France 13 9 Biochemistry & Chemical biology and beyond Chair: 5:00 pm 6:05 pm Katarina MIKUSOVA 35 Amino acid sensing by protein kinase G in metabolic regulation and virulence 5:00 H. O'Hare Dept of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom 36 Targeting topology modulators and topoisomerases of Mycobacterium 5:25 tuberculosis V. Nagaraja Jawaharlal Nehru Centre for Advanced Scientific research & Department Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India 37 In Search of Drugs to Shorten Treatment of TB 5:40 C. Nathan Depts. of Microbiology & Immunology and Medicine, Weill Cornell Medicine, New York, United States 8:00 Conference Dinner - Cité Universitaire de Paris Thursday, 22 nd September 2016 10 NTM & Emerging mycobacterial pathogens Chair: 8:30 am 10:15 am Caroline DEMANGEL 38 The distinct fate of smooth and rough mycobacterium abscessus variants inside 8:30 macrophages J.L. Herrmann UMR1173, Université de Versailles Saint Quentin en Yvelines, Versailles, France 39 Global spread of Mycobacterium abscessus clones amongst cystic fibrosis 8:55 patients J. Bryant Department of Medicine, University of Cambridge, Cambridge, United Kingdom 40 A multidisciplinary approach to decipher the epidemiology of Mycobacterium 9:10 ulcerans infection: where do we stand? S. Eyangoh Head of Mycobacteriology Service, Centre Pasteur du Cameroun, Yaounde, Cameroon 41 NOD2, mycobacteria and chronic enteritis 9:35 D. Montamat-Sicotte Research Institute of the McGill University Health Centre, Montreal, Canada 14 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 42 New Approaches to Target Mycobacterium abscessus Infections 9:50 L. Kremer Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Centre National de la Recherche Scientifique FRE3689, Université de Montpellier, France 10:15 Coffee break Table on (socio economic) impact of 11 Round mycobacterial research 10:45 am 12:30 pm 10:45 Gilla Kaplan (Bill & Melinda Gates Foundation) 11:00 Brigitte Gicquel (Institut Pasteur Paris and Institut Pasteur Shangai) 11:15 Hannu Laang (European Commission, Brussels, Belgium) 11:30 René Coppens (TBVI, Lelystad, The Netherlands) 11:45 Christian Lienhardt (WHO, Switzerland) 12:00 General Discussion 12:30 Lunch buffet (Hall of CIS + Espace Congrès) 1:30 Poster Session 3 - No. 232 - 308 12 Mycobacteria and Host Interaction / Responses Chair: 3:00 pm 4:45 pm Laleh MAJLESSI 43 C-type lectin receptor DCIR modulates immunity to tuberculosis by sustaining 3:00 type I interferon signaling in dendritic cells O. Neyrolles Institute of Pharmacology & Structural Biology, CNRS-University of Toulouse, Toulouse, France 44 Immuno-metabolic regulation of M. tuberculosis infection by HIF-1α and nitric 3:25 oxide S. Stanley School of Public Health, Molecular and Cell Biology, University of California, Berkeley, United States Tuberculosis 2016 – September 19-23, 2016 – Paris, France 15 45 T cells and TB - do we know what kind of T cell works best? 3:40 A.M. Cooper Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom 46 Type I interferon production by B cells controls the balance between 4:05 immunopathology and pathogen burden in lungs during Mycobacterium tuberculosis infection D. Hudrisier CNRS, IPBS, Toulouse, France 47 Anti-microbial effector pathways used by T cells to restrict intracellular M. 4:20 tuberculosis replication S. Behar Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, United States 4:45 Coffee break 13 Host Pathogen interaction and beyond Chair: 5:15 pm 7:00 pm Peter PETERS 48 Title to be confirmed 5:15 J. Cox Berkeley University, San Francisco, United States 49 CD36-mediated uptake of surfactant lipids by human macrophages promotes 5:40 intracellular growth of M. tuberculosis C.E. Dodd Microbiology, The Ohio State University, Columbus, United States 50 Infecting Amoebae with Mycobacteria to Study Conserved Mechanisms of Innate 5:55 Immunity T. Soldati Department of Biochemistry, University of Geneva, Geneva, Switzerland 51 Insights into the host-pathogen and microbiome interactions from dual RNA6:20 sequencing of tuberculous sputum R. Lai The Francis Crick Institute, London, United Kingdom 52 The minimal unit of infection: M. tuberculosis in the macrophage 6:35 D. Russell Microbiology and Immunology, Cornell University, Ithaca, New york, United States 16 Tuberculosis 2016 – September 19-23, 2016 – Paris, France rd Friday, 23 September 2016 14 Drug design-new developments Chair: 8:30 am 10:25 am Giovanna RICCARDI 53 Reseting acquired antibiotic resistance in Mycobacterium tuberculosis 8:30 A. Baulard Center for Infection and Immunity, Institut Pasteur de Lille et INSERM, Lille, France 54 Mycobacterium tuberculosis CTP synthetase and pantothenate kinase: two 8:55 promising targets for the development of multitargeting drugs M.R. Pasca Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy 55 Antitubercular profile of GSK070 a Mycobacterium tuberculosis leucyl-tRNA 9:10 synthetase inhibitor A. Mendoza-Losana TbDPU, GSK, Tres Cantos, Spain 56 First clinical trial with the novel drug candidate PBTZ169: expected results and 9:35 surprises V. Makarov Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia 57 Griselimycins, a new class of anti-TB drugs 10:00 S. Lagrange Infectious Diseases Unit, Sanofi, Marcy L'étoile, France 10:25 Coffee break 15 From animal models to man Chair: 11:00 am 12:55 pm Ludovic TAILLEUX 58 Animal Models of Tuberculosis for Efficacy Testing 11:00 A. Rawkins National Infections Service, Public Health England, Salisbury, United Kingdom 59 Tuberculosis and One Health: comparative analyses of the human and bovine 11:25 tubercle bacilli S. Gordon School of Veterinary Medicine, University College Dublin, Dublin, Ireland Tuberculosis 2016 – September 19-23, 2016 – Paris, France 17 60 Comparative Efficacy of New Oxazolidinones in a NHP Model of Tuberculosis 18 11:50 evaluated by [ F]FDG PET/CT and Bacterial Burden L.E. Via Tuberculosis Research Section, NIAID, NIH, Bethesda, United States 61 Evolution of Drug Resistance in Mycobacterium tuberculosis 12:05 A. Pym Research Laboratory, AHRI (African Health Research Institute), Durban, South Africa 62 Severe childhood tuberculosis as a genetic disorder 12:30 S. Boisson-Dupuis INSERM Hôpital Necker/Rockefeller University, United States 12:55 Lunch buffet (Hall of CIS + Espace Congrès) 1:45 Poster Session 4 - No. 309 - 359 16 Chair: Closing Keynote Lectures Session 3:15 pm 5:00 pm Pedro ALZARI 63 Interactions of mycobacterial phenolic glycolipids with host macrophages 3:15 L. Ramakrishnan University of Cambridge, Cambridge, United Kingdom 64 Tuberculosis drug development: the leaking pipeline 4:00 S. Cole EPFL, Lausanne, Switzerland 4:45 Closing comments / End of congress 18 Tuberculosis 2016 – September 19-23, 2016 – Paris, France ORAL PRESENTATIONS SUMMARY Page Opening Keynote Lecture Session ............................................. 21 Mycobacterial Genomics and Evolution ..................................... 23 Mycobacterial Biology & Physiology I ......................................... 28 Transport systems & Secretion in Mycobacteria ........................ 33 Mycobacterial Biology & Physiology II ........................................ 37 Biomarkers, Profiling and Beyond .............................................. 42 Whole cell vacines and beyond .................................................. 47 Biochemistry & Chemical biology ............................................... 51 Biochemistry & Chemical biology and beyond ........................... 55 NTM & Emerging mycobacterial pathogens ............................... 58 Mycobateria and Host Interaction/Responses ............................ 63 Host Pathogen interaction and beyond ...................................... 68 Drug design-new developments ................................................. 73 From animal models to man ....................................................... 78 Closing Keynote Lectures Session ............................................. 83 1 Opening Keynote Lecture Session 19/09/2016 Cellular and molecular insights on the survival of Mycobacterium tuberculosis in phagosomes P. Brodin Center for Infection and Immunity, Institut Pasteur de Lille - INSERM U1019, Lille, France The highly successful pathogen Mycobacterium tuberculosis persists and replicates in macrophages. It is then crucial to decipher the host-pathogen cross-talk which allows M. tuberculosis to establish a niche favorable to the infection within such professional phagocytic cells. More precisely the arsenal deployed by the pathogen consists of various cellular mechanisms, which need to be elucidated in detail, including inhibition of phagosome maturation, block of phagosome acidification, vacuolar rupture, activation of the inflammasome and resistance to killing by oxygenated metabolites. To identify the molecular and cellular players involved in these multiple intracellular mechanisms, we have implemented high content screening approaches, using a combination of RNAi screens and dynamic visual phenotypic assays (relying on automated confocal fluorescence microscopy) to monitor the trafficking and replication of M. tuberculosis inside host cells. We thus identified novel key host signaling pathways, which will be presented here. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 21 2 Opening Keynote Lecture Session 19/09/2016 Pursuing Edward Jenner’s Revenge: Novel Strategies for Sterilizing Mycobacterium tuberculosis Infections W.R. Jacobs Howard Hughes Medical Institute, Bronx Microbiology and Immunilogy, Albert Einstein College of Medicine, New York, United States In 1789, when Edward Jenner inoculated a child with material from a cowpox lesion to prevent smallpox, he was using a related but milder infectious agent to protect against a deadly pathogen, an approach that ultimately led to smallpox eradication. Notably, Jenner’s family members died from consumption (tuberculosis, TB), also prevalent at that time and a disease that continues to have devastating consequences. Researchers Calmette and Guérin followed Jenner’s inspiration of using a related bovine disease as a vaccine source, developing the BCG vaccine for TB. However, this vaccine has failed to eradicate TB, and despite 30 years of genetically engineering BCG and Mycobacterium tuberculosis, the resulting vaccine candidates are not substantially improved over BCG. Short-course chemotherapies for TB were a great advancement of the last century, but sterilization of the bacillus within human hosts requires prolonged treatment. I hypothesize that vaccine ineffectiveness and the need for prolonged chemotherapies result from M. tuberculosis cells entering a state that resists killing, a property called persistence. New strategies are required to study and kill persistent M. tuberculosis cells. This talk will describe laboratory approaches for observing persistent cells, including new reporter mycobacteriophages that enable visualization of persisters. Through genetic manipulation of M. tuberculosis and improved knowledge of how isoniazid works, novel strategies have been developed to sterilize cultures of M. tuberculosis and overcome persistence in vivo. I will propose how a greater knowledge of persister cell killing could lead to better TB chemotherapies. Lastly, in line with Jenner’s model of testing radically different approaches for vaccines, I will describe how years of failure to develop recombinant BCG vaccines against herpes simplex virus (HSV)-1 and HSV-2 led to the testing of a genetically engineered HSV2 ΔgD construct as a vaccine candidate. Serendipitously, this HSV-2 mutant induces sterilizing immunity against HSV-1 and HSV-2, even though, contrary to the accepted dogma for an effective herpes vaccine, it fails to elicit neutralizing antibodies. Rather, this vaccine induces non-neutralizing antibodies that mediate antibody-dependent, cell-mediated cytotoxicity (ADCC). The implications of this new HSV vaccine strategy for developing more efficacious TB vaccines will be proposed. 22 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 3 Mycobacterial Genomics and Evolution 20/09/2016 TB under the sea: re-thinking the origins of Mycobacterium tuberculosis S. Pidot2, H. Izumi1, N. West1, J. Fuerst1, I. Monk2, J. Porter2, K. Mangas2, T. Seemann3, R. Brosch4, T. Stinear2 1 School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 2Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity 3Victorian Life Sciences Computation Initiative, University of Melbourne, Melbourne, Australia 4Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France During a study of microbial diversity in marine sponges, a mycobacterium (strain name: FSD4b-SM) was isolated in pure culture from tissue of a Fascaplysinopsis sp. sponge, taken from waters on the Great Barrier Reef in Queensland, Australia at a depth of 26 metres. FSD4b-SM replicates slowly (doubling every 10 days), growing preferentially in liquid sea-water media up to 30°C. Initial 16S rRNA gene sequence comparisons suggested that it was closely related to the M. tuberculosis complex (MTBC). We established the complete genome of FSD4b-SM. With a single circular 5,575,519 bp chromosome harbouring 4624 protein coding DNA sequences (CDS), it is larger than the MTBC genomes. However, DNA and protein sequence comparisons of 2500 core CDS confirmed this mycobacterium is the most closely related species yet discovered to the tubercle bacilli. Despite the unusual origin of the bacterium, the FSD4b-SM genome revealed extensive conservation of genes associated with M. tuberculosis pathogenesis, including ESX-1, ESX-3, ESX5 secretion system and associated effectors, PE/PPE proteins, and signature MTBC cell wall glycolipids (e.g. PGLs, mycoketides). There were 418 CDS specific to FSD4b-SM with predicted functions likely related to growth within the marine environment, including membrane antiporters and permeases, production of osmoprotectants and novel secondary metabolite loci. There were also 900 CDS present only in the MTBC. The discovery of FSD4b-SM (proposed name Mycobacterium spongiae sp. nov.) adds to growing evidence that the recent evolutionary origins of the MTBC were likely marine and it provides an additional important resource as we refine our understanding of the genetic factors that shaped the evolution of M. tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 23 4 Mycobacterial Genomics and Evolution 20/09/2016 Comparative genomics and phylogenetics of Uruguayan Mycobacterium bovis isolates provide insights into their genetic variability M. Lasserre4, G. Greif4, M. Castro-Ramos1, A. Juambeltz1, H. Naya3, C. Robello4-2, L. Berná4 1 Departamento de Bacteriología, División de Laboratorios Veterinarios (DI.LA.VE.) 2Dpto. de Bioquímica, Facultad de Medicina 3Unidad de Bioinformática 4Unidad de Biología Molecular, Institut Pasteur de Montevideo, Montevideo, Uruguay Bovine tuberculosis is one of the most important diseases in cattle as well as being a zoonotic risk. Cattle slaughter as means of contingence results in losses in the production of dairy and meat products. The existence of strain-specific phenotypes of Mycobacterium bovis, characterized by having different ranges of incidence, prevalence and/or severity, highlights the necessity of strainlevel discrimination of M. bovis by means of molecular typing approaches. We selected 37 local strains isolated for spoligotyping, and ten of these strains for whole genome sequencing. Genomic DNA was paired-end sequenced and assembled, and comparative analysis were performed. Amongst the studied strains five spoligotype patterns were detected, belonging to the British clonal complex European 1, which is rarely found in mainland Europe and the most frequent in South America. Spoligotypes identified (SB0274, SB0145, SB0130, SB0140, SB1072) had a frequency and distribution pattern showing local traits concordant with cattle movement and different from what is found in Brazil and Argentina. SNP calling against the reference AF2122/97 revealed homogeneous SNP distributions along the genome. Among the SNP-densest regions, lppl and pks12 showed the highest mutations, the latter being widely associated with pathogenesis and host immunomodulation in Mycobacterium tuberculosis. MntH, arsB2 and moeB1 showed to be truncated in all strains, suggesting a potential alteration against the reference that could preview a bigger genomic difference. Comparative analysis split these strains into two groups: those with spoligotypes SB0145/SB0130, and SB1072/SB0274. The former showed an absence of the region of difference RD3 that contains ORFs from phiRv1 prophage, not absent in the latter. Truncated genes evidenced a pattern of incidence between these groups, commonly belonging to strains of the same group. SNP phylogenetics supported the segregation between these groups. Nonetheless, SNP PCA nor genome multiple alignment showed a clustering adequate to these groups or locality of origin. Our comparative study shows that there is a bigger pattern of genomic variability than the provided with a spoligotype classification, the latter being correlated to SNPs but not to genomic structure. This analysis sheds light on M. bovis genomic variability as well as the nature of Uruguayan strains. 24 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 5 Mycobacterial Genomics and Evolution 20/09/2016 Generalists and Specialists in Mycobacterium tuberculosis S. Gagneux Medical Parasitology & Infection Biology, Swiss Tropical & Public Health Institute, Basel, Switzerland Generalist and specialist species differ in the breadth of their ecological niche. Little is known on the niche width of obligate human pathogens. We analyzed a global collection of Mycobacterium tuberculosis Lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that Lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses revealed that generalists exhibited a higher diversity in human T cell epitopes, and support a European origin for the most common generalist sublineage. Hence, the global success of Lineage 4 reflects distinct strategies adopted by different sublineages and human migration. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 25 6 Mycobacterial Genomics and Evolution 20/09/2016 Origin of M. tuberculosis Beijing family and its possible hypervirulent determinants Q. Gao Fudan University, Shanghai, China Mycobacterium tuberculosis Beijing family is the most successful genotype and responsible for more than a quarter of the global tuberculosis epidemic. As the predominant genotype in East Asia, the Beijing family has been emerging in various areas of the world and is often associated with disease outbreaks and antibiotic resistance. We characterized the global diversity of this family based on whole-genome sequences of 358 Beijing strains and show that the Beijing strains endemic in East Asia are genetically diverse, whereas the globally emerging strains mostly belong to a more homogenous subtype known as “modern” Beijing. Phylogeographic and coalescent analyses indicate that the Beijing family most likely emerged around 30,000 y ago in southern East Asia, and accompanied the early colonization by modern humans in this area. By combining the genomic data and genotyping result of 1,793 strains from across China, we found the “modern” Beijing sublineage experienced massive expansions in northern China during the Neolithic era and subsequently spread to other regions following the migration of Han Chinese, supporting a parallel evolution of the Beijing family and modern humans in East Asia. The dominance of the “modern” Beijing sublineage in East Asia and its recent global emergence are most likely driven by its hypervirulence, which might reflect adaption to increased human population densities linked to the agricultural transition in northern China. We further analyzed online available sequencing data of 1082 M. tuberculosis Beijing isolates and determined the genetic changes that were commonly present in modern Beijing strains but absent in ancient Beijing strains. These genetic changes include 44 single nucleotide polymorphisms (SNPs) and 2 short genomic deletions, which significantly enriched in the function category of regulatory proteins. We are now applying RNA-Seq and Lipidomics analysis to further elucidate the mechanisms that underlie the successful expansion of modern Beijing strains. 26 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 7 Mycobacterial Genomics and Evolution 20/09/2016 Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform J. Rock1, F. Hopkins1, A. Chavez2, M. Diallo1, M. Chase1, E. Gerrick1, J. Pritchard1, G. Church2, E. Rubin1, C. Sassetti6, D. Schnappinger5, S. Fortune1-3-4 1 Harvard School of Public Health 2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston 3The Broad Institute of MIT & Harvard 4The Ragon Institute of MGH, Harvard and MIT, Cambridge 5Department of Microbiology and Immunology, Weill Cornell Medical College, New York 6University of Massachusetts Medical School, Worcester, United States Mycobacterium tuberculosis (Mtb) is a globally important pathogen for which new drugs and drug regimens are desperately needed. Treatment of Mtb infection requires multidrug therapy both to minimize the evolution of drug resistance and to ensure complete clearance of the pathogen. However, in the process of drug development, it has been difficult to identify drug target combinations that are likely to display therapeutic synergy, largely as a result of laborious genetic manipulations in Mtb and the inability to interrogate multiple genes simultaneously. CRISPR interference (CRISPRi) repurposes the CRISPR/Cas9 immune system for the sequence-specific control of gene expression and, in model bacteria, has allowed robust and regulated knockdown of gene expression. However, in Mtb, the existing Streptococcus pyogenes-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. We hypothesized that orthologous Cas9 alleles from other bacterial species might allow more robust control of gene expression in Mtb with less toxicity. We screened eleven diverse Cas9 alleles in vivo and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these alleles, the CRISPR1 allele from Streptococcus thermophilus (dCas9Sthe#1), typically achieves 20-100 fold knockdown of endogenous gene expression. In contrast to other CRISPRi systems, we show that gene knockdown in mycobacteria is robust when targeted far from the transcriptional start site, thereby allowing high resolution phenotyping in the context of bacterial operons. Finally, we demonstrate the utility of this system by investigating synthetic genetic and chemical interactions within the mycobacterial folate synthesis pathway, an important but controversial drug target for drug resistant Mtb. We anticipate that this CRISPRi system will have broad utility for functional genomics, drug target screening, and genetic interaction mapping. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 27 8 Mycobacterial Biology & Physiology I 20/09/2016 Cell envelope remodeling and evolution of tuberculosis bacilli virulence C. Guilhot CNRS, IPBS, Toulouse, France The etiologic agent of tuberculosis (TB) are bacteria from the Mycobacterium tuberculosis complex which gathers sub-species each able to cause disease in humans; the most efficient of them in humans is M. tuberculosis, which remains a major public health problem worldwide. Persistence and efficient transmission are the hallmark of this disease and determine the TB pandemics. However, the bacterial functions required for these key steps of the infectious cycle are mostly unknown. M. tuberculosis is thought to have emerged from a pool of mycobacteria resembling Mycobacterium canettii, a group of bacterial strains suspected to be more adapted to environment and which caused few cases of human TB, mostly in East Africa. An intriguing question is what adaptations were associated with the enhanced capacity of M. tuberculosis to invade humans and to become the highly successful pathogen actually circulating worldwide. Later a phylogenetic lineage of the M. tuberculosis complex specialized on non–human mammals and lost the ability to sustain in humans. The key determinants of these evolutions of tubercle bacilli virulence are still mysterious. However, recent findings have suggested that discrete rearrangements of the cell envelope composition have been key determinant to these evolutions. These results will be presented and discussed. 28 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 9 Mycobacterial Biology & Physiology I 20/09/2016 Mycobacterium tuberculosis periplasmic protease MarP activates the peptidoglycan hydrolase RipA during acid stress H. Botella4, J. Vaubourgeix4, W. Xu4, S. Song4, H. Makinoshima2-1, M.H. Lee3, M.S. Glickman2, S. Ehrt4 1 Division of Translational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Kashiwa, Chiba, Japan 2Immunology program, Memorial Sloan Kettering Cancer Center 3Medicine, Weill Cornell Medical college 4Microbiology and Immunology Department., Weill Cornell Medical College, New York, United States Mycobacterium tuberculosis (Mtb) can persist in the human host in a latent state for decades, in part because it has the ability to withstand numerous stresses imposed by host immunity. Former studies have established the essentiality of the periplasmic protease MarP for Mtb to survive in acidic media and establish and maintain infection in mice. Here, we document that—when subjected to acidic stress—MarP cleaves the peptidoglycan hydrolase RipA, a process required for RipA’s activation. Failure of RipA processing in MarP-deficient cells leads to cell elongation and chains formation, a hallmark of progeny cell separation arrest. To our knowledge, this is the first example of a protease that activates a peptidoglycan-degrading enzyme by proteolytic cleavage. Our results suggest that sustaining peptidoglycan hydrolysis may be essential to Mtb’s survival in acidic conditions. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 29 10 Mycobacterial Biology & Physiology I 20/09/2016 The tailoring modifications of Mycobacterium tuberculosis polysaccharides M. Jackson2, S. Angala2, M. Mc Neil2, G. Larrouy-Maumus1, M. Gilleron1, L. Shi2, H. Pham2, H. Škovierová2, J. Nigou1, W. Wheat2 1 IPBS-CNRS, Toulouse, France 2Mycobacteria Research Laboratories, Colorado State University, Fort Collins, United States The covalent modification of (lipo)polysaccharides with discrete substituents plays important roles in the physiology of Gram-positive and Gram-negative bacteria and their interactions with the host. This presentation will review what is known of the tailoring modifications of the major cell envelope polysaccharides of Mycobacterium tuberculosis, arabinogalactan and lipoarabinomannan, their biosynthetic origin and biological functions. 30 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 11 Mycobacterial Biology & Physiology I 20/09/2016 Drug use on a single cell level H. Rego2, E. Rubin1 1 Harvard School of Public Health, Boston 2Microbial Pathogenesis, Yale, New Haven, United States Even in a genetically identical population, bacterial cells display a remarkable amount of physiological diversity on a single cell level, which can lead to important phenotypic differences. This seems especially true for the human pathogen Mycobacterium tuberculosis, which causes tuberculosis (TB). TB is notoriously difficult to treat because small subpopulations die more slowly despite being genetically identical to the vast majority of bacteria that are quickly killed by antibiotics. While we have known about this phenomenon since antibiotics were first discovered, little is understood about the underlying mechanisms, especially outside of the well-studied organism E. coli. Using a combination of fluorescence single-cell methods and bacterial genetics I have been investigating this question in mycobacteria. Mycobacteria create asymmetry from the very beginning – at cell division – and I show that this asymmetry propagates to differential antibiotic killing in the population. I then identify a gene – lamA - which upon deletion creates a more homogenous population of cells that die more rapidly with antibiotic treatment. These results suggest that mycobacteria create heterogeneity at a very high frequency with specificity and that subversion of this heterogeneity is a viable option toward improved TB therapy. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 31 12 Mycobacterial Biology & Physiology I 20/09/2016 Single-cell approaches to unravel mycobacterial individuality and persistence G. Manina Junior Group Microbial Individuality and Infection, Institut Pasteur, Paris, France Clonal cells grown under identical conditions exhibit strikingly different behavior, also known as phenotypic heterogeneity. While part of this heterogeneity is caused by inherent randomness of intracellular processes, most of cell-to-cell variation grounds on deterministic bases. Both social context and surrounding environment can greatly impact the phenotypic spectrum of individual cells, possibly resulting in beneficial variation. We found that also Mycobacterium tuberculosis displays phenotypic heterogeneity at steady state with respect to growth potential and metabolic activity, and this variation rises when cells are subjected to environmental perturbations. The ability of M. tuberculosis to explore a wide phenotypic landscape may be associated with its tendency to persist not only to protracted antibiotic therapy but also to the multifaceted host milieu. We are currently exploring whether and how phenotypic heterogeneity contributes to mycobacterial persistence in its broadest sense. The combination of fluorescently-labelled bacteria, real-time microfluidic microscopy and multiparametric analysis enables quantitative and predictive understanding of the behavior of individual cells over time, under tightly controlled environmental conditions and within hostbiomimetic platforms. By studying mycobacteria at the single-cell and subpopulation level, we aim to discriminate stochastic noise from robust phenotypic switches and their correlation with bacterial fitness. We strive to elucidate the molecular bases of phenotypic heterogeneity and its implications for pathogenesis and adaptation, giving special attention to the quiescent bacterial reservoirs and their signatures of persistence. We postulate that subverting phenotypic heterogeneity, by finetuning the behavior of discrete subpopulations, may aid us to weaken bacilli and tackle the hurdle of persistent tuberculosis. In conclusion, the identification of subpopulation-specific instead of averagepopulation biomarkers will be instrumental to develop enhanced disease control strategies and to possibly shorten the treatment duration. 32 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 13 Transport systems & Secretion in Mycobacteria 20/09/2016 The role of the ESX-5 secretion system, from nutrient acquisition to immune modulation L. Ates5, E. Houben4, R. Ummels5, C. Jiménez5, R. Warren3, A. Abdallah2, A. Pain2, R. Hernández-Pando1, W. Bitter5-4 1 Nat Inst of Med Sciences and Nutrition Salvador Zubiran, Mexico City, Mexico 2KAUST, Thuwal, Saudi Arabia 3Stellenbosch University, Stellenbosch, South Africa 4VU university 5VU university medical center, Amsterdam, The Netherlands Mycobacteria are well protected from effectors of the immune system and from antibiotics by their unusual diderm cell envelope. Nonetheless, pathogenic mycobacteria require a large number of secreted proteins for nutrient acquisition and for survival inside the host. Therefore, specialized secretion systems must be in place to transport proteins to the cell surface or into the environment, without affecting membrane permeability. Most of the known effector proteins are transported via the different type VII secretion (T7S) systems. The most recently evolved T7S system is ESX-5, which is unique for the slow-growing mycobacteria. ESX-5 is responsible for the secretion of dozens of substrates belonging to the PE and PPE families. A number of pathogenic mycobacteria, including M. tuberculosis and M. marinum, produce an extraordinary large number of these PE and PPE proteins, suggesting that they play a role in virulence. We study the secretion of PE and PPE proteins by ESX-5 and the role of these proteins in mycobacterial survival and virulence. We have shown that ESX-5 is essential for M. marinum, but that this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Therefore, we propose that ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient acquisition. These proteins probably compensate for the lack of MspA-like porins in slow-growing mycobacteria. In our search for novel ESX-5 components we identified two independent transposon mutants in M. marinum that were deficient in PE_PGRS secretion but situated outside of the esx-5 locus. Both mutants had a transposon insertion in a single specific ppe gene. Although secretion of many different PE_PGRS proteins was blocked, this mutation had no effect on bacterial growth. Deletion of the orthologous gene in M. tuberculosis also blocked PE_PGRS secretion. Proteomic analysis showed that in fact the two largest subsets of ESX-5 substrates, i.e. PPE-MPTR and PE-PGRS, are not secreted by this mutant. Interestingly, hypervirulent clinical M. tuberculosis isolates of the Beijing lineage have a natural mutation in this gene and a concomitant loss of PE/PPE secretion. Restoration of this secretion defect reverted the hypervirulence phenotype of such a Beijing strain. Therefore, these specific PE/PPE substrates seem to play an important role in virulence attenuation. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 33 14 Transport systems & Secretion in Mycobacteria 20/09/2016 Intercellular communication links ESX-1 to ESX-4 in mycobacterial conjugation T. Gray1, R. Clark2, N. Boucher2, P. Lapierre2, C. Smith2, K. Derbyshire1 1 Department of Biomedical Sciences, University at Albany 2Division of Genetics, New York State Department of Health, Wadsworth Center, Albany, United States ESX (Type 7) secretion systems in mycobacteria have many diverse roles. ESX-1 alone appears to play three roles in the process of Distributive Conjugal Transfer (DCT), involving the transfer of chromosomal DNA from donor to recipient strains of Mycobacterium smegmatis. Loss-of-function studies have shown that ESX-1 suppresses DNA transfer in the donor, but the recipient requires ESX-1 activity to receive DNA. GWAS-like analyses of transconjugants further showed that genes within ESX-1 determine the mating identity of the conjugal strains. Reasoning that DCT requires coordination between participating strains, we hypothesized that the diverse functions exhibited by ESX-1 secretion are consistent with intercellular communication networks. We developed a SNPguided RNA-seq approach to examine the strain-specific transcriptional responses to co-culture under mating conditions to identify the effects of communication. One of the most highly induced transcripts was esxUT, encoding the hetero-dimeric secretion substrate EsxUT. ESX-4 is the evolutionary progenitor for the other mycobacterial ESX secretion systems, but has no described function to date. The co-culture transcriptional response we observed was specific to the recipient strain, and to esx4, since the other esx systems (1 and 3) remained transcriptionally unchanged. This recipient ESX-4 response required a functional recipient ESX-1 system, indicating a dependency not previously seen between paralogous ESX systems. Importantly, ESX-1 mutant donors elicited a greater esxUT transcription response in the co-cultured recipients, consistent with intercellular communication. Mutation of several genes in the seven-gene esx4 locus in the recipient strain prevented DNA transfer, but did not affect DCT when mutated in the donor. These data identify a biological role and provide a quantifiable assay for the enigmatic ESX-4 secretion apparatus in M. smegmatis. Collectively, this study supports a model wherein ESX-1 coordinates intercellular communication between co-cultured conjugal donor and recipient strains, leading to an activation of ESX-4, and culminating in DCT. Our findings begin to explain some of the disparate functions of ESX-1 for DCT in M. smegmatis, and suggest that the many ESX systems of actinobacteria may also play an unexpected novel role: mediating intercellular communication. 34 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 15 Transport systems & Secretion in Mycobacteria 20/09/2016 Structure of the ESX-5 Type VII Secretion System K. Beckham1, L. Ciccarelli2, C. Bunduc3, A. Parret1, W. Bitter4, E. Houben3, T. Marlovits2, M. Wilmanns1 1 EMBL - Hamburg 2University Medical Center Eppendorf, Hamburg, Germany 3Vrije Universiteit 4VU University Medical Center, Amsterdam, The Netherlands Bacteria utilize a diverse repertoire of secretion machineries to facilitate the transport of a range of substrates in and out of the cell. The structure and function of these systems from Gram-negative bacteria have been the subject of investigation for many years, furthering our understanding of hostpathogen interactions mediated by these systems. However, the secretion machineries from Grampositive bacteria are less well understood, despite their importance in the virulence in several pathogens. Arguably the most the important human pathogen, Mycobacterium tuberculosis, relies on the activity of the T7SS to infect the human host. Pathogenic mycobacteria encode up to five T7SSs within ESX loci (ESX-1 to 5) which are structurally similar but functionally distinct as these systems have been shown to play defined roles within the cell. This functional diversity is thought to be mediated partially by the specific range of substrates that are secreted by each ESX-system, for example the ESX-5 system secretes a diverse range of PE/PPE substrates that modulate the infection process whereas the ESX-3 system has a role in iron transport. Currently, little is known about the structure of the T7SS and the mechanism by which it translocates substrates across the diderm mycomembrane. Previous studies have shown that the T7SS complex comprises four conserved components, which assemble to form a 1.5 MDa complex (Houben et al., 2012). However, the stoichiometry and arrangement of the core components within this complex is unknown. To investigate this further, we have reconstituted a functional ESX-5 system in Mycobacterium smegmatis, which allowed for the analysis of the role of specific substrates in ESX-5 functioning and their interactions (see also the abstract of Bunduc et al.). Using the reconstituted system we optimized the purification protocol of the ESX-5 membrane complex. Characterization of the complex using DLS, small-angle X-ray scattering and electron microscopy has provided novel insights into this integral-membrane machinery. Here we report the first threedimensional reconstruction of the structure of the T7SS using electron microscopy. These novel insights into the structure of this nanomachine further our understanding of the molecular mechanisms of substrate translocation across the mycomembrane. Houben et al. (2012) Composition of the type VII secretion system membrane complex. Molecular Microbiology, 86: 472–484. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 35 16 Transport systems & Secretion in Mycobacteria 20/09/2016 The Tuberculosis Necrotizing Toxin M. Niederweis Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. The Mtb protein CpnT consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted C-terminal domain. This C-terminal domain causes necrotic cell death in eukaryotic cells and was named Tuberculosis Necrotizing Toxin (TNT). Infection experiments revealed that TNT gains access to the macrophage cytosol and constitutes the main cytotoxicity factor of Mtb in macrophages. The purified TNT protein hydrolyzes the essential coenzyme NAD+. A non-catalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, thus demonstrating that the NAD+ glycohydrolase activity is required for TNTinduced cell death. The crystal structure of TNT revealed a new NAD+ glycohydrolase fold. TNT homologs of previously unknown functions are found in more than 300 bacterial and fungal species. Thus, TNT represents the founding member of a toxin family widespread in pathogenic microorganisms. 36 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 17 Mycobacterial Biology & Physiology II 20/09/2016 Stress resistance, cell division and in vivo persistence S. Ehrt, R. Wang, H. Botella, K. Kreutzfeldt Department of Microbiology & Immunology, Weill Cornell Medicine, New York, United States Among the factors that contribute to the success of M. tuberculosis as a pathogen is its ability to withstand potentially bactericidal host defenses and to resist elimination by an activated immune system. Acidification of the macrophage phagosome represents one such antimycobacterial defense mechanism. We identified acid hyper-susceptible M. tuberculosis mutants and report their phenotypic and mechanistic characterization that highlights a link between stress resistance, cell division and pathogenesis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 37 18 Mycobacterial Biology & Physiology II 20/09/2016 Inherited cell-surface wave-troughs mark future division sites in mycobacteria H.A. Eskandarian Global Health Institute, UPKIN, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Cell division is tightly controlled in space and time in order to maintain cell size and ploidy within narrow bounds. In bacteria, the canonical Minicell (Min) and nucleoid occlusion (Noc) systems together ensure that division is restricted to midcell after completion of chromosome segregation. It is unknown how division site selection is controlled in bacteria that lack homologs of the Min and Noc proteins, including mycobacteria responsible for tuberculosis and other chronic infections. We use correlated time-lapse optical and atomic force microscopy to demonstrate that morphological landmarks (waveform troughs) on the undulating surface of mycobacterial cells correspond to future sites of FtsZ contractile ring formation and cell division. The manifestation of these features are only visible by long-term time-lapse AFM, for which we have developed to our knowledge the first system capable of continuous imaging for up to one week. Newborn cells inherit wave-troughs from the (grand) mother cell and ultimately divide at the center-most wave-trough, making these morphological features the earliest known landmark of future division sites. Wavetroughs are established at subpolar regions, as cell material in mycobacteria is added at the poles. Off-center wave-troughs are shifted towards the cell center by the successive addition of new wavetroughs. In cells lacking the chromosome partitioning (Par) system, missegregation of chromosomes is accompanied by asymmetric cell division at off-center wave-troughs, resulting in the formation of anucleate cells. Our observations suggest a bipartite model of division site selection, in which inherited cell-surface wave-troughs are “licensed” sites for cell division and symmetrically segregated chromosomes suppress division at off-center wave-troughs. Our study focuses our search for a molecular mechanism determining division-site selection in mycobacteria to a point much earlier in time and spatially far from midcell. 38 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 19 Mycobacterial Biology & Physiology II 20/09/2016 The inosine monophosphate dehydrogenase, GuaB2, is a vulnerable new bactericidal drug target for tuberculosis V. Singh4, S. Donini3, A. Pacitto6, C. Sala5, R. Hartkoorn5, N. Dhar5, G. Keri2, D. Ascher6, G. Mondesert1, A. Vocat5, A. Lupien5, R. Sommer5, H. Vermet1, S. Lagrange1, J. Buechler7, D. Warner4, J. Mckinney5, J. Pato2, S. Cole5, T. Blundell6, M. Rizzi3, V. Mizrahi4 1 Sanofi-Aventis Research & Development, Marcy L'etoile, France 2Vichem Chemie, Budapest, Hungary 3University of Piemonte Orientale, Novara, Italy 4Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Cape Town, South Africa 5Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 6University of Cambridge, Cambridge, United Kingdom 7Alere, San Diego, United States VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15,344-compound library. Sequencing of a VCC234718resistant mutant identified a Tyr487Cys substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently confirmed to be the primary target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki value of 100 M and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is uncompetitive. VCC234718 forms strong pi interactions with the Tyr487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC2344718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 µM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new TB drug target. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 39 20 Mycobacterial Biology & Physiology II 20/09/2016 Functional, regulatory, and evolutionary aspects of the phage-shock-protein response of Mycobacterium tuberculosis P. Datta2, J. Ravi2, A. Barton2, R. Khan2, R. Manganelli1, M. Gennaro2 1 University of Padova, Padova, Italy 2PHRI, Rutgers University, Newark, United States The phage shock protein (Psp) response, which is enacted by a multi-gene system extensively studied in Gram-negative organisms, protects bacteria from envelope stress through a cascade of protein interactions that stabilize the cell membrane. In Enterobacteria, the Psp response participates in membrane polarization maintenance, divalent metal transport, and bacterial virulence. A key component of the Psp response is PspA, a protein that stabilizes the inner membrane when the cell envelope structure/composition is altered. PspA orthologs are widely distributed in eubacteria, archea, cyanobacteria, and higher plants. In Gram-positive bacteria, the Psp system has been studied in Bacillus subtilis (where it is called Lia) and, more recently, discovered in Mycobacterium tuberculosis. In these two organisms, the PspA ortholog is associated with gene modules that differ from each other and from the E. coli paradigm. We will present functional and regulatory characteristics of the Psp system of M. tuberculosis, an envelope-stressresponsive four-gene operon that includes clgR (transcriptional regulator), pspA, a third gene encoding an integral membrane protein, and a fourth gene of unknown function. Moreover, we will compare and contrast regulatory and functional properties and gene-expression dynamics observed in M. tuberculosis with those of the E. coli and B. subtilis systems. We will also review conservation and genomic context of the Psp system in Firmicutes and Actinobacteria, and contrast the four-gene operon structure uniquely found in tuberculous mycobacteria with the gene configuration identified in nontuberculous mycobacteria and other Actinobacteria. Since Psp functions have been linked to bacterial virulence, susceptibility to membrane-perturbing antibiotics and associated phenotypes such as biofilm formation, understanding the Psp system of M. tuberculosis might help identify novel targets for antimicrobial therapeutics. 40 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 21 Mycobacterial Biology & Physiology II 20/09/2016 Playing nice together - making cell walls in mycobacteria E. Rubin Immunology and Infectious Diseases Department, Harvard TH Chan Scholl of Public Health, Boston, United States Biosynthesis of the mycobacterial cell wall requires the coordination of a complex network of enzymes that both synthesize and degrade different components. Many of these enzymes appear to have redundant functions. However, while some overlap, many have been adapted to have unique functions. Some only interact with specific partners while others have evolved to fulfill completely new roles. This complexity offers mycobacteria new opportunities for regulation. Conversely, however, it also represents a set of vulnerabilities that can be exploited therapeutically, both new targets and new chances to alter killing by existing drugs. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 41 22 Biomarkers, Profiling and Beyond 21/09/2016 Human CD8+ T-cells recognizing peptides from Mycobacterium tuberculosis presented by HLA-E have an unorthodox Th2-like, multifunctional, Mtb-inhibitory phenotype and represent a novel human T-cell subset T. Ottenhoff2, K.E. Meijgaarden2, M.C. Haks2, N. Caccamo1, F. Dieli1, S.A. Joosten2 1 Central Laboratory for Advanced Diagnostic and Biomedical Research (CLADIBIOR), Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Università di Palermo, Palermo 90129, Italy 2Leiden University Medical Center, Leiden, The Netherlands Mycobacterial antigens are not only presented to T-cells by classical HLA-class Ia and HLA-class II molecules, but also through alternative antigen presentation molecules such as CD1a/b/c, MR1 and HLA-E. We recently described mycobacterial peptides that are presented in HLA-E and recognized by CD8+ T-cells. Using T-cell cloning, phenotyping, microbiological, functional and RNA-expression analyses, we found that these T-cells can exert cytolytic or suppressive functions, inhibit mycobacterial growth, yet express GATA3, produce Th2 cytokines (IL-4,-5,-10,-13) and activate Bcells via IL-4. In TB patients, Mtb specific cells were detectable by peptide-HLA-E tetramers, and IL4 and IL-13 were produced following peptide stimulation. The frequencies were highest in untreated TB patients and declined following successful treatment. These results identify a novel human T-cell subset with an unorthodox, multifunctional Th2 like phenotype and cytolytic or regulatory capacities, which is involved in the human immune response to mycobacteria and demonstrable in active TB patients’ blood. The results challenge the current dogma that only Th1 cells are able to inhibit Mtb growth and clearly show that Th2 like cells can strongly inhibit outgrowth of Mtb from human macrophages. These insights significantly expand our understanding of the immune response in infectious disease. 42 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 23 Biomarkers, Profiling and Beyond 21/09/2016 Profiling persistent tubercule bacilli from patient sputa during therapy predicts early drug efficacy I. Honeyborne5, T. Mchugh5, I. Kuittinen1, A. Cichonska1, D. Evangelopoulos5, K. Ronacher2, P. Van Helden2, S. Gillespie7, D. Fernandez-Reyes6, G. Walzl2, J. Rousu1, P. Butcher4, S. Waddell3 1 Helsinki Institute for Information Technology, Aalto University, Espoo, Finland 2Department of Science and Technology, Stellenbosch University, Stellenbosch, South Africa 3Brighton and Sussex Medical School, University of Sussex, Brighton 4Institute for Infection and Immunity, St George's University of London 5Centre for Clinical Microbiology 6Department of Computer Science, University College London, London 7Medical and Biological Sciences Building, University of St Andrews, St Andrews, United Kingdom Background: New treatment options are needed to maintain and improve therapy for tuberculosis, which caused the death of 1.5 million people in 2013 despite potential for an 86% treatment success rate. A greater understanding of Mycobacterium tuberculosis (M.tb) bacilli that persist through drug therapy will aid drug development programs. Predictive biomarkers for treatment efficacy are also a research priority. Methods and Results: Genome-wide transcriptional profiling was used to map the mRNA signatures of M.tb from the sputa of 15 patients before and 3, 7 and 14 days after the start of standard regimen drug treatment. The mRNA profiles of bacilli through the first two weeks of therapy reflected drug activity at 3 days with transcriptional signatures at days 7 and 14 consistent with reduced M.tb metabolic activity similar to the profile of pre-chemotherapy bacilli. These results suggest that a pre-existing drug-tolerant M.tb population dominates sputum before and after early drug treatment, and that the mRNA signature at day 3 marks the killing of a drug-sensitive subpopulation of bacilli. Modelling patient indices of disease severity with bacterial gene expression patterns demonstrated that both microbiological and clinical parameters were reflected in the divergent M.tb responses; evidence that factors such as bacterial load and disease pathology influence the host-pathogen interplay and the phenotypic state of bacilli. Transcriptional signatures were also defined that predicted measures of early treatment success (rate of decline in bacterial load over three days, TB test positivity at 2 months, and bacterial load at 2 months). Conclusions: This study defines the transcriptional signature of M.tb bacilli that have been expectorated in sputum after two weeks of drug therapy, characterizing the phenotypic state of bacilli that persist through treatment. We demonstrate that variability in clinical manifestations of disease are detectable in bacterial sputa signatures, and that the changing M.tb mRNA profiles 0-2 weeks into chemotherapy predict the efficacy of treatment 6 weeks later. These observations advocate assaying dynamic bacterial phenotypes through drug therapy as biomarkers for treatment success. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 43 24 Biomarkers, Profiling and Beyond 21/09/2016 Understanding and Intervening in HIV-1 associated Tuberculosis R. Wilkinson University of Cape Town, Cape Town, South Africa Imperial College London The Francis Crick Institute, London, United Kingdom The commonest opportunistic infection worldwide that occurs in HIV-1 infected persons is tuberculosis (TB). HIV-1 co-infection predisposes to both infection by, and reactivation of TB, and modifies its natural history and clinical presentation. An increase in extrapulmonary disease is wellrecognized, and early or subclinical TB disease characterised by very few or no symptoms is also common. Immunodiagnostic methods to ascertain TB sensitisation in HIV-1 infected persons are compromised in sensitivity. The obvious immune defect caused by HIV-1 is a progressive reduction in CD4 T cell numbers that correlates with both increasing risk of TB and the likelihood of extrapulmonary dissemination. However, and unlike most opportunistic conditions in HIV-1 infected persons, the risk of TB is increased from the time of acquisition of HIV-1 before CD4 deficiency is profound. This raises the question of whether there is additional qualitative defect in CD4 function. Antiretroviral therapy (ART) reduces susceptibility to TB in HIV-1 infected persons via viral suppression thus allowing partial immune restoration. Work has shown ART is associated with expansion of both terminally differentiated and effector memory M. tb. antigen-specific CD4. Conversely the occurrence of ART-induced inflammatory reactions in the presence of a high antigen load in active tuberculosis (TB-IRIS) brings into focus the importance of pathological immunity in TB and how poorly it is understood in humans. Recent interest has been in interaction between reconstituting innate and acquired responses in the induction of TB-IRIS. There is evidence of myeloid activation, in particular a signature of inflammasome activation with downstream inflammatory cytokine elevation. This presentation will review recent work in these areas and the translational consequences of those findings. 44 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 25 Biomarkers, Profiling and Beyond 21/09/2016 Condensation of DNA as a generic stress response in Mycobacterium tuberculosis: apoptosis in bacteria? N. Van Der Wel1, E. Scutigliani1, D. Picavet1, E. Scholl1, A. Den Hertog2, H. Van Veen1 1 Electron Microscopy Center Amsterdam, Cell Biology and Histology, AMC 2Biomedical research, KIT, Amsterdam, The Netherlands Multidrug-resistant tuberculosis is a grave threat to public health worldwide. Even in Europe, about 10% of the Mycobacterium tuberculosis cases are confronted with resistance. Recently we have discovered that M. tuberculosis condenses its DNA in response to antibiotic stress. Condensation is visualized using high resolution fluorescence and electron microscopy (Figure 1). DNAcondensation of is an established mechanism to survive antibiotic stress, and in developing multidrug resistance. Here, we demonstrate that condensation appears already 1 hour after antibiotic stress and does not affect the viability of M. tuberculosis. Interestingly patient derived, multidrug-resistant M. tuberculosis strains are more capable of condensing their DNA than drugsusceptible strains. These results imply that by condensing their DNA, mycobacteria can avoid the effects of antibiotics, which facilitates resistance development. Our study provides the first principal evidence that DNA-condensation is a reversible, generic stress response of viable M. tuberculosis, and understanding this mechanism could be crucial for the development of new therapeutic approaches against latent and multidrug resistant TB. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 45 26 Biomarkers, Profiling and Beyond 21/09/2016 TB Subunit Vaccine Immunity Expressed in The Lung P.L. Andersen Statens Serum Institut, Copenhagen, Denmark The capacity of CD4 T-cells to protect against Mycobacterium tuberculosis (Mtb) is governed by their ability to localize to the lung site of infection. The subunit vaccine H56/CAF01 confer durable protection and elicits polyfunctional CD4 T-cells that are preferentially localized to the lung parenchyma. These lung-resident T-cells have an intermediate state of Th1 differentiation and are also found in the lung vasculature and peripheral circulation of vaccinated animals, but not controls. This population efficiently trafficks into the Mtb-infected lung parenchyma after adoptive transfer. Thus, durable immunity elicited by H56/CAF01 vaccination is associated with the maintenance of circulating less differentiated CD4 T-cells that selectively home to the lung. I will discuss these findings in relation to recent data from the analysis of the immune profile obtained with the H56 subunit vaccine in ongoing clinical trials in QFT+ and QFT-individuals. 46 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 27 Whole cell vacines and beyond 21/09/2016 VPM1002 as a TB prime vaccine, but also as post-exposure vaccine on the horizon L. Grode, M.Cotton, A. Hesseling, J. Knaul, S.H.E Kaufmann and B.Eisele Business Development, Vakzine Projekt Management GmbH, Honnover, Germany VPM1002 is a live recombinant vaccine against tuberculosis (TB). As BCG is not sufficiently effective to stop the spread of TB, two modifications have been implemented in VPM1002 (rBCG∆ureC::Hly+) to improve its immunogenicity. Two phase I studies in healthy adult volunteers and one phase IIa study in healthy newborn infants were performed. All clinical Phases were randomized, controlled studies which evaluated safety and immunogenicity of VPM1002 in comparison with BCG, including multiparameter flow cytometry to characterize the quality of the T cell response following immunization. Safety and tolerability results from all clinical trials showed no serious adverse reactions after VPM1002 vaccination. VPM1002 is currently in a Phase II doubleblinded clinical trial in newborn infants and a Phase III clinical trial in adults evaluating VPM1002 as a TB post-exposure vaccine will commence by the end of this year in India. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 47 28 Whole cell vacines and beyond 21/09/2016 Mycobacterium bovis BCG Δzmp1 – a promising, novel live vaccine candidate P. Sander Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Immunization with the live vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG) confers significant but still incomplete protection against tuberculosis (TB). We hypothesized that interference with pathogen-mediated subversion mechanisms of the host’s immune response may provide a rationale for TB vaccine development and the improvement of BCG. Zmp1 (Rv0198c) is a mycobacterial zinc metallopeptidase which shows a high degree of structural and biochemical conservation to human proteases Neprilysin and ECE-1. Genetic inactivation of zmp1 interferes with mycobacterial induced phagosome maturation arrest, induces innate immune mechanisms upon mycobacterial infection of macrophages and attenuates Mycobacterium tuberculosis. Relief from Zmp1-mediated phagosome maturation arrest facilitates presentation and enhances immunogenicity of mycobacterial antigens in in vitro and in vivo BCG infection models. In a guinea model, vaccination with BCG Δzmp1 strain confers improved protection over BCG against a low dose aerosol challenge with M. tuberculosis. Furthermore, BCG Δzmp1 deletion mutants exhibit decreased virulence in a SCID mouse model, as compared to parental strains. The improved safety profile and the enhanced protective efficacy indicate that BCG Δzmp1 is a promising, novel live vaccine candidate. 48 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 29 Whole cell vacines and beyond 21/09/2016 ESX-1 type VII secretion, cytosolic pattern recognition and consequences for vaccine design R. Brosch Institut Pasteur, Paris, France Recent insights into the implication of ESX-1 type VII secretion in cytosolic pattern recognition of Mycobacterium tuberculosis, the etiologic agent of human tuberculosis and its bacterial products, inside the cytosol of host phagocytes, open novel prospects for rational design of whole cell vaccines. Mycobacterium bovis BCG (Bacille Calmette-Guérin) and Mycobacterium microti, the only two vaccines used in large-scale vaccination programs against tuberculosis, are both ESX-1 deletion mutants that are unable to induce ESX-1-mediated vacuole-to-cytosol communication and downstream innate immune signaling. After previous ESX-1 complementation of BCG with the ESX1 system of M. tuberculosis has shown an increase of protective efficacy but also an increase in virulence, here we have used the ESX-1 from Mycobacterium marinum to complement BCG. We found that heterologous expression of the ESX-1 from M. marinum, a class II organism, in BCG, led to a recombinant strain (BCG::ESX-1Mmar) that was able to induce phagosomal rupture and consequent innate immune signaling by activating the cGas/STING/TBK1/type I interferon axis and enhancing inflammasome activity. Consistently, the BCG::ESX-1Mmar induces higher proportions of CD8+ T-cell effectors against mycobacterial antigens shared with BCG and generates polyfunctional CD4+ Th1 cells specific to ESX-1 antigens, ultimately displaying superior protective efficacy against a M. tuberculosis challenge, relative to parental BCG in a mouse model. This strain can be seen as an example of a promising rBCG candidate that combines low virulence with enhanced protection abilities. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 49 30 Whole cell vacines and beyond 21/09/2016 MTBVAC: from Discovery to Evaluation in High-Burden Countries B. Gicquel1, J. Gonzalo-Asensio2, N. Aguilo2, D. Marinova2, E. Broset2, S. Alvarez2, I. Perez2, C. Martin2 1 Institut Pasteur, Paris, France 2University of Zaragoza, Zaragoza, Spain The only available vaccine in use against TB in humans today is BCG, a live attenuated vaccine derived from the bovine pathogen Mycobacterium bovis. BCG presents variable protection against pulmonary forms of TB. Genomic comparative studies have shown loss of a number of major mycobacterial antigens and about 23% of M. tuberculosis-specific human T-cell epitopes in BCG relative to the human pathogen Mycobacterium tuberculosis. MTBVAC is a new live tuberculosis vaccine based on a genetically attenuated phoP-fadD26-deletion mutant of M. tuberculosis. The presence of stable deletion mutations in two independent virulence genes abrogates the risk of reversion to virulence. MTBVAC has shown a comparable or superior safety and immunogenicity profile to BCG in different preclinical animal models including new-born mice model (Arbues et al Vaccine 2013 and Broset el al mBio 2015, Aguilo et al Tuberculosis 2016). A first-in-human MTBVAC clinical trial was recently completed successfully in healthy adults in Lausanne, Switzerland sponsored by Biofabri (NCT02013245) (Spertini et al LRM 2015). In this trial, when MTBVAC was given at the same dose as BCG (5x10⁵ CFU), there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. MTBVAC is the first live-attenuated M. tuberculosis vaccine to enter clinical trials and to date has shown a comparable safety profile to BCG. A notable finding in the first trial was the absence of ESAT-6 and CFP-10-specific T cell responses at the end of the study, suggesting that interferon-g release assays (IGRAs) could be utilized as study endpoints in future efficacy trials to test efficacy against M. tuberculosis infection. The immunogenicity data show that MTBVAC is at least as immunogenic as BCG. Altogether these data supported the advanced clinical development in high-burden countries where TB is endemic. A dose-escalation safety and Immunogenicity study to compare MTBVAC to BCG in newborns with a safety arm in adults is currently ongoing in South Africa sponsored by Biofabri (NCT02729571). MTBVAC is developed with the goal to provide improved efficacy over BCG for use in new-borns, adolescents and adults as a preventive strategy against tuberculosis in high-burden countries (Arregui et al PeerJ 2016). Funding: Biofabri, TuBerculosis Vaccine Initiative (TBVI). The Norwegian Agency for Development Cooperation (NORAD). BIO2014 5258P and TBVAC2020 643381 Spanish and European grants. References: • Aguilo N, Uranga S, Marinova D et al MTBVAC vaccine is safe, immunogenic and confers protective efficacy against Mycobacterium tuberculosis in newborn mice. Tuberculosis 2016; 96:71-4. • Arbues A, Aguilo JI, Gonzalo-Asensio J, et al. Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials. Vaccine 2013; 31(42):4867-73 • Arregui S, Sanz J, Marinova D, et al. On the impact of masking and blocking hypotheses for measuring the efficacy of new tuberculosis vaccines. PeerJ. 2016 Feb 11;4:e1513. • Broset E, Martín C, Gonzalo-Asensio J.Evolutionary landscape of the Mycobacterium tuberculosis complex from the viewpoint of PhoPR: implications for virulence regulation and application to vaccine development. MBio. 2015 Oct 20;6(5):e01289-15. • Spertini F, Audran R, Chakour R, et al. Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial. The Lancet Respiratory Medicine 2015; 3:953-62. 50 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 31 Biochemistry & Chemical biology 21/09/2016 The dynamics of TB lesions revealed by PET/CT scanning using [F-18] trehalose C. Barry Tuberculosis Research Section - LCID, NIH, Bethesda, United States PET/CT scanning is receiving increasing attention as a useful means of evaluating TB disease status and response to therapy however current scans are limited in that the most commonly used probe - 2-[18F]-fluorodeoxyglucose (FDG) - reports only on host inflammatory processes and not on bacterial viability. To circumvent this problem we have synthesized trehalose derivatives containing [18F] and applied these to imaging TB-infected rabbits and non-human primates. Serially scanning with FDG followed by FDT (fluorodeoxytrehalose) revealed that sites of rapid trehalose incorporation and sites of high levels of inflammation are largely non-overlapping within individual animals. These results suggest that local areas with high levels of inflammation effectively suppress rapid bacterial growth while new lesions rapidly develop that are not subject to inflammatory pressure. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 51 32 Biochemistry & Chemical biology 21/09/2016 Flipping trehalose monomycolate across the inner membrane of mycobacteria Z. Xu2, G. Poce1, S.S. Chng2 1 Sapienza University of Rome, Rome, Italy 2National University of Singapore, Singapore The mycobacterial outer membrane (OM) is characterized by the presence of mycolic acids, which are C60-C90 long, branched chain fatty acids, either existing in the forms of trehalose mono- and diesters, or covalently attached to peptidoglycan via arabinogalactan polysaccharides. Being the major component in the OM, mycolic acids make this bilayer extremely hydrophobic and render the membrane impervious to many antibiotics. Mycolic acids are synthesized in the cell as trehalose monomycolates (TMMs), and have to be translocated across the inner membrane (IM) and the aqueous periplasm before being functionalized onto the cell wall. While the biosynthetic pathway of TMM and the final steps of assembly at the OM have been well characterized, how TMM is transported across the IM and periplasm are still not clear. Recently, an essential IM protein MmpL3 has been implicated in TMM transport across the cell envelope and is believed to be inhibited by multiple pharmacophores. Here, we present direct biochemical evidence for the function of MmpL3 in flipping TMM across the IM. Furthermore, we demonstrate that a couple of potential MmpL3 inhibitors tested directly target the TMM flippase. Our work provides fundamental insights into mycolic acid transport and validates MmpL3 as a viable target for the development of new antibiotics against mycobacterial infections. 52 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 33 Biochemistry & Chemical biology 21/09/2016 Mechanisms of high fidelity DNA replication in Mycobacterium tuberculosis J. Rock2, U. Lang1, S. Fortune2, M. Lamers1 1 Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom 2Harvard School of Public Health, Boston, United States Antibiotic resistance in Mycobacterium tuberculosis (Mtb) is increasing at an alarming rate. Resistance in Mtb is predominantly caused by point mutations, yet the mechanisms that govern the mutation rate in this bacterium are poorly understood. Importantly, mycobacteria lack DNA mismatch repair, an evolutionary conserved mechanism that removes replication errors from the DNA. However, they do not show increased mutation rates when compared to E. coli or S. typhimurium, suggesting that in mycobacteria, the DNA replication machinery itself may be capable of unusually high fidelity DNA synthesis. To understand how Mtb controls its mutation rate, we have characterized the replicative DNA polymerase from Mtb, DnaE1. We show that high fidelity DNA replication relies on a non-canonical exonuclease, the PHP domain, that is distinct from the replicative exonuclease in E. coli as well as the eukaryotic exonucleases. We further show that inhibition of the PHP exonuclease renders mycobacteria hypersensitive to nucleotide analogs that are used in antiviral therapy, indicating that the replicative DNA polymerase from Mtb is an attractive target for novel antimicrobial drugs. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 53 34 Biochemistry & Chemical biology 21/09/2016 Structural basis of phosphatidylinositol mannosides biosynthesis in mycobacteria M. Guerin CICbioGUNE, Spain Many cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of cytoplasm and organelles. Enzymes performing this type of reaction are required to access a lipophilic substrate located in the membranes and to catalyze its reaction with a polar, water-soluble compound.[1] Here we focus on two enzymes involved in the early steps of the phosphatidylinositol mannosides (PIMs) biosynthetic pathway, unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidylinositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the precursors of the lipoglycans lipomannan and lipoarabinomannan, important molecules implicated in host-pathogen interactions in the course of tuberculosis. Of particular relevance, we demonstrate the occurrence of a conformational switch during the catalytic cycle of the retaining glycosyltransferase PimA, the enzyme that start the pathway, involving both β-strand– to–α-helix and α-helix–to–β-strand transitions. [2] These structural changes seem to modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane surface. Although scant structural information is currently available on protein catalysis at the lipid-water interface, our studies demonstrate that protein-membrane interactions might entail unanticipated structural changes in otherwise well conserved protein architectures, and suggests that similar changes may also play a functional role in other membrane enzymes. Finally, we report the crystal structures of PatA, an essential membrane associated acyltransferase that transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring added by PimA, in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl–CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design. References: 1. Forneris, F.; Mattevi, A. Science 2008, 321, 213-216. 2. Giganti et al., Nat. Chem. Biol. 2015, 11, 16-18. Highlighted in the News and Views Section: Brodhun F, Tittmann K. Nat. Chem. Biol. 2015, 11, 102-103. 3. Albesa-Jove et al., Angew. Chem. Int. Ed. Engl. 2015, 54, 9898-9902. 4. Albesa-Jove et al., Nat. Commun. 2016, 7, 10906. 54 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 35 Biochemistry & Chemical biology and beyond 21/09/2016 Amino acid sensing by protein kinase G in metabolic regulation and virulence H. O'Hare Dept of Infection, Immunity and Inflammation, University of Leicester, United Kingdom Protein kinase G (PknG) has previously been implicated in metabolic regulation and virulence of Mycobacterium tuberculosis. This multi-domain kinase has been the subject of diverse studies. Amongst the various putative functions, evidence is accumulating that the link to metabolic regulation is likely to be through its substrate GarA. Together these proteins form a molecular switch to control the tricarboxlic acid cycle and glutamate synthesis/degradation. Phosphorylation of GarA by PknG inactivates it, thus genetic disruption of garA or pknG should have opposing effects on metabolism, while GarA variants lacking phosphorylation sites should mimic the effects of pknG deletion. Both the kinase and substrate (with intact phosphorylation sites) were required in infection models, highlighting the importance of responsiveness of this regulatory system, but the garA mutant was markedly more attenuated than pknG deficient M. tuberculosis. Combined with the growth defects of these strains, this hints that M. tuberculosis may experience glutamate starvation in vivo. Glutamate itself was a stimulus for rapid phosphorylation of GarA in bacterial cells. We propose that M. tuberculosis and other actinobacteria may sense external amino acids via a novel complex of PknG with transmembrane GlnX and lipoprotein GlnH. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 55 36 Biochemistry & Chemical biology and beyond 21/09/2016 Targeting topology modulators and topoisomerases of Mycobacterium tuberculosis V. Nagaraja Jawaharlal Nehru Centre for Advanced Scientific research & Department Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India DNA topoisomerases catalyze the topological reactions that are essential for cell survival. Contrasting activities of different kinds of topoisomerases ensure to regulate the supercoiling, relaxation and other topological reactions. Most mycobacteria possess only one topoisomerase I and a DNA gyrase as representatives of type I and II group. Our earlier studies have revealed that mycobacterial topoisomerase I and DNA gyrase are distinct from other prokaryotic and eukaryotic topoisomerases in many characteristics. Topoisomerase I is site specific and a unique carboxy terminal domain is involved in strand passage reaction. Mycobacterial gyrase also exhibits distinct features and is a strong decatenase unlike the E.coli enzyme. In order to maintain topological homeostasis of the genome, topoisomerases catalyse DNA cleavage, strand passage and rejoining of the ends. Thus, although they are essential housekeeping enzymes, they are the most vulnerable targets; arrest of the reaction after the first transesterification step leads to breaks in DNA and cell death. We have targeted both DNA gyrase and topoisomerase1 from mycobacteria. The latter, although essential, has no inhibitors described so far. We have now characterized the first set of small molecule inhibitors that inhibit the enzyme with bactericidal effect. In addition to topoisomerases, nucleoid associated proteins(NAPs), are the key topology modulators responsible for DNA compaction and genome organization. We have targeted HU, an essential protein from M. tuberculosis to develop small molecule inhibitors by structure based design. Overexpression of HU leads to alteration in the nucleoid architecture. The crystal structure of the Nterminal half of HU reveals a cleft that accommodates duplex DNA. Based on the structure, we have designed inhibitors which bind to the protein and affect its interaction with DNA, de-compact the nucleoid and inhibit cell growth. Chemical probing using the inhibitors reveal the importance of HU regulon in M.tuberculosis. 56 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 37 Biochemistry & Chemical biology and beyond 21/09/2016 In Search of Drugs to Shorten Treatment of TB C. Nathan, B. Gold, G. Lin, J. Vaubourgeix, T. Lupoli, K. Saito, S. Somersan-Karakaya, T. Warrier Depts. of Microbiology & Immunology and Medicine, Weill Cornell Medicine, New York City, New York, United States Pyrazinamide’s ability to shorten treatment of TB while being active in vitro largely against nonreplicating (NR) Mycobacterium tuberculosis (Mtb) points to the clinical value of finding new drugs active against NR Mtb. Yet, the prolonged course even of PZA-containing regimens suggests poor activity against some other subpopulation of Mtb. We approach these challenges by distinguishing three forms of phenotypic tolerance1, including a form in which stressed Mtb no longer forms colonies on agar but retains disease-causing potential: differentially detectable (DD) Mtb. We take three routes to search for compounds active against Mtb showing phenotypic tolerance to most TB drugs: (1) whole cell screening under NR conditions; (2) target-based screening against enzymes that Mtb needs to survive under stresses that block replication; and (3) testing active agents from (1) and (2) against DD Mtb in vitro. Screens (1) and (2) are directed against what was called class II phenotypic tolerance1, now called class IIa because under those NR conditions, without drug, the number of CFU neither increases nor decreases during the assay, in contrast to conditions that reduce CFU but leave DD Mtb, whose phenotypic tolerance is termed class IIb. Corresponding actives are (1) novel cephalosporins, (2) species-selective inhibitors of the Mtb proteasome and (3) a natural product-based nitrofuranylcalanolide. We hypothesize that rapid cure of TB will require achieving four goals in each infected physical compartment. Overcoming heritable resistance will require drug(s) for which 1/(product of their frequencies of resistance) > number of viable Mtb. Overcoming class I phenotypic tolerance1 (displayed by a small fraction of cells in a replicating population) will require n drugs where n > number of mechanisms of class I phenotypic tolerance displayed by different individual bacteria toward those drugs. Overcoming class IIa phenotypic tolerance will require a drug that kills NR Mtb detectable as CFU. Finally, overcoming class IIb phenotypic tolerance will require a drug that kills DD Mtb. The number of drugs required to meet all four goals could in theory be as few as 1 but an upper bound on the number is undefinable— a daunting prospect. 1. Nathan, C. Bacterial pathogenesis: Fresh approaches to anti-infective therapies. Science Translational Medicine 4: 1-13, 2012 We acknowledge for support: Tri-Institutional TB Research Unit (NIH grant U19 AI1111043); Bill & Melinda Gates Foundation TB Drug Accelerator; Tri-Institutional Therapeutics Discovery Institute; Milstein Program in Chemical Biology and Translational Medicine. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 57 38 NTM & Emerging mycobacterial pathogens 22/09/2016 The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages J.L. Herrmann UMR1173, Université de Versailles Saint Quentin en Yvelines, Versailles, France Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). M. abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. M. abscessus S and R variants are able to grow inside macrophages but are present in morphologically distinct phagosomes. The S forms are found as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. In contrast, infection with the R form leads to phagosomes containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable to restrict the intraphagosomal acidification and induces less apoptosis and autophagy. Unexpectedly, the membrane of phagosomes enclosing the S forms showed signs of alteration such as breaks or partial degradation. Although such events were not frequently encountered, they suggest that the S form is capable to provoke phagosome-cytosol communications. The mycobacterial constituents and molecular mechanisms involved in the rupture, or at least alteration, of the M. abscessus Scontaining phagosome membrane remain as yet unknown. It has been shown clearly that M. abscessus is not equipped with the ESX-1 apparatus, which mediates establishment of cytosol contact for slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium marinum and Mycobacterium kansasii. Ongoing work with a panel of defined mutants, will hopefully allow us to identify the putative membrane-damaging constituents of the S strain, which are obviously mediated by an ESX-1-independent mechanism. To summarize, it seems to be rather difficult to strictly compare the behaviors of S and R morphotypes within in vitrogrown macrophages cultures and patients, because in patients they might occur at dedicated stages during progression of the infection and disease. S and R variants can most likely be regarded as two representatives of the same isolate, which can co-exist and/or evolve differently in response to host immunity. Nonetheless, we provide compelling evidence that, at the cellular scale, M. abscessus S imitates phenotypic traits of pathogenic SGM and that the loss of cell-wall associated lipids, namely GPL, can result in the acquisition of an RGM intracellular behavior with a peculiar extracellular state characterized by a very high replication capacity. 58 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 39 NTM & Emerging mycobacterial pathogens 22/09/2016 Global spread of Mycobacterium abscessus clones amongst cystic fibrosis patients J. Bryant3-4, D. Grogono3, D. Rodriguez-Rincon3, I. Everall4, K. Brown2-3, P. Moreno1, D. Verma5, E. Hill5, J. Drijkoningen3, C. Haworth2, S. Harris4, D. Ordway5, J. Parkhill4, R.A. Floto2-3 1 EMBL-EBI 2Cambridge Centre for Lung Infection, Papworth Hospital 3Department of Medicine, University of Cambridge, Cambridge 4Wellcome Trust Sanger Institute, Hinxton, United Kingdom 5Mycobacteria Research Laboratory, Colorado State University, Fort Collins, United States Lung infections with Mycobacterium abscessus have increased in frequency worldwide, emerging as an important global threat to individuals with cystic fibrosis (CF) where they cause accelerated inflammatory lung damage and death. M. abscessus was previously thought to be independently acquired by susceptible individuals from the environment. However, using whole genome sequencing and detailed epidemiological analysis of a cohort of patients attending the CF centre at Papworth Hospital, we found strong evidence for transmission between patients. This observation is supported by our subsequent whole genome analysis of a global collection of 1,080 clinical isolates from 517 patients, showing that the majority of infections are from densely clustered M. abscessus genotypes with low levels of diversity, indicating a high level of human associated spread. Moreover, the phylogeny reveals the presence of three recently emerged dominant circulating clones that have global spread. We found that these clones are associated with worse clinical outcomes and show increased virulence in both cell-based and mouse infection models. Within patients we found evidence of significant genetic diversity and evolutionary adaptation through the processes of convergent evolution and hypermutation, demonstrating the propensity of M. abscessus to evolve from an environmental organism into a transmissible human pathogen. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 59 40 NTM & Emerging mycobacterial pathogens 22/09/2016 A multidisciplinary approach to decipher the epidemiology of Mycobacterium ulcerans infection: where do we stand? S. Eyangoh Head of Mycobacteriology Service, Centre Pasteur du Cameroun, Yaounde, Cameroon Buruli ulcer (BU) is a severe human debilitating skin and soft tissue disease caused by the bacterium Mycobacterium ulcerans. BU has been reported in 33 countries mainly located in Central and West Africa. Amidst the rising number of BU cases, in 1998, the World Health Organization declared BU as an emerging neglected tropical disease. Since then, several scientific studies to better understand BU have been conducted. Despite increase in knowledge about the disease, the mode of disease transmission is still unclear. As a result, it has been difficult to propose and effective prevention strategy for BU. Here, we present the results of a multidisciplinary approach to describe the dynamics of M. ulcerans in the aquatic environment, and to understand the links between M. ulcerans transmission, human activities and their evolutions, in anthropologically modified environments. This approach allowed; to obtain an array of results. 1. Age and sex were identified as risk factors for BU and population was classified in three main groups: Children 5-15 years (especially males) Women 15-49 years Adults over 60 years; 2. individual risk factor analysis (habits/ activities) showed the protection effect of bed nets, good care of wounds and wearing shoes during activities and contact with stagnant water identified as risk factor 3. Spatial analysis of time aggregated incidence rate of BU in Akonolinga- a BU endemic site, from 2002–2012 identified the Nyong river as a major risk factor for BU; 4. BU incidence in Akonolinga varies significantly by season and this seasonal variation is linked to the fluctuation of M. ulcerans occurrence in the environment. Fluctuation in the occurrence of M. ulcerans in the environment is probably driven by the dynamics of freshwater ecosystems of the Nyong River; 5. M. ulcerans dynamic was shown geographically, in aquatic communities and in freshwater ecosystem; 6. Seasonal fluctuations of M. ulcerans in freshwater ecosystems; 7. Potential biotic and abiotic factors as drivers of M. ulcerans 8. The ecological traits (living on aquatic vegetation or bottom of water column and macropredator) may predispose water bug taxa to acquire M. ulcerans from the aquatic environment. These results improve our understanding of the epidemiology of BU and could have direct public health implication in improving early detection, guiding treatment decentralization and implementation of prevention strategies. 60 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 41 NTM & Emerging mycobacterial pathogens 22/09/2016 NOD2, mycobacteria and chronic enteritis D. Montamat-Sicotte2, J.D. Lalande2, F. Mcintosh2, M. Paquet3, L. Kreitmann2, D. Houle2, M. Divangahi1, M. Behr2-1 1 Department of Microbiology and Immunology, McGill International TB Centre 2Research Institute of the McGill University Health Centre 3Université de Montréal, Faculté de médecine Vétérinaire, Montreal, Canada Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that preferentially recognizes N-glycolyl MDP produced by mycobacteria. In humans, loss-of-function mutations in NOD2 predispose to Crohn’s disease. We set out to study the outcome of gastrointestinal infections as a function of NOD2, using Nod2-/- mice that phenocopy the human mutations. To do so, we tested bacteriologic, histopathologic and immunologic outcomes in Nod2+/+ and Nod2-/- mice following infection with Listeria monocytogenes (LIST), E. coli strain LF82 and Mycobacterium avium paratuberculosis (MAP). Following intrajejunal infection with 109 bacteria, LIST and E. coli resulted in acute gastroenteritis, with Nod2-independent mortality in the first week following infection. With a lower dose of 108, infected mice that survived acute infection cleared the pathogens by 8 weeks after infection and there was no pathology in their small intestine at this time-point. In contrast, mice infected with 109 MAP survived the full 8 weeks of infection without gross morbidity (normal weight compared to PBS sham infections) and no mortality. Bacteriologic analysis revealed that MAP-infected Nod2-/- mice had greater numbers of bacteria in the small intestine, the liver and the spleen, compared to Nod2+/+ infected controls. Histopathologic analysis revealed granulomatous inflammation in the serosa of the ileum. Responses were quantified via FACS analysis, which revealed increased numbers of neutrophils and monocytes in the small intestine of Nod2/- mice; by co-staining, we determined that the Nod2-/- group had more TNFα producing neutrophils. To understand the potential role of these cells in the host response, we conducted ex vivo studies of MAP infection. Nod2-/- macrophages showed reduced recognition and clearance of MAP infection. Nod2-/dendritic cells showed reduced recognition of MAP infection, and when loaded with Ovalbuminexpressing MAP, reduced OVA-specific T-cell proliferation. Nod2-/- neutrophils showed reduced recognition and clearance of MAP infection. Together, our results show that of the three intestinal pathogens tested, only MAP resulted in a chronic infection and produced Nod2-dependent outcomes. The recruitment of cells to the small intestine that had impaired responses to MAP infection may contribute to a climate of chronic inflammation in the gut that drives ongoing pathology. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 61 42 NTM & Emerging mycobacterial pathogens 22/09/2016 New Approaches to Target Mycobacterium abscessus Infections L. Kremer Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Centre National de la Recherche Scientifique FRE3689, Université de Montpellier, France Mycobacterium abscessus (Mabs) is a rapid growing pathogenic species capable to induce severe and chronic pulmonary infections and is particularly harmful for cystic fibrosis patients. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence, involving the formation of massive serpentine cords and abscesses. The intrinsic resistance of Mabs to most commonly available antibiotics seriously limits chemotherapeutic treatments. Therefore, new targets and drugs are urgently needed. Until recently, the identification of new chemotypes and the description of their mechanism of action have been largely hampered by our restricted knowledge of the physiopathological events characterizing Mabs infections and by the lack of genetic tools and appropriate animal models for in vivo drug assessments. To better understand the natural resistance of Mabs to antiTB drugs, a deletion of MAB_4780, encoding a putative dehydratase, was created in the R variant. Unexpectedly, the mutant exhibited alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype in wild-type and in immunocompromised zebrafish. This indicates that MAB_4780 is required for Mabs cording and for the successful establishment of acute and lethal infections. Thereby, targeting MAB_4780 may represent an attractive anti-virulence strategy to control Mabs infections. The combination of our dehydratase assay with a highresolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors. In a different approach, we demonstrated the usefulness of a phenotypic screen for bactericidal compounds against Mabs using a previously validated library of compounds active against M. tuberculosis. A new compound exhibiting potent activity against Mabs in vitro and in infected macrophages and zebrafish was identified. This lead compound, PIPD1, targets the mycolic acid transporter MAB_4508 of the MmpL family. Interestingly, multiple single mutations in MAB_4508 conferring high resistance to PIPD1 defined a potential PIPD1-binding pocket when mapped on a MAB_4508 structural model. This emphasizes a yet unexploited structure class with promising translational development possibilities against Mabs. 62 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 43 Mycobateria and Host Interaction/Responses 22/09/2016 C-type lectin receptor DCIR modulates immunity to tuberculosis by sustaining type I interferon signaling in dendritic cells A. Troegeler, D. Hudrisier, O. Neyrolles Institute of Pharmacology & Structural Biology, CNRS-University of Toulouse, Toulouse, France Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals, but can become detrimental if not finely tuned. Here, I will discuss our recent results that show that C-type lectin dendritic cell (DC) immunoreceptor (DCIR) is required to modulate lung inflammation and bacterial burden in TB. In mice, we found that DCIR deficiency impairs STAT1mediated type I interferon (IFN) signaling in DCs, leading to increased production of interleukin-12 (IL-12) and increased differentiation of T helper-1 (Th1) cells during infection. As a consequence, while DCIR-deficient animals control M. tuberculosis better than wild-type animals, they also develop more inflammation. Altogether, our study reveals a novel pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen’s control by sustaining type I IFN signaling in DCs. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 63 44 Mycobateria and Host Interaction/Responses 22/09/2016 Immuno-metabolic regulation of M. tuberculosis infection by HIF-1α and nitric oxide J. Braverman, K. Sogi, S. Stanley School of Public Health, Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States IFN-γ is critical for immunity to Mycobacterium tuberculosis. We show that the transcription factor Hypoxia Inducible Factor 1-α (HIF-1α) is a master regulator of IFN-γ dependent signaling that promotes inducible antibacterial immunity. Mice lacking HIF-1α in the myeloid lineage are extremely susceptible to infection with M. tuberculosis. HIF-1α is required for optimal production of a large number of critical immunity genes and promotes the production of inflammatory cytokines, chemokines and eicosanoids. In addition, HIF-1α mediates IFN-γ dependent metabolic transitions in macrophages that promote control of infection by enhancing immune responses and influencing nutrient availability to M. tuberculosis. We demonstrate that HIF-1α and inducible nitric oxide synthase (iNOS) are linked in a positive feedback loop in which each promotes optimal expression of the other. Thus, nitric oxide plays a major immunomodulatory role by promoting a broad array of antibacterial defense mechanisms. Further, we provide evidence that the interconnected regulation of HIF-1α and iNOS is important to achieve proper inflammatory balance during infection. 64 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 45 Mycobateria and Host Interaction/Responses 22/09/2016 T cells and TB - do we know what kind of T cell works best? A.M. Cooper Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom Mycobacterium tuberculosis (Mtb) has been evolving with its human host for many generations and has developed the capacity to manipulate the human immune response. One working model of the interaction between Mtb and the human host is that the bacterium induces both a strong inflammatory and a strong acquired immune response and then actively regulates these responses at the primary lesion site. This induction and subsequent regulation then results in an infectious lesion in the lung while maintaining a relatively ambulatory host. CD4 T cells play a critical yet contradictory role in this process by both controlling disseminated disease while promoting the development of the lesion in the lung that mediates transmission. It is not surprising that our ability to vaccinate against tuberculosis (TB) has not been totally successful as our attempts have largely mimicked what the bacterium does in vivo. In order to overcome the current impasse in vaccine development we need to define the factors which impact the phenotype and function of protective CD4 T cells. Vaccination using a variety of T cell epitopes can impact the long term protetction mediated by T cells, the nature of these T cells will be discussed. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 65 46 Mycobateria and Host Interaction/Responses 22/09/2016 Type I interferon production by B cells controls the balance between immunopathology and pathogen burden in lungs during Mycobacterium tuberculosis infection A. Bénard5-9, I. Sakwa10, P. Schierloh1, M. Del Carmen Sasiain1, I. Mercier5-6-7-9-4, L. Jouneau2, P. Boudinot2, L. Tailleux3, T. Al-Saati8, R. Lang12, J. Rehwinkel15, S.H. Kaufmann11, V. Anton-Leberre9-7-4-6, A. O'garra13-14, B. Gicquel3, S. Fillatreau10, O. Neyrolles5-9, D. Hudrisier5-9 1 IMEX-CONICET, Caba, Argentina 2Virologie et Immunologie Moléculaires, INRA, Jouy-EnJosas 3Institut Pasteur, Paris 4CNRS UMR5504 5CNRS, IPBS 6UMR792, INRA 7INSA 8INSERM, US006 9Université de Toulouse, Toulouse, France 10DRFZ 11Max Planck Institute of Infection Biology, Berlin 12Institute of Clinical Microbiology, Erlangen, Germany 13MRC National Institute for Medical Research 14NHLI, Imperial College, London 15MRC Human Immunology Unit, Oxford, United Kingdom The type I interferon (IFN) pathway decisively influences the outcome of Mycobacterium tuberculosis (Mtb) infection. Here, we show that B lymphocytes, which accumulate in the lungs of Mtb-infected animals and patients, play a significant role in the type I IFN response associated with tuberculosis (TB). Through global transcriptome analyses, we discovered that B cells were activated by type I IFN during disease, and produced this cytokine upon direct stimulation with Mtb. The production of type I IFN by B cells involved the dinucleotide sensor STING and the C-type lectin Mincle, and was markedly antagonized by intrinsic MyD88 signaling. Thus, B cells from mice with a B cell-restricted deficiency in Myd88 displayed an enhanced type I IFN production. This resulted in reduced inflammation-driven tissue damage, while providing a favorable environment for Mtb multiplication. B cells from the pleural fluid of TB patients similarly displayed a striking expression of type I IFN, implicating this pathway in TB pathogenesis in human. Altogether, our findings identify type I IFN-producing B cells as key modulators in the balance between host tissue protection and bacterial burden during TB, and further emphasize the critical roles of cytokine production by B cells in health and disease. 66 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 47 Mycobateria and Host Interaction/Responses 22/09/2016 Anti-microbial effector pathways used by T cells to restrict intracellular M. tuberculosis replication S. Behar Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, United States Identifying anti-microbial effector pathways used by T cells to restrict intracellular M. tuberculosis replication could help improve vaccine efforts and offer new immunotherapy targets. Apart from IFNγ and TNF, there are several other T cell functions that have been described to contribute to an antimicrobial response. Early during the immune response, IL-17 enhances control of M. tuberculosis, in part by promoting granuloma formation. CD8+ T cell cytolytic activity also contributes to protection in both mice and humans. We have recently discovered that IL-21 and GM-CSF are produced by T cells and contribute to host resistance. IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection. IL-21 augments CD8+ T cell priming, promotes T cell accumulation in the lungs, and enhances T cell cytokine production. IL-21 is produced predominantly by activated CD4+ T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (γc) cytokine receptor family. IL-21R-/- mice have increased lung bacterial burden and earlier mortality compared to WT mice. To causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R-/- T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. GM-CSF-/- mice are highly susceptible to infection with Mycobacterium tuberculosis and clinical data has shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis. However, the general role of T cell-derived GM-CSF during infection has not been defined. We find that non-conventional and conventional T cells produce GM-CSF during M. tuberculosis infection. Early during infection, non-conventional iNKT cells and γδ+ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4+ T cells as the infection progresses. Although we find that T cell production of GM-CSF can be important for host defense, production of GM-CSF by radioresistant cells is quantitatively more important in the mouse model. Thus, beyond the major contribution of GM-CSF production by radioresistant cells, we identify T cell production of GM-CSF to be potential modifiable effector function that contributes to host resistance against M. tuberculosis. These data show that IL-21 and GM-CSF are T cell products that modify the outcome of tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 67 48 Host Pathogen interaction and beyond 22/09/2016 Title to be confirmed J. Cox Berkeley University, San Francisco, United States 68 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 49 Host Pathogen interaction and beyond 22/09/2016 CD36-mediated uptake of surfactant lipids by human macrophages promotes intracellular growth of M. tuberculosis C.E. Dodd4-1, C.J. Pyle2-1, M. Rajaram1, L.S. Schlesinger3-1-4 1 Center for Microbial Interface Biology 2College of Pharmacy 3Department of Microbial Infection & Immunity 4Microbiology, The Ohio State University, Columbus, United States Mycobacterium tuberculosis (M.tb) is an extremely salient pathogen and causes a human death every twenty seconds. M.tb is also capable of enduring for decades inside of the human body and therefore a substantial reservoir of latently infected individuals exists as a lurking threat to global health. A historical scourge of the human species, M.tb is now highly adapted to the lung environment and is capable of intracellular survival within alveolar macrophages (AMs). As the immunological guardians of the alveolar airspace, AMs not only phagocytose inhaled microbes and particulate matter but are also crucial in the catabolism of lung surfactant, a lipid-protein complex which lines the tissue. Macrophage phenotype and behavior is regulated by surfactant and M.tb can utilize host lipids as a carbon source during infection. We therefore investigated which receptor(s) contribute to surfactant uptake and whether the presence of those surfactant lipids within human macrophages prior to infection with M.tb enhances bacterial survival. We show that preformed scavenger receptor CD36 is redistributed from an intracellular pool to the cell membrane following exposure to lipids and proteins found in lung surfactant. Over days in culture with surfactant components, CD36 transcript and protein levels are increased relative to resting macrophages. siRNA knockdown of CD36 inhibits the ability of human macrophages to acquire the most abundant lipid species in surfactant, dipalmitoylphosphatidylcholine (DPPC). DPPC uptake is specifically mediated by CD36, as acquisition of another surfactant lipid, phophatidylglycerol, is unaffected by CD36 knockdown. Furthermore, Scavenger Receptor-A knockdown macrophages retain the ability to acquire DPPC. Finally, when human macrophages are cultured in surfactant lipids prior to infection with M.tb, bacterial growth is increased significantly. This surfactant-mediated growth advantage does not occur if CD36 is knocked down prior to culturing macrophages in surfactant. We conclude that CD36 contributes to surfactant lipid uptake by human macrophages and that M.tb is able to exploit this function in an endogenously lipid-filled AM. Ongoing experiments are investigating whether surfactant lipids are readily available as a carbon source for M.tb early during infection. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 69 50 Host Pathogen interaction and beyond 22/09/2016 Infecting Amoebae with Mycobacteria to Study Conserved Mechanisms of Innate Immunity T. Soldati Department of Biochemistry, University of Geneva, Geneva, Switzerland The first line of defence against bacteria infections are phagocytic cells of the innate immune system. These cells kill bacteria via oxygen-dependent (e.g. ROS) and oxygen-independent (e.g. chemicals, enzymes and microbicidal peptides) mechanisms. While multicellular organisms use phagocytosis to kill microbes and initiate a sustained immune response, phagocytic amoebae internalise bacteria as nutrients, via mechanisms of recognition, signalling and killing that are surprisingly conserved. In particular, at the transition to multicellularity, eukaryotic organisms acquired NOX enzymes to generate ROS. Dictyostelium is a social amoeba that feeds by phagocytosis and has a rudimentary but highly conserved cell-intrinsic immune system. It is genetically and biochemically tractable and has emerged as a powerful and experimentally versatile host model organism. In particular, we study the mechanisms of infection by Mycobacterium marinum, a close cousin of M. tuberculosis, which uses similar virulence strategies to re-program and proliferate inside macrophages. We use this Dictyostelium/M. marinum model to study the interrelationships between host and pathogen in terms of nutritional immunity, acquisition of metabolites, interference with the membrane trafficking and cytsokeletal processes. For example, recently, we have used this system to study the role of iron, zinc and their transporters during mycobacterial infection. In addition, we have focused on the monitoring of the impact of the TOR pathway and the interplay between membrane damage and autophagy. Furthermore, we discovered that M. marinum can use fatty acids derived from both host phospholipids and triacyglycerols and that this lipidbased metabolism is not directly linked with dormancy. We also used this complex system to identify anti-infective compounds that impact on the fate of the infection without significant antibiotic activity. 70 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 51 Host Pathogen interaction and beyond 22/09/2016 Insights into the host-pathogen and microbiome interactions from dual RNA-sequencing of tuberculous sputum R. Lai5, T. Cortes4, S. Marais1, M. Burke5, N. Rockwood2-1, A. Garza-Garcia5, D. Young3-5, R. Wilkinson2-5-1 1 Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town, South Africa 2Department of Medicine 3MRC Centre for Molecular Bacteriology and Infection, Imperial College London 4Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine 5The Francis Crick Institute, London, United Kingdom Background: Transmission of Mycobacterium tuberculosis (Mtb) is via aerosol droplets of infectious sputum. Understanding the phenotypes of the tubercle bacilli and their microenvironment during this transition phase may illuminate new strategies to reduce transmission. Methods: Sputum samples were collected from 17 patients with active TB disease, among which 8 were co-infected with HIV-1. Additional sputa were collected from 8 patients who were respiratory symptomatics with diseases other than TB. Total RNA was extracted from sputa as well as reference laboratory Mtb (H37Rv) cultures grown to exponential and stationary phase. We performed a one-step, ultra-deep dual RNA-Seq on tuberculous and control sputa to survey the global transcriptome of the host, Mtb and the resident microbiota. Result: The transcriptional profile of Mtb most closely related that of actively replicating bacteria in laboratory culture, with significantly increased expression of transcripts linked to cholesterol metabolism and zinc deprivation, and reduced expression of the PhoP regulon implicated in control of virulence determinants. Approximately 1% of the total bacterial reads in sputum mapped to Mtb. Transcriptional profiling of commensal bacteria in the sputum provided further support for a low zinc environment and suggested an integrated, interdependent metabolic network. The ability to utilize cholesterol may reduce pressure on Mtb to compete for common nutrients with the established microbiota in the respiratory tract. In the host transcriptomes, sputa from TB patients displayed a strong type I and II interferon and TNF-mediated inflammatory responses compared to those from non-TB controls, similar to whole blood signatures already reported. We also observed transcriptional activity corresponding to the Warburg effect in TB sputa, implicated by increased expression of genes associated with glycolysis and decreased expression of genes associated with oxidative phosphorylation. Conclusion: RNA-sequencing allows simultaneous characterization of host, Mtb and commensal bacteria and gives a comprehensive snapshot on the oral and respiratory microenvironment. The knowledge acquired offers insight into pathogenesis and on future strategies to better prevent Mtb transmission. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 71 52 Host Pathogen interaction and beyond 22/09/2016 The minimal unit of infection: M. tuberculosis in the macrophage D. Russell Microbiology and Immunology, Cornell University, Ithaca, New York, United States The interaction between Mycobacterium tuberculosis (Mtb) and its host cell is highly complex and extremely intimate. Were it not for the disease, one might regard this interaction at the cellular level as an almost symbiotic one. The metabolic activity and physiology of both cells are shaped by this co-existence. We believe that where this appreciation has greatest significance is in the field of drug discovery. Evolution rewards efficiency, and recent data from many groups indicate that Mtb has evolved to utilize the environmental cues within its host to control large genetic programs or regulons. But these regulons may represent chinks in the bacterium’s armor because they include off-target effects, such as the constraint of Mtb’s metabolic plasticity. A prime example is how the presence of cholesterol within the host cell appears to limit the ability of Mtb to fully utilize or assimilate other carbon sources. We believe firmly that to understand the physiology of Mtb, and to identify new drug targets, it is imperative that the bacterium be interrogated within the context of its host cell. The constraints induced by the environmental cues present within the host cell need to be preserved and exploited. The Mtb-infected macrophage truly is the “minimal unit of infection”. 72 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 53 Drug design-new developments 23/09/2016 Reseting acquired antibiotic resistance in Mycobacterium tuberculosis A. Baulard Center for Infection and Immunity, Institut Pasteur de Lille et INSERM, Lille, France Antibiotic resistance is one of the biggest threats to human health. Amongst the scariest worldwide progressions of this scourge is that of multi- and extensively-drug resistant Mycobacterium tuberculosis. For some key antituberculosis pro-antibiotics, resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules waking-up a cryptic alternative bio-activation pathway of ethionamide in M. tuberculosis to circumvent the classic bioactivation pathway where mutations are observed in resistant clinical strains. The first of its kind molecule, named SMARt-1 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and successfully treats ethionamide-resistant infection in mice, but also increases the basal sensitivity of bacteria to ethionamide. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 73 54 Drug design-new developments 23/09/2016 Mycobacterium tuberculosis CTP synthetase and pantothenate kinase: two promising targets for the development of multitargeting drugs L.R. Chiarelli4, M. Esposito4, B.S. Orena4, G. Mori4, N. Buttari4, G. Degiacomi3, F. Gosetti2, M. Manfredi2, S. Ekins7, K. Mikusova5, M. Bellinzoni1, R. Manganelli3, E. Marengo2, L. Ballell-Pages6, G. Riccardi4, M.R. Pasca4 1 Structural microbiology Unit, Pasteur Institut, Paris, France 2Department of Science and Technological Innovation, University of Piemonte Orientale, Alessandria 3Department of Molecular Medicine, University of Padova, Padova 4Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Italy, Pavia, Italy 5Department of Biochemistry, Comenius University of Bratislava, Bratislava, Slovakia 6Tres Cantos Medicines Development Campus, GlaxoSmithKline, Madrid, Spain 7Collaborative Drug Discovery, Burlingame, United States Tuberculosis is responsible for about 1.5 million deaths/year, and the emergence and spread of Mycobacterium tuberculosis multidrug-resistant strains has prompted the discovery for new drugs. In a previous work (Mori et al., 2015), we validated the CTP synthetase PyrG as the target of thiophene-carboxamide (82) and carbamothioyl-propanamide (88) prodrugs activated by EthA, and of the (026) sulfonic active metabolite of 82. In order to confirm PyrG as valuable TB target, testing the possibility of cross-reactivity of the inhibitors with the human enzyme, we expressed and purified the human CTP synthetase 1. The enzymatic inhibition assays of CTP synthase of PyrG inhibitors are in progress. Recently, by genetic and biochemical approaches, we found a secondary target of these three PyrG inhibitors. In particular, biochemical studies confirmed that panthotenate kinase PanK is inhibited by the EthA-activated compounds, as well as by the active metabolite 026. These evidences suggested that PyrG and PanK enzymes could be successfully used for the screening of compound libraries, in order to identify potential multitargeting hits. For this reason, the publically available GSK TB-set library of antitubercular compounds was firstly assayed against PyrG activity, identifying 3 pyridine-thiazoles as effective, ATP-competitive inhibitors (Ki=3-20 μM). PyrG was also genetically validated as target of these 3 GSK compounds, while metabolic labeling studies demonstrated that the compounds affect different biosynthesis pathways involving PyrG. In order to verify if these compounds could be possible multitargeting hits, they were assayed against recombinant PanK enzyme activity, finding that one of them, the N-(4-(pyridin-2-yl)thiazol-2yl)-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]thiazol-6-amine, affected PanK. Biochemical analysis demonstrated that the compound inhibits PyrG and PanK with a similar competitive inhibition mechanism, and the molecular docking confirmed that it is able to interact with the ATP binding site of both proteins. In conclusion, the two targets PyrG and PanK have been demonstrated to be suitable tools for identification of new potential multitargeting antitubercular compounds. 74 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 55 Drug design-new developments 23/09/2016 Antitubercular profile of GSK070 a Mycobacterium tuberculosis leucyl-tRNA synthetase inhibitor A. Mendoza-Losana2, E. Perez-Herran2, A. Palencia1, S. Cusack1, X. Li3, Y. Zhou3, M.R.K. Alley3, D. Barros-Aguirre2 1 EMBL, Grenoble, France 2TbDPU, GSK, Tres Cantos, Spain 3Anacor Phamaceuticals Inc, Palo Alto, United States Background: Protein synthesis inhibitors play an important role in the treatment of tuberculosis. The essential protein synthesis enzyme leucyl-tRNA synthetase (LeuRS) has two catalytic sites, an aminoacylation site, which charges tRNALeu with leucine and an editing or proof-reading site that hydrolyses incorrectly charged tRNALeu. The application of the oxaborole tRNA trapping (OBORT) mechanism (Rock & Mao et al., 2007 Science 316: 1759-1761) to Mycobacterium tuberculosis LeuRS led to GSK070, a new anti-tubercular agent with excellent potency and selectivity. Material/methods: MIC determination, frequency of spontaneous resistance and other microbiological experiments were performed as described by Parish & Roberts, 2015 (Mycobacteria Protocols 3, Methods in Molecular Biology). Clinical isolates were obtained through the Spanish National Institute of Health and antibacterial activity determined using the BACTEC MGITTM 960 System (Becton Dickinson). The GSK070 co-crystal structure was obtained with the editing domain of M. tuberculosis LeuRS using amino acid residues from Gly309 to Ile515 in the presence of AMP and the inhibitor. Results: GSK070 was co-crystalised with the editing domain of M. tuberculosis LeuRS and was found in the typical OBORT binding mode with the 4-chlorobenzoxaborole core occupying the amino acid substrate site, while the boron atom formed two covalent bonds with the cis-diol from the ribose of AMP. The 3-aminomethyl group from the inhibitor also plays a critical role as it interacts with two negatively charged residues (Asp447, Asp450) and participates in an H-bond with the carbonyl of Met441. These interactions yield a potent inhibitor of the enzyme that retains activity against sensitive, MDR and XDR M. tuberculosis clinical isolates, exhibiting an MIC90 of 0.16 μM with an MIC range of ≤0.02-0.31 µM. The frequency of spontaneous resistance is in the range of other antituberculars and no cross-resistance in vitro has been observed with other antituberculars that are in the clinic or in development. Further confirming GSK070’s unique mode of action, we observed mutations in the leuS gene from these resistant mutants. Conclusions: GSK070 has excellent activity against a set of 98 strains including drug-sensitive, multidrug and extremely drug-resistant strains of M. tuberculosis. Its frequency of spontaneous resistance is in the range of other clinically relevant antituberculars and most of the resistant mutations map to the editing site of M. tuberculosis LeuRS. Finally, GSK070 does not show crossresistance with other antituberculars. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 75 56 Drug design-new developments 23/09/2016 First clinical trial with the novel drug candidate PBTZ169: expected results and surprises V. Makarov1, S. Cole2 1 Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia 2Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland PBTZ169 is novel drug candidate with high efficacy in animals models and its combination treatment of PBTZ169 with BDQ and pyrazinamide was shown to be more efficacious than the standard treatment for tuberculosis in a mouse model. The target of PBTZ169 is famous DprE1, an essential enzyme in cell wall biosynthesis. Furthermore this drug candidate demonstrated during preclinical research “drug like” properties what made it an attractive drug candidate to treat TB in humans. During first clinical trials several cohorts of the healthy volunteers were treated by the single doses of PBTZ169 as well as two weeks repeated treatment was chosen for two maximal doses. As expected PBTZ169 was well tolerated and no significant toxicity effects was observed during the trials. The study of the metabolism shown that human metabolism of PBTZ169 is very different from microbial or animals compound transformation. So main pathway of microbial, mice and less rats metabolism connected with reduction processes, but human metabolism mainly connected with oxidation processes. Due this difference we observed several new active metabolites of PBTZ169 in human and now we can conclude that animal antituberculosis activity of PBTZ169 is a result not only activity of the drug itself but it is a result of the sum activity of the drug and its metabolites. Direct antimicrobial plasma activity was studied and such activity was observed for 24 hours after human treatment for some doses. This data gets high chance for good efficacy of PBTZ169 in human for treatment TB infection. 76 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 57 Drug design-new developments 23/09/2016 Griselimycins, a new class of anti-TB drugs S. Lagrange Infectious Diseases Unit, SANOFI, Marcy L'etoile, France Griselimycin is a natural cyclic peptide isolated from Streptomyces species. Lead optimization of Griselimycin resulted in identification of a novel synthetic analog derivative Cyclohexylgriselimycin (CGM). DNA Polymerase sliding clamp (DnaN) has been identified as the target. CGM profiling confirmed the interest of this new class for TB treatment. An update on this program will be done and the selection of the most promising advanced lead for further late preclinical studies will be presented. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 77 58 From animal models to man 23/09/2016 Animal Models of Tuberculosis for Efficacy Testing A. Rawkins National Infections Service, Public Health England, Salisbury, United Kingdom Pre-clinical testing of novel therapies and vaccines in animal models of tuberculosis provides essential information on safety, immunogenicity and efficacy which supports the development of new products towards use in humans. The species most commonly used to test the efficacy of vaccines are mice, guinea pigs and nonhuman primates. Efficacy is measured by comparing the relative severity of disease in terms of bacterial burden, survival and/or analyses which describe the severity of the key pathological features. Bacterial burden can be measured robustly in small animal species and enables relatively rapid screening of large numbers of candidate vaccines in order to select the most promising for testing in more relevant and complex animal models such as non-human primates or models which more closely replicate human TB. These include models of HIV/TB co-infection, latent or reactivated TB, simulation of neonatal or adolescent vaccination strategies and the use of more relevant strains or doses of M. tuberculosis for challenge, including natural transmission between animals. Studies are on-going at PHE with funding by the Bill and Melinda Gates Foundation to establish a guinea pig model of naturally acquired infection using experimentally infected macaques as the source of aerosols. Data generated in a pilot study have demonstrated the feasibility of transmission between macaques and guinea pigs. An increase in the complexity of the animal model can result in greater variation between animals with regards to the measurements of disease severity and there is a balance to be struck between the relevance of the model to humans and the ability to control variables and interpret the data. Sophisticated methods of measuring bacterial burden and disease pathology, in-life by advanced imaging have been developed and these significantly reduce this variation. With scarce resources and for ethical reasons, it is imperative that data generated in animals are informative. Evaluation of the same vaccine in early stage clinical trials and in animals, in parallel, is feasible and greatly enhances the value of the animal studies and offers the potential to identify correlates of protection which would significantly improve and accelerate the development of an improved TB vaccine. 78 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 59 From animal models to man 23/09/2016 Tuberculosis and One Health: comparative analyses of the human and bovine tubercle bacilli S. Gordon School of Veterinary Medicine, University College Dublin, Dublin, Ireland The One Health concept seeks to improve human, animal and environmental health by building on synergies across these areas. Comparative studies across human and animal TB offer a unique area in which to explore such One Health synergies; indeed in his classic studies in the late 19th century on the differentiation of human and bovine tubercle bacilli, Theobald Smith already recognised the potential for such comparative work to “lead eventually to more light on the whole subject of tuberculosis from the preventive as well as the therapeutic side” (Smith 1898). In this presentation I will discuss work on comparative analyses of M. tuberculosis and M. bovis as the exemplar human- and animal-adapted members of the M. tuberculosis complex, and look to what these studies can teach us about the virulence, evolution and host adaptation of the complex as a whole. The presentation will encompass comparative virulence studies of M. tuberculosis and M. bovis in bovine experimental infections, the use of transcriptomics to define differential responses of bovine alveolar macrophages to infection with M. tuberculosis and M. bovis, and exploration of potential host tropic virulence mechanisms across the bacilli. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 79 60 From animal models to man 23/09/2016 Comparative Efficacy of New Oxazolidinones in a NHP Model of Tuberculosis evaluated by [18F]FDG PET/CT and Bacterial Burden L.E. Via1, G. Marriner1, Y. Park1, K.K. Robbins1, J. Sarathy2, M. Zimmerman2, B. Prideaux2, C. Chen2, H. Boshoff1, V. Dartois2, J. Flynn3, C. Barry1 1 Tuberculosis Research Section, NIAID, NIH, Bethesda 2PHRI, Rutgers University, Newark 3Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, United States The number of new cases of multidrug resistant tuberculosis (MDR-TB) is increasing worldwide. New chemotherapeutics are needed to augment treatment for MDR-TB, with the ultimate goal of a new drug regimen useful for both drug susceptible and resistant disease. Linezolid (LIN) is used in salvage treatment of MDR-TB, but it has an unfavorable toxicity profile with long-term use. Therefore, other potentially less toxic oxazolidinones are being evaluated for TB. The common marmoset, a small, non-human primate (NHP) model, is proving useful in assessing Mycobacterium tuberculosis (MTB) drug efficacy. Five oxazolidinones were evaluated in the marmoset and mouse models using in vivo imaging and terminal assays. Marmosets were infected with MTB for approximately 7 weeks, given a pre-treatment 18F-fluorodeoxyglucose (FDG) PET/CT scan, and randomized into treatment groups. The NHP were administered oral daily doses of LIN, AZD5847 (AZD), Radezolid (RAD), Sutezolid (SUT), or Tedizolid (TED) adjusted to match the T>MIC of the expected human dose. Serial PET/CT scans were performed during the 1 or 2 months of treatment and computationally evaluated to assess TB-associated abnormalities. C57BL/6 mice were similarly infected and treated for 1 or 2 months with oxazolidinone doses equivalent to the T>MIC achieved in the marmoset. After treatment, the animals were euthanized and the bacterial burden was assessed. Marmosets treated with the oxazolidinones showed reduced or stabilized lung disease by PET/CT and increased survival post-infection, compared to controls. Animals treated with LIN, AZD, SUT and TED experienced the most consistent response showing reductions in both abnormal lung volume and FDG uptake. Lesion and organ bacterial burdens were significantly reduced in marmosets administered LIN, AZD and SUT. Quantitative reductions in abnormal lung volumes and FDG uptake were highly associated with lower bacterial burdens. Studies of drug distribution into epithelial lining fluid, penetration into caseum, and in vitro PK and PD assays partially predicted the activity of these agents. In the mice, SUT promoted the most significant reduction in bacterial burden followed by LIN with the other agents showing only modest activity. These results support further investigation of SUT, AZD, and possibly TED as components in new drug regimens. 80 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 61 From animal models to man 23/09/2016 Evolution of Drug Resistance in Mycobacterium tuberculosis A. Pym Research Laboratory, AHRI (African Health Research Institute), Durban, South Africa The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug resistant (XDR) tuberculosis (TB) identified at Tugela Ferry, KwaZulu-Natal, South Africa, in 2005. It is unclear whether the emergence of XDR-TB was due to recent inadequacies in TB control or other factors. Using whole genome sequencing on clinical and historical isolates we demonstrate the evolution of drug resistance over a 50 year period and identify a common mutational pathway responsible for multiple episodes of de novo evolution. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we were also able to identify new mutations associated with resistance. One of these was a loss-of-function mutations in ald (alanine dehydrogenase) which conferred resistance to D-cycloserine and emerged independent of antibiotic selection in the evolution of animal adapted strains of the M. tuberculosis complex. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 81 62 From animal models to man 23/09/2016 Severe childhood tuberculosis as a genetic disorder S. Boisson-Dupuis INSERM Hôpital Necker/Rockefeller University, United States Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and has long been considered exclusively as an infectious disease. Up to one third of the world population is infected by Mtb, however, only 10% of infected people develop clinical TB, including about 5% within two years after infection (known as primary tuberculosis). This form predominantly affects children, is clinically subacute and often spreads via the blood to tissues outside the lungs. The remaining 5% of infected individuals develop TB later in life, years or decades after infection, typically due to microbial reactivation from latency, which results in the chronic pulmonary form of adult TB. The genetic factors of the host have been shown to play an important role in humans. The investigation of a rare syndrome designated as Mendelian susceptibility to mycobacterial disease (MSMD), which is characterized by susceptibility to poorly virulent mycobacteria (the vaccine strain, BCG, and environmental mycobacteria), has led to the identification of a set of 10 human genes essential for antimycobacterial immunity, all involved in IFN-γ-mediated immunity (upstream or downstream from IFN-γ). In this context, unsurprisingly, several MSMD patients were also shown to be susceptible to TB. In addition, children with complete IL-12Rβ1 or TYK2 deficiency were reported to display severe TB, providing the first proof of principle that severe childhood TB may result from single-gene inborn errors of immunity. They all display impaired production of IFN-γ in response to IL-12/IL-23, probably accounting for their susceptibility to TB. These results strongly support the hypothesis that childhood TB may result from a collection of single-gene inborn errors of immunity, at least in a fraction of patients. However, most children with TB lack a genetic etiology. The laboratory has recruited 200 children from Turkey, Iran and Morocco with severe tuberculosis and completed Whole Exome Sequencing on each. Some of the results obtained will be presented during the seminar, highlighting that TB of childhood results from a collection of diverse single-gene inborn errors of immunity in a significant proportion of children. 82 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 63 Closing Keynote Lectures Session 23/09/2016 Interactions of mycobacterial phenolic glycolipids with host macrophages L. Ramakrishnan University of Cambridge, Cambridge, United Kingdom We have developed the zebrafish model of tuberculosis where we can exploit the genetic tractability and optical transparency of the larval stage to monitor and modulate host-pathogen interactions in real-time. More recently, we have developed the zebrafish as a model for leprosy. I will present work showing the interactions of mycobacterial phenolic glycolipids with host macrophages that contribute to pathogenesis in both tuberculosis and leprosy. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 83 64 Closing Keynote Lectures Session 23/09/2016 Tuberculosis drug development: the leaking pipeline S. Cole EPFL, Lausanne, Switzerland Tuberculosis (TB) was one of the first infectious diseases to be rationally treated and the golden age of TB drug discovery led not only to new antibiotics but also to the development of combination therapy. Until recently, no new TB drugs had been developed since the 1960s but, in response to the HIV-pandemic and widespread resistance to first- and second-line drugs, a pipeline of candidate TB drugs has now been established. I will review the scientific progress that fed this pipeline and emphasize the important role played therein by public-private-partnerships. I will also highlight the slow progress in clinical trials and extensive attrition rate, both compounded by lack of funding and industrial commitment. As a result of these factors and poor coordination the pipeline has many leaks. 84 Tuberculosis 2016 – September 19-23, 2016 – Paris, France POSTER SESSION 1 Tuesday, September 20 SUMMARY Mycobacterial Genomics and Evolution ........................................ 87 Mycobacterial Biology & Physiology ............................................ 117 Transport systems & Secretion in Mycobacteria ......................... 184 65 Mycobacterial Genomics and Evolution 20/09/2016 Detection of genomic mutations in KatG, inhA and rpoB genes of Mycobacterium tuberculosis isolates using polymerase chain reaction and multiplex allele-specific polymerase chain reaction A.D. Khosravi1, H. Goodarzi1, S.M. Alavi1, M. Afzali Behbahani2 1 Infectios and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz 2Tehran University of Medical Sciences, Tehran, Iran Background: Isoniazid (INH) and Rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis (MTB). Mutations in several genes are determining the resistance of MTB to INH, with the most common gene target of KatG, and resistance to RIF is due to mutation in rpoB gene.[1] The aim of present study was to determine the mutations in the regions related to RIF and INH resistance in MTB strains isolated from tuberculosis patients in Ahvaz, Iran. Methods: We characterized 80 clinical isolates of confirmed MTB to analyze the most commonly observed INH and RIF mutations.[2,3] PCR analysis with subsequent sequencing were used to detect mutations related to RIF and INH INH resistance. The multiplex allele-specific-PCR, was performed as a comparative assay and for evaluation of this method. [4] Results: The sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7℅ of the MTB isolates. The sequencing of the 270-bp central region of the rpoB gene, revealed point mutations at 7 different codons in 12 (15%) of the MTB isolates. The results obtained with multiplex allele-specific-PCR assay, were in concordance to PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526). Conclusions: The result of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of MTB. Both DNA sequencing and multiplex allele-specific -PCR technique gave a high value sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases. Key words: M. tuberculosis, resistance, Isoniazid, Rifampin, mutation Tuberculosis 2016 – September 19-23, 2016 – Paris, France 87 66 Mycobacterial Genomics and Evolution 20/09/2016 Whole Genome Analysis of Clinical Mycobacterium bovis in Ghana Contradicts its Classification Among Ancient Lineages of the Mycobacterium tuberculosis Complex I.D. Otchere2-1, S. Harris4, S.L. Busso4, A. Asante-Poku2, S. Osei-Wusu2, K. Koram2, J. Parkhill4, S. Gagneux3, D. Yeboah-Manu2 1 Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Accra 2Noguchi Memorial Institute for Medical Research, University of Ghana, Accra 3Swiss TPH, Basel 4Wellcome Trust Sanger Institute, Genome Campus, Cambridge University, UK, Cambridge, Ghana Background: Tuberculosis caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. With the scare of drug resistant strains, it imperative that an understanding of the causative agent is realized for proper control of M. bovis associated TB. Methods: Mycobacterium bovis (15) among 1740 Mycobacterium tuberculosis complex (MTBC) isolates from Northern and Greater Accra regions of Ghana were screened for susceptibility to INH and RIF. Five of the M. bovis strains were whole-genome sequenced and added to a pool of L4, L5, L6 and global collection of MTBC genomes for comparative analysis. Results: Mycobacterium bovis was isolated from 3/560 females and 12/1380 males. The average age of the patients infected with M. bovis was 46.8 years (7 to 72 years). Four out of 212 (1.9%) TB patients from the Northern compared to 11/1714 isolates from the Greater Accra regions were infected with M. bovis. Two of the 15 (13.3%) bovine strains were isolated from HIV positive patients whereas 6 (37.5%) were from patients who were in constant contact with livestock. The bovine strains were susceptible to the 2 drugs except 2 INH (with katG S315T) and 1 RIF (with Q432P and I1491S) mono-resistant strains. Maximum likelihood phylogenetic tree with 100 bootstraps and rooted on M. canettii showed clustering of the 5 bovine TB genomes with the animal strains of the global collection and were closer to MAF L6 than L5. Pan-genome analyses of the 5 M. bovis genomes showed shared 3824 core-genes and 320 shell-genes. Comparing the presence and/or absence of genes among the genomes, we found the bovine strains have relatively smaller genome similar to L6 as compared to L4 and L5. We found the density of SNPs within the ESAT6 secretion system was higher (1.4) than L6 (0.9), L5 (1.0) and L4 (0.1). We observed a number of mutations within some house-keeping genes essential for in vitro growth in the bovine strains. Conclusion: The bovine strains cluster with L6 and share relatively smaller genome compared to L5 and L4. This challenges the classification of M. bovis as ancient and L4 as modern since the MTBC evolves by genome deletions. 88 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 67 Mycobacterial Genomics and Evolution 20/09/2016 Transcriptional and proteomic profiling of diverse clinical isolates of Mycobacterium tuberculosis A. Banaei-Esfahani2-4, B. Collins2, O. Schubert2-5, A. Trauner1, S. Borrell1, S. Gygli1, M. Coscolla1, S. Gagneux1, R. Aebersold2-3 1 Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel 2Department of Biology, Institute of Molecular Systems Biology, ETH Zurich 3Faculty of Science, University of Zurich 4PhD Program in Systems Biology, University of Zurich and ETH Zurich, Zurich, Switzerland 5Departments of Human Genetics, University of California, Los Angeles, United States Mycobacterium tuberculosis (Mtb) as one of the most successful human pathogens still has remained a major source of mortality worldwide. Increasing number of drug resistant strains as well as its ability in co-infection with HIV/AIDS place a massive burden on health care systems. Mycobacterium tuberculosis complex (MTBC) comprises seven human-adapted lineages. Three lineages (2-4) have been referred to as evolutionarily “modern” due to a deletion in genomic region namely TbD1. Advent of next-generation sequencing and advanced proteomics in addition to new computational approaches has paved the way for studying and curing tuberculosis more systematically. Although Mtb is considered as highly clonal specie, genomic data show that two human adopted MTBC strains differ by about 1200 SNPs on average while two thirds of SNPs in coding regions are non-synonymous. This observation can propose that majority of these SNPs are functional. In this study we profiled proteome of six fully sequenced clinical isolates from lineages one (ancient lineage) and two (modern lineage). Quantification of about 80% of expressed genes (~2500 proteins) using SWATH-MS allowed us to characterize the strains effectively. RNA sequencing also enabled analysis of expression across the transcriptomes. PCA analysis admits that the strains stand near to each other based on their relationship. We have corroborated that DosR regulon has higher expression in lineage two at proteome level. It can be an appropriate explanation for phenotypic behavior of modern lineages embedding more virulent strains. Around one fourth of proteins including several enzymes and transcription factors, show significantly differential expression between lineage one and two. Surprisingly number of changes at proteome level as function of genomic differences (SNPs) behaves linearly. It’s also noted that every 7-8 SNPs is responsible for each protein change approximately. Overall these findings elucidate the value of an integrated multilayered omics approach to characterize the underlying mechanisms involved in Tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 89 68 Mycobacterial Genomics and Evolution 20/09/2016 Identification of mutations associated with resistance to the novel anti-tuberculosis drug delamanid by a Whole Genome Sequencing approach S. Battaglia5, E. Schena5, L. Nedialkova2, A.M. Cabibbe5, E. Borroni5, A. Trovato5-4, S. Niemann1, C. Utpatel1, M. Merker1, S. Hoffmann-Thiel2-3, H. Hoffmann2-3, D.M. Cirillo5 1 Research Center Borstel, Molecular Mycobacteriology, German Center for Infection Research (DZIF), Partner Site Borstel, Borstel 2Institute of Microbiology and Laboratory Medicine, IML red GmbH, WHO Supranational Reference Laboratory of TB 3Institute of Microbiology and Laboratory Medicine, IML red GmbH, WHO Supranational Reference Laboratory of TB, Germany Synlab MVZ, Gauting, Germany 4Center for Translational Genomics and Bioinformatics (CTGB), San Raffaele Scientific Institute 5Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation, and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy Among the new anti-TB drugs, delamanid (DLM) has been conditionally approved for the therapy of MDR-TB in 2014. This compound impairs the biosyntesis of mycolic acids and requires activation by F420-dependent nitroreductase encoded by ddn gene. F420 cofactor is synthetized by enzymes coded by fbiA, fbiB, fbiC and fgd1 genes. Polymorphisms in these genes were shown to be involved in in vitro DLM resistance (DLM-R). We characterized the mutations leading to DLM resistance in a panel of M. tuberculosis complex clinical isolates. A total of 250 MTBC strains representing eleven different lineages and all drug resistance profiles were included in this study. Minimal inhibitory concentrations (MICs) of DLM were determined by Bactec 960 MGIT system and resazurin microtiter assay (REMA). Genomic DNA was extracted by CTAB method for Whole Genome Sequencing (WGS) analysis. Paired-end (101 bp) libraries were prepared using Nextera XT DNA Sample Preparation kit and sequenced on HiSeq 2500 platform (Illumina Inc). Samples showing a coverage ≥ 20X were considered for SNPs calling by PhyResSE web-tool. DLM susceptible isolates showed MICs between 0.03 and 0.0005 mg/L in REMA and between 0.06 and 0.0005 ml/L in MGIT. WGS analysis on MTB isolates revealed fifteen non-synonymous mutations in the five genes screened. Two polymorphisms were previously reported as lineage-specific for Harlem and M. africanum 2. WGS analysis of DLM-S strains revealed the presence of nine new non-synonymous mutations, while analysis of the five DLM-R strains (REMA MIC ≥ 32 mg/L, MGIT MIC ≥ 16 mg/L) revealed the presence of three ddn W88Stop mutations and one fbiA K250Stop change in four clustered Beijing strains (W148 clone), and absence of mutations in the last DLM-R isolate. Moreover, NGS technique allowed us to rapidly analyse others potentially DLM-R related genes, possibly involved in the emergence of DLM-R phenotype. It is worth noting that the population of strains analysed were isolated from patients never exposed to DLM showing that mutations inducing DLM-R phenotype are pre-existing in MTBC clinical strains and potentially associated to specific clones. 90 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 69 Mycobacterial Genomics and Evolution 20/09/2016 Genetic characterization of a non-canonical MMR-like pathway in mycobacteria A. Castañeda-García2, A. Prieto3, A. Rojas3, J. Rodriguez-Beltran3, T. Tønjum1, S. Waddell4, A. Doherty4, J. Blazquez2-3 1 Oslo University Hospital, Oslo, Norway 2Centro Nacional de Biotecnologia, Madrid 3Instituto de Biomedicina de Sevilla (IBIS)-CSIC, Seville, Spain 4University of Sussex, Brighton, United Kingdom Mismatch Repair (MMR) is a near ubiquitous pathway that removes mismatched nucleobases from DNA. MMR is essential for the maintenance of genome stability. Despite its major importance, an apparent MMR mechanism is absent in mycobacteria. However, these bacteria exhibit rates and spectrum of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative MMR pathway. To identify novel mutation avoidance components in mycobacteria, a Mycobacterium smegmatis insertion mutant library was generated and screened for spontaneous mutation to rifampicin resistance, used as a hypermutator hallmark. As a result, a novel gene, named mmrE, was identified as a key anti-mutator gene in Mycobacterium. MmrE, a putative DNArepair enzyme, is required for mutation avoidance and anti-recombination, hallmarks of canonical MMR, and bears no structural homology to known MMR factors. Indeed the ΔmmrE phenotypes are almost identical to those produced by the MMR deficiency in other bacteria (very high mutation rates with transition-biased mutational spectrum and increased homeologous recombination rates). Furthermore, mmrE and known MMR genes are mutually exclusive in their distribution among organisms, as expected for alternative pathways. Finally, naturally occurring mmrE polymorphisms in clinical M. tuberculosis isolates with diminished MmrE activity produce hypermutable phenotypes, as occurs with MMR-bearing bacteria. In summary, our results strongly suggest that a novel MMRlike mechanism operates in organisms as mycobacteria that possess MmrE, but lack MutS-MutL. Therefore, MmrE appears as an essential piece that, together with the high-fidelity DNA-polymerase DnaE1 and its PHP domain-proofreader, maintain low mutation rates (~10-10 mutations per base per generation) in mycobacteria, ensuring genome stability and DNA fidelity. Understanding the mechanisms and pathways that influence mutation rates may unveil new strategies to predict and to combat the development of drug resistance. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 91 70 Mycobacterial Genomics and Evolution 20/09/2016 Impact of the M. tuberculosis Genetic Background on the Acquisition of Drug Resistanceconferring Mutations S. Borrell1-2, J. Feldmann1-2, R. Castro1-2, S. Gagneux1-2 1 Swiss Tropical and Public Health Institute 2University of Basel, Basel, Switzerland The extent of genetic diversity in M.tuberculosis (Mtb) is more pronounced than was traditionally believed, and can be classified into seven different geographically distributed lineages. The average inter lineage genetic distance is approximately 2000 single nucleotide polymorphisms (SNPs). The impact of this diversity on the baseline metabolism is still unknown. One of these lineages; the Beijing lineage has repeatedly been associated with drug resistant tuberculosis (DR-TB). The reason for this association is still not clear. To evaluate the influence of genetic background on the acquisition of specific Isoniazid (INH) resistance-conferring mutations, and to explore whether the Beijing lineage is better adapted to the potential physiological effects than other phylogenetical distant lineages, associations between lineage and INH-DR were sought. We performed Lüria-Delbruck fluctuation assay (LDFA), and found lineage-specific differences in the baseline INH-resistance acquisition rate, with Beijing showing a higher rate compared to the other lineages. Lineage specific INH-associated mutations were also explored. In addition, since Beijing intra lineage diversity shows a phylogeographical structure and epidemiological studies have evidenced differences on the Beijing-DR association depending on the strain geographical origin, we further explored the impact of intra lineage diversity on the basal INH-DR mutation rate performing LDFA of a collection of clinical Beijing strains globally distributed. Overall, our findings support a role of Mtb lineage diversity in the emergence of global drug resistance and consequently on the specific evolution of drug-resistance within each lineage. 92 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 71 Mycobacterial Genomics and Evolution 20/09/2016 HIV/TB coinfection reduces selective constraints on Mycobacterium tuberculosis D. Brites1-2, E. Wampande3, J. Hattendorff1-2, K. Eisenach5, H. Boom4, M. Joloba3, S. Gagneux1-2 1 Tuberculosis Research Unit, Swiss Tropical and Public Health Institute 2University of Basel, Basel, Switzerland 3Department of Medical Microbiology, Makerere University, Kampala, Uganda 4Tuberculosis Research Unit, Case Western Reserve University and University Hospitals of Cleveland, Cleveland 5Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, United States The incidence of tuberculosis (TB) has increased in many parts of the world, partially fueled by HIV coinfections. Given the importance of T-cell responses both in providing protective immunity against tuberculosis, but as well as drivers of lung pathology and ultimately mediators of transmission, the immunocompromised host environment is likely to affect the life cycle of M. tuberculosis. We therefore hypothesized that the immunocompromised host environment affects the fitness of M. tuberculosis and tested whether and how HIV/M. tuberculosis coinfections impact the genetic structure of a M. tuberculosis population. To this end, we apply molecular population genomics to whole-genome polymorphism data from a population of 180 M. tuberculosis strains circulating in HIV infected and uninfected individuals sampled across 12 years in Kampala, a city with a high burden of TB/HIV coinfections. Our results suggest that HIV coinfections have not subdivided this M. tuberculosis population significantly. The HIV status of the host seems however to affect the fate of mutations of M. tuberculosis strains significantly; M. tuberculosis from HIV co-infected hosts harbor an excess of mutations present at low frequencies. This excess is significant both for synonymous and nonsynonymous mutations pointing to an exacerbated role of genetic drift in M. tuberculosis from HIV/TB coinfections. This could reflect that M. tuberculosis from HIV infected patients is less transmitted. Supporting this, we show that M. tuberculosis from patients with lower CD4+ T-cells (≤250 cells/ul) also tend to have more mutations which are uniquely found in the population (i.e. where not transmitted), than M. tuberculosis strains which infect patients with higher CD4+ T-cell numbers. Our study supports findings from epidemiological studies suggesting that transmission of M. tuberculosis from HIV coinfected hosts is reduced, and suggests that M. tuberculosis fitness is reduced by HIV/TB coinfections. However, it also provides evidence that the continuous progression of M. tuberculosis/HIV syndemics can suply M. tuberculosis with genetic diversity and hence provide opportunities for bacterial adaptation. The latter might facilitate the evolution of resistance to anti-TB drugs. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 93 72 Mycobacterial Genomics and Evolution 20/09/2016 Comparison of phenotypic and genotypic methods for the detection of resistance to second line injectable drugs in multidrug-resistant clinical isolates of Mycobacterium tuberculosis in France F. Brossier, A. Pham, C. Bernard, V. Jarlier, A. Aubry, N. Veziris, W. Sougakoff French NRC for Mycobacteria, Pitié-Salpêtrière Hospital, Paris, France Background: The second line injectable drugs (SLID, i.e. the aminoglycosides amikacin (AMK) and kanamycin (KAN) and the cyclic peptide capreomycin (CAP)) are key drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). SLID act by binding to 16S rRNA and inhibiting protein synthesis. Mutations at positions 1401, 1402 and 1484 in the 16S rRNA gene (rrs) have been associated with cross-resistance to these drugs. In addition to rrs modifications, mutations in the promoter region of the eis gene, which encodes an aminoglycoside acetyltransferase, and in the tlyA gene, which encodes a putative rRNA methyltransferase, are associated with resistance (R) to KAN and CAP in Mycobacterium tuberculosis, respectively. The goal of the present study was to compare the sequencing data of SLID resistance-associated genes to the corresponding results of phenotypic drug susceptibility testing (DST) by the proportion method. Methods: 206 MDR M. tuberculosis clinical isolates collected in 2010-2014 at the French Reference Center for Mycobacteria were included, 153 susceptible to AMK/KAN/CAP, and 53 resistant to at least 1 of the 3 SLID (23 R-AMK/CAP/KAN, 22 monoR-KAN, 3 R-AMK/KAN, 2 R-CAP/KAN, 3 monoR-CAP) including 29 XDR. DST was performed on Lowenstein-Jensen medium. Results: Among the 53 strains resistant to at least 1 of the SLID, mutations in rrs accounted for resistance to AMK, CAP and KAN for 81%, 75% and 44% isolates, respectively, while mutations in eis promoter were detected in 44% of the isolates resistant to KAN. By contrast, no mutations in tlyA were observed in the isolates resistant to CAP. Among the 153 isolates susceptible to the 3 SLID, 145 showed no mutation, 1 harbored unknown T1404C plus G1473A mutations in rrs and 7 had an eis promoter mutation. Conclusions: The discrepancies observed between the genotypic (on the primary culture) and phenotypic drug susceptibility testing could be explained by i) a low level of resistance not detected by phenotypic testing (n=8 strains), ii) a low percentage of resistant mutants not detected by sequencing of drug resistance-associated genes on the primary culture (n=8 strains), and iii) to other resistance mechanisms not yet characterized (n=7 strains). 94 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 73 Mycobacterial Genomics and Evolution 20/09/2016 Genetic diversity of Mycobacterium tuberculosis strains spread in the Northwestern region of Russia E. Chernyaeva2, M. Rotkevich2, A. Yurchenko2, V. Zhuravlev1, N. Solovieva1, M. Shulgina1, A. Lapidus2, S. Obrien2 1 St. Petersburg Research Institute of Phtisiopulmonology 2Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, St. Petersburg, Russia The principal goal of the project is to develop a genome-wide variations catalogue of Mycobacterium tuberculosis strains spread in the Russian Federation for clinical and epidemiological purposes. For whole genome sequencing we chose DNA samples obtained from strains revealed in St. Petersburg (n=20), Leningrad region (n=18), Republic of Karelia (n=11) and Kaliningrad region (n=26). Whole genome sequencing (WGS) were performed using Illumina MiSeq platform. M. tuberculosis H37Rv reference genome (NC_000962.3) was used for SNP and Indel calling using bowtie2 and samtools packages. RaxML package was used for phylogenetic analysis, Mycobacterium canettii genome was used as outgroup. R-package was used for statistical analysis. The majority of sequenced isolates formed 5 known phylogenetic groups corresponding to Beijing, LAM, Ural, Haarlem and T spoligotypes. More than a half of sequenced isolates in each region belonged to Beijing family. The second highest prevalence group in St. Petersburg, Leningrad region and Karelia was LAM famity, however in Kaliningrad it was a T family which was rearly identified in Leningrad region and was not detected in St. Petersburg and Karelia. Ural genetic family was identified in 15% of M. tuberculosis isolates from Kaliningrad and only few isolates from St. Petersburg, Leningrad region and Karelia belonged to this genetic family. Thus, genome-wide analysis of M. tuberculosis isolates obtained in the Northwestern region of the Russian Federation, revealed a difference in population structure of M. tuberculosis isolates obtained in Kaliningrad region compared to other regions. However, all studied geographical regions are characterized by prevalence of Beijing genetic family. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 95 74 Mycobacterial Genomics and Evolution 20/09/2016 Genome-wide analysis of Mycobacterium tuberculosis strains isolated from patients with tuberculous spondylitis E. Chernyaeva2, M. Rotkevich2, A. Yurchenko2, V. Zhuravlev1, N. Solovieva1, M. Shulgina1, S. Obrien2 1 St. Petersburg Research Institute of Phtisiopulmonology 2Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, St. Petersburg, Russia The aim of the study is to find nucleotide polymorphisms (SNPs and InDels) associated with different types of pulmonary tuberculosis (TB) and tuberculous spondylitis (TS) based on whole genome sequencing data of Mycobacterium tuberculosis strains, collected in different regions of the Russian Federation. Genomic DNA of 146 M. tuberculosis strains was sequenced using Illumina MiSeq platform. M. tuberculosis H37Rv reference genome (NC_000962.3) was used for SNP and Indel calling using bowtie2, samtools and GATK packages. RaxML package was used for phylogenetic analysis, Mycobacterium canettii genome was used as outgroup. R-package was used for statistical analysis. Studied M. tuberculosis isolates were collected from patients with different clinical TB outcome: 55 were isolated from patients with TS; 68 – from patients with pulmonary TB (dissiminated, infiltrative and fibro-cavernous TB); 23 – from patients with mixed TB (pulmonary and extrapulmonary). To perform genome association study a phylogenetic analysis was conducted. Pulmonary and extrapulmonary M. tuberculosis isolates were found in all phylogenetic clusters. Beijing genotype was prevalent in all groups of M. tuberculosis isolates, however the prevalence of Beijing genotype among TS isolates was higher than among pulmonary TB isolates (76% and 67%, respectively). Meanwhile pulmonary TB isolates belonged to LAM family that TS isolates (16% and 5%, respectively). SNPs and InDels associated with phylogenetic clusters were removed and the rest genetic variants were used for associations study. Statistical analysis allowed to identify SNPs and InDels associated with clinical patterns (p ≤ 10-5). Over 40 SNPs discriminating infiltrative and fibrocavernous TB and 15 SNPs discriminating TS and fibro-cavernous TB were found in CDS and intergenic regions. However, there were no reliable SNPs found discriminating pulmonary TB in general and extrapulmonary TB. InDels discriminating pulmonary and extrapulmonary TB were identified in PE_PGRS genes and need to be validated by Sanger sequencing. 96 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 75 Mycobacterial Genomics and Evolution 20/09/2016 Predicting the impact of global genomic variation of Mycobacterium tuberculosis in the bacterial regulatory network Á. Chiner-Oms4, F. González-Candelas4-3-2, I. Comas1-2 1 Tuberculosis Genomics Unit, Biomedicine Insitute of Valencia 2CIBER in Epidemiology and Public Health 3Infection and Public Health, Foundation for the Promotion of Health and Biomedical Research of Valencia Region 4Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, Valencia, Spain We have constructed a set of computational models to predict the expression of Mycobacterium tuberculosis genes from a previously published regulatory network and a very large expression dataset. The accuracy of the models was assessed by testing them versus a set of random models. The final models were also tested with two different expression datasets. We use these models to predict the expression of a mutated strain of M. tuberculosis lacking an important transcription factor called phoP. Combining the information derived from these robust models obtained we propose a new, more robust regulatory network. We have also tested a set of genome-wide single nucleotide polymorphisms and deletions from a large collection of clinical strains. We noted that some transcription factors from the regulatory network were mutated or deleted in several of the seven M. tuberculosis lineages. These transcription factors are embedded in regulatory subnetworks involving several metabolic, transcription and transport related processes. We speculate that the regulatory network may vary, albeit only slightly, among lineages and these differences could be related to some of the phenotypic and epidemiological differences observed among M. tuberculosis strains of different lineages. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 97 76 Mycobacterial Genomics and Evolution 20/09/2016 SMRT Genome Assembly Corrects Reference Errors, Resolving the Genetic Basis of Virulence in Mycobacterium tuberculosis A. Elghraoui, S. Modlin, F. Valafar biomedical Informatics Research Center, San Diego State University, San Diego, United States The genetic basis of virulence was investigated with the publication of the Mycobacterium tuberculosis H37Ra genome, the avirulent counterpart to the M. tuberculosis reference strain H37Rv. Variants with respect to H37Rv affecting 56 genes were identified as H37Ra-specific and potentially causal to its attenuated virulence. These genomic differences have been subsequently explored in at least 19 studies. Such analysis, however, heavily relies on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. Here, we report the sequencing, assembly, and finishing of the H37Ra genome with single-molecule sequencing, a platform capable of exceeding the accuracy of Sanger-based technologies. Our assembly reveals that the number of H37Ra-specific variants is less than half of what was determined based on the current H37Ra reference sequence. PE/PPE family genes, which are intractable to typical short-read sequencing platforms because of their repetitive and GC-rich nature, were over-represented in the set of genes with all reported H37Ra-specific variants contradicted. Furthermore, we identified a sequencing error in H37Ra that masked a true variant which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome. By identifying sequencing errors in the H37Ra reference genome, the conclusions of several studies are undermined and in some cases invalidated. Through the reduction of genes with variants of unknown effect on virulence, our assembly allows for a more focused and pertinent inquiry into the genetic factors that may contribute to the difference in virulence between H37Ra and H37Rv. Our assembly also illustrates the power and utility of single-molecule sequencing in producing accurate and fully resolved de novo assembled genomes of mycobacteria. Funding: This work has been supported by grants from National Institute of Allergy and Infectious Diseases (NIAID) grant RO1AI05185 and National Science Foundation (NSF) grant 0966391. 98 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 77 Mycobacterial Genomics and Evolution 20/09/2016 HADTB: Hub for Aggregated Data in Tuberculosis, An Aggregated Database for Sharing, Annotating, and Analyzing Tuberculosis (TB) Genomic Data D. Oh, Y. Kim, A. Elghraoui, F. Valafar biomedical Informatics Research Center, San Diego State University, San Diego, United States Recent advances in sequencing have generated vast amounts of genomics data. Existing databases are either not reliably maintained or do not integrate well with existing analysis tools. As such, it is difficult to use this data for reliable knowledge generation that could be used in bedside practice or in public health efforts in controlling outbreaks. To promote collaboration and global analysis, a single database that can aggregate publicly available (genomic and meta) data, provide multi-facetted dissemination interface, and be easily incorporated in bioinformatics pipelines is essential. HADTB is an integrated database that allows users to query, upload, and annotate TB data. Its multi-modal interface allows easy incorporation into custom analysis pipelines. HADTB is community driven and self-sustaining. The backend of HADTB is built on a MySQL database and mapped using SQLAlchemy. The Flask framework is used to create REST API calls where users can access the database through a simple URL. The front end is built using AngularJS framework. This provides more dynamic views than PHP or other alternatives. The web-interface of HADTB is divided into three unique groups; genes, variants and isolates. All the fields in each of these groups are relationally mapped to one another which allows users to retrieve all associated genomic and metadata in a single table view. Moreover, built in report function provides users with broad range of powerful statistical and graphical methods for easy visualization. In addition to community contribution, HADTB automatically aggregates publicly available raw sequencing reads from published papers and uniformly processes them for variant calling. Along with independent gene, variant, isolate, and literature data, the database also features relational mapping where each entity can be viewed with its interactive counterparts for pathway and regulatory analysis. Curation status promotes database integrity by differentiating user uploaded experimental data from verified published data. HADTB provides multiple access points: a web interface, a console client, a Python library, and a REST API. Funding: This work has been supported by grants from National Institute of Allergy and Infectious Diseases (NIAID) grant R01AI05185 and National Science Foundation (NSF) grant 0966391. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 99 78 Mycobacterial Genomics and Evolution 20/09/2016 Identification of Conditionally-Essential Genes of the M. kansasii Genome to Understand the Evolution of Mycobacterial Virulence M. Ghanem1-3-4, F. Mcintosh1-4, J. Wang5, M. Behr1-2-3-4 1 McGill International TB Centre 2Department of Medicine 3Department of Microbiology and Immunology, McGill University 4Research Institute of the McGill University Health Centre, Montreal, Canada 5University of Michigan, Ann Arbor, United States Mycobacterium kansasii, commonly isolated from tap water, is an NTM that can cause a pulmonary TB-like disease. However, there are no records showing person-to-person transmission and infection is usually cleared in healthy individuals. Phylogenetic analyses performed in our lab have shown M. kansasii to be the most closely related environmental species to the M. tuberculosis complex (MTC). Genomic comparisons revealed a genome 2-Mbp larger than that of M. tuberculosis, with orthologues in M. kansasii for at least 65% of all M. tuberculosis antigens. From this, we infer that certain M. tuberculosis virulence genes are not specific to the MTC, but instead are generic factors that contribute to the pathogenesis of disease by multiple mycobacteria. To identify genes that contribute to M. kansasii survival, we use transposon sequencing; a random genome-wide mutagenesis technique that will identify which mutants thrive (anti-virulence genes) or succumb (virulence genes) following pulmonary infection. This method makes use of MycoMarT7; a mycobacteriophage engineered to integrate transposons into mycobacterial genomes in a TAdinucleotide-specific manner. The M. kansasii genome includes ~ 95,000 TA sites dispersed throughout its genome, making MycoMarT7 an ideal mutagenic agent for our purposes. We have created 4 mutant libraries with the goal of covering a large portion of the M. kansasii genome. We have also validated two high-dose infection models in C57/BL6 mice, enabling us to ensure that a greater number of bacteria are studied than the number of genes in the M. kansasii genome. Following a 6-week infection, Illumina HiSeq Next Generation Sequencing will then be used to quantify two output pools, contrasting in vitro growth on Middlebrook agar and the lung homogenate. Both output pools will be contrasted with the input (initial inoculum). This study aims to identify genes that have played important roles during the evolution of different mycobacterial pathogens. These genes are classified into 4 categories: (1) shared amongst all slow-growing mycobacteria, (2) unique to M. kansasii, (3) shared between M. kansasii and other NTMs excluding the M. tuberculosis complex and (4) shared between M. kansasii and M. tuberculosis excluding other NTMs. 100 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 79 Mycobacterial Genomics and Evolution 20/09/2016 Complementation of Mycobacterium tuberculosis-specific genes in M. kansasii to investigate the evolution of TB as a human pathogen M. Ghanem2-4-1, J. Wang6, F. Mcintosh3-1, M. Reed2-4-1-3, P. Domenech4-1, D. Young5, B. Moody5, M. Behr2-4-1-3 1 McGill International TB Centre 2Department of Microbiology and Immunology, McGill University 3Department of Medicine, McGill University Health Centre 4Research Institute of the McGill University Health Centre, Canada 5Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School 6Department of Microbiology and Immunology, University of Michigan, United States Our group has recently executed a genomic comparison between Mycobacterium tuberculosis and M. kansasii, an environmental species that causes a TB-like non-transmissible lung disease. These findings support the hypothesis that the latter can be used as a pertinent model to assess the genetic requirements that led to the emergence of M. tuberculosis as a professional human pathogen. In the present project, we are using M. kansasii as a surrogate host, via gain-of-function studies, to better understand the role of M. tuberculosis-specific genes on the behavior of the microorganism during infection. The genes of interest in this study, Rv3377-8c, are implicated in the synthesis of 1-tuberculosinyladenosine (1-TbAd), a lipid found exclusively in M. tuberculosis. 1-TbAd makes up 1% of all lipids produced by the pathogen, is constitutively synthesized and its absence leads to impeded growth of M. tuberculosis in macrophages, at least partially due to problems with phagosome acidification. We hypothesize that the production of 1-TbAd will provide M. kansasii with the means to become a more robust pathogen. Through heterologous complementation, we have produced a clone of M. kansasii that has successfully gained the ability to produce a denosine-labeled lipids, as s een by TLC chromatography. We have also confirmed the identity of these lipids as 1-TbAd by liquid chromatography–mass spectrometry. Our current efforts focus on assessing the in vitro growth of the complemented M. kansasii strain as well as its abilities to survive and replicate in macrophages and in vivo murine infection models. This study provides proof-of-concept for the role of horizontal gene transfer in the emergence of M. tuberculosis and provides a tool to study the specific contribution of M. tuberculosis- specific genes in the pathogenesis of TB. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 101 80 Mycobacterial Genomics and Evolution 20/09/2016 Comparative genome of Mycobacterium bovis strain 1595 isolated from Korean cattle J.M. Kim, Y.H. Jang, M.H. Hwang, N.R. Kim, H.S. Lee Zoonosis lab, Animal and Plant Quaratine Service, Gyeongsangbuk-Do, South Korea Mycobacterium bovis is the causative agent of bovine tuberculosis and zoonotci and both animal and human health concerns. In this study, we isolated and performed and genomic investigation of M. bovis strain 1595 from laryngopharyngeal lymph node of Korean native cow. M. bovis 1595 was reveled to have SB0140 in spoligotype and 4-2-5-3-2-7-5-5-4-3-4-3-4-3 in MIRU-VNTR typing. Combine these, strain 1595 was categorized the most frequenct patten in Korea. The complete genome sequence of strain 1595 determined using Illumina MiSeq and PacBio single-molecular real-time technology indicated 4,351,712 bp in size with a 65.64% G+C content. And 4,358 proteincoding genes were predicted from this assembly. In addition, we performed brief comparative genomic analysis with genomes of Mycobacterium tuberculosis complex strains which reveled that all genomes have similar size and G+C content. In phylogenetic analysis, all strains located within 0.1% average nucleotide identity value, and MUMmer analysis illustrated all genomes showed positively collinear with strain 1595. Comparison based on BLASTP, the strain that has the most completely matched proteins with M. bovis 1595 was M. bovis AF2122/97. This genome sequence will serve as a valuable reference for improving the understanding of the disparity in the virulence and epidemiologic traits between M. bovis genotypes in South Korea. 102 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 81 Mycobacterial Genomics and Evolution 20/09/2016 The evolution of Mycobacterium tuberculosis in HIV co-infected individuals in an HIV/TB endemic setting A. Koch3-5, D. Brites8-9, D. Stucki8-9, J. Evans6-5, R. Seldon6-5, M. Nicol1-5, T. Oni7, D. Warner6-5, V. Mizrahi6-5, J. Parkhill10, S. Gagneux8-9, D. Martin4-5, R. Wilkinson3-5-12-11 1 National Health Laboratory Service 3Clinical Infectious Diseases Research Initiative 4Division of Computational Biology 5Institute of Infectious Disease and Molecular Medicine 6Molecular Mycobacteriology Research Unit 7School of Public Health and Family Medicine, University of Cape Town, Cape Town, South Africa 8Swiss Tropical and Public Health Institute 9University of Basel, Basel, Switzerland 10The Wellcome Trust Sanger Institute, Hinxton, Cambridge 11Francis Crick Institute 12Department of Medicine, Imperial College, London, United Kingdom Infection with HIV increases the risk of infection with tuberculosis (TB), and co-infection leads to acceleration of both diseases. However, the biological interactions between HIV-1, Mycobacterium tuberculosis (Mtb), and the human immune system are incompletely understood. We applied comparative genomics to investigate the impact of HIV-1 co-infection on the evolution of Mtb strains circulating in Khayelitsha, Cape Town, South Africa, a region with high rates of HIV-associated TB. Evolutionary models, Fast Unconstrained Bayesian Approximation (FUBAR) and Model of Episodic Directional Selection (MEDS), were applied to whole-genome sequence (WGS) data for 169 Mtb strains. These phylogenetically-informed models, not previously applied to Mtb, allowed evaluation of the relative nonsynonymous (dN) and synonymous (dS) substitution rates at individual codons. Natural selection was evaluated by FUBAR, without compartmentilising Mtb sequences according to HIV-1 status. Classification of resultant dN/dS values into functional gene categories revealed differences across several categories. MEDS was then applied to evaluate relative differences in positive selection in strains isolated from HIV-1 co-infected or uninfected individuals. The proportion of codons showing evidence of relative differential positive selection was different in Mtb strains isolated from HIV-1 co-infected individuals, including codons in virulence-associated and epitopeencoding genes. A lack of detailed clinical/epidemiological metadata meant that transmission patterns and, therefore, the evolutionary history of the Mtb strains could not be determined. While this could undermine the attribution of evolutionary differences to a particular environmental variable (HIV-1 infection, in this case), MEDS has been shown to be relatively conservative and robust against these uncertainties. Morevoer, the same differences were not observed in a control dataset where HIV-1 status was randomly assigned to Mtb sequences, indicating that the data are not an artifact of the evolutionary model applied. These data suggest that patterns of evolution of Mtb may differ in HIV-1 co-infected individuals. HIV-1-associated differences in epitope evolution may have important implications for the design of Mtb vaccines intended for use in populations with high rates of HIV-1 infection. This work paves the way both for validation of these findings on larger Mtb WGS datasets, and investigations of the underlying causes of putative differential selection during HIV-1 co-infection. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 103 82 Mycobacterial Genomics and Evolution 20/09/2016 The true origins and evolution of tuberculosis D.E. Minnikin University of Birmingham, Birmingham, United Kingdom Increasingly, genomic studies are being interpreted in various ways to suggest definitive evolutionary pathways for tuberculosis (TB). However, such interpretations are often simply correlations leading to unsupported estimates of the age of the disease, from 6ka to 70ka! Considering practicalities, evolution from environmental Mycobacterium kansasii, through an intermediate “Mycobacterium canettii” taxon, to Mycobacterium tuberculosis sensu stricto is a good working hypothesis, supported by strong phylogenetic links in cell envelope lipid composition. The transformation from “smooth” morphology “M. canettii” to “rough” M. tuberculosis correlates with significant changes in cell envelope surface lipids1,2. Indeed in original studies1, it has been shown that such differences in lipid profiles correspond to a dramatic change in overall cell hydrophobicity, with M. tuberculosis being orders of magnitude more hydrophobic than “M. canettii”. Increased hydrophobicity is closely linked with enhanced aerosol transmission and overall pathogenicity. Although a coherent, plausible pathway for the bacterial aspects of tuberculosis evolution is becoming clear, zoological and paleogeographical aspects need to be established. Using DNA fragment amplification and lipid biomarker detection, tuberculosis in Homo sapiens has been confirmed in skeletons from the “Fertile Crescent” back to 9-11ka BP1. Pleistocene evidence for tuberculosis is principally confined to megafauna exhibiting specific metapodial lesions1. For a 17ka bison metacarpal from Natural Trap Cave, Wyoming, tuberculosis was confirmed by detection of strong lipid biomarkers and DNA amplification1. In ongoing studies, tuberculosis lipid biomarkers have been detected in a ~40ka bison from Kent’s Cavern, Torquay and a range of ~12ka mastodons from Buffalo, NY. Supported by micro-CT scanning, lesions are being pinpointed in widespread bison metacarpals dating back to ~700ka. Such findings suggest a practical venue, in megafaunal stomachs, for transforming environmental M. kansasii into “M. canettii”, the first recognisable tubercle bacillus. Late Pleistocene dramatically enhanced hydrophobicity in tubercle bacilli could well have produced megafaunal pandemics and extinction of bison and other contempory taxa. 1. Minnikin et al. In: Ribón, W. (Ed), Tuberculosis –Expanding Knowledge. InTech-Open Access, Rijeka, 2015 pp. 145-175; Tuberculosis 2015;95:S133-S139; Biofutur no 265, May 2015. 2. Boritsch et al. Nature Microbiology 2016;1:15019. 3. Rothschild & Martin Naturwissenschaften 2006;93:565-569. 104 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 83 Mycobacterial Genomics and Evolution 20/09/2016 Lineage-based analysis of Genomic Methylation and variations within Methyltransferases in Mycobacterium tuberculosis Using Kinetic Data from Single-Molecule Real-Time (SMRT) Sequencing Technology in Clinical Isolates S. Modlin2, A. Elghraoui2, S. Hoffner1-2, F. Valafar2 1 Karolinska Institute, Stockholm, Sweden 2biomedical Informatics Research Center, San Diego State University, San Diego, United States Methylation is a type of epigenetic modification, the effects of which are poorly understood in prokaryotes. It is well-known that many methyltransferases (MTases) combine with a cognate restriction enzyme (RE) to create a primitive immune system termed restriction-modification (R-M) systems. MTases that lack a cognate RE are termed “orphan” Mtases and are believed to serve functions other than R-M-mediated immune defense. Recent work has revealed Mtb has at least three MTases, of which two appear to be orphan Mtases. Using kinetic data, Pacific Biosciences' SMRT -sequencing allows for detection of methylation at single nucleotide resolution concurrently with SMRT-sequencing. Methylation affects gene expression in Mtb in response to hypoxia, presumably allowing phenotypic adaptation to the host-environment. Methylation-mediated phenotypic changes occur relatively quickly allowing the bacterium to survive under the drug/immune system pressure without chromosomal adaptation. Understanding how this capability differs within and between lineages provides important information about lineage-specific adaptation towards resistance, persistence, virulence, etc. Using SMRT-sequencing we sequenced and de novo assembled genomes of 69 clinical isolates of Mtb from 5 countries comprising 4 lineages. We compared activity of three known MTases by examining methylation patterns in each isolate, and compared MTase sequences of each isolate to a functional reference MTase to profile which mutations abrogated MTase activity, and which were not deleterious. We analyzed how these were dispersed within and between lineages, revealing that methylation activity of each MTase was consistent within lineages, but varied between them. We found several missense mutations in each MTase, some of which abrogated methylation, while others did not. MTase SNP patterns were also consistent within lineage, but different across lineages, suggesting significant selective pressure on MTase functionality in Mtb. Future work will examine methylated motif distribution in association with lineage and drug-resistance, to provide a picture of which genes’ expression levels may be affected by methylation. Funding: This work has been supported by grants from National Institute of Allergy and Infectious Diseases (NIAID) grant R01AI05185 and National Science Foundation (NSF) grant 0966391. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 105 84 Mycobacterial Genomics and Evolution 20/09/2016 Mycobacterium tuberculosis population structure in Brazzaville, Republic of Congo – identification of a new dominant H37Rv-like sublineage associated with transmission S. Malm, L.S. Ghoma Linguissi, E.M. Tekwu, T.A. Kohl, P. Beckert, J.C. Vouvoungui, A. Sidibe, S. Rüsch-Gerdes, I.K. Madzou- Laboum, S. Kwedi, V. Penlap Beng, M. Frank, F. Ntoumi, S. Niemann Molecular and Experimental Mycobacteriology Group, Research Center Borstel, Borstel, Germany The Republic of Congo has a tuberculosis (TB) incidence of 381 cases/100,000 inhabitants and thus is considered as a high TB burden country. Its population counts 4 million inhabitants and approximately one fourth of the population agglomerates in its capital city, Brazzaville. Precise data on the population structure and transmission dynamics of Mycobacterium tuberculosis complex (MTBC) strains are key for effective TB control and have not been addressed for the Republic of Congo yet. Thus, we characterized 74 MTBC strains isolated from patients with pulmonary TB in Brazzaville by whole genome sequencing. The population diversity was high, with the majority of strains belonging to the Euro-American lineage which split into LAM, Uganda I, Uganda II, Haarlem, X-type and a new dominant sublineage termed the Congo-type (n = 26). Thirty strains were grouped in 5 genome clusters with a maximum distance of 12 SNPs out of which 23 belonged to the newly described Congo-type sublineage. Our study provides the first insights in the population structure of MTBC strains in the Republic of Congo. High cluster rates and low genome diversities indicate recent emergence and ongoing transmission of the Congo-type strains, a new Euro-American sublineage. 106 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 85 Mycobacterial Genomics and Evolution 20/09/2016 Characterization of the Mycobacterial Integration Host Factor N. Odermatt1, M. Lelli2, T. Herrmann2, N. Dhar1, S. Cole1 1 GHI, SV, UPCOL, EPFL, Lausanne 2ISA CRMN, Université de Lyon, Villeurbanne, Switzerland The nucleoid associated protein (NAP) mycobacterial integration host factor (mIHF, rv1388) is one of the most abundant proteins in Mycobacterium tuberculosis (Mtb). Although there is no sequence similarity to the E. coli homologue, mIHF might not only be responsible for phage DNA integration, but have a similar important role as IHF in E. coli, which acts as a global transcription factor and DNA modelling protein. We found that, in contrast to other NAPs, mIHF is present at the same high level in all growth phases of Mtb. The coding sequence of the mihF gene is only about 60 % of the originally annotated gene sequence, shrinking the protein to a size of 12 kDa. Upon construction of a conditional knockdown mutant in Mtb, we were able to prove its essentiality and observed a severe growth defect when mIHF was reduced to a level of below 10 % compared to the wildtype. Allowing expression of mihF again, the batch culture recovered within three days and grew at a rate comparable to the wildtype. Microfluidic analysis showed that depletion of mIHF led to cell death or lysis, and in contrast to batch culture did not regain growth in permissive conditions. To further understand the mode of DNA binding and gene regulation, we purified the mIHF protein, which cannot be detected by UV absorption, and showed that it is not only highly soluble with a concentration of up to 50 mg/ml but also very stable even at room temperature for several days. Its structure, determined by nuclear magnetic resonance, showed a very high similarity to its monomeric Streptomyces homologue, although we found some evidence that mIHF is forming dimers under certain conditions. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 107 86 Mycobacterial Genomics and Evolution 20/09/2016 The First Population Structure and Comparative Genomics Analysis of Mycobacterium africanum strains from Ghana Reveals Higher diversity of Lineage 5 I.D. Otchere1-2, S. Harris4, S.L. Busso4, A. Asante-Poku1, S. Osei-Wusu1, K. Koram1, J. Parkhill4, S. Gagneux3, D. Yeboah-Manu1 1 Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana 2Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Accra, Ghana 3Swiss TPH, Basel, Switzerland 4Genome Campus, Wellcome Trust Sanger Institute, Cambridge University, Cambridge, United Kingdom Background: Mycobacterium africanum (MAF) remains an important TB causing pathogen in West Africa however, little is known about its population structure and actual diversity which may have implications for diagnostics and vaccines. We carried out comparative genomics analysis of candidate Mycobacterium tuberculosis (MTB) and MAF using whole genome sequencing. Methods: Clinical MTBC strains (187) comprising L4 (22), L5 (126) and L6 (39) isolated over 8 years from the Northern and Southern parts of Ghana were whole genome sequenced. The reads were mapped unto a reference genome for phylogenetic and functional genomics analysis. Maximum Likelihood tree with 100 bootstraps was constructed from the SNPs called using RAxML. A total of 147 (18 L4, 36 L6 and 93 L5) of the genomes were de novo assembled and annotated for comparative pan-genome analysis using Roary. Results: The population structure of MAF showed at least 10 sub-lineages of L5 and 3 under L6. There were year specific clusters of L5 in 2013, 2012 and 2008. We also identified a cluster of 3 MDR L5 strains from Southern Ghana in 2013. Among the global collection of MTBC, there were two Ghana-specific L5 clusters of which one exhibited clonal expansion. From the 5947 pan genes extracted from the collection, 3215 (54.1%) were core to all the 147 genomes whereas 719 (12.1%) were found in single genomes. We identified the absence of some unique genes among specific lineages and/or clades with possible clinical implications. For example, mpt64 and mlaD encoding respectively an immunogenic protein and a mammalian cell entry protein were missing from all L6 genomes. Analysis of SNPs within some genes encoding proteins for substrate metabolism, ion transport and secretory systems showed higher proportion of SNPs among L6 compared to L5 and L4. We also identified a number of Lineage/sub-lineage specific SNPs that may be utilized in rapid PCR based genotyping of MTBC. Conclusion: Contrary to the notion that MAF is an ancient species and may show less diversity as compared to MTBSS, MAF especially L5 displayed an extensive diversity similar to what is known about L4. The diversity observed has implications for current and future diagnostic and vaccine prospects. 108 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 87 Mycobacterial Genomics and Evolution 20/09/2016 Understanding the regulation of cytochrome P450s: core enzymes involved in M. tuberculosis metabolism A. Otter, S. Kendall Dept. of Pathology & Pathogen Biology, Centre for Emerging, Endemic and Exotic Diseases, Royal Veterinary College, London, United Kingdom The genome of Mycobacterium tuberculosis encodes for 161 helix-turn-helix DNA binding transcriptional regulators. The most abundant of these (32%) belong to the TetR family. This family of regulators were first described in Escherichia coli and are best known for their control of efflux pumps. However, more recent understanding derived in both Streptomyces and mycobacteria indicates that this family are frequently regulators of oxidoreductases. In M. tuberculosis and Streptomyces, 36-40% of genes located next to TetR regulators are oxidoreducatase enzymes and not membrane proteins as previously thought, with membrane proteins only representing 10-24% of TetR associated genes.. These insights from both genomes suggest that the majority of TetR regulators may be crucial regulators of metabolic pathways [1, 2]. In M. tuberculosis, TetRs have been identified to control enzymes required for branched chain amino acid catabolism and cholesterol catabolism, a pathway that has an established role in virulence [3-5]. TetR regulators often regulate nearby genes and bioinformatic analysis of the M. tuberculosis genome shows that 11 TetR regulators are within close proximity to gene(s) encoding Cytochrome P450s (CYPs). CYPs are absent in many bacteria, but mycobacteria are an exception, with 20 CYP genes located throughout the genome of M. tuberculosis [6]. CYPs have been found to play key roles in metabolism, sterol transformation and synthesis of host lipids, providing attractive drug targets as previous research has shown antifungal drugs, particularly azole based compounds, to be potent CYP inhibitors. Though numerous CYPs have been studied for their function and role in M. tuberculosis, very little is known about their regulation. To further understand TetRs and their role in CYP regulation, we are expressing and purifying candidate TetRs to demonstrate binding to CYP promoters, identifying regulatory motifs within CYP promoters using MEME and searching for motifs on a genome wide scale using FIMO. In the future we will also make deletion mutants of both the TetRs and CYPs in order to better understand their regulation and physiological role in M. tuberculosis. 1. Ahn SK, Cuthbertson L, Nodwell JR: Genome Context as a Predictive Tool for Identifying Regulatory Targets of the TetR Family Transcriptional Regulators. PLoS One 2012, 7(11). 2. Balhana RJC, Singla A, Sikder MH, Withers M, Kendall SL: Global Analyses of TetR Family Transcriptional Regulators in Mycobacteria Indicates Conservation across Species and Diversity in Regulated Functions. BMC Genomics 2015, 16. 3. Balhana RJ, Swanston SN, Coade S, Withers M, Sikder MH, Stoker NG, Kendall SL: bkaR is a TetRtype Repressor That Controls an Operon Associated with Branched-Chain Keto-Acid Metabolism in Mycobacteria. FEMS Microbiology Letters 2013, 345(2):132-140. 4. Kendall SL, Burgess P, Balhana R, Withers M, Ten Bokum A, Lott JS, Gao C, Uhia-Castro I, Stoker NG: Cholesterol Utilization in Mycobacteria is controlled by Two TetR-Type Transcriptional Regulators: kstR and kstR2. Microbiology 2010, 156(Pt 5):1362-1371. 5. Kendall SL, Withers M, Soffair CN, Moreland NJ, Gurcha S, Sidders B, Frita R, Ten Bokum A, Besra GS, Lott JS et al: A Highly Conserved Transcriptional Repressor Controls a Large Regulon Involved In Lipid Degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis. Molecular Microbiology 2007, 65(3):684-699. 6. McLean KJ, Belcher J, Driscoll MD, Fernandez CC, Le Van D, Bui S, Golovanova M, Munro AW: The Mycobacterium Tuberculosis Cytochromes P450: Physiology, Biochemistry & Molecular Intervention. Future Medicinal Chemistry 2010, 2(8):1339-1353. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 109 88 Mycobacterial Genomics and Evolution 20/09/2016 Comparative genomic analysis of hypervirulent Mycobacterium tuberculosis strains and closely related hypovirulent strains reveals novel mutations associated with enhanced growth during macrophage infection R. Rajwani, G. Siu, W.C. Yam Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, China Background: Among clinical strains of Mycobacterium tuberculosis (MTB), the relationship between genetic diversity and disease severity remains inadequately understood which hinders development of new and improved vaccine against adult tuberculosis (TB). We had previously isolated two hypervirulent MTB strains (H107 and H112) from immunocompetent patients suffering from tuberculous meningitis. Both strains demonstrated significant higher bacillary growth in ThP1 macrophage and lower T-cell helper cell 1 (Th1) response when compared to virulent reference strain M. tuberculosis H37Rv and other ordinary pulmonary clinical strains. Here, we describe the whole genome sequencing results of the hypervirulent strains in comparison with other two hypovirulent pulmonary strains (H54 and H83) of same lineage. Methods: Genomic DNA was extracted from late-log phase cultures of MTB strains. 20-Kb single molecule real-time (SMRT)-bell libraries were prepared and sequenced using P6-C4 chemistry. The resulting reads with an average length of 8Kb were assembled through hierarchical genome assembly process (HGAP). Fully closed circular genomes were obtained for all strains which were annotated using Glimmer3. For genome comparisons, raw reads were aligned against reference Mycobacterium tuberculosis H37Rv genome (refseq accession no. NC_000962.3) and variants were called using Quiver. Variants were annotated using ensembl variant effect predictor (VEP). Further analysis and comparisons were performed in SAMtools./BCFtools. Literature was reviewed to identify variants associated with hypervirulence. Result: A total of 1297 single nucleotide polymorphisms and 1004 indels that are specific to hypervirulent strains were identified, in which two frame-shift deletions and seven missense variants in genes previously shown to confer hypervirulence in MTB. These include mutations in mammalian cell entry 1 (mce1) operon including a frameshift deletion in mce1D (H107) and misense mutations in lipoprotein LprK (H107) and integral membrane protein YrbE1B (H112). We also identified a glutamic acid to lysine amino acid substitution in sensor kinase domain of KdpD (H112). Conclusion: The current study identifies possible mechanisms of enhanced virulence in H107 and H112. In our laboratory, we are developing allele complementation based functional assays to prove that these variants are indeed the underlying reason of hypervirulence in these strains. 110 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 89 Mycobacterial Genomics and Evolution 20/09/2016 M. tuberculosis whole genome sequence analysis reveals cell wall-associated genes as targets of convergent evolution in tuberculous meningitis C. Ruesen4, L. Chaidir2, A. Van Laarhoven4, S. Dian1, A. Rizal Ganiem1, M. Huijnen3, B. Alisjahbana2, B. Dutilh3, R. Van Crevel4 1 Department of Neurology, Faculty of Medicine, Padjadjaran University/Hasan Sadikin Hospital 2Health Research Unit, Faculty of Medicine, Padjadjaran University/Hasan Sadikin Hospital, Bandung, Indonesia 3Centre for Molecular and Biomolecular Informatics, Radboud university medical center 4Department of Internal Medicine, Radboud university medical center Nijmegen, Nijmegen, The Netherlands Background: Meningitis is the most severe manifestation of tuberculosis. It is largely unknown why certain people develop pulmonary TB (PTB) and others TB meningitis (TBM), but we hypothesize that genetic diversity of infecting M. tuberculosis strains plays a role. Methods: M. tuberculosis strains isolated from HIV-negative patients, 102 from cerebrospinal fluid of TBM patients, and 176 from sputum of PTB patients were whole-genome sequenced using Illumina HiSeqTM2000 and aligned to the reference genome M. tuberculosis H37Rv. A maximum likelihood phylogenetic tree was constructed based on multiple alignment of the common variable positions. M. tuberculosis genetic variation was linked to clinical TB phenotype by looking for phylogenetic convergence: the repeated appearance of phenotype-related changes at specific loci or genes in phylogenetically unrelated branches. Enrichment scores for the TB phenotype with associated p-values were calculated for each SNP. Candidate SNPs were validated in an independent set of isolates. Results: Strains belonged to the East-Asian Beijing lineage (35.6%), East-Asian non-Beijing lineage (0.7%), Euro-American lineage (61.2%), and Indo-Oceanic lineage (2.5%). We found no association between lineage and phenotype (Chi-square = 5.095; p=0.165). Large genomic differences were observed between the isolates; the minimum genetic distance varied from 5 to 952 SNPs. The phylogenetic tree, based on 25198 common variable positions, revealed 104 isolates in terminal branch pairs with distinct phenotypes, with 38 to 544 SNPs difference between paired isolates. Mutations in Rv1396c (PE-PGRS25) and Rv1899c (LppD) were enriched for TBM in paired isolates (p=0.025 and p=0.045 respectively), and validated in the set of 174 unpaired isolates (p=0.025 and p=0.011). Conclusion: Using phylogenetic convergence analysis we identified mutations in 2 possibly immunoregulatory genes that might be important in TBM pathogenesis. These cell wall-associated genes play a role in host-pathogen interaction, possibly through pathogen recognition and antigen presentation. Further validation may come from independent cohorts, or experimental or in-vitro studies. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 111 90 Mycobacterial Genomics and Evolution 20/09/2016 Dynamics of Bovine Tuberculosis Disease Transmission in Egypt: The “Real” Jewel of the Nile A. Talaat, H. Abdelaal Pathobiological Sciences, University of Wisconsin-Madison, Madison, United States Bovine tuberculosis (BTB) is a chronic infectious disease characterized by the formation of granulomatous lesions in organs, mainly lungs and lymph nodes. BTB is caused by slowly growing bacilli, mainly, M. bovis. In the developing countries, BTB represent a major problem with a prevalence that could reach up to 10-15% of cattle herds in some parts of Africa. In Egypt, BTB transmission among animals and from animals to humans represents a major problem because of the complexity of animal husbandry and animal trade. In this project, our teams in both USA and Egypt used Single Intradermal Comparative Skin Test (SICST) and whole genome sequence analysis (WGSA) to examine the dynamics of BTB transmission among 5 different regions within the Nile Delta of Egypt. During the years of 2012-2015, several farms were visited and M. bovis isolates from the same herds were analyzed. As expected, farms with a known history of risky management behavior were associated with high level of BTB prevalence that reached up to 45% in some herds. Interestingly, WGSA was able to trace dynamics of BTB transmissions among herds when source and timing of infection were verified with available historical data. Moreover, isolates belong to M. bovis BCG lineage or those with drug resistance phenotypes were identified. In this presentation, we will discuss the key outcomes of this project highlighting the importance of ecological combined with genomic (EcoGenomic) analyses of BTB, especially in enzootic countries. Currently, we are investigating the virulence of M. bovis isolates with different genomotypes. 112 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 91 Mycobacterial Genomics and Evolution 20/09/2016 Prevalence of PZase, PanD, and RpsA mutations in multidrug- and extensively-drug resistant Mycobacterium tuberculosis Clinical Isolates S. Ramirez-Busby2, L. Fink2, T. Rodwell3, M. Pettigrove3, L. Jackson3, D. Catanzaro1, A. Goodmanson2, A. Amallraja2, R. Shanmugan2, A. Catanzaro3, F. Valafar2 1 Department of Biology, University of Arkansas, Fayetteville 2Biomedical Informatics Research Center, San Diego State University 3Medicine, University of California, San Diego, San Diego, United States Pyrazinamide (PZA) is an important first-line drug recommended by the World Health Organization (WHO) and included in all new treatment regimens to treat the highly infectious M. tuberculosis. However, prevalence of resistance to PZA continues to rise while reliable phenotyping has yet to be established. Three tests were performed on our isolates: DST, Wayne’s enzymatic assay, and whole-genome sequencing. Coding and promoter regions of three genes—pncA, rpsA, panD—were investigated for markers of PZA resistance. Enzyme activity was used when there was phenotypegenotype discordance. Two hundred and thirty-five resistant (PZAR) and 80 susceptible (PZAS) isolates were included in this study. High concordance was observed between DST and enzymatic assay but less so with the genotype. 86% of PZAR and 30% of PZAS isolates had a mutation in PZase or its promoter. Fourteen percent of PZAR isolates lacked a genetic explanation for the observed resistance phenotype. In our population, only 6 (2%) PZAR isolates did not have a PZase mutation but had a mutation in RpsA or PanD. Assuming that all RpsA and PanD mutations cause resistance, the combination of PZase, RpsA, and PanD potentially offer a combined sensitivity of 88% for prediction of PZA resistance phenotype. Overall, this highlights the need for discovery of alternative mechanisms of resistance for PZA, at least in resistant isolates that do not have a PZase mutation. Funding: This work has been supported by grants from National Institute of Allergy and Infectious Diseases (NIAID) grant R01AI05185 and National Science Foundation (NSF) grant 0966391. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 113 92 Mycobacterial Genomics and Evolution 20/09/2016 MIRU-Heuristics for Evaluation of Repeats and their Ordinal (MIRU-HERO): MIRU analysis on Genomic Sequencing Data L. Fink, F. Valafar biomedical Informatics Research Center, San Diego State University, San Diego, United States A technique regularly used by public health agencies to distinguish between strains of Mycobacterium tuberculosis (Mtb) in an outbreak is MIRU-VNTR typing. While the technique is relatively fast, it does not offer the resolution that whole genome sequence (WGS) analysis provides. Since WGS technologies are quickly becoming more accessible and less costly, it is probable that they will eventually be commonly used in contact tracing and analysis of Mtb outbreaks. In an effort to correlate the public health efforts of the PCR era to those of the WGS era, MIRU-HERO has been developed as a stand-alone, command-line accessible program for Linuxbased systems to quickly and efficiently analyze 24-locus MIRU-VNTR data in reference-based or de novo-assembled genomes. The benefits of this system are three-fold: privacy, transparency and resolution. MIRU-HERO uses a set of subject MIRU-VNTR sequences to BLAST against a query genome in fasta format. Sequences in the genome which match up to 97% identity of a subject sequence are appended to a list. Sequential matches from the list are correlated to a MIRU region, and the results are output to a file including the miru name, number of copies detected, and the specific regions of detection in the genome (denoted by base pair position number). The program was run successfully on a set of 75 in-house denovo-assembled genomes with 97.6% correlation to laboratory conducted MIRU-typing. MIRU-HERO is both functional and applicable in the determination of MIRU-VNTR information for mycobacterial genomes. MIRU-HERO resolves privacy issues that web-based programs face as it is a locally installed program not needing to transmit sensitive medical information over the internet. Additionally, compared to conventional PCR based approaches in MIRU typing, there is an increased level of detail which can be used to resolve differences between mycobacterial strains. Funding: This work has been supported by grants from National Institute of Allergy and Infectious Diseases (NIAID) grant R01AI05185 and National Science Foundation (NSF) grant 0966391. 114 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 93 Mycobacterial Genomics and Evolution 20/09/2016 Whole genome phylogenetic analysis of clinical isolates from high-burden countries C. Chan2, R. Shanmugan2, S. Hoffner1, F. Valafar2 1 Karolinska Institute, Stockholm, Sweden 2biomedical Informatics Research Center, San Diego State University, San Diego, United States Lineage-specific differences in MTBC play an important role in virulence and emergence of drugresistance. Most recent research indicates that genome-wide single nucleotide polymorphisms (SNPs) are good markers for lineage analysis compared to previous systems (like spoligo/MIRU). This study includes 368 mostly MDR/XDR clinical isolates collected from India, Moldova, South Africa, the Philippines, and 7 mono-PZA resistant isolates from Sweden. We used Pacific Biosciences’ single molecule sequencing technology for our WGS approach. Based on our analysis, we identified 36,634 unstable loci in Mtb genomes that could harbor a SNP. Nearly 12K of these positions are more prone to indicate heterogeneity. RAxML was used to create a high-resolution phylogenetic tree. Using the clustering patterns obtained from our tree, five isolates were identified as discordant with lineages predicted using MIRU12/spoligo patterns. Bootstrap values for these isolates were high indicating high-confidence grouping, and their lineages confirmed using PhyTB. Using our tree, we were also able to resolve some sub-lineages within the four discovered lineage groups. Ancestral reconstruction of SNPs identified 6247 potential lineage markers and 498 genes under convergent selection. Comparison of our lineage-associated SNPs with those determined by Coll et al. (2014), identified 2028 common SNPs, while 11 SNPs were associated with different lineages than those reported by Coll et al. Six of the 11 SNPs are harbored by PE/PPE genes. 236 SNPs identified by Coll et al. as associated with a single lineage were associated with multiple lineages in our isolates. On examining convergent evolution patterns in the tree, it was noted that almost a third of the SNPs indicative of convergent evolution occurred within PE/PPE genes. This suggests the constantly changing nature of the PE/PPE gene family and the need for further investigation of PE/PPE genes, which may only be elucidated using sequencing with longer reads. Funding: This work has been supported by grants from National Institute of Allergy and Infectious Diseases (NIAID) grant RO1AI05185 and National Science Foundation (NSF) grant 0966391. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 115 94 Mycobacterial Genomics and Evolution 20/09/2016 Current perspectives on the glycoside hydrolases of Mycobacterium tuberculosis: their importance and prospects on assigning function to unknowns N. Van Wyk3, L. Kremer3-4, M. Drancourt2, B. Henrissat1-5 1 Architecture et Fonction des Macromolécules Biologiques (AFMB), Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257 CNRS, Université Aix-Marseille 2Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Centre National de la Recherche Scientifique (CNRS), Institut de Recherche pour le Développement, Faculté de Médecine, Aix-Marseille Université, Marseille 3Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Centre National de la Recherche Scientifique FRE3689, Université de Montpellier 4INSERM, Montpellier, France 5Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia Glycoside hydrolases (GHs) are the enzymes that catalyze the hydrolysis of glycosidic bonds in glycoconjugates, oligo- and polysaccharides. A classification of these enzymes based on conserved sequence and structure motifs initiated by the Carbohydrate Active Enzyme (CAZy) database has proven useful in the systematic groupings of similar enzymes into families. The human pathogen Mycobacterium tuberculosis employs 30 GHs to perform a variety of different functions which can be divided into four broad categories: alpha-glucan metabolism, peptidoglycan remodelling, betaglycan hydrolysis and α-demannosylation. The poster presented here provides an overview of how the GHs that have been characterized play a role in each category. Expanding the genomic analysis of GH presence to other Mycobacterium species has highlighted the importance of certain families – most notably GH13 and GH23 – in the general genomic make-up of mycobacteria. Since many GHs are still uncharacterized and considered as “conserved hypothetical”, the grouping of them into respective families provides a strong prediction on their putative biological functions. 116 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 95 Mycobacterial Biology & Physiology 20/09/2016 Development of an innovative Point-Of-Care (POC) device for Tuberculosis Diagnosis E. Ressami2, B. Lakssir2, M. Abid3, H. Ait Benhassou1 1 Medical Biotechnology Center 2Microelectronics Packaging Center, Moroccan Foundation for Advanced Science, Innovation and Research, Rabat 3Laboratoire de Génétique Mycobactérienne, Institut Pasteur du Maroc, Tangier, Morocco Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. It is caused by bacteria called Mycobacterium tuberculosis that infects a new person every second in the world. Each year, about 1% of the world population is infected and about 9 million people develop the disease, resulting in approximately 2 million deaths per year. In Morocco, as in most developing countries in North Africa and the Middle East, where the timely and accurate diagnosis of TB remains a great challenge, TB constitutes a major public health threat with 30,000 new cases each year. Recently, Morocco and other countries in the MENA region have become a primary destination of new waves of refugees from Syria and Sub-Saharan countries, which increased the difficulty in the control and the management of TB. Currently, the conventional methods routinely used to diagnose TB such as smear microscopy and bacterial culture are complex, unreliable, technically challenging and time-consuming at point-of-care settings. The goal of this project is to contribute in reinforcing the implemented national and regional public health strategy in the effective management of tuberculosis among local populations, Syrian and Sub-Saharan refugees living in Morocco and in the MENA region. Indeed, through this proposal, we aim to reduce susceptible and resistant tuberculosis transmission, morbidity, and mortality by enabling early TB screening and diagnosis. The idea of the project is to establish an innovative platform combining new advanced high-tech approaches, tools and techniques in Molecular biology, Microelectronics and Photonics. Thus, we developed, a mobile phone-based, easy-to-use readout device to perform a LAMP (< $US 2.0) in less than 15 min reaction. This LAMP assay have several advantages that are relevant and attractive as a diagnostic platform for resource-poor settings: it is specific, highly sensitive, fast and generates a result that can be directly detected by an automated phone camera and remotely transmitted to clinical centers. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 117 96 Mycobacterial Biology & Physiology 20/09/2016 DrrS, a DosR regulated small RNA in Mycobacterium tuberculosis A. Moores3, A. Riesco3, S. Waddell1, D. Young2, K. Arnvig3 1 University of Sussex, Brighton 2Francis Crick Institute 3Structural and Molecular Bilogy, University College London, London, United Kingdom M. tuberculosis (Mtb) depends on an ability to adjust to stresses delivered by a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. Our aim is to elucidate this aspect of Mtb gene regulation by applying a multidisciplinary approach complementing an RNA-seq systems level analysis with more traditional techniques including Northern blotting, qRT-PCR and 5’/3’ RACE. We have previously identified several sRNAs in Mtb, and their sheer abundance, and the fact that many are part of pathogenesis-associated regulons suggest that they make a significant contribution to the regulation of gene expression during infection. This study is focussed on the sRNA DrrS, which is regulated by the dormancy master regulator DosR. We have investigated DrrS expression under different growth conditions and used reporter gene assays to define its promoter elements. We have found that DrrS is highly stable and generated by rapid processing of a longer transcript, DrrS+. Moreover, we have investigated the turnover of the mature DrrS and identified RNA determinants involved in its extraordinary stability. In order to elucidate the biological function of DrrS, we have combined reverse genetics with microarray analysis and identified >200 genes that are differentially expressed in a ΔdrrS strain compared to the parental Mtb H37Rv with significant enrichment of genes belonging to information pathways. By subsequently interrogating this data using in silico target prediction, we have identified a small subset of genes that are potentially direct targets of DrrS; among these are genes associated with information pathways, amino acid biosynthesis and two major secretion pathways in Mtb. In summary, our study is a comprehensive analysis of the Mtb sRNA, DrrS in terms of expression, processing, stability and target identification, which makes an important contribution to our growing knowledge of Mtb RNA biology. 118 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 97 Mycobacterial Biology & Physiology 20/09/2016 The identification of novel proteins involved in iron-sulphur cluster biogenesis in mycobacteria J. Arries, S. Sampson, R. Warren, M. Williams Department of Biomedical Sciences, Stellenbosch University, Cape Town, South Africa Mycobacterium tuberculosis remains a public health threat. Given the emergence of drug resistance, it is imperative that we improve our understanding of the pathogenesis of Mtb, in order to develop new anti-TB drugs and strategies for shortening drug treatment. Iron-sulphur clusters are ubiquitous cofactors required for the maturation of various proteins, many of which are involved in essential biological processes. Multiprotein complexes are required for the in vivo assembly of Fe-S clusters, and the SUF system, encoded by the Rv1460-Rv1461-Rv1462Rv1463-csd-Rv1465-Rv1466 operon in Mtb, is thought to be the major Fe-S cluster biogenesis machinery. This process is poorly understood in mycobacteria, and it is currently unclear if proteins outside of this operon are involved in Fe-S cluster biogenesis. In this study, we sought to identify novel proteins involved in Fe-S cluster assembly. We employed affinity purification and mass spectrometry to identify proteins that interact with the SUF machinery. Utilising this method, we were able to demonstrate affinity enrichment for Fe-S cluster assembly proteins, and Fe-S cluster-containing enzymes, as well as identify potential novel interacting partners. This work has established the methodology for identifying novel protein-protein interactions, and lays the foundation for elucidating the process of Fe-S cluster assembly in mycobacteria. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 119 98 Mycobacterial Biology & Physiology 20/09/2016 Ergothioneine is essential for Mycobacterium tuberculosis growth during starvation and long term infection M. Richard-Greenblatt, A. Steyn, H. Bach, Y. Av-Gay Infection and Immunity Research Centre, University of British Columbia, Vancouver, Canada Ergothioneine (EGT), an antioxidant obtained from the diet in mammalian systems, is synthesized by M. tuberculosis (Mtb). EGT is derived from histidine and contains sulfur bound to its imidazole ring, imparting unique chemical properties to this amino acid. Unlike other sulfur-containing molecules, such as the low molecular weight (LMW) thiol, and glutathione (GSH), EGT exists primarily as a thione under physiological conditions. The chemical structure of EGT, which provides the molecule with enhanced stability to oxidation. Nonetheless, the knockdown of the EGT transporter in mammalian cells leads to augmented oxidative damage and cell death in the presence of exogenous stressors. Similar to other microorganisms, the physiological role and regulation of EGT biosynthesis in Mtb is unknown. We identified an Mtb gene (Rv3701c) to encode for EgtD, the enzyme that catalyzes the methylation of the amino acid histidine. Through the generation of an ΔegtD mutant we found that this methyltransferase is essential for EGT biosynthesis. We further demonstrated that EgtD activity is regulated by an Mtb protein-serine/threonine kinase in response to nutrient starvation. Under starvation, the Mtb ΔegtD mutant was unable to maintain viability compared to its parental wild type strain. As, starvation is associated with induction of a non-replicative state in Mtb, these findings indicate that EGT plays a role in mediating persistent infection or disease latency. Indeed, macrophage infection showed minimal impact on the survival of the ΔegtD mutant compared to its parental strain during initial stages (replicative phase) of intracellular infection. Further metabolic analysis identified Mtb intracellular EGT levels to be directly correlated with carbon source type and availability pointing to a role for EGT in energy storage. 120 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 99 Mycobacterial Biology & Physiology 20/09/2016 Identifying mechanisms for environmental adaptation in CMNR bacteria: a systems analysis of gene regulation of alternative substrate utilization and cell envelope biosynthesis R. Bailo Vergara1, E. Peterson2, A. Singh1, N. Baliga2, A. Bhatt1 1 IMI - Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom 2Institute for Systems Biology, Seattle, United States The genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus form the so-called CMNR group that belongs to the order Actinomycetales. The core common characteristic of this group is the presence of a waxy cell envelope which is mainly composed of peptidoglycans, arabinogalactans, and mycolic acids. CMNR bacteria are pathogens, soil inhabitants, plant commensals, and industrial microbes that are of pharmaceutical, environmental, and biotechnological interest. The diversity of habitats colonized by CMNR bacteria is based on their ability to adapt to new environments by dynamically altering the composition of mycolic acids in their cell envelope. The hypothesis of our research is that depending on the environment and available substrates, the CMNR bacteria selectively alter the composition of their cell envelope by using expanded families of uptake and biosynthesis enzymes in different combinations. Our initial experiments are being carried out using M. smegmatis because it is a model organism that maintains the unique and complex cell envelope organization that is common to the CMNR bacteria. Later, we will use other model representatives of this group such as Corynebacterium glutamicum, Rhodococcus jostii RHA1. So, firstly, we are phenotypically characterising changes in the cell envelope composition of M. smegmatis during growth on ecologically-relevant substrates and environments monitoring the OD600 and analysing its lipid content by 2-D chromatography. We will then characterise the growth dynamics and cell envelope changes during transitions between selected growth conditions. Once we have revealed those transitions in which M. smegmatis underwent significant changes in its cell-envelope composition, we will measure genome-wide gene expression changes over a time course during these adaptations by RNA-seq. The compendium of transcriptome measurements along with publically available gene expression data from microarray experiments will be used to generate our gene regulatory network model. Finally, we will develop algorithms to interrogate the model to discern modular architecture (i.e., conditional co-regulation of specific combinations of genes) and topology (i.e., promoter architecture and regulatory influences of transcription factors) that are conserved or unique across the CMNR bacterial genomes. Model predictions will be tested by analysing consequences of the deletion of specific genes related to the cell envelope composition of representative CMNR bacteria under relevant environmental transitions. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 121 100 Mycobacterial Biology & Physiology 20/09/2016 Understanding the role of malic enzyme in Mycobacterium tuberculosis infections P.S. Basu, R. Balhana, I. Gobe, D. Beste Microbial Sciences, FHMS, University of Surrey, Guildford, United Kingdom The PEP-pyruvate-oxaloacetate or anaplerotic node represents a critical metabolic cross-road for central metabolism controlling the distribution of flux between anabolism, catabolism and energy supply to the cell. In Mycobacterium tuberculosis this node consists of the enzymes PEP carboxykinase (PCK), pyruvate carboxylase (PCA), malic enzyme (MEZ) and pyruvate phosphate dikinase (PPDK). Aside from PCK these enzymes remain uncharacterised. Here we show that mez encodes a functional malic enzyme which has a high affinity for malate and therefore preferentially functions in the gluconeogenic direction, however this enzyme can also function glycolytically. M. tuberculosis strains deficient in MEZ produce small shiny colonies with a very sticky texture suggesting changes in the cell wall lipids. MEZ was not required for growth on a range of glycolytic and gluconogenic substrates in vitro. However, we demonstrate that MEZ in collaboration with PPDK provide M. tuberculosis with an alternative gluconeogenic route in the absence of PCK. Although MEZ was not required for intracellular growth of M. tuberculosis in macrophages this strain was attenuated for the initial invasion. We are currently investigating the hypothesis that that this is due to alterations in the cell wall lipids. Our studies further demonstrate the metabolic plasticity of central carbon metabolism in M. tuberculosis. 122 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 101 Mycobacterial Biology & Physiology 20/09/2016 Aspartate amino acid biosynthesis in Mycobacterium tuberculosis - characterization of a new drug target space M. Berney1, L. Berney-Meyer1, E. Hasenoehrl1, D. Rae Sajorda1, W.R. Jacobs1-2, F. Wang2 1 Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx 2California Institute for Biomedical Research (CaliBR), La Jolla 3Howard Hughes Medical Institute, Maryland, United States A major constraint for developing new TB drugs is the limited target space. We are in the process of exploiting a largely untapped drug target space that is unique to M. tuberculosis. Intracellular pathogens are faced with a host-induced process called nutritional immunity that depletes essential nutrients from the pathogen-containing vacuole. Emerging evidence suggests that many intracellular pathogens manipulate the phagocytes to regain access to those nutrients. In contrast, M. tuberculosis is not able to scavenge essential amino acids in the host and relies on its own biosynthetic machinery. We have recently shown that inactivation of methionine and Sadenosylmethionine biosynthesis leads to unusually rapid culture sterilization and complete clearance from host tissues (Berney et al., 2015, PNAS). The underlying unprecedented multi-target inhibition mechanism is optimal for drug development. We have now expanded our focus to characterize the regulation and metabolic flux in the whole aspartate amino acid biosynthesis pathway. Using metabolomics, transcriptomics, animal experiments and conditional knockdowns we could identify several new vulnerable enzymes in the aspartate pathway that have the potential to become new drug targets. Berney, M., Berney-Meyer, L., Wong, K.W., Chen, B., Chen, M., Kim, J. et al. (2015) Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 112: 10008-10013. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 123 102 Mycobacterial Biology & Physiology 20/09/2016 13 C isotopomer analysis identifies an essential pathway for the intracellular survival of Mycobacterium tuberculosis P.S. Basu2, K. Noh1, R. Balhana2, T. Mendum2, I. Gobe2, J. Mcfadden2, D. Beste2 1 Institute of Bio- and Geosciences 1: Biotechnology 2, Forschungszentrum Jülich, Jülich, Germany 2 Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom Metabolic reprogramming in response to the stresses of the host niche during both the acute and chronic phase of tuberculosis infections is a crucial determinant of virulence. Experimental evidence has identified central carbon metabolism as instrumental in this pathogenic strategy. Using mutant strains, 13C isotopomer analysis and mathematical modelling we demonstrate that intracellular M. tuberculosis uses a novel pathway which requires the enzymes phosphate dikinase (PPDK), PEP carboxykinase (PCK). Disruption of this pathway impacts on the intracellular survival of M. tuberculosis. Strains deficient in either pyruvate phosphate dikinase (PPDK) or PEP carboxykinase (PCK) are similarly attenuated for intracellular growth and M. tuberculosis lacking both pyruvate carboxylase (PCA) and PCK do not survive within human macrophages. As PCA, PPDK and PCK were also required for optimal survival in the presence of reactive nitrogen intermediates we hypothesise that this pathway generates pyruvate to thwart nitrosative stress and generates the high-energy metabolite phosphoenolpyruvate (PEP) which allows for ATP synthesis via substratelevel phosphorylation. This work identifies a novel pathway required for intracellular survival which could be targeted with therapeutics. Funding: MRC -MR/K01224X/1. 124 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 103 Mycobacterial Biology & Physiology 20/09/2016 Proteomic Analysis of Mycobacterium tuberculosis Membrane Vesicles A. Birhanu2, S. Yimer1, T. Riaz1, T. Tønjum1-2 1 Microbiology, Oslo University Hosiptal 2Microbiology, University of Oslo, Oslo, Norway Secreted membrane vesicles (MVs) are abundant in Mycobacterium tuberculosis (Mtb) and are suggested to have important roles in mycobacterial physiology and pathogenesis. MVs may contain varied cargo, including nucleic acids, toxins, lipoproteins and enzymes. In particular, MVs have been shown to serve as a vehicle for the selective packaging and release of virulence factors, such as toxins and immunomodulatory molecules. However, how and why MVs escape the thick cell walls of mycobacteria is still unknown. The aim of this project is characterize the proteomic profile of MVs from selected Mtb lineages and to unveil the role of MVs in mycobacterial adaptation to environmental factors. Mtb cells were cultured with and without oxidative and nitrosative stress. The Mtb cell free culture supernatant was used as a source to isolate enriched MVs by density gradient ultra-centrifugation. The purified membrane vesicles were investigated by mass spectrometry. The MaxQuant and Perseus softwares were used for peptide search, data analysis and quality control, respectively. The functional categories of the proteins identified were grouped using the KEGG pathway database. The mass spectrometry proteomic data revealed that MVs contain enriched packaging of virulence associated mycobacterial proteins. The majority of the MV-specific components detected were secreted proteins which are reported to be major virulence factors (antigen-85 complexes, ESAT-6 and PE/PPE, etc). Based on the KEGG pathway functional analysis, the identified proteins were found to be involved in two-component systems, ABC transporters, amino acid and fatty acid metabolism, as well as catalytic and hydrolase activity and hypothetical proteins. This study generated new knowledge on the proteomic profile of Mtb MVs. We have characterized Mtb membrane vesicles, which are enriched with surface/secreted antigenic proteins and virulence factors. Further in silico and in vitro/in vivo studies are required to investigate the adaptive and clinical relevance of the MV proteins identified, including the newly discovered un-characterized proteins. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 125 104 Mycobacterial Biology & Physiology 20/09/2016 The rv0805 gene in M. tuberculosis encodes a metallophosphoesterase with unique structural features that determine its biological functions P. Biswas1, N. Matange1, G. Larrouy-Maumus2, S. Visweswariah1 1 Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India 2Department of Life Sciences, Imperial College, London, United Kingdom Cyclic AMP-mediated signalling in mycobacteria plays an important role in the physiology of the organism and survival of pathogenic mycobacteria within host macrophages. Rv0805 was initially identified as a class III cNMP PDE, and orthologs are found only in slow growing mycobacteria.The crystal structure of Rv0805 reveals that it has a β𝛂β𝛂β MPE core fold with Fe3+ and Mn2+ present at its active site which are co-ordinated via aspartate, histidine and asparagine residues. These metal ions contribute to dimerization of Rv0805. The C-terminal cap domain of Rv0805 modulates its expression, and helps in its cell envelope localization. Interestingly, a strain of M. bovis BCG deleted for the rv0805 gene shows a stark growth defect when glucose or propionate was provided as the sole carbon source. The growth defect could be complemented by expression of the full length protein but not following deletion of the C-terminal 40 residues.Acetate, succinate, glutamate or citrate supplementation did not rescue the growth defect observed in glucose. In contrast, metabolomic analysis indicated differences in aspartate and ⍺-ketoglutarate levels when the bacteria were grown in propionate as the sole carbon source. This observation suggests a role for Rv0805 in the biosynthesis of intermediates involved in cell wall generation in slow-growing mycobacteria under conditions where glucose or propionate serve as the primary carbon source. 126 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 105 Mycobacterial Biology & Physiology 20/09/2016 Building genome-wide mutant resources in slow-growing mycobacteria by Cartesian Pooling-Coordinate Sequencing (CP-CSeq) K. Vandewalle3, K. Borgers3, N. Festjens3, E. Plets3, M. Vuylsteke1-3, Y. Saeys2, N. Callewaert3 1 Gnomixx 2IRC, Unit for Immunoregulation and Mucosal Immunology 3MBC, Unit for Medical Biotechnology, VIB - Ghent University, Ghent, Belgium Scientific mycobacterial research requires the availability of efficient molecular and genetic tools. The genetic manipulation of slow-growing mycobacteria has long been problematic due to their slow growth, the low efficiency of classical transformation methods and the low frequency of homologous recombination. The introduction of conditionally replicating mycobacteriophages changed the field: specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria. But the slow growth of mycobacteria remains a problem and applying these new methods in a gene-by-gene approach is laborious and costly. However, transposon mutagenesis makes it possible to make a wide variety of mutants in an isogenic mycobacterial background in a quick and efficient manner. Our lab developed a method to create characterized ordered transposon insertion mutant libraries, namely Cartesian Pooling-Coordinate Sequencing or CP-CSeq, combining pooling steps of transposon mutants along Cartesian coordinates with multiplex transposon sequencing of the resulting pools. In this manner a database containing positional information about each individual mutant present in a sequence-tagged mutant library can be obtained. We demonstrated this approach in the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. The ordered library contains 9.216 clones (96 x 96-well plates) for which 77% of the mutants could be positioned in the library, targeting 64% of the nonessential genes for in vitro growth. CP-CSeq has the potential to dramatically speed up hypothesis testing in Mycobacterium biology and can be extended to any entity with difficult genetics and/or slow growth for which sequencetagged identification is possible. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 127 106 Mycobacterial Biology & Physiology 20/09/2016 Key experimental evidence of chromosomal DNA transfer among selected tuberculosiscausing mycobacteria E. Boritsch7, V. Khanna4, A. Pawlik7, N. Honoré7, V.H. Navas5, L. Ma6, C. Bouchier6, T. Seemann2, P. Supply3, T.P. Stinear1, R. Brosch7 1 Department of Microbiology and Immunology, University of Melbourne, Parkville 2Monash University, Clayton, Victorian Bioinformatics Consortium, Victoria, Australia 3Center for Infection and Immunity, Inserm U1019, CNRS UMR8204, Université de Lille, Institut Pasteur de Lille, Lille 4Hub Bioinformatique et Biostatistique, C3BI, USR 3756 IP CNRS 5Lymphocyte Cell Biology Unit 6PF1-Plate-Forme Génomique 7Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France Horizontal gene transfer (HGT) is a major driving force of bacterial diversification and evolution. For tuberculosis-causing mycobacteria, the impact of HGT in the emergence and distribution of dominant lineages remains a matter of debate. Here, by using fluorescence-assisted mating assays and whole genome sequencing, we present unique experimental evidence of chromosomal DNA transfer between tubercle bacilli of the early-branching Mycobacterium canettii clade. We found that the obtained recombinants had received multiple donor-derived DNA fragments in the size range of 100 bp to 118 kbp, fragments large enough to contain whole operons. Even though the transfer frequency between M. canettii strains was low and no transfer could be observed among classical Mycobacterium tuberculosis complex (MTBC) strains, our study provides the proof of concept for genetic exchange in tubercle bacilli. This outstanding, now experimentally validated phenomenon presumably played a key role in the early evolution of the MTBC towards pathogenicity. Moreover, our findings also provide important new information for the risk-evaluation of potential transfer of drug resistance and fitness mutations among clinically-relevant mycobacterial strains. 128 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 107 Mycobacterial Biology & Physiology 20/09/2016 A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis C. Boutte2, C. Baer3, C. Sassetti3, T. Ioerger1, E. Rubin2 1 Texas A&M University, College Station 2Immunology and Infectious Disease, Rubin Lab, Harvard TH Chan School of Public Health, Somerville 3UMass Worcester Medical School, Worcester, United States Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 129 108 Mycobacterial Biology & Physiology 20/09/2016 Differences in the expression profile of the phoP locus among laboratory Mycobacterium tuberculosis strains in acidic stress response N. Bresciani, D.M. Cirillo, P. Miotto Emerging bacterial pathogens unit, Division of mmunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy The phoPR two-component system is involved in the regulation of relevant pathogenic features in M. tuberculosis (MTB). Despite its relevant role, the environmental signals triggering the phoPR machinery are still unclear. In addition, small regulatory RNAs (sRNAs), that regulate several key bacterial processes, control stress response and virulence even if their role in tuberculosis (TB) pathogenesis remains poorly understood. The aim of this study is to characterize the expression profile of ncRv0757c, a recently identified mycobacterial sRNA cis-encoded to the phoP gene, in two MTB laboratory strains (CDC1551 and H37Rv) during acidic pH stress response in vitro. We compared strains in early logarithmic growth phase grown at pH 7.0 with bacteria grown at pH 5.0. At selected time points (24 and 48 hours), we extracted the total RNA using the mirVana miRNA isolation kit (Thermo Fisher Scientific) and checked the RNA purity on Agilent 2100 Bioanalyzer (Agilent Technologies). We assessed the expression profile of ncRv0757csRNA, phoP and phoR genes by qRT-PCR using SYBR Green RT-PCR reagents kit (Life Technologies) and rrs gene as endogenous control. The sRNAncRv0757c was found to be significantly downregulated at 24 and 48 hours in CDC1551 at pH 5 compared to the reference strain at pH 7: its expression resulted 5-fold change lower than H37Rv (p-value <0.001). At the same time, the phoP (p-value24H <0.05; p-value48H<0.01) and phoR (p-value24H <0.01; p-value48H<0.001) genes are downregulated more than 5-fold at pH 5. The phoP gene and ncRv0757c showed no response to acidic pH in H37Rv. The phoP locus was modulated in response to acidic stress only in in the CDC1551 strain, but not in H37Rv, thus suggesting that the response to acidic pH is more complex than thought and additional factors might be involved. Despite the sRNA ncRv0757c could play a role in the regulation of the phoP gene, as suggested by a similar expression profile, our results do not support its direct involvement in the differential modulation of the phoP gene during acidic conditions observed between CDC1551 and H37Rv strains. 130 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 109 Mycobacterial Biology & Physiology 20/09/2016 THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria J. Cox3, K. Abrahams3, D. Barros2, K. Futterer3, L. Ballell2, G. Drewes1, G. Besra3 1 Cellzome - a GSK Company, Heidelberg, Germany 2GSK, Tres Cantos, Spain 3University of Birmingham, Birmingham, United Kingdom Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target M. tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 131 110 Mycobacterial Biology & Physiology 20/09/2016 Several Mycobacterial species are capable of forming spore-like state J. Dahl Department of Biology, University of Minnesota, Duluth, United States Recent reports have argued both for and against the capability of mycobacteria to form heatresistant spores. The existence of a spore-like state would help explain mycobacteria persistence and dormancy in infected hosts and in the environment. Our laboratory has recently identified several mycobacterial species that can survive conditions exceeding classic pasteurization. Two of these mycobacterial species remain viable even after 6 hours exposure to wet heat of 65oC. Production of the heat-resistant state does not appear to be dependent upon production of dipicolonic acid nor upon the action of the stringent response. This is in spite of these two features being vital for gram-positive bacterial sporulation. Heat resistance of these two mycobacteria correlates with an ability to survive in a desiccated state for upwards to 3 years. Collectively our data shows that mycobacterial are capable of much more stress-resistant physiological states than was previously realized. 132 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 111 Mycobacterial Biology & Physiology 20/09/2016 Mycobacterium tuberculosis cyclophilins show chaperone like activity in-vitro and in-vivo S. Pandey1-3, D. Tripathi2, A. Sharma2, T.K. Chaudhary2, S.E. Hasnain2, N.Z. Ehtesham1 1 Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, Allahabad 2Kusuma School of Biological Sciences, Indian Institute of Technology, Delhi 3School of Life Sciences, University of Hyderabad, Hyderabad, India Tuberculosis, named after its causative agent the tubercle bacillus or Mycobacterium tuberculosis is one of the major causes of mortality and morbidity all over the world. Approximately 2 million people die of this deadly disease every year. PPIase peptidyl-prolyl cis-trans isomerases commonly known as cyclophilins are ubiquitously expressed enzymes that (PPIase, EC 5.2.1.8) assist in protein folding by isomerization of Xaa-Pro peptide bonds, which otherwise can act as folding bottleneck and will limit the rate of final folding steps. Mycobacterium tuberculosis (M.tb) is known to possess two cyclophilins (Ppiases), PpiA (Rv0009) and PpiB (Rv2582). This study describes chaperone-like activity of mycobacterial Ppiases and their role in stress adaptation. We show that recombinant rPpiA and rPpiB can bind to nonnative proteins in vitro and can prevent their aggregation. Purified rPpiA and rPpiB exist in oligomeric form as evident from gel filtration chromatography. E. coli cells overexpressing PpiA and PpiB of M.tb could survive thermal stress as compared to plasmid vector control. HEK293T cells transiently expressing M.tb PpiA and PpiB proteins show increased survival as compared to control cells in response to oxidative stress and hypoxic conditions, thereby pointing to their likely role in adaption under host generated oxidative stress and conditions of hypoxia. The chaperone-like function of these M.tuberculosis cyclophilins may possibly function as a stress responder and consequently contribute to virulence. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 133 112 Mycobacterial Biology & Physiology 20/09/2016 The role of glycolysis in the activation of human monocyte-derived dendritic cells following exposure to bcg K. Elayati, D. Triglia, S. O'leary, J. Keane, M. O'sullivan Clinical Medicine, TCD, Dublin, Ireland Background: Dendritic cells(DCs) play an essential role in both innate and adaptive immunity. Recent advances have indicated a role for monocyte-derived DCs (moDCs) in the production of IL1α and IL-1β in a murine model of Mycobacterium tuberculosis (Mtb) infection. In addition, moDCs are essential for transport of live mycobacteria from the lung to the draining lymph nodes to initiate an adaptive immune response. A metabolicswitch from oxidative phosphorylation to glycolysis occurs in DCs stimulated with TLR agonists and is required for activation. The aim of this study is to investigate the role of glycolysis in human moDCs activated with mycobacteria. Methods: Human monocyte-derived DCs were infected with different ratios of BCG-GFP (MOI 50 – 250) in the present and absence of glycolysis inhibitor 2-deoxyglucose (2DG) for 24 hr. Changes in expression of maturation markers, chemokine receptors and cell viability were assessed by flow cytomety. Statistical analysis was performed using standard statistical software (Prism 5.0).Significance level was set at p˂0.05. Results: Infection of moDCs resulted in increased expression of the maturation marker CD86.There was no change in the percentage of HLA-DR positive cells but surface expression was increased in DCs infected at an MOI of 100 compared to uninfected cells.There was a decrease in surface expression of CCR7(% and MFI) in cells infected at an MOI of 100 compared to uninfected but this did not reach statistical significance.Additionally, there was a dose-dependent increase in cell death of infected moDCs. Inhibition of glycolysis had no significant effect on expression of CD86, HLA-DR or CCR7. However, there was a significant increase in the viability of moDCs infected at MOI 100 and 250 BCG-GFP following treatment with 2DG.Annexin V staining revealed that DCs did not undergo apoptosis with or without 2DG treatment. Conclusion: BCG-GFP infection of moDCsmay cause a metabolic switch towards glycolysis and away from oxidative phosphorylation.However, aerobic glycolysis does not appear to be essential for DC maturation following infection with BCG andappears to compromise cell viability.Future experiments will investigate whether DCs overcome blocking of glycolysis by generating energy through alternate methods. 134 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 113 Mycobacterial Biology & Physiology 20/09/2016 A new mechanism of programmed cell death in M. tuberculosis by a TA toxin hydrolyzing intracellular NAD+ D. Freire3, C. Gutierrez1, A. Grabowska4, V. Pogenberg3, T. Schneider3, M. Cianci3, A. Sala2, K. Nukdee2, P. Genevaux2, M. Wilmanns3, O. Neyrolles1, A. Parret3 1 Institute of Pharmacology and Structural Biology 2Laboratory of Microbiology and Molecular Genetics, University of Toulouse, Toulouse, France 3European Molecular Biology Laboratory, Hamburg, Germany 4Department of Pathogen Molecular Biology, London School for Hygiene and Tropical Medicine, London, United Kingdom Toxin-antitoxin (TA) modules are two-gene operons that are widely prevalent in bacterial genomes. They are essential to various cellular processes, such as cell wall synthesis and gene regulation, as well as protection against phages and programmed cell death. TA modules encode a toxic protein which infers growth arrest of the producer cell by interfering with cellular replication and translation processes in a similar way as antibiotics do. The antidote is provided in the form of an antitoxin which very efficiently inhibits the toxic activity in normal growth conditions. In M. tuberculosis (Mtb), TA systems are found in usually high numbers and are associated with pathogenesis and persistent infections. The vast majority of TA systems in Mtb belong to class II, systems, where the antitoxin is a labile protein which inhibits toxin activity through complex formation. Here, we present the structure-function analysis of a new type of class II TA system from M. tuberculosis H37Rv encoded by Rv1990c-Rv1989c. The crystal structure of the TA complex reveals unexpected structural homology of the toxin Rv1989c to bacterial exotoxins with NAD+ hydrolase activity. The antitoxin inactivates the toxin by occupying the active site through substrate mimicry. This TA complex forms a compact heterododecameric complex in which the hypothetical DNAbinding domain of each antitoxin moiety is sterically blocked. We established NAD+-hydrolysis activity in cellular extracts of M. smegmatis and E. coli upon induction of the toxin. Expression of active site mutants in M. tuberculosis no longer conferred the killing phenotype. Our findings suggest that this TA module represents a new subclass of type II TA systems where the cognate toxin directly confers bactericidal activity by rapid depletion of cellular NAD+. As a result, constitutive activation of Rv1989c by blocking interaction with Rv1990c would trigger bacterial suicide. These findings open up new avenues for the development of highly effective antituberculosis drugs. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 135 114 Mycobacterial Biology & Physiology 20/09/2016 Mycobacterium tuberculosis Dormancy proteins: Key players in cell wall integrity, stress tolerance and virulence R. Garg3-4, D. Tripathi3, S. Kant2, V. Gupta3, A. Seth1, R. Bhatnagar3, N. Banerjee3 1 Department of Bio-Science and Biotechnology, Banasthali University, Banasthali, Rajasthan 2International Centre for Genetic Engineering and Biotechnology 3BSL3 Laboratory, School of Biotechnology, Jawaharlal Nehru University, Delhi 4Amity Institute of Biotechnology, Amity University Gurgaon Haryana, New Delhi, India Dormancy is a physiological phenomenon of Mycobacterium tuberculosis, which has still remained an enigma for scientists across the globe. M. tuberculosis (Mtb) has two stages of disease pathogenesis, the first being active stage involves replication of bacteria in the macrophages and the second is the dormancy stage, which the present drug regimen fails to target. It has been seen that a two component system DosRS is responsible for inducing 48 member dormancy regulon in Mtb, using signals like hypoxia, NO and CO. Diversity of functions associated with dormancy regulon genes makes it important to characterize them, so that new avenues in drug research can be opened. In an attempt to discover role of various dormancy genes, we studied locus Rv0574c which codes for polyglutamate synthase like protein, contributing to virulence of Mtb, by virtue of synthesizing poly-α-L-glutamine layer in mycobacterial cell wall. Rv0574c gene showed highest expression in late log phase with concomitant accumulation of poly-α-L-glutamine in the cell wall. Rv0574c was activated under conditions prevalent in the tubercular granuloma e.g. hypoxia, nitric oxide and CO2. For functional characterization, a deletion mutant of the gene was produced by allelic exchange. The mutant produced lower amounts of poly-α-L-glutamine in the cell wall compared to the wild type bacteria. Additionally, increased sensitivity of the mutant to antitubercular drugs, SDS, lysozyme and mechanical stress was accompanied with drastic reduction in ability to form biofilm. Growth of the ΔRv0574c strain was normal under in vitro conditions but was retarded in THP-1 macrophages and in the lungs and spleen of BALB/c mice. This was in agreement with histopathology of the lungs showing slow growth and less severe pathology than the wild type strain. Therefore, Rv0574c locus by virtue of modulating PLG content in the cell wall, helps in maintaining cellular integrity under hostile host environment. The second gene which we are studying is cation transporter protein F (ctpF), which belongs to the under explored P-type ATPase family in Mycobacteria. ctpF gene expresses maximally in stationary phase and shows upregulation in acidic conditions, NO stress, CO2 as well as in hypoxia. Creation of a deletion mutant will help us further assess the contribution of this gene in virulence of Mtb. These two proteins as well as other conserved but functionally uncharacterized dormancy proteins pose themselves as attractive anti-tubercular drug targets. 136 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 115 Mycobacterial Biology & Physiology 20/09/2016 Discovery and characterization of a model mycobacterial sRNA involved in the iron-sparing response E. Gerrick, S. Fortune IID, Fortune Lab, Harvard School of Public Health, Boston, United States An emerging theme in bacteria is a reliance on small regulatory RNA molecules (sRNAs) for rapidly modulating gene expression. sRNAs, which in most bacteria rely on the sRNA accessory factor protein Hfq to bind to their target mRNAs, play a vital role in virtually all stages of growth. However, despite the increasingly well-recognized importance of bacterial sRNAs, very little is known about these regulators in the important human pathogen Mycobacterium tuberculosis (Mtb) or any of its relatives. While a number of putative sRNAs have been identified in Mtb, not a single sRNA target has been experimentally validated and mycobacteria lack every known sRNA accessory factor protein. In order to remedy this, we have performed bacterial sRNA sequencing (bsRNA-Seq) on Mtb grown under iron starvation conditions to identify potential iron-sparing sRNAs. We then created a computational pipeline to identify novel sRNAs within bsRNA-Seq datasets and discovered one novel sRNA that is highly abundant only during iron starvation. This sRNA, which we have named MyhB, is highly conserved across all mycobacterial lineages, and we have confirmed its role in the iron sparing response in M. smegmatis. We then used RNA-sequencing to identify 14 genes as putative members of the MyhB regulon and confirmed regulation of one target, bfrA, by RT-qPCR and using a luciferase reporter fusion. In addition, we have identified a 6nt putative seed region of MyhB, which is predicted to bind to its mRNA targets. These results represent the most in-depth characterization of a mycobacterial sRNA to date and provide valuable insights into mycobacterial sRNA-mediated riboregulation. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 137 116 Mycobacterial Biology & Physiology 20/09/2016 The role of nitric oxide in the production of Rpf-dependent Mycobacteria S. Glenn1, O. Turapov1, B. Kana3, P. Andrew1, V. Makarov2, G. Mukamolova1 1 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, France 2Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia 3DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa Tuberculosis (TB) is a major global health problem, causing 1.5 million deaths annually. One third of the world’s population are thought to be latently infected with Mycobacterium tuberculosis (Mtb) which has specialised mechanisms for long-term persistence in vivo and in vitro. Past studies have shown that a high number of Mtb bacilli isolated from human TB sputum or from murine infected tissues are only able to grow in the presence of resuscitation promoting factors (Rpf). Furthermore Rpf-dependent Mtb are more tolerant to first line tuberculosis drugs rifampicin and isoniazid and apparently enriched in sputum of treated tuberculosis patients, indicating the clinical importance of these bacilli for TB management and treatment. The mechanisms behind the formation of these Rpf-dependent bacilli are still poorly understood at both a host and pathogen level, and the lack of a reliable model has made investigations into this problematic. We hypothesised that nitric oxide, an important component of the host immune defences, produced in vivo might be responsible for the generation of Rpf-dependent bacilli. Using several novel NO donors, we investigated the induction of Rpf-dependency in mycobacteria in vitro. Treatment of mycobacteria with novel NO donor resulted in rapid generation of Rpf-dependent bacilli, which maintained integrity of cellular membrane and could be resuscitated by addition of Rpf-containing culture supernatant. By application of DAF-FM staining we confirmed that the NO donors release a low level of nitric oxide, which we hypothesise stimulates signaling pathways within the bacteria, resulting in Rpfdependency. Current investigation of transcriptomics and proteomics profiles of NO-treated Mtb will aid establishment of molecular mechanisms underlying generation of RPF-dependent mycobacteria in vivo. 138 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 117 Mycobacterial Biology & Physiology 20/09/2016 Evaluation of selective translation in Mycobacteria using reporter strains A. Grabowska, N. Andreu, T. Cortes Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London, United Kingdom Mycobacterium tuberculosis differs from other bacterial pathogens in expressing an unexpected number of leaderless mRNA transcripts. Leaderless mRNAs lack a 5’ untranslated region (UTR) that usually harbours the Shine-Dalgarno (SD) sequence, the dominant signal for canonical translation initiation in bacteria. In Escherichia coli, only a few leaderless transcripts have been described and they are translated with low efficiency by E. coli ribosomes, when the 70S monosome directly binds to the ATG start codon. The scenario changes during stress conditions, where a translational reprogramming takes place generating specialised ribosomes that selectively translate leaderless transcripts. We have previously demonstrated that proteins with secondary adaptive functions are generally leaderless in M. tuberculosis. Furthermore, in a starvation model of growth arrest, we detected a significant increase in the overall ratio of leaderless to SD transcripts. Here, we have engineered bioluminescent mycobacterial reporter strains representative of leaderless and SD transcripts to study differences in their translational efficiencies. First, reporter constructs were assessed in E. coli. As expected, we detected basal levels of luminescence for the leaderless reporter in E. coli during exponential growth. In contrast, leaderless and SD transcripts were translated with similar efficiencies in exponentially growing mycobacteria, suggesting the mycobacterial ribosome differs from the E. coli model in being able to translate both types of transcripts. Secondly, reporter strains were used to study changes in translation efficiencies during different phases of growth. Whereas translation of leaderless and SD transcripts was maintained at similar rates during exponential growth, upon entrance into growth arrest, translation of leaderless transcripts was maintained whilst translation of SD transcripts significantly declined. Overall, our results suggest that preferential translation of leaderless transcripts may have particular importance in the physiology of non-replicating M. tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 139 118 Mycobacterial Biology & Physiology 20/09/2016 Mycobacterium tuberculosis H37Rv encoded ParE2 toxin induces persister cell formation through SOS response M. Gupta1-2, R. Sitaraman2, R. Bhatnagar1, N. Banerjee1 1 Molecular and Cell Biology Laboratory, Jawaharlal Nehru University 2Department of Biotechnology, TERI University, New Delhi, India Mycobacterium tuberculosis H37Rv (Mtb) escapes host-generated stresses and antibiotics by entering a dormant, persistent state. Activation of toxin-antitoxin modules is one of the mechanisms known to trigger such a non-replicative state. We have earlier investigated the parDE2 operon of Mtb, which encoded the MParE2 toxin, an inhibitor of its own DNA gyrase. Here we scrutinize the toxicity of MParE2 in RecA1 and RecA+ E. coli backgrounds to study the role of SOS response as a survival strategy and examine different phenotypes generated due to MParE2 expression. Ectopic expression of parE2 affected growth and viability of both the E. coli strains to different degrees. Live-dead staining (SG-I/PI) revealed that in a short span of 4h, the toxin killed ~54% of the RecA1 strain compared to ~27% cells of the RecA+ cells. In both cases, the majority of the live cells ~99.99% were viable but non-culturable (VBNCs) and only 0.01% population were colony formers. The results suggested that though more cells survived from the toxic effect in the RecA+ strain, VBNC formation was not solely dependent on SOS regulation. MParE2 toxicity caused DNA damage, triggering activation of SOS response, which led to inhibition of cell division and formation of multi-nucleoid, long filamentous cells. A massive increase in sulA and tisB transcript levels, and loss in membrane potential in the RecA+ cells and not in the RecA1 strain, all pointed to SOS mediated regulation of these phenotypes. Severe morphological aberrations observed under the electron-microscope in the parE2 expressing RecA1 E. coli strain were largely mitigated when the toxin was expressed in the RecA+ cells. The latter cells were extensively filamentous with smooth and intact membranes compared to the RecA1 cells, which were wrinkled and corrugated with hyper-hydrated periplasmic space and large electron-lucent ‘vacuoles’, reiterating the SOSmediated rescue of MParE2-toxicated E. coli cells. As the effect of toxin waned with time, the cells resumed division and recovered in colony forming ability. The MParE2 expressing RecA+ cells produced significantly higher number of persisters than the RecA1 strain, upon exposure to different antibiotics. The MParE2 triggered VBNC phenotype and persistence are hallmarks of dormancy, potentially relevant in Tuberculosis. 140 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 119 Mycobacterial Biology & Physiology 20/09/2016 Regulatory circuits in the mycobacterial Pup-proteasome system Y. Elharar, M. Korman, S. Schlussel, E. Gur Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel Pup is a bacterial ubiquitin analog that is conjugated to lysine side chains of protein targets, thereby tagging them for proteasome-mediated degradation. The Pup-proteasome system (PPS) was discovered in Mycobacterium tuberculosis, where it was shown to be essential for both long term persistence under starvation conditions, and for resistance to reactive nitrogen intermediates. Indeed, PPS-defective M. tuberculosis mutants present attenuated virulence as compared to the wild type strain. Being an intracellular proteolytic system, the PPS is tightly regulated to prevent excessive protein degradation and unnecessary destruction of proteins. However, the mechanisms that control PPS function and are likely to be targets for the development of anti-TB medicine, are currently poorly characterize. In the PPS, the single Pup-ligase, PafA, shows exceptionally broad specificity in tagging hundreds of protein targets for degradation. This promiscuity of PafA target selection is well suited to the role of the PPS as an amino acid recycling factory under starvation conditions. However, such PafA promiscuity also poses a threat to proper cell function, as irreversible damage would be caused by excessive non-specific protein degradation without strict control of the PPS. Our studies indicate that the PPS is, indeed, highly regulated, such that at the maximum, protein pupylation only affects about 2% of the cell’s protein mass. While our previous findings indicated that the PPS is auto-regulated by the tagging and degradation of its own components, the mechanisms that control the expression of PPS genes remained unknown. Furthermore, how these levels are coordinated at the system level remains poorly understood. We now report that the level of PafA transcription is negatively regulated by PafB and PafC, two PPS components encoded by the paf operon whose functions were unknown until now. Translational control allows high PafA expression under starvation conditions, yet maintains low levels of this enzyme when nutrients are in excess. We also find that levels of pupylated proteins in the cell are limited by the concentration of Pup. Finally, our current understanding of the regulatory circuits that control the PPS allow us to now to simulate the levels of PPS components and their effects on the level of pupylated proteins, with the results of such simulation being consistent with experimental observations made in both exponentially growing and starving cells. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 141 120 Mycobacterial Biology & Physiology 20/09/2016 Single-cell mycobacteria analysis reveals variability in the division time inherited J.E. Hernandez, N. Ma, S. Hingley-Wilson, J. Mcfadden University of Surrey, Guildford, Surrey, United Kingdom Introduction: Variable outcomes observed in tuberculosis treatment could be attributed to mycobacteria variable growth and division patterns that create deterministic population diversity at a very high frequency [1]. Advances in microfluidics technology have allowed to study cell at singlecell level, and persistence phenomenon of bacterial populations has been analysed [2]. Tuberculosis treatment was unsuccessful in 14% of cases for 2013 [3] that suggest that new drugs are necessaries to achieve the infection control. Studies of population heterogeneity can contribute to find new therapeutic targets Aim: To examine growth and division in mycobacteria using microfluidics and time-lapse microscopy, and to track these cells to analyse division time Material and Method: We studied Mycobacterium smegmatis mc2 155 at single cell level by the use of microfluidics technology CellASIC ONIX Microfluidic Platform with B04A Microfluidic Bacteria Plate (Millipore Corporation). Media flow was controlled via the CellASIC system at a flow rate of ~10 µl/hr. Imaging was performed under a Nikon confocal microscope (Nikon A1M on Eclipse Ti-E) equipped with an environmental chamber, motorized stage and perfect focus system. Automated multi area imaging was carried out using a 40X air objective lens (Nikon Apo λ) with a numerical aperture of 0.95 Time-lapse videos of growing cells were recorded. Nikon Elements software was used to obtain crops from the videos, and cells were manually tracked until 5st generation with. Division time was calculated, and Pearson correlation between mother-daughters and sisters-sisters division times were established Results: Time-lapse video of M. smegmatis growth were obtained. After three independent experiments we tracked an average 1000 cells each time. Correlation coefficient for mother and daughters division time was around -0.2 which suggest non-correlation between them. Interestingly, a positive correlation, coefficient correlation around 0.5 was observed between sisters. Conclusion: Inheritance of division on time in M. smegmatis suggested that a factor inherited at birth determines the division time and that this factor is rearranged during each generation. Additional studies to identify factor(s) responsible of that behaviour are required to design new strategies that reduce the variability in the mycobacteria population References 1. Bree B. Aldridge, M.F.-S., Danielle Heller,Vijay Ambravaneswaran, and M.T.a.S.M.F. Daniel Irimia, Asymmetry and Aging of Mycobacterial Cells Lead to Variable Growth and Antibiotic Susceptibility. Science. 335(100): p. 4. 2. Balaban N. Q., J Merrin, J., Chait R., Kowalik L. and Leibler S., Bacterial Persistence as a Phenotypic Switch. Science, 2004. 305: p. 4. 3. WHO, Global Tuberculosis report. 2014 142 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 121 Mycobacterial Biology & Physiology 20/09/2016 The ParA-Wag31 interaction provides the link between chromosome segregation and other cell cycle processes in Mycobacteria M. Pióro1, T. Małecki2, K. Ginda3, D. Jakimowicz2-1 1 Institute of Immunology and Experimental Therapy 2Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland 3University of Oxford, Oxford, United Kingdom Bacterial chromosome segregation depends on the ParA and ParB proteins. ParB binds DNA and forms large nucleoprotein complexes which organise newly replicated chromosomes. The ParB complexes are positioned in the specific locations of the cell due to interactions with dynamic ATPase, ParA. It was suggested that ParA is essential in M. tuberculosis (1).We have shown that elimination of ParA inhibits growth of Mycobacterium smegmatis and disturbs chromosome segregation (2). The dynamic structures of ParA segregate ParB complexes soon after the initiation of the chromosome replication (3). Moreover, we have shown that mycobacterial ParA interacts with polar growth determinant Wag31, the homologue of DivIVA protein (2). This interaction is unique to Mycobacteria and taking into account indispensability of both proteins in M. tuberculosis it provides a potential drug target. Here, we further explored interaction between ParA and Wag31 using M. smegmatis as model species. To determine the biological significance of the interaction, we have identified the mutant ParA protein that does not interact with Wag31. We have constructed the M. smegmatis strain expressing mutated parA and analysed its growth in different conditions. We have also analysed the localisation of mutated ParA and its function in chromosome segregation. Our studies indicate that interaction between ParA and Wag31 provides the link between the chromosome segregation and the other processes of cell cycle. We suggest this interaction allows the coordination of the cell cycle and may be crucial during persistence in M. tuberculosis. 1. Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Molecular microbiology 48(1):77–84. 2. Ginda K, et al. (2013) ParA of Mycobacterium smegmatis co-ordinates chromosome segregation with the cell cycle and interacts with the polar growth determinant DivIVA. Molecular microbiology 87(5):998–1012. 3. Trojanowski D, et al. (2015) Choreography of the Mycobacterium Replication Machinery during the Cell Cycle. 6(1):1–12. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 143 122 Mycobacterial Biology & Physiology 20/09/2016 Biosynthesis of mycobacterial lipoarabinomannan: disruption of aftB results in complete loss of terminal β(1→2) arabinofuranosyl residues M. Jankute3, L. Alderwick3, S. Noack2, N. Veerapen3, J. Nigou1, G. Besra3 1 Institute of Pharmacology and Structural Biology, The French National Centre for Scientific Research, Toulouse, France 2Institute for Bio- and Geosciences: Biotechnology, Research Centre Jülich, Jülich, Germany 3Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom Mycobacterium tuberculosis, the etiological agent of tuberculosis, remains a highly successful bacterial pathogen. Its persistence is associated with the thick, carbohydrate and lipid rich cell wall with distinct lipoglycans that is highly impermeable to hydrophilic drugs. This highly complex and unique structure is crucial for the growth, viability and virulence of M. tuberculosis, thus representing an attractive target for vaccine and drug development. It contains a large macromolecular structure known as the mycolyl–arabinogalactan– peptidoglycan complex (mAGP), as well as phosphatidylmyo-inositol derived glycolipids with potent immunomodulatory activity, notably lipomannan and lipoarabinomannan (LAM). AG and LAM (lipo)polysaccharides share similar arabinan domains with a well-defined structure, however, the biosynthesis of both remains somewhat incomplete. Mycobacterial glycosyltransferases with dual functionalities have been previously identified. Here, we have investigated the potential role of known arabinosyltransferase AftB in the biosynthesis of LAM. Deletion of aftB in Mycobacterium smegmatis could only be achieved in the presence of a rescue plasmid carrying a functional copy of a gene, strongly suggesting that aftB is essential. Lipid analysis of the conditional mutant strain demonstrated an increase in cell wall associated lipids and a significant decrease in cell-wall bound mycolic acid methyl esters. A monoclonal antibody generated against arabinan motif of LAM no longer recognized the LAM structure extracted from the conditional mutant strain. Subsequent structural characterization confirmed a complete lack of β(1→2) arabinofuranosyl residues. Furthermore, we demonstrated that truncated LAM display proinflammatory activity, which is due to its ability to activate Toll-like receptor 2. Altogether, our results indicate that Ms-AftB is an essential mycobacterial ArafT that carries dual functionality and is responsible for the synthesis of the arabinan domain of both LAM and AG. 144 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 123 Mycobacterial Biology & Physiology 20/09/2016 Mycolic acid processing in Mycobacterium tuberculosis A. Javid, A. Bhatt, R. Kalscheuer University of Birmingham, Birmingham, United Kingdom Mycobacterium tuberculosis the causative agent of tuberculosis (TB) infects upto 9 million people per year resulting in approximately 1.5 million deaths due to the disease. With the emergence of multi drug resistant (MDR) and extremely drug resistant (XDR) strains arises the need for novel targets for anti-TB therapy. Mycolic acids are essential components of the unique, lipid rich cell wall of M. tuberculosis. However, enzymes involved in the biosynthesis of mycolic acids remain under exploited as drug targets despite one of the early and hallmark anti-TB drug isoniazid which inhibits mycolate biosynthesis. Previous studies from our laboratory identified mycolate processing enzymes and transporters. Using gene knockdowns we have now extended these studies to slow growing mycobacteria like Mycobacterium tuberculosis. Furthermore using BLAST-P alignments and predictions of a 3D structure we identified unique domains in the mycolate processing enzymes, and present functional studies on the same. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 145 124 Mycobacterial Biology & Physiology 20/09/2016 Mycobacterial HspX is both necessary and sufficient for the unipolar sorting of misfolded protein aggregates J.H. Zhu, Y.W. Zhang, X.H. Zheng, Y. Wu, B. Javid Tsinghua University School of Medicine, Beijing, China Protein misfolding is an inevitable consequence of protein synthesis and therefore all cells have evolved mechanisms with which to deal with misfolded proteins, which have a tendency to form aggregates known as inclusion bodies (IBs) in bacteria. In mycobacteria, in common with other model organisms such as Escherichia coli, inclusion bodies tend to form at the poles of the cells. Recent work has highlighted the importance of the heat shock proteins/ chaperones DnaK and ClpB in dealing with misfolded and irreversibly oxidized proteins respectively at the poles of mycobacteria, but how inclusion bodies get to the poles in mycobacterial cells has, until now, not been known. We decided to investigate the role of the small heat shock protein, HspX in mycobacterial inclusion body formation. HspX is significantly upregulated in tuberculous granulomata and under conditions of hypoxia, and is an immunodominant antigen, but its precise role in mycobacterial physiology is not known. Here, with the use of novel inclusion body fluorescent protein reporters, we show that the formation of large inclusion body aggregates and their sorting to predominantly to a single pole of the mycobacterial cell are both critically dependent on HspX. HspX also localizes at the poles and IBs tend to form at the pole with greater HspX expression. In an hspX null mutant of Mycobacterium smegmatis, small aggregates still form, but no longer coalesce into inclusion bodies and are found throughout the cell. Importantly, the siting of IBs is dependent on HspX: when HspX is relocalised to the septum, IBs now form, but are septally located, verifying the causal role of HspX in IB localization. Our work identifies the cellular function of the important mycobacterial antigen HspX and determines the mechanism for unipolar IB sorting – one of the many mechanisms that mycobacteria employ for the generation of phenotypic diversity. 146 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 125 Mycobacterial Biology & Physiology 20/09/2016 Epigenetic control of Tuberculosis persistence: The role of the different sigma factors L. Kaminski3, A. Benard1, N.R. Thomson1, G. Dougan2, W.R. Jacobs3 1 Microbial Pathogenesis 2Pathogen Genetics, The Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom 3Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States Mycobacterium tuberculosis (Mtb) infects 30% of the human population with the typical outcome of causing a latent infection. The persistence phenotype in Mtb is a phenomenon of cells where a fraction is resistant to a killing assault as a consequence of a transient gene expression or protein modifications. This phenomenon of persisters is being unmasked at the presence of bactericidal drugs or host adaptive immunity and it is an important impediment to tuberculosis control. While several genes were previously proposed as contributing to the emergence of persistence, the complete regulatory network is still unknown and it is clearly not attributed to one 'persister gene'. To identify the specific genetic network causing the emergence of persistent cells we made a null deletion mutant library of the twelve non-essential sigma factor transcription initiation factors in Mtb and tested the effect of bactericidal drugs using these mutants as a screen for persister cells. We have identified one of these sigma factor mutants to be sterilized in the presence of the first line drug Isoniazid (INH). The RNA sequencing analysis showed differential expression of several additional genes involved in transcription regulation and some genes in the oxidative regulation. Other ongoing efforts are aimed to confirm the sterilization of this mutant in vivo in the presence of the first line INH as a monotherapy. Screening of the transcription factor mutants yields a future way in understanding their role in vivo in the face of other drugs and host immunity. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 147 126 Mycobacterial Biology & Physiology 20/09/2016 Proteolytic control of the mycobacterial cell cycle J. Kester2, O. Kandror1, T. Akopian1, A. Goldberg1, S. Fortune2 1 Cell Biology, Harvard Medical School 2Immunology & Infectious Diseases, Harvard University, Boston, United States Tuberculosis (TB) is the primary infectious killer worldwide, in part owing to increased resistance of Mycobacterium tuberculosis—the etiologic agent of TB—to the current suite of antibiotics. Bactericidal drugs typically target growth and division events, yet regulators of the mycobacterial cell cycle remain undiscovered. In this work, we seek to identify key players in mycobacterial cell cycle control, potentially leading to new antibacterial targets. Mycobacteria share a unique pattern of growth and division with the model bacterium Caulobacter crescentus. Caulobacter initiates its cell cycle through the highly controlled proteolysis of key cell cycle regulators using the housekeeping protease complex, ClpXP. Therefore, we postulated that a ClpX-dependent mechanism might underlie cell cycle control in mycobacteria as well. To test this hypothesis, we depleted the essential AAA+ ATPase ClpX in the nonpathogenic model Mycobacterium smegmatis (Msm) and used dynamic live cell imaging to assay for growth and division effects. Loss of ClpX results in cell cycle arrest, supporting a role for ClpX and one or more of its substrates in cell cycle regulation. To identify the set of ClpX substrates, we used a small molecule inhibitor in whole cell lysates to trap potential substrates within the barrel of ClpX hexamers. We used tandem MS/MS, biochemical examination, and molecular techniques to validate cell cycle factors as substrates of ClpX. In addition to providing a framework for understanding the unique essentiality for ClpX in mycobacteria, this study also suggests potential cell cycle targets for new antibiotics. Additionally, this work provides a methodology for biochemical identification of high-confidence substrates of essential ATPases/chaperones. JCK supported by NIH F31 AI120616-01 and NSF GRFP DGE-1144152 148 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 127 Mycobacterial Biology & Physiology 20/09/2016 The role of glycosyltransferase Rv1459c in mycobacteria M. Šarkan2, M. Gilleron1, J. Nigou1, K. Mikušová2, M. Jackson3, J. Korduláková2 1 Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Toulouse, France 2Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia 3Department of Microbiology, Mycobacteria Research Laboratories, Immunology, and Pathology, Colorado State University, Fort Collins, United States The cell envelope of mycobacteria is a complex structure, which is important not only for the integrity of mycobacterial cell, but also for pathogenicity and virulence of mycobacteria. It is composed of plasma membrane, mycolyl-arabinogalactan-peptidoglycan core intercalated by variety of lipids and glycolipids and polysaccharide rich capsule. Despite intense research investigating the machinery of its biogenesis during last decades, several steps in the individual pathways still need to be clarified. Much knowledge, especially regarding the processes essential for mycobacteria, arose from the study of its family member corynebacteria. However, functions of some homologous mycobacterial enzymes studied in corynebacteria are still uncertain. The corynebacterial protein MptB was characterized as alpha(1→6) mannosyltransferase involved in the initiation of core mannan synthesis of corynebacterial phosphatidylinositol-based lipomannan and of multi-mannosylated lipopolysaccharide based on glucosyluronic acid-diacylglycerol, which was not detected in mycobacteria (1). While its homologues Rv1459c from M. tuberculosis and MSMEG_3120 from M. smegmatis demonstrated similar activity in vitro, deletion of msmeg_3120 gene in M. smegmatis did not affect the production of mycobacterial lipopolysaccharides lipomannan and lipoarabinomannan (1). To address the function of mycobacterial proteins we generated conditional knock-down mutant of msmeg_3120 gene using TET-PIP OFF system (2) and examined the cell envelope composition of this strain grown in different conditions. 1. Mishra, A. K., et al., (2008), Mol. Microbiol., 68, 1595-1613 2. Kolly, G. S., et al., (2014), Mol. Microbiol., 92, 194-211 Acknowledgements: This work was supported by the Slovak Research and Development Agency under the contract No. APVV-15-0515 and NIH-NIAID grant AI064798. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 149 128 Mycobacterial Biology & Physiology 20/09/2016 Mycofactocin and cellular metabolism in Mycobacteria G. Krishnamoorthy3, P. Kaiser3, R. Hurwitz2, H.J. Mollenkopf1, S.H. Kaufmann3 1 Core Facility Microarray/Genomics 2Core Facility Protein Purification 3Immunology, Max Planck Institute for Infection Biology, Berlin, Germany “Mycofactocin” (Rv0691A, Rv0692) is a short peptide clustered together with a radical S-adenosyl methionine enzyme (Rv0693) and a lactate dehydrogenase (Rv0694) in Mycobacterium tuberculosis (Mtb). This uncharacterized gene cluster is found in many actinobacteria and it is predicted that mycofactocin requires post-translational modification by Rv0693 for its functioning that is still unknown. In this study, we found that the transcript levels of mycofactocin components (Rv0692, Rv0693, Rv0694) increased up to 30 folds under restricted oxygen condition, although these genes were not essential for survival under hypoxic/anoxic conditions. Intriguingly, both under normoxic and hypoxic conditions, deletion of Rv0693 prevented Mtb growth in the presence of fermentable carbon source, whereas deletions of Rv0692 and Rv0694 accelerated the growth rate under similar conditions. Furthermore, targeted metabolic measurements revealed that at least under low oxygen growth conditions, ∆Rv0692 and ∆Rv0694 produced increased abundance of lactate, while ∆Rv0693 exhibited increased extracellular pyruvate. These findings imply that mycofactocin could partner with Rv0694 which mediates the interconversion of pyruvate or lactate and thus regulates glycolytic flux. In addition, the growth of mycofactocin mutants in cholesterol was poor with concomitant decrease in intracellular ATP levels and increased ratio of NADH/NAD+. We found that the observed cholesterol growth defect in the mycofactocin mutants was due to added ethanol, a solvent that is used to solubilize cholesterol. Transcriptome analysis revealed the presence of significant induction (>3 folds) of genes associated with oxidative stress as well as those regulated by DosS/T/R in ∆Rv0693 treated with ethanol, suggesting their inability to resist redox imbalance. Taken together, these results demonstrated that mycofactocin can act as an alternative electron carrier to possibly enable the function/s of dehydrogenases and/or associated redox enzymes, and could potentially contribute to Mtb pathogenesis and persistence in human host. 150 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 129 Mycobacterial Biology & Physiology 20/09/2016 A new class of anti-tuberculosis drug : a Griselimycin targeting DnaN P. Laboudie2, E. Fontaine2, S. Klieber3, I. Blanc2, C. Lair2, X. Boulenc2, C. Cantalloube3, M. Magnon3, S. Sans2, M. Doubovesky2, M. Geslin2, L. Collière1, A. Bauer1, G. Zech1, S. Lagrange2, E. Bacqué2, L. Fraisse2, A. Kling5, R. Müller5, A. Upton6 1 LGCR, Chilly-Mazarin, Frankfurt 2TSU ID, Marcy L'etoile 3DSAR, Montpellier, Vitry Sur Seine, France 4LGCR, Sanofi R&D, Frankfurt 5Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany 6TB Alliance, New York, United States Novel anti TB (tuberculosis) agents with mechanisms of action distinct from current TB drugs are urgently needed due to the increasing prevalence of Drug Resistant (DR) TB. As part of our TB drug discovery strategy, we revisited forgotten antibiotics with high anti-TB potential and re-discovered griselimycin (GM). GM is a natural cyclic depsipeptide, identified in the 1960’s, structurally unrelated to any known TB drug and active against sensitive and DRMycobacterium tuberculosis. We developed a total synthesis to access to GM analogues and to be able to prosecute a lead optimization program. Following an optimization program, we discovered an outstanding TB compound, Cyclohexylgriselimycin (CGM) which was selected for in depth profiling. This compound displayed excellent anti-TB properties comprising in-vivo activity alone and in combinations. We discovered that resistance to griselimycins, occurring at very low frequency, was associated with amplification of a chromosomal segment containing DnaN. Our results demonstrated that griselimycins had high translational potential for tuberculosis treatment, validated DnaN as an antimicrobial target. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 151 130 Mycobacterial Biology & Physiology 20/09/2016 Anti-tuberculosis in vitro and in vivo activity of a new Sequanamycin-like Macrolide C. Lair2, Y. Benedetti3, I. Blanc2, X. Boulenc1, C. Briot1, L. Fraisse2, S. Lagrange2, M. Magnon1, E.L. Nuermberger4, S. Sans2, S. Silve2, A. Upton5, T. Kaneko5, T.J. Yang5, J. Zhang3 1 Disposition Safety and Animal Res, Chilly, Montpellier 2TSU ID, Marcy L'etoile 3LGCR/ Chemistry, Sanofi R&D, Vitry, France 4Johns Hopkins University, Baltimore 5TB Alliance, New York, United States Medicinal chemistry optimization of Sequanamycin A, a 14-membered ring macrolide related to Erythromycin, has lead us to a new class of macrolides with potential for the treatment of Tuberculosis (TB). Here, we present the in vitro and in vivo activity profile of SEQ-9, an optimized Sequanamycin that has demonstrated a promising contribution to anti-TB drug combinations in the infected TB mouse models. SEQ-9 is active on the replicative and non-replicative Mycobacterium tuberculosis (M.tb) phenotypes. It is also potent against M.tb within mouse macrophages. Its even activities across different M.tb lineages and strains mono-resistant to anti-TB drugs are in line with a new mode of action in the TB area. Like classical macrolides, SEQ-9 targets the bacterial ribosome thereby inhibiting protein synthesis but unlike macrolides, it displays a different mechanism of resistance. In the case of classical macrolides, the mechanism of resistance is well known and is linked to the methylation of the ribosome following expression of the Erm37 gene. Contrary to Clarithromycin and Erythromycin, the over expression of this gene in M.bovis or M.tb strains, doesn’t confer resistance to SEQ compounds. On the other hand, mutants resistant to SEQ display an unprecedented deletion in rRNA 23S in M.bovis BCG Pasteur strain. Mouse PK parameters for SEQ-9 have been shown to be suitable for once-daily oral dosing. After single administration of 300 mg/kg, Cmax of 3.1 µg/mL, t1/2 of 5.6 hours and AUC of 21 µg.h/mL were obtained along with a lung/plasma ratio around 14 fold and oral bioavailability at 23%. The minimum effective dose (MED) was reached at 300 mg/kg in the acute TB mouse model. In the chronic TB model, SEQ-9 displayed a dose-dependent cidal efficacy from 75 mg/kg onward with a 1.7 log CFU reduction at 300mg/kg. In combination studies, SEQ-9 increased the bactericidal activity of Pyrazinamide, Linezolid, Pa824 and Bedaquiline. 152 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 131 Mycobacterial Biology & Physiology 20/09/2016 Bioenergetic rewiring: turning the respiratory flexibility of Mycobacterium tuberculosis against itself D. Lamprecht1, P. Finin1, M. Rahman1, B. Cumming1, S. Russell1, S. Jonnala3, J. Adamson1, A. Steyn1-4-5 1 KwaZulu Natal Research Institute for Tuberculosis and HIV (K-RITH), Durban, South Africa 2University of Michigan Medical School, Ann Arbor 3NIAID, Tuberculosis Research Section, NIH, Bethesda 4Centers for AIDS Research and Free Radical Biology 5Department of Microbiology, University of Alabama at Birmingham, Birmingham, United States The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as drug target, however its vulnerability may be affected by its flexibility in response to chemical disruption. To determine the effect of the ETC inhibitors bedaquiline and Q203 on the Mtb ETC and the value of the ETC as a drug target, we measured Mtb’s respiration upon inhibitor addition using existing eukaryotic extracellular flux technology. We found that Mtb’s ETC rapidly reroutes election flux around the sites of inhibition by these inhibitors and increases its total respiration to maintain ATP levels, showing to greater metabolic flexibility than has previously been appreciated. Our data shows that, unlike in eukaryotes, back pressure does not substantially impede ETC activity and allows for the increase respiration in the presence of these ETC inhibitors. The lack of back pressure suggests that an energy spilling pathway modulates PMF in Mtb to prevent excessive accumulation, which would ultimately stop ETC flux. We propose a model whereby both BDQ and Q203 increase total ETC flux and respiration due to loss of feedback inhibition of carbon catabolism and the TCA cycle by ATP. Rather than suggesting that the ETC is an undesirable drug target, however, our results have demonstrated a manner to turn this plasticity against Mtb. In combination targeting of the Mtb ETC by using respiratory inhibitors to rewire energy metabolism, we potentiate CFZ’s production of bactericidal reactive oxygen species (ROS) which lead to enhanced killing in vitro. The efficacy of ETC targeting drug combinations in vitro was validated in a macrophage infection model, which showed no toxicity towards host cell lines. Overall, our findings revealed novel insights into the Mtb ETC as a drug target that can be effectively exploited for new therapeutic intervention strategies. We discovered an example of combination-targeting that denies Mtb of the ATP generation benefits of the ETC, while also exploiting Mtb’s metabolic flexibility to potentiate ETC-mediated ROS toxicity leading to enhanced killing both in vitro and in a macrophage infection model. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 153 132 Mycobacterial Biology & Physiology 20/09/2016 Sodium chloride alters mycobacterial physiology and antibiotics tolerance G. Larrouy-Maumus2, L. Marino1, A. Madduri4, T. Ragan3, D. Hunt3, L. Bassano2, M. Gutierrez3, B. Moody4, F. Pavan1, L. De Carvalho3 1 School of Pharmaceutical Sciences, São Paulo State University, Araraquara, Brazil 2Life Sciences, Imperial College London 3The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom 4Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, United States Deciphering the mechanisms and host clues utilized by the bacterium to adapt and resist to pharmacological sterilization is essential for improved development of novel therapeutics. Among the factors that affect Mtb within host, sodium chloride appeared to be one of the key molecules identified as a novel environmental cue. Hypotonic cultures of Mycobacterium tuberculosis are widely used in basic research and to determine the effects of antibiotics. However, natural infection and persistence within intra- and extracellular host compartments involve exposure to varying salt concentrations. Using kinetic, organism-wide multi-omic measurements we show that exposure of M. tuberculosis to high salinity markedly restricts growth and rapidly generates an adaptive response, known as halotolerance. Our results demonstrate a broad remodelling of metabolism and the cell envelope that occurs in two steps: a fast metabolic response independent of a late response involving gene regulation and lipid remodelling. Together, these remodelling responses render M. tuberculosis less sensitive to saline stress and, importantly, also to several clinically used antibiotics. These findings broadly define the dynamic process of halotolerance in M. tuberculosis, providing a new perception of envelope remodelling and drug action, as well as improved strategies for antimicrobial sensitivity testing. 154 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 133 Mycobacterial Biology & Physiology 20/09/2016 Elucidating a new mechanism of drug activation E. Meiler1, D. Blanco-Ruano1, K. Abrahams2, L. Guijarro-Lopez1, J. Rullas-Trincado1, A. MendozaLosana1, G. Besra2, C. Alemparte1, E. Perez-Herran1 1 Diseases of the Developing World, TB DPU, GlaxoSmithKline, Tres Cantos, Madrid, Spain 2Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom Drug-resistant tuberculosis (TB) is a major concern from a global health perspective. Thus new treatments are needed to combat multidrug and extensively drug resistant TB strains that mainly affect patients in developing countries with limited treatment success. The current treatment against TB consists of combinations of drugs and can take longer than two years for drug-resistant strains. Over the last couple of years, a big effort in phenotypic screening was carried out to identify hits against Mycobacterium tuberculosis (Mtb), the causative bacterium of TB. One series of compounds was discovered to efficiently kill Mtb in such a screen and its biological and pharmacological properties were examined. In spite of the promising in vitro potency and favorable pharmacological properties, one disadvantage of this series appeared to be a relatively high frequency of resistance. Its mechanism of action studies established that it is a prodrug and needs to be activated by a nonessential bacterial enzyme AmiB2. Since the enzyme is not essential, it can be mutated in Mtb causing a rather high level of resistance. This poster describes the profile of the hit and our targetidentification effort. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 155 134 Mycobacterial Biology & Physiology 20/09/2016 Disruption of carbonic anhydrase increases the intracellular virulence of Mycobacterium tuberculosis and alters glycolysis T. Mendum, R. Balhana, J. Mcfadden University of Surrey, Guildford, United Kingdom Carbon dioxide fulfils a number of universally important metabolic and physiological roles in many bacteria, including M. tuberculosis. These include, amongst others, the biosynthesis of lipids, the production of a number of small molecules, parts of important connections within central carbon metabolism, as well as roles in pH homeostasis. In cells, CO2 exists in equilibrium with carbonic acid (H+ + HCO3-), a distinction that is important for many of these enzymatic and physiological functions. The flux between CO2 and HCO3- needs to be sufficient to supply these processes, however, as the rate constant for the hydration of CO2 is relatively low many organisms produce a carbonic anhydrase. This requirement for CO2/HCO3 flux means that carbonic anhydrases have been investigated as potential drug target. The M. tuberculosis genome encodes three such carbonic anhydrases. In this study one of these carbonic anhydrases, Rv1284c, was knocked out, to give a mutant that exhibited a series of unexpected phenotypes: 1. The ΔRv1284 mutant was hyper-virulent in the THP-1 macrophage model of infection. 2. The ΔRv1284 mutant had an enhanced ability to grow on six carbon sugars. 3. The amount of CO2 incorporated into the cell, as measured by C13 analysis of proteogenic amino acids, rose in ΔRv1284 when compared to wild type. All these phenotypes could be complemented, and genomic analysis did not identify any SNP likely to result in such behaviour. These seemingly unrelated phenotypes of macrophage virulence, glycolysis and CO2 incorporation suggest a hitherto unrecognised connection in M. tuberculosis physiology. 156 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 135 Mycobacterial Biology & Physiology 20/09/2016 Molecular Basis of Heme Acquisition by Mycobacterium tuberculosis A. Mitra2, A. Speer1, M. Niederweis2 1 Vrije University Amsterdam, Amsterdam, The Netherlands 2Microbiology, University of Alabama at Birmingham, Birmingham, United States Mycobacterium tuberculosis (Mtb) is primarily transmitted through aerosols which are taken up in the lung by alveolar macrophages. However, Mtb efficiently inhibits macrophage maturation and phagolysosome formation. Acquisition of iron within the macrophage is essential for Mtb survival and virulence. Although iron is abundant it is efficiently sequestered in the human host, e.g. 90-95% of iron is bound in heme and hemoproteins making heme the major iron source in humans. Mtb can acquire iron through production of siderophores called mycobactins, which can solubilize iron from host transferrin or other iron proteins but not from heme. While siderophore-mediated iron acquisition is well studied in Mtb, knowledge about utilization of heme by Mtb is limited. In order to identify molecular components involved in heme acquisition by Mtb we exploited the toxicity of Gallium (III). Ga-PIX was synthesized and a high-density Mtb transposon library was screened for resistance to Ga-PIX. Deletion of two canonical Mtb ppe genes resulted in complete resistance to Ga-PIX relative to wild-type Mtb. Deletion of one ppe gene partially decreased heme utilization, while deletion of the other ppe gene completely abrogated growth of Mtb on heme. Surface plasmon resonance (SPR) spectroscopy indicated that both ppe genes encode heme-binding proteins. Subcellular localization and surface detection experiments indicated that these PPE proteins are cell surface-accessible membrane proteins. These experiments provide the first evidence for cell surface heme-receptors of Mtb and establish a novel role of PPE proteins in Mtb physiology. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 157 136 Mycobacterial Biology & Physiology 20/09/2016 Towards understanding the molecular basis of PknB essentiality in mycobacteria O. Turapov3, F. Forti1, D. Ghisotti1, P. Ajuh4, S. Waddell2, W. Vollmer5, G. Mukamolova3 1 Università degli Studi di Milano, Milano, Italy 2University of Sussex, Brighton 3University of Leicester, Leicester 4Gemini Biosciences Ltd, Liverpool 5Newcastle University, Newcastle, United Kingdom Tuberculosis is a global infectious disease affecting 8 million people and resulting in 1.5 million deaths annually. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has evolved mechanisms for evasion of host immune system and can persist in vivo without causing an active disease. Eleven serine/threonine protein kinases control Mtb growth, persistence and cell wall biosynthesis. PknB is an essential serine/threonine protein kinase required for mycobacterial growth, however the molecular basis of its essentiality remains unknown. We have designed a special medium for supporting growth of PknB-depleted Mtb and conducted omics analyses of PknB-depleted and PknB-producing Mtb bacilli. Our phosphoproteomics data indicate that 47 proteins were up-phosphorylated in the PknB producing strain compared to the PknB-depleted strain and 9 out of them have been previously annotated as essential proteins. Our transcriptome analysis showed that the depletion of pknB altered expression of 48 genes. Based on our results we propose that PknB is dispensable for growth and division itself but it plays a critical role in coordinating cell wall biosynthesis and cell growth. We are currently validating the biological significance of phosphorylation of PknB substrates identified in our study. Elucidation of the molecular mechanism of PknB essentiality will offer novel opportunities for treatment and prevention of tuberculosis. 158 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 137 Mycobacterial Biology & Physiology 20/09/2016 Novel method to improve recovery of mycobacteria from difficult samples T. Munshi, P.M. Lopez-Perez, J. Garcia, S. Hofmann, K. Hilpert, T. Bull Institute for infection & immunity, St. George's, University Of London, London, United Kingdom Isolation of mycobacteria is the gold standard diagnostic technique. Slow division rates combined with stringent decontamination methods drastically slow down mycobacterial growth and require long incubation times. In children, respiratory sampling can be difficult and faecal/ urine samples are prone to high contamination rates. New more efficient methods are called for to decontaminate and stimulate mycobacterial growth from samples. Here, we describe an innovative approach to decontaminate and culture mycobacteria from difficult samples using sequential combinations of antimicrobial compounds that kill contaminating flora while promoting mycobacterial growth. We screened for a complex combination of antimicrobial compounds (TiKa-Kic) against bacteria (Gram positive and negative) and fungi whilst having no deleterious effect on mycobacterial species. We evaluated the overnight TiKa-Kic treatment of faeces spiked with Mycobacterium tuberculosis (MTB), followed by culture in MGIT 320 detection system supplemented with a different growth stimulating compound (TiKa-B08). This sample preparation method was highly effective at decontaminating samples and gave rapid MTB growth with time to positivity in ~10 days for 1x106 organisms, ~16 days for 1x104 organisms, ~25 days for 1x102 organisms with no background contamination. As a pilot study, we also used this system to culture mycobacteria from human and animal (tissue, sputum, faeces and urine) samples with known mycobacterial disease. We were able to isolate M. tuberculosis, M. bovis and M. avium subspecies paratuberculosis from these samples in MGIT system as well as on solid media plates without any contamination. The method provides an innovative and convenient technique that offers significant progress in isolation of mycobacterial species from difficult samples and improving timely confirmatory diagnoses. The technique is especially relevant for testing faecal samples from young children with suspected MTB infection who tend to swallow rather than expectorate their sputum. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 159 138 Mycobacterial Biology & Physiology 20/09/2016 Supercoiling sensitive transcription and topology –transcription coupling in Mycobacterium tuberculosis W. Ahmed1, C. Sala2, S. Hegde1, S. Cole2, V. Nagaraja1 1 Department Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India 2Global Health Institute, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland Transcription relies on DNA topology at different steps of the process. During initiation, the promoter melting is facilitated by negative supercoiling of the template. Next, during transcription elongation, positive and negative supercoils generated ahead and behind the moving elongation complex have to be removed by the action of two different topoisomerases, namely DNA topoisomerase I and DNA gyrase respectively. Thus, to maintain the topological homeostasis of the genome and to maintain genome architecture, DNA topoisomerases catalyse DNA cleavage, strand passage and rejoining of the ends. The transcription of the genes encoding topoisomerases are in turn regulated in response to alterations in genome supercoiling. Gyrase expression is up-regulated by relaxation stimulated transcription (RST) by distinct mechanisms in E.coli, Mycobacterium tuberculosis and Mycobacterium smegmatis. More recently, we have uncovered additional regulatory mechanism of gyr operon regulation in M.tuberculosis involving a process termed as reiterative transcription. However, the molecular mechanism of transcriptional regulation of Topoisomerase I is not studied so far. We describe supercoiling sensitive transcription (SST) of the topoI gene. The expression is regulated by high transcription of the upstream gene and the promoter architecture of the topoI. Perturbation of topology induced in the conditional knock down strains of these essential enzymes affect transcription initiation and elongation leading to several pleiotropic downstream consequences. To understand further the in vivo roles of DNA gyrase and Topoisomerase I in chromosome dynamics and gene expression, we have carried out a genome-wide occupancy studies of these enzymes along with RNA polymerase. These data provide the first glimpse of topoisomerase traffic on the transcription units in vivo. The pattern of the distribution profile of RNA polymerase and the topoisomerases suggests the operation of twin - supercoiled domain during transcription validating the long standing model. 160 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 139 Mycobacterial Biology & Physiology 20/09/2016 Iron sensing via a catalytically divergent rhomboid protease S. Nambi, C. Sassetti Umass medical school, Worcester, United States Environmental sensing in prokaryotes can be accomplished by the release of regulatory proteins from the membrane following two sequential proteolytic events. In Mycobacterium tuberculosis (Mtb), Rip1, a zinc metalloprotease similar to other described site-2 proteases mediates the final release of anti-sigma factors as part of this process. However, site-1 protease(s) that sense environmental signals and initiate the cascade have not been identified in Mtb. We report that a functionally non-annotated and highly divergent member of the rhomboid family of intramembrane proteases performs this activity. Despite lacking a canonical rhomboid active site, Rv3193c (RomA) uses distinct catalytic residues to perform the site 1 cleavage of the anti-sigma factor (RsmA), priming it for processing by Rip1. A physical interaction between the PDZ domain of Rip1 and a proline-rich region in extracytoplasmic domain of RomA facilitate the signaling cascade. Structural analysis of the extracytoplasmic domain revealed a conserved iron coordination site, indicating a likely environmental trigger for this system. Indeed, we found that RsmA processing was altered by extracellular iron concentration, and RomA-deficient bacteria were unable to replicate in low iron conditions due to altered levels of iron homeostatic genes. The unique topology, active site, and sensory domains of catalytically divergent rhomboid family (cdRhom) impart an environmental sensing function that is unprecedented for this family of proteins. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 161 140 Mycobacterial Biology & Physiology 20/09/2016 Fishing for bortezomib mechanisms of resistance in mycobacteria G.J.Y. Ngan, W. Moreira, T. Dick Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore We previously identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth in mycobacteria. Here we aimed to elucidate mechanism of resistance of bortezomib in mycobacteria. We selected spontaneous resistance mutant of bortezomib in Mycobacterium smegmatis and characterized them. We ruled out the genetic modification and changes in expression level of the target ClpP1P2, and the adaptor proteins and the regulator of Clp (ClpC1, ClpX, clgR). Phenotypic characterization of bortezomib mutants suggests correlations between resistance level, growth, ATP level, membrane potential and resistance to a panel of other antibiotics (Rifampicin, Vancomycin, Isoniazid, Bedaquiline and Ethionamide). Based on these observations, we speculate that alteration of cell envelope permeability may contribute to resistance. Currently, we employ whole genome sequencing to determine the mechanism of resistance of mutants. Acknowledgements: This work was supported by the Singapore Ministry of Health’s National Medical Research Council grant (NMRC/ CBRG/0022/2012) and its Centre grant MINE / Research core no. 4 (NMRC/CG/013/2013) to T.D., and is part of the Singapore Programme of Research Investigating New Approaches to Treatment of Tuberculosis (SPRINT-TB; www.sprinttb.org) led by Nick Paton. G.N. receives a SMA3 fellowship from Singapore MIT Alliance for Research and Technology (SMART). Reference: Moreira W, Ngan GJY, Low J. L, Poulsen A, Chia BCS, Ang MJY, Yap A, Fulwood J, Lakshmanan U, Lim J, Khoo AYT, Flotow H, Hill J, Raju RM, Rubin EJ, Dick T. 2015.Target mechanism-based whole-cell screening identifies bortezomib as an inhibitor of caseinolytic protease in mycobacteria. mBio 6(3):e00253-15. doi:10.1128/mBio.00253-15. 162 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 141 Mycobacterial Biology & Physiology 20/09/2016 Significant under expression of the DosR regulon in M. tuberculosis complex Lineage 6 in sputum B. Ofori-Anyinam1-2, G. Dolganov6, T. Van6, L. Davis5, N. Walter4, B. Garcia4, M. Voskuil4, K. Fissette1, M. Diels1, M. Driessen1, C. Meehan1, M. Coscolla3, S. Gagneux3, M. Antonio2, G. Schoolnik6, F. Gehre1-2, B. De Jong1 1 Institute of Tropical Medicine, Antwerpen, Belgium 2Medical Research Council Unit, The Gambia, Fajara, Gambia 3Swiss Tropical and Public Health Institute, Basel, Switzerland 4University of Colorado Denver, Aurora, Colorado 5Yale University, New Haven, Connecticut 6Stanford University, Stanford, California, United States Background: Mycobacterium africanum L6 is associated with HIV-infection, extrapulmonary disease, and with slower progression to active tuberculosis disease as compared with Mycobacterium tuberculosis sensu stricto. The biology underlying these clinical differences remains poorly understood. Within the host, the DosR regulon of M. tuberculosis complex allows for quick adaptation to oxygen limitation and genetic mutations within the regulon leads to growth and virulence attenuation. Early literature indicated a preference of M. africanum for microaerobic growth. Therefore, we suspected that the dormancy regulon would be differentially induced between M. tuberculosis and M. africanum L6. Methods: We compared the ex vivo expression of 2179 genes of M. tuberculosis and M. africanum L6 in sputa from 9 HIV-negative TB patients from The Gambia, six with M. tuberculosis and 3 with M. africanum L6. Further, we compared polymorphisms in the differentially expressed genes, as well as related genes, in 44 M. africanum L6 genomes from TB patients from The Gambia and Ghana. Lebek’s test was used to confirm differences in oxygen requirements for growth. Results: All genes within the DosR regulon had lower expression in M. africanum L6 compared to M. tuberculosis with half (n=26) being significantly under expressed (Modified Fisher’s exact p-value =9.59E-13, Bonferroni Adjusted p-value = 1.06E-11). Lineage 6 defining nonsynonymous mutations were detected in PhoR as well as the intergenic region of Rv0080 and the foremost gene regulator in M. tuberculosis during hypoxia, Rv0081. In Lebek’s test, M. tuberculosis grew only at the aerobic surface while M. africanum L6 grew throughout the medium. Conclusions: Based on the relative under expression of DosR regulon genes, M. africanum L6 appears to have adapted to growth under hypoxic conditions potentially through DosR-independent mechanisms or to different biological niches. These findings fit with genomic changes in related genes and a preference for microaerobic growth as well as the association with extrapulmonary disease. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 163 142 Mycobacterial Biology & Physiology 20/09/2016 Evaluation of Genotype MTBDRsl version 2 for rapid detection of second-line antituberculosis drug resistance in Ghana identifies the first pre-XDR TB case S. Osei-Wusu2-3, A. Asante-Poku2, I. Otchere Darko2, S. Omari1, A. Forson1, D. Yeboah-Manu2 1 Department of Chest Diseases, Korle-Bu Teaching Hospital 2Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana 3West Africa Centre for Cell Biology of Infectious Pathogens, University of Ghana, Legon, Ghana, Accra, Ghana Background: The emergence of drug resistance TB strains requires rapid but sensitive and specific tools for control. We evaluated the genotype MTBDRsl v2 for detecting second-line anti-TB drug resistance in Ghana. Method: We screened 117 resistant TB isolates against moxifloxacin (MOX), streptomycin (STR) and amikacin (AMK) using the Epsilometer test (Etest) and with the MTBDRsl rapid kit. Drug resistant conferring genes (gyrA, gyrB, eis, rrs, tap, whiB7 and tlyA) of strains resistant to MOX or AMK were PCR amplified and sequenced for mutation analysis. Results: Isolates phenotypically resistant to STR, AMK and MOX were 38 (32.5%), 13 (11.1%) and 1 (0.9%) respectively. Among the isolates, only 1 multidrug resistant (MDR) isolate was diagnosed resistant to both MOX (MIC=3µg/mL) and AMK (MIC =16µg/mL) by both the phenotypic assay and MTBDRsl LPA. Gene sequencing analysis led to the identification of gyrA (G87C) and rrs (A514C and A1401G) in this isolate. Whereas none of the remaining isolates were resistant to MOX (MIC<0.125µg/mL), 9/76 (11.8%) MDR, 1/14 (7.1%) rifampicin resistant and 3/27 (11.1%) isoniazid resistant isolates were resistant to AMK with MICs ranging from 1 to 8µg/mL. However, none of these were identified as resistant to AMK by the MTBDRsl LPA. Except one MDR isolate harbouring a tlyA mutation (N236K), no other mutation was detected. The agreement between the phenotypic and the LPA was 87.1% and 100% whereas the sensitivity/specificity was 7.7%/100% and 100%/100% for AMK and MOX respectively. Conclusion: We report the first instance of a pre-XDR-TB case in Ghana. Our findings support the use of the MTBDRsl v2 for detecting second anti-TB drug resistance in Ghana. 164 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 143 Mycobacterial Biology & Physiology 20/09/2016 Single-cell growth and antibiotic tolerance in mycobacteria M. Priestman1, B. D. Robertson1, V. Shahrezaei2 1 Centre for Molecular Bacteriology and Infection 2Department of Mathematics, Imperial College London, London, United Kingdom Drug tolerance is the phenomenon by which bacteria that are otherwise genetically susceptible to an antibiotic are able to survive extended treatment. This is usually associated with growth arrest and shutdown of protein synthesis, to form 'persister' cells. Persister cells form a small subset of genetically homogenous populations, with a phenotypic switch triggering the entry and exit into persistence. Persistence in mycobacteria remains relatively poorly characterised, and given the lengthy treatment regime required to completely eradicate M. tuberculosis infections, understanding how mycobacteria enter into this drug-tolerant state may be of great value for drug development and strategies. Microfluidics and timelapse microscopy allows the tracking of single cells and their subsequent fate upon exposure to antibiotics. Growth conditions can be rapidly changed, and antibiotics added at specific timepoints, allowing the precise control of the environment, revealing how growth affects survival to antibiotics at the single cell level. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 165 144 Mycobacterial Biology & Physiology 20/09/2016 Quiescence survival in Mycobacterium tuberculosis requires a cAMP mediated metabolic switch E. Rittershaus, S. Baek, S. Nambi, C. Sassetti Microbiology & Physiological Systems, Sassetti Lab, University of Massachusetts Medical School, Worcester, Massachusetts, United States Upon exposure to respiratory stress imparted by the host, e.g. nitric oxide or hypoxia, M. tuberculosis slows its growth and is relatively unresponsive to antibiotic treatment. In this study, we utilized a combination of metabolite profiling in tandem with comprehensive mutant fitness analysis to identify critical regulatory or metabolic pathways that could be targeted to kill quiescent cells. Metabolomics revealed alterations in abundance of TCA intermediates that correlated with increased cAMP. The parallel genetic study identified the responsible adenylate cyclase (Rv1359) and a cAMP-binding protein acetyl-transferase (Pat/Rv0998) that were conditionally essential for survival in hypoxia. 13C-labeled carbon tracing in hypoxia revealed that this cAMP pathway mediates a switch in the TCA cycle from oxidative to reductive reactions. Inhibition of Rv1359 and Pat to make this metabolic shift resulted in over-reduction and reduced viability. Genetic inhibition of TCA enzymes verified the importance of reductive reactions in hypoxic cultures and murine lung. Under both conditions inhibition of reductive TCA resulted in rapid cell death, suggesting a new strategy for eradication of quiescent bacteria. 166 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 145 Mycobacterial Biology & Physiology 20/09/2016 Identifying the critical oxygen response regulators using a novel regulator overexpression mutant pool screen T. Rustad1, J. Farrow-Johnson1, K.J. Minch1, D.R. Sherman1-2 1 Center for Infectious Disease Research 2Department of Global Health, University of Washington, Seattle, United States Hypoxia is one of the stresses encountered by M. tuberculosis (MTB) during the course of infection and is associated with latent disease. In response to lowered oxygen tension, MTB alters the expression of hundreds of genes, including dozens of transcription factors (TFs), any of which could be participating in generating the hypoxic response. The initial transcriptional adaptation to hypoxia is mediated by the TF DosR, but little is known about the regulators of the much larger subsequent transcriptional signature. To identify which TFs are critical for the adaptation to hypoxic stress, we developed a regulator overexpression mutant pool (ROMP) assay to identify TFs with stress-specific phenotypes. This method involves a pool of 207 MTB mutants that each contain a TF under control of a tetracycline inducible promoter. This approach allowed us to screen the regulons associated with all known TFs for growth phenotypes in response to reduced oxygen tension, using high throughput sequencing of mutant-specific tags to follow all 207 mutants in a single experiment. This ROMP assay identified five critical oxygen responsive regulators (CORRs) with inhibited growth when recovering from hypoxic stress. Using our previously generated transcriptional regulatory map we found that these CORR TFs coregulate a significant number of genes, suggesting that these TFs operate as a tightly knit regulatory subnetwork. Finally, we show that the majority of the genes that change expression in response to hypoxia interact with one or more of the CORRs. In summary, this work presents a novel approach that can be used to screen for stress-specific TF phenotypes as well as a previously undescribed hypoxic response regulatory network. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 167 146 Mycobacterial Biology & Physiology 20/09/2016 A two-stress model for the generation of viable M. tuberculosis cells that do not form colonies K. Saito2, T. Warrier1, S. Somersan-Karakaya2, J. Mi1, S. Park1, X. Jiang1, B. Gold1, J. Roberts1, C. Nathan1 1 Department of Microbiology and Immunology, Weill Cornell Medicine 2Department of Medicine, Division of Infectious Diseases, Weill Cornell Medicine/NY Presbyterian Hospital, New York, United States Despite the need for more rapidly effective therapy for tuberculosis, limited understanding of phenotypically drug-tolerant states of Mycobacterium tuberculosis (Mtb) hampers the development of new drugs. Non-replicating Mtb becomes relatively or absolutely tolerant to most TB drugs. Among phenotypically tolerant and temporarily non-replicating Mtb populations are cells that have lost the ability to grow as CFU on agar and yet are proven viable in other ways, marking them as “differentially detectable” (DD). Some DD Mtb can be detected and quantified by culturing replicate limiting dilution (LD) series in liquid medium, with or without fresh Mtb culture filtrate (CF). The highest dilution that produces growth is used to back-calculate the original quantity. A statistical formula can adjust for differences in the dilution limit seen in each replicate series to identify the most probable number (MPN). LD will exaggerate the MPN if clumps of cells break up during dilution. This artifact handicaps study of DD Mtb despite its potential clinical importance, as does the inconsistent impact of CF, the divergent identifications of effector molecules in CF, and the lack of confirmation of reported methods for generating DD Mtb in vitro. In an effort to minimize such obstacles, we studied numerous technical variables—first with manual handling and then with robotics—until quantification of Mtb by CFU and by LD produced indistinguishable numbers, except after certain treatments. We then tested nutrient starvation, hypoxia, acidification, human plasma, neutrophils or their lysates, reactive nitrogen species, TB drugs and prolonged stationary phase for their ability to induce DD Mtb. We could only reproducibly generate populations of >=90% DD Mtb with two stresses in sequence: starvation followed by exposure to rifampin. The proportion of DD Mtb increased with the duration of starvation. Longer drug exposure and longer starvation allowed DD Mtb generation with less rifampin. CF had no effect. This model supports two lines of inquiry. First, we are trying to identify genes required for entering or sustaining the DD Mtb state. Second, we are testing compounds that can kill Mtb in other states and classifying whether they generate, kill or have no effect on DD Mtb. 168 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 147 Mycobacterial Biology & Physiology 20/09/2016 Non-replicative in vitro tests for Tuberculosis drug research S. Sans1, B. Gold3, H. Boshoff2, L. Gouilleux1, C. Couturier1, K. Kissane1, I. Blanc1, K. BurnsHuang3, L. Lopez Quezada3, E. Ballinger3, J. Roberts3, Y. Ling3, D. Zhang3, G. Mondesert1, E. Bacqué1, L. Fraisse1, C. Nathan3, S. Lagrange1, C. Roubert1 1 TSU Infectious Disease, Sanofi R&D, Marcy L'etoile, France 2Tuberculosis Research Section, NIAID, Bethesda 3Departments of Microbiology & Immunology, Weill Cornell Medical College, New York, United States Tuberculosis (TB) is the first leading infectious cause of death worldwide and WHO estimates that one third of the world population is silently carrying M. Tuberculosis (Mtb), the etiological agent of tuberculosis. This pathogen is highly adapted to humans, its exclusive host. Humans and Mtb have co-evolved for more than 70000 years. In most cases of primary infections, the human immune system is able to clear the bacteria or at least “trap” the bacteria, thereby controlling the disease. However, Mtb has developed a “persister” state in which its metabolism is minimized in order to escape the host immune system, to tolerate drug treatment and to adapt to different micro-environments. These socalled Mtb persisters are believed to be the potential source of reactivation of active TB, years after primary infection. As a consequence, a complete cure of tuberculosis requires a combination of drugs that not only inhibit growth of replicating bacteria but also kill the persister bacteria. For characterizing new compounds with potential against the persister sub-population, multiple nonreplicative (NR) assays using different stress factors as well as multiple readouts have been reported. In this poster, we will describe, compare and highlight pros and cons of these in vitro models of non-replicative bacteria, using standard anti-tuberculosis drugs. Furthermore, in order to develop new anti-tuberculosis drugs active on NR Mtb, we have screened a 80 K compound library against a Mtb strain under different conditions designed to mimic four physiological stresses encountered by Mtb in one or more of its microenvironments in the host: acidity, reactive nitrogen intermediates, hypoxia, and fatty acid as a carbon source (Gold et al.; 2015). Following active hit triaging, we have been able to identify several series that display specific patterns under the different NR conditions. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 169 148 Mycobacterial Biology & Physiology 20/09/2016 EspR-dependent ESAT-6 secretion of Mycobacterium tuberculosis requires the presence of virulence-associated response regulator PhoP V. Anil Kumar, R. Goyal, R. Bansal, N. Singh, A. Kumar, D. Sarkar Microbiology and biochemistry laboratory, CSIR - Institute of Microbial Technology, Chandigarh, India M. tuberculosis (Mtb) uses the ESX-1 secretion system to transport virulence factors in host cell. ESX-1 secretion system is encoded by the genes of the esx-1 locus, which is highly conserved in members of the Mtb complex and in other pathogenic mycobacteria. Attenuation of M. bovis BCG strain is related to the loss of RD1-encoded ESX-1 secretion system. M. tuberculosis ESX-1 system secretes virulence factor ESAT-6 which plays a critical role in modulation of host immune system for establishment of productive infection. Previous studies suggest that among the reasons for attenuation of M. tuberculosis H37Ra is a mutation in the phoP gene that interferes with the ESX-1 secretion system and inhibits secretion of ESAT-6. Here, we identify a totally different and distinct regulatory mechanism involving PhoP and transcription regulator EspR on transcriptional control of the espACD operon, which is required for ESX-1-dependent ESAT-6 secretion. While both these regulators are capable of influencing espACD expression, we show that activation of espACD requires direct recruitment of both PhoP and EspR within the promoter. The most fundamental insights derive from the inhibition of EspR binding at the espACD regulatory region of phoP mutant strain owing to PhoP-EspR protein-protein interactions. Based on these results a model is proposed suggesting how PhoP and EspR protein-protein interactions contribute to activation of espACD expression, and in turn, controls ESAT-6 secretion, an essential pathogenic determinant of M. tuberculosis. Together, these results have significant implications on the mechanism of virulence regulation of M. tuberculosis. 170 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 149 Mycobacterial Biology & Physiology 20/09/2016 Investigating the cost of rifampicin-resistance in mycobacteria B. Sewgoolam, A. Koch, V. Mizrahi, D. Warner Dept. Pathology, Molecular Mycobacteriology Research Unit, University of Cape Town, Cape Town, South Africa Rifampicin (RIF), the front-line anti-tubercular drug, targets the rpoB-encoded β-subunit of RNA polymerase (RNAP), blocking transcription. In Mycobacterium tuberculosis (Mtb), mutations in rpoB are primarily associated with RIF resistance (RIFR), which poses a major threat to tuberculosis (TB) control. Some rpoB mutations have been associated with fitness costs, however the observed phenotypes are often dependent on strain background and the assay system used to measure them. Therefore, determining the implications of RIFR-associated rpoB mutations for mycobacterial physiology is essential for understanding the development and maintenance of RIFR, as well as the spread and pathogenesis of drug resistant strains of Mtb. In other bacterial systems, mutations in rpoB have been associated with alterations to core bacterial functions and physiology. Similarly, in Mtb, recent reports have identified changes to components of the cell wall, the proteome, and fatty acid metabolism; increased expression of dnaE2, encoding a DNA damage-inducible mutagenic repair polymerase; and increased ofloxacin minimum inhibitory concentrations (MICs). In all studies to date, RIF exposure was used to select the resulting rpoB mutant strains; therefore, it is unknown whether secondary mutations, or potentially even epigenetic effects, might have influenced the assay outcome. To address this potential limitation, we have applied site-directed mutagenesis and two-step allelic exchange to insert a RIFR SNP into the native Mtb rpoB gene in the absence of RIF selection. Whole-genome sequencing will be used to determine whether the defined rpoBS531L (TCG->TTG) mutant contained any secondary mutations. Thereafter, this RIF naïve strain will be applied in standard microbiological and immunological assays to investigate the impact of a clinically relevant rpoB mutation on functions unrelated to drug resistance. Priority assays include characterization of the transcriptome via RNA-seq, and macrophage survival assays. In addition, a flow cytometry assay is being optimized in parallel for corresponding mutants of M. smegmatis that will allow culture free determination of cell numbers in growth-based assays. This will allow for accurate determination of the fitness of the rpoB mutant strain in competition with the wild-type parental strain, under a range of growth conditions that are considered relevant to host infection. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 171 150 Mycobacterial Biology & Physiology 20/09/2016 Serine/threonine protein phosphatase of Mycobacterium tuberculosis is necessary for precise cell division A.K. Sharma2, D. Arora3, L.K. Singh2, A. Sajid2, V. Molle1, Y. Singh2, V.K. Naandicoori3 1 CNRS-Universite de Montpellier II, Montpellier, France 2CSIR-Institute of Genomics and Integrative Biology 3National Institute of Immunology, Delhi, India Signal sensing and transduction leads to a wide range of cellular responses, and thus must be tightly regulated in pathogenic bacteria to allow optimal survival under variable conditions. A common mode of regulation of the cell’s response to external cues is via phosphorylation/dephosphorylation of specific target proteins that lead to cellular responses like altered subcellular localization of proteins, protein turnover rates, and protein-protein interactions. The analysis of the M.tuberculosis whole genome sequence identified the presence of 11 eukaryotic-like STPKs, one Ser/Thr phosphatase (PstP), one tyrosine kinase (PtkA), and two tyrosine phosphatases (PtpA and PtpB). All these kinases and phosphatases have now been characterized and found to be catalytically active. The lone serine/threonine phosphatase of M. tuberculosis, PstP belongs to Metal- dependent family of phosphatase whose members strictly require Mn2+ ion for their activity. While two metal centers are found in the catalytic core in most PP2C phosphatases, crystal structural of PstP showed presence of three metal-binding centers. The conserved active site residues in D38 and D229 are involved in metal binding and R20 involved in binding with the phosphate moiety in the target proteins, bridging together to stabilize the active structure. Previous data from our laboratory have shown that PknA and PknB phosphorylate PstP on specific residues in its cytosolic domain, and this phosphorylation is influenced by the presence of Zn2+ ions and inorganic phosphate (Pi). The phosphatase-dead PstP mutant PstPcD38G is more efficiently phosphorylated by PknA and PknB. Importantly, we found that the phosphorylated PstP is more active compared with its unphosphorylated counterpart, suggesting a possible reverse regulation mediated through phosphorylation cascades by STPKs. Investigations carried out thus far are in-vitro studies using purified PstP. The present study aims to investigate the impact of PstP overexpression or depletion scenario on the growth of the pathogen and its survival within the host. Using these mutants we investigated the role of various domains of PstP in modulating cellular events, cell division and host adaptation. Also we identified mechanistic pathway important for cell division of M.tuberculosis. 172 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 151 Mycobacterial Biology & Physiology 20/09/2016 Survival strategies of Mycobacterium tuberculosis: Insights from the vitamin C dormancy model K. Sikri, S. Batra, M. Nandi, J. Tyagi TB Molecular Biology Lab, Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that successfully resides in the hostile phagosomal environment by resisting multiple stresses including hypoxia, low pH, nutritional deficiency, and oxidative and nitrosative stresses. The success of tubercle bacilli comes from their ability to employ strategies that enable their adaptation to a state of ‘dormancy’/ persistence. A serendipitous discovery was made in our laboratory that vitamin C (vit C) triggered the induction of the DevR ‘dormancy’ regulon in axenic cultures of Mtb, owing to its O2 scavenging properties. Vit C also induced bacteriostasis and an isoniazid-tolerant phenotype, an accepted indicator of bacterial adaptation to hypoxia. Further, vitC-treated cultures developed a ‘viable but non-culturable’ antibiotic-tolerant state, which was reversed on withdrawal of vit C. Vit C is reported to have a significant effect on macrophage physiology and on the outcome of tuberculosis infection. Therefore, a global gene expression analysis was undertaken to decipher the molecular events underlying the adaptive response of Mtb to vit C. A massive transcriptional response was noted in axenic cultures that included the DevR ‘dormancy’ regulon, triacylglycerol synthases, efflux pumps and oxidative stress response genes. These responses were consistent with development of a state of ‘dormancy’/ persistence in Mtb. Notably, the transcriptional response to vit C mimicked bacterial responses to (1) multiple intracellular stresses, thus generating a multiple stress environment for the bacterial cell, and more importantly (2), an activated macrophage-like condition. These findings signify that vitamin C has wide-ranging regulatory effects on Mtb and offers a new perspective on the possible contribution of vit C to Mtb adaptation. In the current scenario of TB disease, vit C could either modulate the efficacy of anti-tubercular drugs or contribute to TB control among immunocompromised individuals, as in HIV subjects. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 173 152 Mycobacterial Biology & Physiology 20/09/2016 Investigating the role of toxin-antitoxin systems in physiology and persistence of Mycobacterium tuberculosis P. Tiwari, S. Agarwal, S. Kidwai, R. Singh Vaccine and Infectious Disease research centre, Translational Health Science and Technology Institute, Haryana, India Toxin-antitoxin (TA) systems are highly conserved in members of the Mycobacterium tuberculosis (Mtb) complex and have been postulated to play an important role in physiology and virulence. By overexpressing each of the putative toxins in M. bovis BCG using inducible systems, we have identified functional toxins. We report that overexpression of these TA systems is bacteriostatic and few of these also disrupt the membrane integrity. We have constructed various TA deletion mutants of Mtb to understand their role in physiology and persistence. The construction of various single-, double- and triple-mutant strains revealed that these ribonucleases contribute synergistically to the ability of Mtb to adapt to conditions such as oxidative stress, nutrient depletion and drug exposure. The genes induced in strains overexpressing these ribonucleases were identical to those induced in persister, hypoxic and nutritionally starved bacteria. Moreover, guinea pigs infected with TA systems deficient strains exhibits significantly reduced bacterial loads and pathological damage in infected tissues in comparison to parental strain-infected guinea pigs. Currently, experiments are in progress to identify cellular targets for these TA systems and to understand the cellular mechanisms by which these TA systems are activated. The present study highlights the importance of TA systems in Mtb stress adaptation, drug tolerance and virulence. 174 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 153 Mycobacterial Biology & Physiology 20/09/2016 Genetic geography of mycobacterium tuberculosis beijing genotype V. Slizen1, L. Surkova2, A. Zalutskaya2 1 Microbiology, Virology, Immunology, Belarusian State Medical University 2Republican ScientificPractical Center of Pulmonology and Tuberculosis, Minsk, Belarus High disease rate of extended and multi-drug resistant tuberculosis (XDR, MDR) in some countries may be associated with the spread of highly virulent genetic variants, such as Beijing, the proportion of which in the TB structure can vary from depending on the geographical region. The aim was to identify genotypes of pre-XDR strains of MTB, isolated from patients in Belarus. Methods: Genotypes of sixty five pre-XDR strains of MTB were detected by MIRU-VNTR typing method based on the evaluation of differences in 15 VNTR-loci. Mutations in the gene gyrA of MTB were detected by sequencing as well as by GenoType MTBDRsl (HAIN), qPCR with hydrolysis probes. Results: It was shown the genotype Beijing to be dominant, comprising 58% of all pre-XDR MTB. MTB, belonging to genotype Beijing, carried nucleotides ACC but not AGC in codon 95 of gene gyrA. Haarlem and TUR genotypes were detected in 23 and 16% of isolates, respectively. Pre-XDR strains of MTB belonging to genotype Beijing demonstrated sufficient genetic homogeneity and had identical MIRU-VNTR profile 2-3-5-3-3-5-4-4-4-2-3-4-6- 7-5 (sequence loci MIRU04- MIRU40MIRU26- MIRU10- MIRU16- MIRU31- ETRA- ETRC- Mtub04- QUB4156- MTUB39- MTUB30QUB11b- QUB26- MTUB21). MTB, belonging to genotype Haarlem and TUR were mostly with 2-35-5-3-3-3-3-2-3-3-4-3-3-3 and 1-2-1- 5-1-3-3-4-4-3-3-4-2-6-2 MIRU-VNTR profiles, respectively. Out of 65 pre-XDR isolates, 83% were found to harbour mutations within gyrA. The most common mutations were observed at codon 94 (n =35), where the wild-type aspartic acid was replaced with glycine (Asp94Gly; n =19), alanine (Asp94Ala; n = 7), or tyrosine (Asp94Tyr; n = 7) or histidine (Asp94His; n=1). Conclusion: In Belarus, as in other post-Soviet countries, the epidemic process of TB is mainly sustained by genotype Beijing. Continuous circulation of genotype Beijing results in the accumulation of mutations and increases the proportion of MDR and XDR. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 175 154 Mycobacterial Biology & Physiology 20/09/2016 Investigation of the spatio-temporal dynamics of peptidoglycan synthesis in Mycobacterium tuberculosis J. Vaubourgeix2, H. Botella2, G. Yang1, O. Ouerfelli1, C. Nathan2, S. Ehrt2 1 Organic Synthesis Core Facility, Memorial Sloan-Kettering Cancer Center 2Microbiology and Immunology, Weill Cornell Medicine, New York, United States Peptidoglycan (PG), a complex polymer of glycan strands cross-linkeded by short D-amino acidcontaining peptides, is an essential component of the bacterial cell wall. PG biosynthesis is the target of beta-lactam antimicrobials, some of which have recently been found to be cidal for Mycobacterium tuberculosis (Mtb), including for non-replicating populations (e.g., Gold et al., J. Med. Chem. in press). We used supravital fluorescent D-alanine analogs (FDAAs) that incorporate into peptidoglycan to investigate the spatio-temporal dynamics of PG synthesis in Mtb in axenic culture and in macrophages. FDAA incorporation into Mtb is predominantly polar, but with striking variations in polar dominance as a function of cell cycle. Immediately after cytokinesis, FDAAs are incorporated chiefly at the old pole. In contrast, just before cytokinesis, FDAAs are incorporated comparably at both poles. These observations suggest that specific enzymes involved in PG synthesis are localized in functional compartments at the poles and further, that Mtb possesses a mechanism for maturation of the new pole. Deeper knowledge of the cell and molecular biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process. 176 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 155 Mycobacterial Biology & Physiology 20/09/2016 Plasticity of M. tuberculosis NADH dehydrogenases and their role in virulence C. Vilcheze2, L. Leung3, G. Cook1, W.R. Jacobs Jr2 1 Dept of Microbiology and Immunology, University of Otago, Dunedin, New Zealand 2Microbiology and Immunology, Howard Hughes Medical Institute at AECOM, Bronx, New York 3Grayboxbiology, New York, United States The NADH dehydrogenases, which oxidize the electron donor NADH into NAD+, are the first enzymes in the electron transport chain. Mycobacterium tuberculosis possesses two types of NADH dehydrogenases: the proton-pumping NdhI, a 15704 bp operon composed of 14 subunits (NuoA-N, Rv3145 to Rv3158), and the non-proton pumping, single polypeptide NdhII, which is represented by two enzymes, Ndh (Rv1854c) and NdhA (Rv0392c). To determine the role of each NADH dehydrogenase in M. tuberculosis, we deleted them and studied their in vitro and in vivo phenotypes. The nuoA-N gene cluster and ndhA have been shown to be non-essential genes while ndh is considered essential. Surprisingly, we were able to obtain a ndh deletion mutant in both M. tuberculosis H37Rv and CDC1551, along with the nuoA-N and ndhA deletion mutants. We also constructed the double ndh nuoA-N knockout and ndhAnuoA-N knockout but were unable to obtain the ∆ndhndhA double knockout suggesting that M. tuberculosis requires at least one NADH dehydrogenase type II enzyme. In these knockouts, we determined the following in vitro phenotypes: level of NADH dehydrogenase activity, NADH/NAD+ ratios, expression level of nuoA-N, ndh, and ndhA, growth under aerobic and hypoxic conditions, and susceptibility to tuberculosis drugs and NADH dehydrogenase inhibitors. We also measured their growth in cultured macrophages as well as their growth and survival in immunocompromised and immunocompetent mice. The following observations were made. The nuoA-N, ndhA and ndhAnuoA-N deletion mutants showed no growth defect either in vitro or in vivo. In contrast, a longer lag phase was observed in the ndh and the ndhnuoA-N knockouts in vitro cultures. Furthermore, mice infected with the ndh knockout survived longer despite granulomatous inflammation and high number of lymphocytes observed in the lungs. Interestingly, although, the nuoA-N knockout had no phenotype in vivo, the ndhnuoA-N double knockout was the most severely attenuated strain in mice. These experiments allow us to conclude that Ndh is the main NADH dehydrogenase of M. tuberculosis and that compounds that could target both type I and type II NADH dehydrogenases could be good candidates for tuberculosis drug development. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 177 156 Mycobacterial Biology & Physiology 20/09/2016 Recruitment and regulation of the mycobacterial mutasome M. Reiche2, Z. Martin2, D. Lang1, N. Dhar3, J. Mckinney3, V. Mizrahi2, D. Warner2 1 Confocal and Light Microscope Imaging Facility, Dept. of Human Biology 2Dept. of Pathology and IDM, University of Cape Town, Cape Town, South Africa 3Global Health Institute, Swiss Federal Institute of Technology in Lausanne (EPFL), Lausanne, Switzerland Previous work in our laboratory identified a DNA damage-inducible pathway that is responsible for adaptive mutagenesis in Mycobacterium tuberculosis (Mtb), including the development of drug resistance during in vivo infection. In subsequent genetic studies, we have subsequently established that this mycobacterial “mutasome” comprises a minimum of three essential components: a dnaE2-encoded DNA Polymerase IIIα subunit, which potentially serves as an errorprone translesion polymerase, and imuB- and imuA’-encoded accessory factors, whose roles within the mutasome remain cryptic. In order to gain molecular insight into mutasome function, we investigated the recruitment dynamics and sub-cellular localization of ImuA’ and ImuB following exposure of bacilli to genotoxic stress. To this end, we constructed a panel of M. smegmatis (Msm) reporter mutants encoding fluorescently tagged mutasome proteins, and confirmed the functionality of the recombinants in DNA damage survival and induced mutagenesis assays. Fluorescence profiles were visualized and quantified utilizing both standard confocal and microfluidic imaging techniques, and population-wide expression characteristics assessed by flow cytometry. Our results reveal differential recruitment and localization of ImuA’ and ImuB in bacilli exposed to genotoxic stress: ImuA’ diffuses rapidly throughout the mycobacterial cell immediately post exposure to DNA damage, whereas ImuB forms distinct foci that co-localize with the dnaN-encoded β processivity factor. In addition, multiple ImuB-β foci are observed at discrete intracellular locations throughout the Msm filaments which form following arrest of cell-division in DNA-damaged cells. These observations reinforce a mutasome model in which ImuB functions as adaptor protein through its interaction with the β clamp and, simultaneously, provide novel insight into the potential for multiploid filaments to accelerate emergence of drug resistance during chronic exposure to sublethal genotoxic stress. Ongoing work is directed towards establishing the temporal order of recruitment of ImuB and β to stalled replication forks, as well as elucidating the function of ImuA’ within the mutasome. Notably, the approach adopted here confirms the utility of combining fluorescence imaging with functional genetics to elucidate the mechanisms regulating expression and activity of a major error-prone damage tolerance pathway in a human pathogen that is increasingly associated with drug resistance. 178 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 157 Mycobacterial Biology & Physiology 20/09/2016 Extremely Drug Resistant Small-Cell Survival Morphotype in Mycobacteria M.L. Wu4, M. Gengenbacher6, C.L. Chan4, J.C.S. Chung3, S.L. Chen3-5, H.J. Mollenkopf1, S.H. Kaufmann2, T. Dick4 1 Core Facility Microarray/Genomics 2Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany 3Infectious Diseases, Genome Institute of Singapore 4Antibacterial Drug Discovery Laboratoy, Department of Microbiology and Immunology 5Department of Medicine, Division of Infectious Diseases 6Tuberculosis Research Laboratory, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Mycobacteria, generally believed to be non-sporulating, are well known to survive shock starvation in saline for extended periods of time in a non-replicating state without any apparent morphological changes. Here we uncover that mycobacteria can undergo cellular differentiation by exposing Mycobacterium smegmatis to mild starvation conditions. Traces of various carbon sources in saline triggered the development of a novel small resting cell (SMRC) morphotype. Development of SMRCs could also be observed for other mycobacteria, suggesting evolutionary conservation of this differentiation pathway. Fluorescence microscopic analyses showed that development of SMRCs progresses via septated multi-nucleoided cell intermediates, which divide to generate mononucleoided SMRCs. Intriguingly, saline shock-starved large resting cells (LARCs), which did not show cell size or surface changes when observed by scanning electron microscopy, remodeled their internal structure to septated multi-nucleoided cells, similar to the intermediates seen during SMRC development. Comparative transcriptome analyses of SMRC and LARC development showed large consistency with our cellular observations. These results suggest that mycobacteria harbor a starvation-induced differentiation program that differs from the canonical cell division cycle, in which at first septated multi-nucleoided cells are generated. Under zero-nutrient conditions bacteria terminate development at this stage as LARCs. In the presence of traces of a carbon source, these multi-nucleoided cells continue differentiation into mono-nucleoided SMRCs. Both SMRCs and LARCs exhibited extreme antibiotic tolerance. SMRCs showed increased long-term starvation survival, which was associated with the presence of lipid inclusion bodies. Interestingly, the stringent response regulator relA appeared to play the role of cellular differentiation other than a starvation survival factor in this process. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 179 158 Mycobacterial Biology & Physiology 20/09/2016 Mycobacterium komossense: A surrogate organism for screening new antibiotic against tuberculosis and for studying the emergence of antibiotic resistance mutation H. Xiao, K. Oravcova, M. Scott, H. Stacey, R. Hammond, S. Gillespie, A. Gupta School of Medicine, University of St.Andrews, St.andrews, United Kingdom Antibiotic resistance of tuberculosis is a significant cause of morbidity and mortality worldwide. However, the long generation time and pathogenicity of Mycobacterium tuberculosis have been obstacles for researches investigating mechanisms of resistance in M. tuberculosis. This study aimed at exploring whether, Mycobacterium komossense, a non-pathogenic fast growing mycobacterium, can serve as a surrogate to study antibiotic resistance in M. tuberculosis and have the potential to be a drug screening tool. MIC test against some first and second-line anti-tuberculosis drugs found that M.Komossense share similar level of susceptibility as susceptible M. tuberculosis. The antibiotics include Rifampicin, ciprofloxacin, isoniazid, ethambutol and bedaquiline. Mutants that are resistant to these antibiotics were subsequently generated by fluctuation assay and validated by exposing the microorganism to 4×MIC of the antibiotics. Fitness costs associated with the development of rifampicin resistance were investigated by competition assay. Through DNA sequencing and MIC measurement, characterization and degree of resistance are mapped to specific mutations. We found that M. komossense and M. tuberculosis resistant strains shared similar mutations in the RRDR of rpoB. Two commonly reported RIFr mutants in M. tuberculosis, S531L and H5265Y, were observed in M. komossense. Results of relative fitness of three isolates with S531L mutation and one isolate with H526Y mutation suggest that resistance mutations are most commonly associated with a fitness cost; the degree of which is mutation specific. The fitness deficit was similar with to that previously reported in M. tuberculosis. These data suggest that M. komossense can be a suitable model to study the evolution of resistance in M. tuberculosis. Due to the lack of published information of M.komossense’s genome sequence, whole genome sequencing of M.komossense and annotation of the sequence is going to be carried out to assist us to characterize mutants against other antibiotics more accurately and effectively. 180 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 159 Mycobacterial Biology & Physiology 20/09/2016 Caseinolytic protease gene regulator (clgR) is a substrate for the mycobacterial Clp protease Y. Yamada1, T. Dick2 1 Department of Medicine 2Department of Microbiology and Immunology, National University of Singapore, Singapore, Singapore Recent discoveries of anti-mycobacterial compounds which target the caseinolytic protease (Clp) demonstrated that Clp inhibition is one of the promising approaches to battle drug-resistant tuberculosis. To further explore potential drug targets associated with mycobacterial Clp protease, we introduced a promoter-reporter library into M. bovis BCG Pasteur (BCG) and measured the effect of Clp inhibition. From a sub-set for the Clp-related gene promoters we found that exposure to the Clp inhibitor Bortezomib (BZ) activated the genes under control of transcriptional activator ClgR (such as ClpP1, Acr2 and ClgR) in a dose-dependent manner. Via ClgR-mCherry fusion proteins, we showed that ClgR is a substrate of Clp and that the C-terminal APVVSLAVA is required for degradation. Taken together, our findings revealed that Clp inhibition activated the ClgR signaling pathway by inhibiting degradation of ClgR and caused ClgR dysregulation. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 181 160 Mycobacterial Biology & Physiology 20/09/2016 Phenotypic and genotypic analyses of pyrazinamide resistance in Mycobacterium tuberculosis clinical isolates M.M.K. Yee1-2, P. Gopal1, R.T.H. Ong2, Y.Y. Teo2, M. Gengenbacher1, T. Dick1 1 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine 2Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapour Pyrazinamide (PZA) was discovered in the 1950s and has since been playing an essential role as a key treatment-shortening drug in tuberculosis (TB) chemotherapy. Yet, the mechanism of action/resistance of PZA (a pro-drug) and its active form, pyrazinoic acid (POA) remains poorly understood. This translates into challenges in accurately diagnosing PZA resistance in the clinical setting. Phenotypic methods, the gold standard in diagnosing TB drug resistance, are limited for PZA because of the low potency of the drug in vitro and the uncertain predictive value of the current growth assays. Molecular methods (detecting mutations in pncA gene as PncA is involved in conversion of PZA into POA) lack sensitivity as some clinical isolates are shown to be PZA resistant with wildtype pncA, suggesting alternative mechanism(s) of PZA/POA resistance. In order to address these shortcomings in current diagnostic methods, we study the correlation between various growth / growth inhibition assays and genetic polymorphisms of PZA resistant and susceptible Mycobacterium tuberculosis clinical isolates. The various growth / growth inhibition assays include classical broth dilution method vs. BACTEC MGIT 960. The broth dilution assays include media with different pH. Furthermore, MICs are determined for PZA and POA. First results of our ongoing analyses will be discussed. 182 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 161 Mycobacterial Biology & Physiology 20/09/2016 Mycobacterial rifampicin exposure leads to a semi-heritable accumulation of RNA polymerase that is correlated with a survival programme J.H. Zhu1-2, M.M. Pan2, E.H. Rego3, B. Javid2 1 PKU-Tsinghua-NIBS Graduate Program, School of Life Sciences, Peking University 2Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, China 3Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, United States Rifampicin (RIF) is a first-line anti-tuberculous antibiotic and still the most important antimicrobial in the fight against tuberculosis. Unlike most anti-tuberculous agents, which kill in a dose-independent manner, rifampicin-mediated killing is strongly dose-dependent, both in vitro an in vivo. This suggests that there is a broad susceptibility landscape to RIF in the mycobacterial population at the single-cell level. Here, we show that upon RIF exposure a significant subpopulation of mycobacterial cells continue to grow. The proportion of ‘growers’ (phenotypic resistors) varies inversely with rifampicin concentration, but is still easily detectable at 20XMIC. Using time-lapse microscopy, we find that there is a strong correlation between survivors and their daughters, suggesting a degree of semi-heritability. Rifampicin exposure leads to a binary decision tree for mycobacterial individuals: death or survival. Survivors in each case strongly upregulate expression of the RNA polymerase beta subunit (RpoB) through a specific transcriptional programme shortly after encountering RIF, which allows further survival and growth in the context of ongoing drug exposure. Importantly, survivors and their daughters exhibit a greater degree of rifampicin-specific phenotypic resistance over time. This was mirrored in (drug-sensitive) M. tuberculosis isolated from sputum from patients started on standard therapy: over time, the proportion of mycobacteria surviving drug-treatment was progressively more phenotypically resistant to rifampicin. Our findings have important implications for understanding tolerance and phenotypic resistance to rifampicin, and the reason why prolonged antibiotic therapy is required for tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 183 162 Transport systems & Secretion in Mycobacteria 20/09/2016 Understanding the role of Rv3921c, a preprotein translocase, in Mycobacterium tuberculosis P. Thakur1-2, M.Z. Abdin2, N. Agarwal1 1 Translational Health Science and Technology Institute, Faridabad 2Jamia Hamdard, New Delhi, India Transport of proteins to cell surface or extracellular environment is accomplished by specialized protein translocases, which regulate multiple physiological activities along with secretion of virulence factors or effector molecules in the pathogenic organisms. In human pathogen Mycobacterium tuberculosis (Mtb) three major protein export pathways are known: the general secretion (Sec), the twin-arginine transport (TAT) and the ESX pathway. Recent studies have shown that in several bacteria the Sec and TAT pathways involve a preprotein translocase known as YidC for membrane insertion of the transported protein, whereas in mitochondria and chloroplasts insertion of membrane proteins is solely accomplished by YidC orthologues, Oxa and Alb proteins, respectively. Analysis of mycobacterial genome sequences reveals the conserved occurrence of yidC homologues, which is encoded by a gene Rv3921c in Mtb. Importantly, not only the yidC but the entire locus, surrounded by cluster of genes involved in protein synthesis and membrane biogenesis is conserved in different mycobacterial species, thus suggesting an essential role of YidC in mycobacteria. In an earlier study we observed that yidC is essential for in vitro growth of Mtb, however, an exact function of this gene in the pathophysiology of Mtb remains to characterize. Here, we show that YidC is expressed in both fast-growing M. smegmatis and slow-growing Mtb at all growth stages, and a controlled expression of this protein is absolutely required for maintaining the optimal in vitro growth of Mtb. Interestingly, expression of YidC is modulated by cell envelope stress agents such as detergent and heat shock. Further, we observed that transient overexpression of YidC in Mtb alters membrane integrity and causes upregulation of many genes including SDS-regulons such as sigE and sigB that encode extracytoplasmic function sigma factors. Taken together these results suggest that YidC plays a pivotal role in biogenesis of mycobacterial cell envelope, which can be explored as novel anti-TB drug target. 184 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 163 Transport systems & Secretion in Mycobacteria 20/09/2016 The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Membrane Rupture Through Its Substrate PPE10 L. Ates2-5, A. Van Der Woude4-5, J. Bestebroer5, G. Van Stempvoort4, R. Musters7, J. GarciaVallejo6, D. Picavet3, R. Van De Weerd5, M. Maletta1, C. Kuijl5, N. Van Der Wel3, W. Bitter5-4 1 Eyen SE, Prague, Czech Republic 2Pathogénomique Mycobactérienne Intégrée, Institut Pasteur, Paris, France 3Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam 4Department of Molecular Microbiology, VU University 5Department of Medical Microbiology and Infection Prevention 6Department of Molecular Cell Biology and Immunology 7Department of Physiology and Cardiology, VU University Medical Center, Amsterdam, The Netherlands Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity, as well as reduced amounts of surface localized proteins and glycolipids. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the capsular defect on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis and rupture of the phagosomal membrane. The observed defects in capsular integrity resulted in a reduced recruitment of ubiquitin in cellular infections and intermediate attenuation in zebrafish embryos, confirming the importance of capsular integrity in the early stages of infection. These results provide a pivotal role for PPE10, a substrate of ESX-5, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 185 164 Transport systems & Secretion in Mycobacteria 20/09/2016 Functional interaction between Mycobacterium tuberculosis RipA, an invasion associated secretory protein, and MoxR1 M. Bhuwan2, N. Arora2, A. Sharma1, M. Khubaib2, S. Pandey2, T.K. Chaudhari1, S.E. Hasnain1, N.Z. Ehtesham2 1 Kusuma school of Biological Sciences, IIT Delhi, Delhi 2Inflammation Biology and Cell signaling, National Institute of Pathology, New Delhi, India Mycobacterium tuberculosis (M.tb) is a leading cause of death worldwide. The Tat (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacterium and is known to transport folded proteins and is reported to be essential for many of the important bacterial processes that include cell wall biosynthesis. RipA, a secretion and invasion protein of M.tb has endopeptidase activity. The MoxR1 protein belongs to ATPases family and is associated with various cellular activities. RipA and MoxR1 were expressed and purified to homogeneity from E.coli. Recombinant MoxR1 protein was characterized in terms of its biophysical feature. Protein aggregation assay of recombinant MoxR1 at higher temperature showed resistance to aggregation. Furthermore, recombinant MoxR1 was able to prevent the aggregation of Maltodextrin glucosidase (MalZ) protein at higher temperature which is enhanced in the presence of ATP suggesting its role as a chaperone. Role of MoxR1 was also demonstrated in in vivo folding of RipA in E. coli cells. Insilico analyses pointed to a protein-protein interaction between M.tb RipA and MoxR1 as well as RipA and Mce2B (mammalian cell entry protein). Furthermore, using bimolecular fluorescence complementation (BiFC) assay the interaction of these proteins was confirmed in HEK293T using confocal microscopy. It has been shown earlier that recruitment of the autophagy protein to sites of bacterial entry leads to lysosomal degradation of the invading bacteria. Our bioinformatics analysis of RipA protein using eukaryotic linear motif resources (www.elm.eu.org) showed the presence of canonical LIR motif that binds to Atg8/LC3 protein family members to mediate processes involved in autophagy. The conserved sequence for this “SFAGV” was found at 390394 amino acid in RipA protein. Overall our study demonstrates M.tb MoxR1 as novel molecular chaperone involved in maturation or refolding of specific protein complexes with likely role in pathogenesis. 186 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 165 Transport systems & Secretion in Mycobacteria 20/09/2016 Exploiting the reconstituted ESX-5 system to understand the role of substrates in system functioning C. Bunduc5, K. Beckham4, L. Ciccarelli2-1-3, A. Parret4, M. Wilmanns4, T. Marlovits1-2-3, W. Bitter5-6, E. Houben5 1 Institute of Molecular Biotechnology GmbH 2Research Institute of Molecular Pathology, Vienna, Austria 3Centre of Structural Systems Biology, University Medical Center Eppendorf 4European Molecular Biology Laboratory, Hamburg, Germany 5Molecular Microbiology, Vrije Universiteit, Amsterdam 6VU University Medical Center, Amsterdam, The Netherlands Pathogenic mycobacteria, including Mycobacterium tuberculosis, use type VII secretion (T7S) systems to secrete important virulence factors across their distinctive cell envelope. These bacteria have up to five of such systems, termed ESX-1 to ESX-5. T7S does not resemble known specialised secretion pathways in other bacteria, reflecting the unique properties of the mycobacterial cell envelope. Although some of these secretion systems are crucial for mycobacterial growth or virulence, the actual mechanism of membrane transport is still largely unknown. As a first step in this analysis, we have previously isolated the ESX-5 membrane complex from Mycobacterium marinum and determined that this macromolecular machinery has a size of 1.5 MDa and consists of four membrane components, i.e. EccBCDE (Houben et al., Mol Microbiol 2012). The aim of this study is to elucidate and further characterize the T7S membrane channel. To more easily analyse the functionality of this membrane complex in secretion, we reconstituted the ESX-5 system in the avirulent and fast-growing mycobacterial species Mycobacterium smegmatis that lacks ESX-5. The reconstituted system proved to be active and was efficiently secreting EsxN and specific PE/PPE substrates. Importantly, we noticed that the different components were relatively overexpressed and the complex was properly assembled in the membrane. This reconstitution therefore not only opened up the possibility to more easily analyse the role of specific Esx and PE/PPE proteins in the secretion process, but also to check possible interactions between substrates and components of the translocation channel. In addition, we purified this ESX-5 membrane complex, determined the molecular composition and examined the structure through negative stain electron microscopy (see also abstract of Beckham et al.). In conclusion, we functionally reconstituted and overproduced the ESX-5 system in M. smegmatis, which opened up the possibility to efficiently and more thoroughly analyse the functioning and structure of this secretion system. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 187 166 Transport systems & Secretion in Mycobacteria 20/09/2016 Outer membrane localization of CpnT and toxin translocation is dependent on type VII secretion in Mycobacterium tuberculosis K. Doornbos1, O. Danilchanka2, J. Sun1, M. Niederweis1 1 Microbiology, University of Alabama at Birmingham, Birmingham 2Epiva Biosciences, Cambridge, United States Mycobacterium tuberculosis (Mtb) secretes a NAD-glycohydrolase which causes necrotic death in host cells. This tuberculosis necrotizing toxin (TNT) constitutes the C-terminal domain of the outer membrane protein (OMP) CpnT and is a major cytotoxicity factor of Mtb. The absence of classical export signals within CpnT raised the question of how CpnT is integrated into mycobacterial outer membranes and how TNT is secreted. In this study we demonstrate that localization of CpnT depends on an N-terminal Esx motif which target proteins to the type VII secretion systems of Mtb, the Esx systems. A separate, C-terminal Esx motif is required for TNT translocation across the outer membrane, but not for the channel activity of CpnT in the outer membrane. Thus, CpnT is the first OMP whose assembly is controlled by type VII secretion. Esx motifs similar to those in CpnT are found in 29 other Mtb proteins including the virulence factor LipY, indicating that this novel OMP assembly mechanism is widespread. Taken together, this study not only advances our mechanistic understanding of toxin secretion by Mtb, but also reveals a previously unknown function of Esx systems and has important implications for OMP assembly and cell permeability of this major human pathogen. 188 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 167 Transport systems & Secretion in Mycobacteria 20/09/2016 Biochemical characterization of the mycobacterial MFS transporter Rv1410 and investigation of its role in the correct assembly of the cell wall Michael. Hohl, F. Arnold, A. Krügel, Markus. Seeger University of Zurich, Institute of Medical Microbiology, Zürich, Switzerland Mycobacterium tuberculosis is surrounded by a complex cell wall that naturally protects the bacilli against noxious substances. The mycobacterial operon rv1410-rv1411 encoding a major facilitator superfamily (MFS) transporter (Rv1410) and a lipoprotein (Rv1411, LprG) is strongly conserved among many mycobacterial species and is critical for virulence in M. tuberculosis. While initial studies on Rv1410 proposed a role in drug efflux, more recent studies showed a connection of the MSF transporter with LprG to play an important role in cell wall assembly. Recent work focused on the lipoprotein alone, showing that cell wall components such as lipoarabinomannans (LAMs) bind to LprG, which transports them to the cell surface. Metabolomic studies using M. tuberculosis rv1410-lprG knockout cells showed the accumulation of triacylglycerides (TAGs) in the cytoplasm, demonstrating a role of this locus in TAG transport. Crystal structures of LprG bound to a LAM precursor and a TAG molecule have been solved. In contrast, biochemical data on the MFS transporter Rv1410 are scarce. We constructed gene deletions of the corresponding operon as well as of the MFS gene alone in M. smegmatis leading to attenuated growth in the presence of ethidium. Introduction of the operons from M. tuberculosis, M. marinum and M. abscessus fully recovered the growth deficit. Further, we were able to complement the MFS gene deletion with rv1410 of M. tuberculosis and identified an aspartate to asparagine (D-to-N) mutation, which fully inhibits the transport activity of Rv1410. To study the molecular basis of the interplay between Rv1410 and LprG we purified these two proteins upon heterologous expression in E. coli as wellfolded, monomeric proteins. Size exclusion chromatography (SEC) and surface plasmon resonance (SPR) analyses, however, did not reveal a physical interaction between these proteins, suggesting a functional interaction between these two transport proteins for the export of LAMs and TAGs. Through a combination of X-ray crystallography, biochemical in vitro assays and functional in vivo experiments we aim to reveal the molecular function of the MFS transporter Rv1410 and thereby may help to design novel TB-drugs. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 189 168 Transport systems & Secretion in Mycobacteria 20/09/2016 Role of specific chaperones in determining system specificity in type VII secretion systems T. Phan1, R. Ummels2, N. Korotkova3, K. Korotkov3, W. Bitter1-2, E. Houben1 1 Vrije Universiteit Amsterdam 2VU University Medical Center, Amsterdam, The Netherlands 3University of Kentucky, Kentucky, United States Type VII secretion (T7S) is used by mycobacteria to export protein effectors across their highly unusual cell envelope. There are five paralogous T7S systems in Mycobacterium tuberculosis, named ESX-1 to ESX-5, each having their own role in viability and/or virulence. The mycobacteriaspecific PE/PPE proteins are one group of proteins that are secreted via these systems, of which at least some are exported as heterodimers. We have previously shown that the cytosolic chaperone EspG specifically interacts with cognate PE/PPE dimers and is required for their successful export1. To further investigate the substrate specific binding of the EspG chaperone we have obtained the crystal structure of the ESX-5 chaperone EspG5 with the ESX-5 substrate pair PE25/PPE412. The structure reveals that the chaperone exclusively interacts with the elongated end of PPE41, a region containing several hydrophobic residues that are conserved in PPE substrates of different secretion systems. Introducing point mutations in these conserved residues of multiple ESX-5 PPE substrates abolished the interaction with the cognate EspG chaperone. Disrupting the interaction with EspG also blocked their secretion by Mycobacterium marinum, indicating that chaperone binding is an important prerequisite for protein secretion. In this study, we show that also for an ESX-1 dependent PPE protein these conserved residues are important for secretion. Subsequently, we exchanged the complete EspG-binding domain between an ESX-1 and an ESX-5 PPE protein and tested their route of export using different M. marinum ESX-1 and ESX-5 mutant strains. These experiments revealed that the ESX-1 substrate containing an EspG5 binding domain is secreted independently on its original ESX-1 system, but is now entirely dependent on the ESX-5 system for secretion. These results suggest that the EspG chaperone is the primary factor for determining system specificity in M. marinum. Rerouting of specific ESX-1 substrates via ESX-5 could be used to understand the role of individual ESX-1 substrates in virulence and to improve the current live vaccine strain BCG, that lacks the ESX-1 system, through modulation of its antigen repertoire. 1. Daleke, M.H., et al. (2012) J Biol Chem 287, 31939-3194710. 2. Korotkova, N., et al. (2014) Mol Microbiol 94, 367-382. 190 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 169 Transport systems & Secretion in Mycobacteria 20/09/2016 Type VII Secretion Pathway Involved in Mycobacterium abscessus Intracellular Survival L. Laencina1, V. Le Moigne1, V. Dubois1, C. Dupont2, N. Van Wyk2, E. Rubin3, L. Kremer2, J.L. Herrmann1, F. Girard-Misguich1 1 U1173 INSERM Infection, Inflammation, Université de Versailles Saint-Quentin-en-Yvelines, Montigny-Le-Bretonneux 2Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé, FRE3689 CNRS / UMR Mycobacterial Pathogenesis and Novel Therapeutic Targets, Montpellier, France 3Department of Microbiology and Immunology, Harvard Medical School, Boston, United States Mycobacterium abscessus represents, with Mycobacterium avium, the major mycobacterial pathogen responsible for pulmonary infections in patients with cystic fibrosis. The M. abscessus complex comprises three subspecies: abscessus, massiliense and bolletii. M. abscessus behaves as an intracellular pathogen, able to grow and survive in macrophages, survival usually not observed for rapid growing mycobacteria (RGM). Genome analysis of M. abscessus showed that this mycobacterium is endowed with several virulence genes, not found in saprophytic RGM, allowing its interaction with free-living amoeba in the environment, and potentially explaining its survival and growth in human macrophages. However, very little is known about the genes helping M. abscessus to grow and survive in antigen presenting cells such as macrophages or in environmental protozoa such as amoeba. A transposon mutant library of a clinical strain of M. abscessus subsp. massiliense was screened based on its ability to persist in amoeba and macrophages. One hundred and thirty-six out of six thousand individually screened mutants exhibited a reduced ability to survive in macrophages and/or amoeba. Among them, sixteen were mutated in genes belonging to M. abscessus esx-4 gene locus, belonging to the type VII secretion system. One gene, eccB, was chosen to create a deletion mutant by replacing eccB by a zeocin resistance cassette in both the smooth and rough variants of the CIP104536T M. abscessus. These mutants are used to confirm the attenuated phenotype linked to a defective ESX-4 secretion system, and to complement this defect by introducing a functional copy of the gene. The ESX-4 locus is conserved in all mycobacteria and is considered as the ancestral gene cluster of the five ESX subtypes. Overall, our results indicate, for the first time, that ESX-4 participates in growth and survival of M. abscessus in macrophages and amoeba. Experiments are currently on going to address whether ESX-4 in M. abscessus shares a biological role similar to the one described for ESX-1 in M. tuberculosis to allow the bacteria to communicate with the cytosol in infected macrophages. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 191 170 Transport systems & Secretion in Mycobacteria 20/09/2016 Colorimetric assay for detecting ESX-3 secretion system disruption in mycobacteria M. Maerk1, T. Strand1, S. Bandyopadhyay2, T. Flo1, M. Steigedal1 1 Dept. of Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Research 2Dept. of Chemical Engineering, Norwegian University of Science and Technology, Trondheim, Norway Tuberculosis (TB) remains one of the major infectious diseases causing morbidity and death in large parts of the world. Various classes of drug-resistant TB strains have emerged over the recent years, which have prompted several initiatives to find new anti-TB drugs. M. tuberculosis, the causative agent of TB, is a persistent bacterium with an unusual and highly impermeable cell envelope, which is part of the reason it is difficult to develop new therapeutic strategies for treating TB infections. Biogenesis and functioning of this unusual cell envelope is however also the target of many new and existing TB drugs. In our current project we aim to identify compounds that target the mycobacterial type VII secretion systems (T7SS), which are located in the cell envelope and secrete effector proteins. Mycobacteria can reside and multiply within infected host cells, and the T7SS secrete proteins across the cell envelope to the bacterial cell surface or into the host environment. Five mycobacterial T7SS have been described; ESX-1 to -5. Three of these (ESX-1, -3 and -5) are essential for virulence or viability. We are developing and evaluating a gold nanoparticle-based colorimetric assay that can detect ESX-3 substrate secretion, and will subsequently use this to carry out high-throughput screening (HTS) of chemical libraries in order to identify potential drug candidates that target this system. 192 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 171 Transport systems & Secretion in Mycobacteria 20/09/2016 Plasmids expressing tagged ESAT-6 and CFP-10 as a tool for immunostaining of ESAT-6 and CFP-10 inside human macrophages J. Raffetseder, V. Loitto, M. Lerm Linköping University, Linköping, Sweden With the global increase of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb), new treatments for tuberculosis are urgently needed, with one of the drug development strategies evolving around Mtb virulence blockers. ESAT-6 is a major virulence factor of Mtb and has been shown to be involved in many processes in the interaction between Mtb and host cells, such as induction of necrotic host cell death, dissemination to other cells and granuloma formation. It has been reported to have membrane-lysing activity, and mycobacteria deficient in ESAT-6 or the ESAT-6 secretion system ESX-1 are unable to translocate from phagosomal compartments into the cytosol. We wanted to investigate the role and the secretion kinetics of ESAT-6 in an established infection model of human monocyte-derived macrophages, both under conditions when Mtb actively replicates inside the macrophage leading to host cell death as well as during Mtb growth restriction with uncompromised macrophage viability. The aim was to better understand how macrophages restrict Mtb growth and under which conditions the secretion of the ESAT-6/CFP-10 heterodimer can be inhibited. This information could be useful for the development of ESX-1 blockers. To facilitate immunostaining of secreted ESAT-6 and CFP-10 after macrophage infection, we transformed Mtb H37Rv strains with plasmids carrying gene sequences for fusion proteins of ESAT6 or CFP-10 and antigenic peptide tags (c-myc or hemagglutinin). Using Western Blotting as well as immunofluorescent staining followed by quantification of fluorescence intensity, we found that tagged ESAT-6 and CFP-10 were expressed and actively secreted during cultivation of Mtb in broth. In contrary, antibody staining of tagged ESAT-6 or CFP-10 inside infected human monocyte-derived macrophages resulted in only weak signals just above background which however correlated with intracellular bacterial growth. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 193 172 Transport systems & Secretion in Mycobacteria 20/09/2016 Cryo-EM Structural characterization of the M. tuberculosis ESX-1 secreted virulence factor EspB R. Ravelli, A. Siroy, G. Tria, D. Chen, P. Afanasyev, N. Iakobachvili, C. Berger, K. Notelaers, R. Bosmans, A. Gisbers, H. Furusho, C. Lopez-Iglesias, P. Peters M4i-Nanoscopy, Maastricht University, Maastricht, The Netherlands Virulent mycobacteria infect their host cells through a Trojan Horse strategy: they get ingested by the normal phagocytosis route but instead of being digested through acidification and maturation of the phagosome to phagolysosome, these bacteria enter the host’s cell cytosol where they replicate freely. Controlled death of the host will allow the spreading of the descendants in the infected organism. A key step in the infectious life cycle of the virulent mycobacteria is their escape from the phagosome compartment. This phenomenon is mediated by the release of different proteins (EsxAB) that will perforate the phagosome membrane. These factors are secreted by the specialized machinery ESX-1 of the recently discovered Type 7 Secretion System family. The ESX1 machinery is a multi-protein complex thought to span the entire mycobacterial cell envelope. It is constituted by a core of structural proteins (EccACabBDE1) and the assembly and/or functioning of the machinery seems to require the recruitment of additional proteins. Among the different ESX-1 substrates, the 48 kDa protein EspB seems to carry multiple roles: this secreted protein carries cytolytic activity on its own; it is co-secreted with EsxA (the main virulence factor) and is required for EsxA secretion; EspB may also play a structural role suggested by its ability to oligomerize as well as its proteolytic maturation by MycP1 during the translocation process. Analysis of EspB primary sequence predicts two main structural domains: similar to other ESX substrates, the N-terminal domain is made of a bundle of α-helices while the C-terminal one seems unfolded. The structure of the N-terminal domain of EspB was recently solved by X-ray crystallography and, combined with negative-stain electron microscopy 2D classes, a heptameric model was computationally generated. Here, we report structural characterization of EspB using transmission electron microscopy. We determined the structure of the N-terminal helix bundle, the mature form as well as the pre-protein EspB at sub-nanometer resolution. Moreover, we were able to identify several distinct oligomeric states of the proteins that were not previously observed, reflecting a possible role of EspB as a structural component of ESX-1. 194 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 173 Transport systems & Secretion in Mycobacteria 20/09/2016 Isolation and structural characterization of native Mycobacterium marinum ESX-1 secretion machinery by cryo-EM A. Siroy, D. Chen, G. Tria, N. Iakobachvili, C. Berger, K. Notelaers, R. Bosmans, A. Gijsbers, H. Furusho, C. Lopez-Iglesias, R. Ravelli, P. Peters M4i-Nanoscopy, Maastricht University, Maastricht, France Pathogenic mycobacteria utilize a common “Trojan Horse” strategy to infect their host cells, i.e. they get phagocytosed and should be killed. Instead, these bacteria are able to rupture the phagosome membrane before its maturation into phagolysosome by secreting different proteins (EsxAB). Set free in the cytosol, those pathogens can replicate and control the cell death of their host. These “Phagosomal Escape” depends on the presence of the protein complex ESX-1 of the Type Seven Secretion System family (T7SS). This ESX-1 machinery is an elaborate protein complex of which not all the components are known to date. Five known structural components of the complex (EccABCDE1) are encoded within the RD-1 genomic region, alongside the substrates EsxAB, EspB and other less characterized ones. More than a decade after the discovery of ESX-1, successful expression of the recombinant machinery has yet to be reported and only partial structural characterization of the core components is available, showing the difficulty of the task at hand. We present here the first results regarding the investigation by cryo-electron microscopy of the structure of native ESX-1 complex produced by Mycobacterium marinum, the fish equivalent to the human pathogen M. tuberculosis. In this regard, we developed a new fluorescent probe that allows the tracking of the protein complex during its isolation and purification, as well as monitoring its location in a correlative fluorescent and electron microscopy workflow in cryogenic conditions. The conditions for the extraction from the cell envelope were screened and the fluorescently-tagged ESX-1 complex was purified by liquid chromatography. The ESX-1 enriched samples were imaged by cryo-transmission electron microscopy and the micrographs analyzed using Single Particle Averaging (SPA) techniques. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 195 174 Transport systems & Secretion in Mycobacteria 20/09/2016 Regulation of ESX-1-mediated Secretion in Mycobacterium tuberculosis P. Soler, M. Zhang, C. Sala, S. Cole GHI, Prof. Cole Lab (UPCOL), EPFL, Lausanne, Switzerland Mycobacterium tuberculosis is an intracellular pathogen that requires a functional ESX-1 system to secrete virulence factors and successfully spread inside the host. The ESX-1 secretory apparatus is predicted to be a multi-subunit system encoded by more than twenty genes. Nowadays, the function of each ESX-1 component and their regulation remain partially unknown. To better understand the virulence mechanisms employed by M. tuberculosis, we are studying the unknown ESX-1 components and trying to assign a functional role to them. To do so, we have constructed an M. tuberculosis mycP1-eccE1 double mutant and an M. tuberculosis whiB6 mutant to analyze their phenotypes in vitro by looking at gene expression, ESX-1 protein secretion and possible interactors as well as their localization in the cell. We also analyzed ex vivo virulence and cytokine production upon macrophage infection and will present the findings. 196 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 175 Transport systems & Secretion in Mycobacteria 20/09/2016 Overexpression of a Mycobacterium abscessus efflux pump of the MmpL family confers resistance to clarithromycin C. Cortesía3, G. López3, A. Pawlik1, R. Brosch1, H. Takiff3-2 1 Integrated Mycobacterial Pathogenomics 2Unité de Genetique Mycobacterienne, Institut Pasteur, Paris, France 3CMBC, Lab. de Genética Molecular, Instituto Venezolano de Investigaciones Científicas, IVIC, Caracas, Venezuela M. abscessus causes soft tissue infections after surgical and cosmetic procedures and respiratory infections in cystic fibrosis patients. We found that the presence of MAB_2275 on a plasmid confers high-level clarithromycin resistance in strains of the M. abscessus family without an inducible methyltransferase. MAB_2275 encodes an efflux pump belonging to the MmpL family. Similar to other MmpL genes, there is an adjacent putative transcriptional repressor. Efflux pump inhibitors decrease the level of resistance of strains both with and without the plasmid based efflux pump. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 197 176 Transport systems & Secretion in Mycobacteria 20/09/2016 MMPL3 is the inner membrane flippase of trehalose monomycolate in mycobacteria Z. Xu1, S. Chng1-2 1 Chemistry Department 2Singapore Centre on Environmental Life Sciences Engineering, National University of Singapore, Singapore, Singapore The mycobacterial cell envelope consists of two membranes, an inner membrane (IM) and a mycolic acid-rich outer membrane (OM). The OM is an exceptional permeability barrier and is essential for virulence. As the major lipid component of the OM, mycolic acids are biosynthesized as trehalose monomycolates (TMMs) in the IM and then transported across the IM, the aqueous periplasm and finally to the OM, where they become functionalized onto the bacterial cell wall. Few proteins involved in TMM transport and assembly into the OM have been identified. In this study, we set out to characterize the function of MmpL3, an essential IM protein belonging to the RND (resistance, nodulation, and cell division) protein family. MmpL3 has been implicated in TMM transport in the IM; however, the exact role of this protein is not known. It has also been hypothesized to be a target of several drug candidates with varied structures, although it is not clear if each of these compounds directly inhibits MmpL3 function or kill mycobacteria through other mechanisms. In this work, we used a cell-based assay to demonstrate that MmpL3 is involved in flipping TMM across the IM in vivo. We first established that newly synthesized mycolic acids accumulated as TMM in the IM of mycobacterial spheroplasts. In fact, almost all accumulated TMM molecules were exposed on the surface of these spheroplasts, indicating they resided in the outer leaflet of the IM. Surface exposure of TMM was partly prevented when MmpL3 was inhibited by selective drug candidates, which did not also affect the pmf (proton motive force). These results define MmpL3 as the flippase for TMM in mycobacteria, and establish it as a direct target for a subset of promising anti-mycobacterial compounds. 198 Tuberculosis 2016 – September 19-23, 2016 – Paris, France POSTER SESSION 2 Wednesday, September 21 SUMMARY Biomarkers & Vaccines................................................................ 201 Biochemistry & Chemical biology ................................................ 228 177 Biomarkers & Vaccines 21/09/2016 Exploration of urinary biomarkers for the detection of paediatric tuberculosis N. Andreas2, L. Tientcheu1, R. Basu Roy2, T. Togun1, M. Gomez-Romero3, N. Harvey3, B. Jiménez3, E. Holmes3, B. Kampmann2-1 1 Vaccines and Immunity Theme, MRC Unit-The Gambia, Fajara, Gambia 2Centre for International Child Health 3Section of Computational and Systems Medicine, Imperial College London, London, United Kingdom Background and aims: The diagnosis of tuberculosis in children is difficult and less than one third of suspected cases are microbiologically confirmed [1]. The aim of this project was to analyse paediatric urine samples, employing a metabonomic strategy, in order to identify novel diagnostic biomarkers for paediatric tuberculosis. The samples analysed were collected longitudinally from children diagnosed with TB (cases) or other respiratory infections (controls) in The Gambia. Methods: 166 urine samples collected at enrolment, two weeks, two months and six months postenrolment from 95 children underwent 1H NMR spectroscopic analysis, in general profiling mode [2]. Spectra collected were normalised [3], aligned [4] and scaled [5]. The subsequent data were analysed using multivariate modelling techniques, including principal components analysis (PCA) and Orthogonal partial least squares-discriminant analysis (OPLS) combining probable and confirmed TB cases. Results: At enrolment, 9 confirmed, 12 probable and 53 non-TB samples were available for analysis. Data from these samples were analysed using OPLS, classifying samples using their clinical diagnosis, Figure 1. Figure 1: OPLS scores plot of non-TB (blue) versus probable (red) and confirmed TB samples (green). R2X=0.19, R2Y=0.61, Q2=0.27, n=70. Samples separated out based on their diagnosis. The identification of metabolites discriminating between the different diagnoses was investigated. Increased concentrations of a mammalianmicrobial co-metabolite, a metabolite associated with muscle metabolism and an as yet unidentified metabolite were observed in the non-TB urine samples, whist several further unidentified Tuberculosis 2016 – September 19-23, 2016 – Paris, France 201 metabolites were increased in probable TB and confirmed TB samples at baseline. TB samples were also seen to separate out from one another based on time of collection post-enrolment. Conclusions: This novel work suggests that several host derived metabolites are altered during TB infection and that the concentrations of these metabolites alters overs the course of infection. However, to employ this approach as a diagnostic tool substantial development would be required prior to implementation. Nevertheless, the data provide insights into the alterations in host metabolism in response to infection with tuberculosis as well as in regard to drug metabolism. References 1. Togun, T.O., et al., Contribution of Xpert (R) MTB/RIF to the diagnosis of pulmonary tuberculosis among TB-exposed children in The Gambia. International Journal of Tuberculosis and Lung Disease, 2015. 19(9): p. 1091-1097. 2. Dona, A.C., et al., Precision high-throughput proton NMR spectroscopy of human urine, serum, and plasma for large-scale metabolic phenotyping. Anal Chem, 2014. 86(19): p. 988794. 3. Dieterle, F., et al., Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in H-1 NMR metabonomics. Analytical Chemistry, 2006. 78(13): p. 4281-4290. 4. Veselkov, K.A., et al., Recursive Segment-Wise Peak Alignment of Biological H-1 NMR Spectra for Improved Metabolic Biomarker Recovery. Analytical Chemistry, 2009. 81(1): p. 5666. 5. Eriksson L, J.E., Kettaneh-Wold N, Wold S, Introduction to multi- and megavariate data analysis using projection methods (PCA & PLS) 1999: Umetrics. 202 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 178 Biomarkers & Vaccines 21/09/2016 Recombinant Attenuated Mycobacterium tuberculosis for Heterologous Expression and Secretion of DTP Antigens: A Rationale to Construct a Multivalent Vaccine E. Broset3-2, A.I. Kanno1, L.C. Leite1, C. Martin3-2, J. Gonzalo-Asensio3-2 1 Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil 2CIBER enfermedades respiratorias, Instituto de Salud Carlos III, Madrid 3Genetica de micobacterias, Universidad de zaragoza, Zaragaoza, Spain Live attenuated BCG vaccine is effective in reducing severe forms of tuberculosis (TB) in children but confers variable protection against pulmonary TB in adults (1). To overcome this handicap of BCG, we have developed the live vaccine MTBVAC, based on the attenuation of Mycobacterium tuberculosis by deletion of the phoP and fadD26 virulence genes (2). MTBVAC is currently in phase Ib clinical trials in South Africa.(NTC02729571) Due to its adjuvant capacity, BCG has been traditionally proposed as vector to deliver viral, bacterial or parasite antigens (3). In this context, we propose the study of MTBVAC as a multivalent recombinant vaccine against different infectious diseases including diphtheria, tetanus and whooping cough. Prior to construct recombinant MTBVAC (rMTBVAC) strains; we studied the possible immune interference between MTBVAC and the current vaccines against diphtheria, tetanus and whooping cough (DTP). Actually two formulations of DTP are administered depending on the region: either the pertussis inactivated toxins (DTaP) or the pertussis whole-cell vaccine (DTwP). In this context, we tested both DTP formulations in mice previously vaccinated with MTBVAC. Results were compared with mice independently vaccinated with MTBVAC or DTP. Mice conserved similar immune response against MTBVAC or DTP in all cases, which proves absence of interference between both vaccines. To construct rMTBVAC, genetically inactivated and codon optimized genes for diphtheria (CRM197), pertussis toxoids (S1), or Fragment C of tetanus toxin (FC) were synthesized. Each DTP antigen was cloned under the control of a new mycobacteriophage derived promoter (4). To lead the secretion of these antigens, the signal sequence of Ag85A (a known mycobacterial secreted protein) was placed in frame before each DTP antigen. Protein expression and secretion of CRM197, S1 and FC was confirmed by Western blot using antigen-specific antibodies. Preliminary experiments in mice showed that vaccination with rMTBVAC expressing the S1 toxoid protects against Bordetella pertussis colonization. On-going experiments aimed to detect anti-DTP antibodies in mice vaccinated with these rMTBVAC. Taking together, these results provide proof-of concept that MTBVAC is an efficient antigenic vector for heterologous antigen expression and put forward rMTBVAC to protect against different infectious diseases. References: 1. Fine, P.E. (1995). Lancet 346(8986):1339-45. 2. Arbués et al. (2013). Vaccine 31(42):4867-73 3. Bastos et al. (2009). Vaccine 27(47): 6495-503. 4. Kanno et al. (2016). Appl. Environ. Microbiol 82(8): 2240-2246 Funding: Esther Broset was recipient of BES-2012-052937 grant by Ministerio de Economía y Competitividad de España. This work was funded by BIO2014-5258P and TBVAC2020 projects Tuberculosis 2016 – September 19-23, 2016 – Paris, France 203 179 Biomarkers & Vaccines 21/09/2016 Differential Diagnosis of Multiplex PCR for Major Mycobacterium species causing Lung Disease H. Chae, S.J. Han, S.J. Shin Brain Korea 21 Plus Project for Medical Science, Yonsei University, Seoul, South Korea Not only distinguishing between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) but also identifying major pathogens causing mycobacterial lung disease at the species level are medically important. In this study, we developed a multiplex PCR assay using novel molecular targets that can differentiate the MTB Beijing family, a major member of MTB in east-Asian countries including Korea, from other MTB lineages (Beijing vs Non-Beijing lineage) and simultaneously identify four clinically important NTM species consisting of M. avium subsp. hominissuis (MAH), M. intracellulare (MI), M. abscessus subsp. abscessus (MAB) and M. abscessus subsp. massilience (MAS). A total of 7 primer sets of multiplex PCR are designed according to the following purposes; 1) Detection of the genus Mycobacterium by 16s rRNA gene (Pan-Mycobacterium detection), 2) Differentiation between MTBC and NTM by rv0577, 3) Differentiation between MTB and other species inside MTBC by the region of difference (RD) 9 region, 4) Specific identification of Beijing family of MTB by mtbk_20680, 5) Identification and differentiation of among Mycobacterium avium complex by IS1311 and DT1, and 6) Identification and differentiation between MAB and MAS by mab_3256c. For the accuracy of the multiplex PCR assay, 37 reference strains (4 MTBC, 4 MTB and 29 NTM) and 83 clinical isolates (5 MTBC, 58 MTB and 20 NTM) were evaluated. Spoligotyping and whole genome sequencing as the standard assays were further employed to validate the multiplex PCR assay in 58 clinical MTB isolates. Assay results demonstrated 100% sensitivity and 97.2% specificity for MTBC, respectively. In addition, multi-locus sequencing analysis of NTM strains revealed 100% sensitivity and 100% specificity, respectively. Collectively, our multiplex PCR assay is a simple, convenient and reliable assay for simultaneous identification of the MTB Beijing lineage and major NTM species causing lung disease in Korea. 204 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 180 Biomarkers & Vaccines 21/09/2016 In depth characterization of a BCG SapM transposon mutant, a potential candidate vaccine against tuberculosis N. Festjens, E. Houthuys, K. Vandewalle, E. Plets, D. Vanderschaeghe, K. Borgers, N. Callewaert MBC, Unit for Medical Biotechnology, VIB - Ghent University, Zwijnaarde, Belgium M. bovis Bacille Calmette Guérin (BCG), the only licensed vaccine against TB, shows variable efficacy in protection against adult tuberculosis (TB). Development of a better vaccine is hampered by the lack of reliable correlates of protection against TB. Earlier, we have described a BCG transposon mutant in the secreted acid phosphatase SapM, which leads to prolonged survival of mice infected with M. tb. We have characterized the genome and transcriptome of this sapM::Tn disruption mutant versus parental BCG Pasteur 1721. Our sequencing data indicate only a few variations compared to the parental M. bovis BCG Pasteur str 1173P2 reference genome, most of which are present in both strains. We also show that transcription of sapM is considerably downregulated and that transcription of sapM’s neighboring genes is directly influenced by the presence of the transposon. By ELISA and Western blot, we detected a substantial decrease in SapM protein for the mutant culture supernatant samples versus the WT. Additionally, the enzyme’s activity was measured by an in vitro phosphatase assay and shown to be significantly reduced. Additionally, we further analyzed host immune response upon vaccination with BCG sapM::Tn versus parental BCG. We show now a better control of BCG sapM::Tn growth compared to WT BCG following vaccination. Previous observations indicated that more effective recruitment of inflammatory dendritic cells (iDCs) towards the draining lymph nodes might account for superior protection by the sapM::Tn mutant in Balb/c mice. Studies in C57BL/6J mice now show that the kinetics of iDC recruitment differ upon BCG sapM::Tn and WT vaccination: iDC recruitment to lymphoid organs starts earlier when mice are vaccinated with the BCG sapM::Tn strain. Intriguingly, vaccination with BCG sapM::Tn induced reduced frequencies of IFNγ-producing CD4+ and CD8+ T cells at early time points post-vaccination. Studies with a sapM::Tn complementation mutant and an M. bovis BCG SapM knock out demonstrate that the observed phenotypes are caused by the decrease in sapM expression, and are not due to the polar transcriptional effects of the transposon on neighboring genes, strengthening the fact that absence of SapM might lead to an improved priming vaccine based on live Mycobacterium. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 205 181 Biomarkers & Vaccines 21/09/2016 Parenteral Adenoviral Boost Enhances BCG Induced Protection, but not Long Term Survival in a Murine Model of Bovine TB C. Garcia-Pelayo3-2, A.K. Daryan3-2, P.R. Webb2, E.E. Wooff2, V.S. Bachy3-2-1, P.J. Hogarth3-2 1 Orbio Laboratoire, BRON, France 2Department of Bacteriology, Animal & Plant Health Agency (APHA) 3Vaccine Immunology Team, Addlestone, United Kingdom Boosting BCG using heterologous prime-boost represents a promising strategy for improved tuberculosis (TB) vaccines, and adenovirus (Ad) delivery is established as an efficacious boosting vehicle. Although studies demonstrate that intranasal administration of Ad boost to BCG offers optimal protection, this is not currently possible in cattle. Using Ad vaccine expressing the mycobacterial antigen TB10.4 (BCG/Ad-TB10.4), we demonstrate, parenteral boost of BCG immunised mice to induce specific CD8+ IFN-γ producing T cells via synergistic priming of new epitopes. This induces significant improvement in pulmonary protection against M. bovis over that provided by BCG when assessed in a standard 4 week challenge model. However, in a stringent, year-long survival study, BCG/Ad-TB10.4 did not improve outcome over BCG, which we suggest may be due to the lack of additional memory cells (IL-2+) induced by boosting. These data indicate BCG-prime/parenteral-Ad-TB10.4-boost to be a promising candidate, but also highlight the need for further understanding of the mechanisms of T cell priming and associated memory using Ad delivery systems. That we were able to generate significant improvement in pulmonary protection above BCG with parenteral, rather than mucosal administration of boost vaccine is critical; suggesting that the generation of effective mucosal immunity is possible, without the risks and challenges of mucosal administration, but that further work to specifically enhance sustained protective immunity is required. 206 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 182 Biomarkers & Vaccines 21/09/2016 A robust genetic marker to identify and quantify Mycobacterium tuberculosis Complex based on evolutionary conservation criteria G. Goig Serrano1, R. Borrás3, A. Gil-Brusola2, I. Comas1 1 Tuberculosis Genomics Unit, Instituto de Biomedicina de Valencia - CSIC 2Microbiology Service, La Fe University Hospital 3Microbiology Service, University Clinic Hospital, INCLIVA Research Institute, Valencia, Spain Background: A number of nucleic acid amplification tests have been developed with the aim of specifically detect Mycobacterium tuberculosis Complex DNA. However, many times these tests rely on the amplification of genetic regions that are not unique to the MTBC, or does not take into account the genetic diversity within the complex. This situation can led to sensitivity problems across the globe. Now, we have the chance to develop diversity-aware diagnostic markers thanks to the availability of large genomic databases representative, not only of the known MTBC diversity, but also of the other non-tuberculosis mycobacteria. In this work, we identify by comparative genome analyses, potential candidate loci specific to the MTBC. Furthermore, we validate our approach by real time PCR with a panel of non-tuberculosis mycobacteria, representative MTBC strains and sputum samples. Material/methods: A list of fully conserved genes among Mycobacterium tuberculosis Complex members was obtained by analyzing genomes of 219 strains representative of the global diversity of the complex. An homology similarity search of these genes in the NCBI non-redundant nucleotide database, was then performed in order to obtain potential markers that are unique to the MTBC. DNA from 18 non-tuberculosis mycobacteria (NTM) species, different representative lineages of the MTBC, and 10 clinical sputum samples, was extracted and then used to validate bioinformatic results by real time PCR (RT-PCR). Sensitivity and specificity of one of these markers was finetuned to explore its potential as a diagnostic tool. Results: We identified 49 genes to be totally conserved within the complex, from which 10 showed to be unique to the MTBC. We tested five of them against non-tuberculosis Mycobacteria DNA collection and representative strains of the MTBC. We further developed one of the markers to be used for quantification and diagnosis of MTBC DNA. All markers tested by RT-PCR showed amplification for MTBC DNA, and consistent amplification of DNA quantities up to 2E-5 ng (4CFU) could be achieved when optimizing the reaction setup for one of them. Furthermore, none of the reactions with non-tuberculosis mycobacteria DNA yielded a positive result, while robust amplification was produced for every MTBC lineage. For clinical sputum samples, 9 out of 10 produced detectable amplification, being the remaining smear and culture negative. Conclusions: We provide a reference set of 10 specific markers for the Mycobacterium tuberculosis complex together with the empirical evaluation of 5 of them. When optimizing the reaction setup for one of this markers, we achieve a 100% specificity for the MTBC, as well as a high sensitivity up to 2E⁻5 ng of template DNA. In addition, since all markers are single copy genes, these can be confidently used in real time PCR experiments to perform absolute quantification of DNA and genome copies, so the bacterial load of a sample can be estimated. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 207 183 Biomarkers & Vaccines 21/09/2016 Analysis of differential expression of MDR and Susceptible Mycobacterium tuberculosis proteins in profiles of INF-ɣ and its regulatory cytokines (IL-10) in MDR-TB patients DC cells as a potential of therapeutic or TB vaccine strategy A. Hadizadeh Tasbiti, S.H. Yari, M. Ghanei, M. Shokrgozar, A. Fateh, S. Siadat, F. Vaziri, A. Bahrmand TB, Pasteur institute of iran, Tehran, Iran Tuberculosis is still a major public health challenge worldwide. One of the major challenges in treating this disease is the widespread development of MDR-TB. The completion of the genomic sequence provides a strong foundation for subsequent identification and characterization of proteins of M.tuberculosis, leading to understanding of protein function and discovery of new drug targets against tuberculosis. Membrane proteins are functionally important and structurally challenging. This study has employed proteomic approach, which is a direct method, to identify proteins from resistant M.tuberculosis isolates compared to sensitive isolates and consequently, we aimed to investigate the cytokine response in MDR tuberculosis patients following stimulation with protein candidate (Rv0379) of Mycobacterium tuberculosis. For this purpose, TB bacilli mechanically disrupted and proteins extracted by Triton X-114 detergent phase separation. Proteins were precipitated by adding refrigerated ethanol or saturated ammonium sulfate to the supernatant. Pellet resuspended in PBS and then dialyzed for 24 h against PBS pH 7.4. IEF was carried out using the Ettan IPGphor 3 isoelecteric focusing system. Proteins were separated in second dimension on 12% SDS-PAGE in a vertical electrophoretic dual gel. 2D gels were analyzed using Image Master, Melanie 7.0 software. Mass spectrometry was performed using Autoflex II TOF/TOF. Protein sequences were retrieved from Tuberculist server hosted by Pasteur Institute, Paris. PBMC were isolated and suspended in RPMI-1640. Six days after starting DC culture, autologous T cells were isolated from PBMC by negative selection. Cells were cultured subsequently in 24 well plates at 37, 5% CO2 and stimulated with 10 µg/ml protein candidate for 72 h. Cell culture supernatants were assayed for cytokine production (IL-10 and INF-ɣ) by ELISA method. Results showed that majority of commonly expressed/upregulated proteins belonged to the cellular metabolism and respiration category (Rv 0946c, Rv 3331, Rv3586, Rv0866, Rv3057c, Rv3248c, Rv1133c, Rv0462, and Rv1876). Rv 0946c: Involved in glycolysis and in gluconeogenesis. Rv 3331: Thought to be involved in transport of sugar across the membrane. Responsible for the translocation of the substrate across the membrane. Rv3586: Conserved hypothetical protein. Another hypothetical protein (Rv2744c) and two membrane and cell wall fraction proteins (Rv0379, Rv1886c) were identified. Analysis revealed increased percentage of INF-ɣ and decreased IL-10 levels in MDR-TB patients compared to normal subjects. Fully matured monocyte derived DC are known to be strong induces of Th1 dominated immune responses. This might be caused the cell wall fraction proteins, Rv0379, induces a Th1 differentiation and committed towards a Th1 response. This present investigation may allow the identification of some valuable vaccine and drug target candidates and this provide basement for future designing of preventive, diagnostic and therapeutic strategies against TB. 208 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 184 Biomarkers & Vaccines 21/09/2016 Attenuated vaccinia virus expressing RpfE of Mycobacterium tuberculosis induces effective immune responses in mice E. Shin, H. Cha, S. Lee, J. Yoo, H. Jeong Division of Vaccine Research, Korea Centers for Disease Control and Prevention, Osong, South Korea There is licensed BCG vaccine but the protective effect against infectious Mycobacterium tuberculosis (M. tuberculosis) is limited in adults, so novel development of tuberculosis vaccines is urgently required. Resuscitation promoting factor E (RpfE) is one of the five Rpf-like proteins in M. tuberculosis. These Rpf-like proteins are secretory, which make them recognize by the host immune system. Vaccinia virus as recombinant vectors and delivery models has several advantages, which can carry many foreign genes and stimulate production of high level cell mediated immunity. In this study, we constructed recombinant vaccinia virus expressing RpfE (VV/RpfE) of M. tuberculosis using the attenuated vaccinia virus KVAC103. We immunized C57BL/6 subcutaneously with BCG or VV/RpfE, then boosted with VV/RpfE. As results, VV/RpfE induced prodection of antigen specific IgG (P<0.001) and CD4 polyfunctional T cell secreting cytokines (IFN-γ, IL-2 and TNF-α) in both prime and boost vaccination. Also, the level of IFN-γ from lung after boost vaccination was significantly increased. In further study, we will analyze protective efficacy through performing challenge test and measuring immune response factors in mice. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 209 185 Biomarkers & Vaccines 21/09/2016 Mycobacterium tuberculosis Rv3112 elicits antigen-specific T cell immune response and confers protective immunity against a predominant Beijing/K strain H. Kim, W.S. Kim, J.S. Kim, S.B. Cha, S.J. Han, K.W. Kwon, S.J. Shin Department of Microbiology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has affected human health for thousands of years and remains a major cause of human death worldwide. Effective vaccination is the most cost-effective measure to achieve long-term control of tuberculosis (TB). To achieve this goal, the identification of vaccine target antigens recognized by the immune system during early phase of infection with a powerful antigen-specific memory Th1 immune response is an essential step in TB vaccine development. Here, we evaluated vaccine efficacy of Rv3112, a molybdenum cofactor biosynthesis protein D (moaD1), against a predominant Beijing/K strain of South Korea. First, we confirmed that host immune system is able to recognize Rv3112 during early phase of Mtb K infection by generating Rv3112-specific T cell response. Second, Rv3112-immunized mice remarkably maintained multifunctional CD4+ T cells co-expressing IFN-γ, TNF-α, and IL-2 in lungs up to 10 weeks post-challenge. Finally, significant reductions of bacterial CFUs and lung inflammation were observed in Rv3112-vaccinated mice. Collectively, our results demonstrate that Rv3112 contributes to the generation of antigen-specific protective CD4+ T cell responses and may be helpful in the design of improved TB vaccines. 210 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 186 Biomarkers & Vaccines 21/09/2016 Structure based epitope mapping of a 28kDa secretary antigen: a probable diagnostic marker from Mycobacterium tuberculosis P. Kundu, R. Biswas, A. Dutta, A.K. Das Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India MTC28 is a proline-rich secreted antigen of Mycobacterium tuberculosis, conserved only in pathogenic strains of mycobacterium. MTC28 have a diagnostic value in TB detection and it increases the immunogenic response when used in combination with other major antigens such as Ag85b, ESAT-6 and CFP-10. Therefore, we characterize this antigen by solving its crystal structure and determine two immunogenic epitopes by both in silico and experimental epitope mapping. The recombinant protein is purified and the structure is solved at 2.8Å (PDB ID: 4OL4) by MIRAS method. Simulation studies are used for epitope prediction. In experimental mapping, trypsin digested peptides of MTC28 are separated and purified by HPLC. The peptides are screened by the ELISA with healthy control and patient sera. Deletion mutagenesis further confirms the epitope part. A total of 30 pulmonary TB patients and 20 healthy individuals are included in the study. The structure possesses a β sandwich like fold and belongs to space group P3121 with one molecule in the asymmetric unit. Simulation studies predict five probable epitope sites in the structure. Two peptides 1240.861 Da (128ALDITLPMPPR137) and 2141.102 Da (138WTQVPDPNVPDAFVVIADR156) found to be reactive against tuberculosis patient sera are experimentally mapped and belong to the predicted probable epitopes. ELISA studies show a significant reduction of reactivity of MTC28 A127_156Rdel against patient serum in comparison to MTC28. In conclusion, The crystal structure of MTC28 is solved and two epitopes of this antigen are mapped experimentally. These epitopes may serve as a diagnostic marker for TB detection in human. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 211 187 Biomarkers & Vaccines 21/09/2016 Engineering a pro-apoptotic BCG strain to improve the immunogenicity of the current TB vaccine A. Lau, V. Singh, H. Soualhine, Z. Hmama Division of Infectious Diseases, Department of Medicine, University of British Columbia, Vancouver, Canada BCG vaccine, introduced almost 100 years ago, is the only option to prevent TB disease. It efficiently protects newborns from meningeal TB but fails to prevent adult pulmonary TB. In fact, TB kills 1.3 million people annually in areas where BCG vaccination is widely practiced. Thus, more efficient TB vaccines are urgently needed. Others and we have shown that BCG mimics features of virulent M. tuberculosis, in particular attenuation of essential macrophage functions such as antigen presentation and apoptosis. One of these studies revealed that defect in antigen presentation is largely due to down-regulation of the cysteine protease cathepsin S (CatS), which prevents MHCII molecule maturation and proper antigen peptide loading. Recent studies also suggested a potential role for cysteine proteases in the regulation of apoptosis, a key cellular process used by the macrophage to i) contain and process ingested bacteria and ii) facilitate cross-talk antigen presentation between the macrophage and dendritic cells. To reverse the phenotype of vaccine-mediated macrophage attenuation, we engineered a novel BCG strain that expresses and secretes active CatS (BCG-CatS). Since caspase-3 plays a central role in the execution of apoptosis, we also constructed a BCG strain secreting an active form of caspase-3 (BCG-C3). Macrophages infected with either recombinant strain elicited a pro-apoptotic phenotype as indicated by increased annexin V staining, PARP degradation, and active caspase-3 production compared to wildtype BCG. Furthermore, transcriptomic profiling of macrophages infected with BCG-CatS revealed upregulation of key pro-apoptotic genes and downregulation of anti-apoptotic genes, which were further confirmed by quantitative PCR analysis. Immunogenicity studies in mice vaccinated with BCG-CatS or BCG-C3 showed increased antigen-specific CD4 and CD8 T-cell response, as well as enhanced cytokine production and proliferation upon ex vivo restimulation. Of particular note, immunogenicity responses from mice vaccinated with BCG-C3 exceeded the effects observed with BCG-CatS, demonstrating that induction of apoptosis is key to achieving high immunogenicity Collectively, this study shows that BCG modifications to promote host cell apoptosis alleviate adverse traits of the wildtype BCG strain, resulting in increased antigen presentation, which leads to highly immunogenic TB vaccines. 212 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 188 Biomarkers & Vaccines 21/09/2016 Improving T-cell assays for diagnosis of latent TB infection: potential role of a diagnostic test based on Interleukin-2 S. Mahmoudi2, S. Mamishi1, B. Pourakbari1 1 Pediatric infectious disease research center 2Tehran University of Medical Sciences, Tehran, Iran Background: Since gamma interferon release assays (IGRAs) cannot differentiate between active tuberculosis and latent tuberculosis infection (LTBI), development of rapid and specific diagnosis tools are essential for discriminating between active TB from LTBI. Both IGRAs are based on Mycobacterium tuberculosis-specific antigens, namely, early secretory antigenic target 6 (ESAT-6) and 10 KD culture filtrate (CFP-10). The aim of this study was to evaluate the potential value of IL-2 secretion by whole blood cells after stimulation with rESAT-6 and rCFP-10 for discriminating between active and latent tuberculosis. Methods: Interleukin-2 and IFN-γ were measured after blood stimulation of 90 cases (30 with active TB, 30 with LTBI and 30 healthy controls) with recombinant ESAT-6 and CFP-10. Receiver operating characteristic (ROC) curve analysis was conducted to determine the best IL-2 and IFN-γ result thresholds in discriminating between cases with active or latent TB, and correspondent sensitivity and specificity were recorded. Results: The IFN-γ release assay demonstrated a good sensitivity and specificity (sensitivity 8384% and specificity 92%) for diagnosis of tuberculosis. The discrimination performance of IL-2 assay (assessed by the area under ROC curve) between LTBI and patients with active TB were 0.75 and 0.8 following stimulation with rESAT-6 and rCFP-10, respectively. Maximum discrimination was reached at a cut-off of 11.6 pg/mL for IL-2 after stimulation with recombinant rESAT-6 with 72% sensitivity and 79% specificity and 10.7 pg/ mL for IL-2 following stimulation with rCFP-10 with 75% sensitivity 79% specificity, respectively. Conclusion: This study demonstrates that rESAT-6 and rCFP-10 can provide a sensitive and specific diagnosis of TB. In addition, it was shown that IL-2 may be serving as a marker for discriminating of LTBI and active TB. Key words: IL-2, discrimination, active TB, LTBI Tuberculosis 2016 – September 19-23, 2016 – Paris, France 213 189 Biomarkers & Vaccines 21/09/2016 Reactivation of a latent mycobacterial infection in adult zebrafish M. Niskanen5, H. Myllymäki5, H. Luukinen5, M. Parikka5-4, M. Rämet5-3-2-1 1 Department of Pediatrics, Medical Research Centre 2Department of Children and Adolescents, Oulu University Hospital, Oulu 3Department of Pediatrics 4Oral and Maxillofacial Unit, Tampere University Hospital 5BioMediTech, University of Tampere, Tampere, Finland An estimated one third of human population have a latentMycobacterium tuberculosis infection, and have a 5 – 10% lifetime risk of reactivation to active tuberculosis. Often reactivation is caused by immunosuppression, but otherwise the mechanisms of reactivation are poorly understood, hence reactivation cannot be prevented. Lack of adequate animal models has hampered tuberculosis research. Zebrafish (Danio rerio) has emerged as a promising model organism for research. Like humans, adult zebrafish has both innate and adaptive immunity. In addition, a natural fish pathogen and a close relative of M. tuberculosis, Mycobacterium marinum, causes a disease that resembles human tuberculosis, having both a natural latency with granuloma structures, and reactivation phases. Our aim was to develop a method to reactivate latent mycobacterial infection in adult zebrafish. Fish were infected with a low dose of mycobacteria, and the following latent infections were reactivated by suppressing their immune system with cortisone feeding. Three different cortisones, prednisolone, methylprednisolone and dexamethasone, were tested. Samples were collected in different time points and bacterial counts of each fish were determined by qPCR. The granuloma structures and dissemination of the bacteria were visualized from histological samples with ZiehlNeelsen and Trichrome staining protocols. Further effectivity of ethambutol was tested in both latent and reactivated infections. To utilize the method in testing effectiveness of potential, new vaccine candidates, latently infected fish were first vaccinated with groups of selected antigens and cortisone feeding was started four weeks later to evaluate effectiveness of the vaccines. Dexamethasone showed to be the most effective and safest cortisone to reactivate the latent mycobacterial infection. Reactivation was seen as increased bacterial burdens: after six weeks, the bacterial counts per fish had increased 50 – 450-fold. Histological staining confirmed increased bacteria counts. Ethambutol was not effective against latent infection, but controlled progression of reactivation. Of the DNA-based vaccine candidates, one showed protection against reactivation of latent M. marinum infection. The developed reactivation method provides a novel way to screen new drug and vaccine candidates and enables the study of reactivation of mycobacterial infection in detail. 214 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 190 Biomarkers & Vaccines 21/09/2016 Construction and characterization of TB vaccines based on MTBVAC in modern lineages of Mycobacterium tuberculosis I. Pérez Sánchez2, C. Martín2-1, J. Gonzalo-Asensio2-1 1 CIBER enfermedades respiratorias. Instituto de Salud Carlos III, Madrid 2Genética de micobacterias, University of Zaragoza, Zaragoza, Spain Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and is one of the infectious diseases that cause more mortality (WHO, 2015). BCG is the only actual vaccine against TB, but its efficacy against pulmonary diseases in adults is variable. To overcome this problem of BCG, it has been constructed a new live attenuated vaccine candidate, MTBVAC, which is in clinical trials (phase Ib, NCT02013245). MTBVAC is based on the deletion in phoP and fadD26 genes, both related with virulence factors (Arbués, 2013). MTBVAC belongs to lineage 4 (Euro-American-Asian) of Mycobacterium tuberculosis (Mtb). The principal objective of this work is to construct the same deletions of MTBVAC in two strains of linages 2 (Asian Beijing) and 3 (African-Indian). These are modern lineages, which together with lineage 4, are highly distributed and responsible for the current transmission of MTB in humans. We have selected clinical strains from lineages 2 and 3 using RFLP and spoligotyping techniques. Vaccines will be constructed in these lineages using the same method that was used to construct MTBVAC. Suicide plasmids which contains the gene (phoP or fadD26) disrupted by an antibiotic resistance gene and a counter-selectable (negative) marker will be used (Arbués, 2013). Mutants will be constructed through a two-step process involving a positive/negative double selection. The antibiotic marker will be eliminated by using a γδ-resolvase which acts on res sites flanking the resistance marker since these constructions will be used as potential vaccine candidates. MTBVAC has been extensively characterized. It has been studied the transcriptome of the transcription factor PhoP, its lipidomics profile by thin-layer chromatography in which it was observed the devoid of cell-wall lipids phthiocerol dimycocerosates (DIM), diacyltrehaloses and polyacyltrehaloses (DAT/PAT) as direct consequence of fadD26 and phoP deletions, respectively (Arbués, 2013). It has also been studied the expression profile of the principal PhoP-regulated genes (pks2, pks3, espA…) by qRT-PCR (Gonzalo-Asensio, 2008; Solans, 2014) and the inability to secrete ESAT-6 (Gonzalo-Asensio, 2014) by Western blot. To study whether these newly constructed vaccines show the same properties as MTBVAC, we plan to deeply characterize these phenotypes prior to conduct to preclinical studies. References: • • • • • • Arbues, A., et al., Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials. Vaccine, 2013. 31(42): p. 4867-73. Gonzalo-Asensio, J., et al., PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence. PLoS One, 2008. 3(10): p. e3496. Gonzalo-Asensio, J., et al., Evolutionary history of tuberculosis shaped by conserved mutations in the PhoPR virulence regulator. Proc Natl Acad Sci U S A, 2014. 111(31): p. 11491-6. Solans, L., et al., The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis. PLoS Pathog, 2014. 10(5): p. e1004183. WHO (2015). Global Tuberculosis Report 2015. Funded by: Diputación General de Aragón (DGA). European Project TBVAC2020. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 215 191 Biomarkers & Vaccines 21/09/2016 The mycobacterial capsule has an impact on the protective efficacy of BCG vaccination against Mycobacterium tuberculosis in mice R. Prados-Rosales CICbioGUNE, Derio, Spain Bacille Calmette-Guérin (BCG) is widely used for the prevention of tuberculosis (TB) throughout the world, despite limited efficacy. Although the production of BCG vaccine for human use involves the growth of the bacilli as a pellicle, most preclinical studies of BCG vaccine or Mycobacterium tuberculosis (Mtb) strains grow bacteria in the presence of detergent to prevent clumping, which also strips the mycobacterial capsule. The impact of the capsule on vaccine efficacy has not been explored by the field. Here, we show that vaccination of mice with encapsulated BCG promotes a more diverse and potent immune response relative to vaccination with unencapsulated BCG, including higher polysaccharide-specific capsule antibody titers, higher IFN-g and IL-17 splenic responses, and more multifunctional CD4+ T cells. When the same group of mice was challenged with unencapsulated Mtb, we observed lower bacterial burden in encapsulated BCG-immunized mice. We also infected mice with encapsulated Mtb. The combination of vaccination and challenge with encapsulated strains resulted in the greatest protection efficacy. The transcriptome of Mtb grown with and without detergent showed a response similar to that of starvation, hypoxia, stationary phase or non-replicating persistence. In summary, the presence of capsule on BCG affects the outcome of vaccination. 216 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 192 Biomarkers & Vaccines 21/09/2016 Control of Mycobacterium tuberculosis extrapulmonary dissemination in mice by an arabinomannan-protein conjugate vaccine R. Prados-Rosales CICbioGUNE, Derio, Spain Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cellmediated immunity (CMI). In contrast, most approved vaccines against bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with mycobacterial capsular AM conjugates to Mtb Ag85b and B. anthracis PA, elicited an AMspecific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice reacted against a broad spectrum of AM structural variants and specifically recognized arabinnan fragments. Conjugate vaccine immunized mice infected with Mtb had lower bacterial numbers in lungs and spleen, and lived longer than control mice. These findings provide strong evidence that humoral immunity can contribute to protection against Mtb and suggest the inclusion of AM in future anti-TB vaccine formulations. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 217 193 Biomarkers & Vaccines 21/09/2016 Early biomarkers associated with progression of Latent Tuberculosis Infection to clinically active disease - a longitudinal cohort study N. Rakotosamimanana3, V. Richard4, T.M. Doherty1, B. Gicquel2, A. Zumla5, V. Rasolofo3 1 GSK, Wavre, Belgium 2Institut Pasteur Paris, Paris, France 3Mycobacteria Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar 4Institut Pasteur Dakar, Dakar, Senegal 5UCL, Londres, United Kingdom Identifying those Mycobacterium tuberculosis (Mtb) latently infected individuals most at risk of developing active tuberculosis (TB) using routine clinical and laboratory tests remains a huge challenge in TB control efforts. We conducted a prospective longitudinal study to assess the risk of developing active TB in latent Mtb infection. HIV-negative household contacts (n=296) of pulmonary TB patients (n=85) and community controls (n=186) monitoring clinical features, full blood cell counts, TST, and chest X-rays were recruited and followed up regularly during 18 months. Paired statistical tests, the Kaplan -Meier method and the Cox proportional hazard modeling were performed on variables between those contacts progressing or not progressing to active TB. The appearance of TB disease symptoms within the contacts was significantly associated with an elevated peripheral percentage of blood monocytes (p = 0.01), a TST response ≥ 14 mm (p = 0.04) and an increased monocyte: lymphocyte ratio (p = 0.03). A strong association was found with both elevated blood monocyte percentage and TST ≥ 14 mm with risk of progression to TB in the contacts (aHR= 11.31 (95%CI 3.29 to 38.85), p<0.001). Elevated percentage of peripheral blood monocytes plus an elevated TST response are potential biomarkers for identifying contacts of TB patients at risk of developing active TB. 218 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 194 Biomarkers & Vaccines 21/09/2016 Novel anti-tuberculosis vaccine carrier development and assessment by in vivo imaging N. Redinger2, A. Zelmer4, D. Christensen1, P. Carroll3, K. Hagens2, P. L. Andersen1, T. Parish3, U. E. Schaible2 1 Adjuvant Research, Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark 2Cellular Microbiology, Research Center Borstel, Borstel, Germany 3Barts and The London School of Medicine & Dentistry, Garrot Building 4Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, United Kingdom The main focus of this project is to combine novel imaging approaches with M. tuberculosis reporter strains and/or QDot labeling of vaccine carrier nanoparticles to assess and monitor efficacy and biodistribution of novel vaccine formulations against tuberculosis. The nanoparticle-based vaccine formulations analysed were based on recombinant fusion protein H56 comprising Ag85B, ESAT-6 and Rv2660c linked to cationic liposomes (CAF01) containing TDB as adjuvant. Apart from accelerating testing vaccines for protective efficacy, this approach will optimize vaccine formulations and candidates. Fluorescent strains of M. tuberculosis were used in the murine tuberculosis model and imaging individual animals and excised lungs over time by IVIS. The detection limit of fluorescent M. tuberculosis in living mice was below detection limit, however, we were able to image excised lungs to assess mycobacterial load. We found that H56-CAF01 used for both, priming and boosting, induced protection against M. tuberculosis but only when given subcutaneously (s.c.) but not via the intranasal (i.n.) route. Fluorescent signals and CFU counts correlated well indicating the usability of the reporter system for vaccine testing. Using H56-CAF01 as a boost for BCG primed immunity, we observed that BCG alone already induced protection leading to reduced mycobacterial loads at the detection limit of the imaging approach, which is less sensitive than the classical CFU assay. In order to reveal the differential efficacy of the s.c. vs. the i.n. vaccination route, we analysed the biodistribution of the vaccine carriers and delivery to draining lymphoid organs, which is a prerequisite for T cell priming. We used QDots as tags to monitor vaccine carrier fate upon i.n. vs. s.c. vaccination. Upon i.n. vaccination, IVIS imaging revealed that fluorescent signals quickly (<4 d) faded from the lungs whereas some fluorescent were retained in the tracheal lymph nodes (LN) up to day 10. Upon s.c. application, the dermal signal faded at around d15 but draining inguinal LN retained the strong vaccine carrier signals at least till day 30 after vaccination. Immunohistology and FACS analysis of draining LN showed a strong influx of CD11b+ macrophages and identified CD11b+/CD11c+ myeloid cells as the predominant vaccine-carrying population. Here we show superiority of the s.c. over the i.n. route for efficient vaccine delivery due to delayed retention in draining LN. In conclusion IVIS imaging can be used to monitor and evaluate the efficacy of new vaccine candidates as well as the course of M. tuberculosis infection in excised lungs and to characterize vaccine delivery and biodistribution in the mouse in order to assess immune defense against TB. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 219 195 Biomarkers & Vaccines 21/09/2016 Activation profile of Mycobacterium tuberculosis-specific CD4+ T cells reflects disease activity, irrespective of HIV status C. Riou3-2, T. Oni3-1-4, H.P. Gideon3-1, R. Goliath3-1, R. Wilkinson3-1-6-5, K.A. Wilkinson3-1-6 1 Clinical Infectious Diseases Research Initiative 2Department of Pathology, Division of Medical Virology 3Institute of Infectious Disease and Molecular Medicine 4School of Public Health and Family Medicine, Division of Public Health Medicine, University of Cape Town, Cape Town, South Africa 5Department of Medicine, Imperial College London 6Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom The diagnosis of pulmonary tuberculosis in HIV-infected individuals is particularly challenging, as HIV-induced alterations of the immune system lead to reduced cavitations, limiting the sensitivity of sputum-based assays. Thus, alternate markers are needed to distinguish between latent (LTBI) and active TB (aTB) in this high-risk group. We therefore compared the potential of the activation and polyfunctional profiles of Mtb-specific CD4+T cells to distinguish between LTBI and aTB in HIV-uninfected and HIV-infected individuals. We analyzed 76 participants divided in four groups according to their TB and HIV status: LTBI/HIV-, aTB/HIV-, LTBI/HIV+ and aTB/HIV+. Cryopreserved PBMCs were stimulated for 16 hours with ESAT-6/CFP-10 peptide pool and stained using a live/dead marker and antibodies towards CD3, CD4, CD8, HLA-DR, Ki67, CD38, IFN-γ, TNF-α and IL-2. As previously shown, in HIV-uninfected persons, the levels of expression of HLA-DR, Ki67 and CD38 on IFNγ+ Mtb-specific CD4+T cells were significantly higher in aTB participants when compared to LTBI. Interestingly, while HLA-DR expression on Mtb-specific CD4+T cells in the LTBI/HIV+ group (median 41.7%) was significantly higher when compared to the LTBI/HIV- group (13.7%), HLA-DR expression on these cells was significantly further increased in HIV-infected individuals with aTB (84%). HLA-DR expression on IFNγ+ Mtb-specific CD4+T cells distinguishes LTBI and aTB in both the HIV- and HIV+ groups (AUC=0.98, p<0.0001; AUC=0.9, p<0.0001, respectively). However, the optimum cutoff values discriminating LTBI from aTB were distinct for HIV-uninfected (40%) and HIV-infected individuals (70%). When assessing the polyfunctional profile of Mtb-specific CD4+ T cells our data show that the proportion of IFNγ+IL2+TNFα+ or IFNγ+IL2TNFα+ Mtb-specific CD4+T cells allowed the distinction between LTBI and aTB in HIV-uninfected individuals (AUC=0.97, p<0.0001 and AUC=0.92, p<0.0001, respectively) but not in HIV-infected persons. Overall, these data show that HLA-DR expression level on Mtb-specific IFNγ+CD4+T cells represents a robust marker to distinguish between LTBI and aTB in both HIV-uninfected and ART naïve HIV-infected individuals. This suggests that despite HIV-induced systemic immune activation, active bacterial replication promotes further up-regulation of HLA-DR on Mtb-specific CD4+T cells. On the contrary, the polyfunctional profile of Mtb-specific CD4+T cells associated with TB status solely in HIV-uninfected individuals. HLA-DR expression could represent an important alternate tool to assess TB status irrespective of HIV status. 220 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 196 Biomarkers & Vaccines 21/09/2016 Blood transcriptomic diagnosis of pulmonary and extrapulmonary tuberculosis J. Roe2, N. Thomas2, E. Gil2, K. Best2, E. Tsaliki2, S. Morris-Jones3, S. Stafford2, N. Simpson2, K. Witt1, B. Chain2, R. Miller2, A. Martineau1, M. Noursadeghi2 1 Queen Mary University of London 2University College London 3University College London Hospitals NHS Trust, London, United Kingdom Background: Novel rapid diagnostics for active tuberculosis (TB) are required to overcome the time delays and inadequate sensitivity of current microbiological tests that are critically dependent on sampling the site of disease. Multiparametric blood transcriptomic signatures of TB have been described, but the number of genes included remains a barrier to their development as a diagnostic tool. We sought to identify the fewest possible blood transcripts that discriminate patients with active TB from healthy individuals, and from those with other infectious diseases. Methods: Support vector machine learning, combined with feature selection, was applied to new and previously published blood transcriptional profiles in order to identify the minimum TB-specific transcriptional signature shared by multiple patient cohorts including pulmonary and extrapulmonary TB, and individuals with and without HIV-1 co-infection. Results: We identified and validated elevated blood BATF2 transcript levels as a single sensitive biomarker which discriminated active TB from healthy individuals, with receiver operating characteristic (ROC) area under the curve (AUC) scores of 0.93-0.99 in multiple cohorts of HIV-1 negative individuals, and 0.85 in HIV-1 infected individuals. In addition, we identified and validated a novel four-gene blood signature comprising CD177, haptoglobin, immunoglobin J chain and galectin 10, that discriminated active TB from other febrile infections giving ROC AUC of 0.94-1. Conclusions: Elevated blood BATF2 transcript levels provide a sensitive biomarker that discriminates active TB from healthy individuals, and a novel four-gene transcriptional signature differentiates active TB and other infectious diseases in individuals presenting with fever. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 221 197 Biomarkers & Vaccines 21/09/2016 Evaluation and Assessment of Tuberculosis Diagnostic Testing Methods in Immigrant Children (Ages 2-14) A. Sharma, A. Lardizabal Global Tuberculosis Institute, New Jersey Medical School, Somerset, United States Objective: Assessment of current CDC recommended tuberculosis screening methods between the United States and foreign countries, both the Mantoux Tuberculin Skin Test and Interferon-Gamma Release Assay, used in immigrant children ages 2-14 years living in countries endemic to tuberculosis. Background: As per the CDC (Center for Disease Control) guidelines, children 2-14 years of age immigrating to the United States from countries endemic to tuberculosis are required to undergo one of two screening tests – tuberculin skin test (TST) or Interferon-gamma release assay (IGRA). CDC defines countries endemic to TB as World Health Organization (WHO) estimated TB incidence rate of greater than or equal to 20 cases per 100,000 people. TST is considered positive with greater than or equal to 10 mm induration. The presence of IGRA in serum is considered positive. If either test is positive, radiological chest tests are performed and appropriate management is undertaken. This study will assess validity of screening tests performed on immigrant children ages 2-14 years by comparing the tests performed in the patient’s respective country of origin and the tests upon immigrating to the United States. Method: Cohort retrospective study of patients seen in affiliation with the New Jersey Medical School Global Tuberculosis Institute will be reviewed. Charts of recent immigrant children, ages 214, seen at the Lattimer Clinic from 2013-2014 will be reviewed. Initial screening methods will be reviewed in association with final diagnosis of these respective patients seen at the Lattimer Clinic. Outcome: Based on cohort retrospective study, there was 38% concordance between the two tests and 62% discordance. This can be due to multiple factors including non-skilled application and interpretation of TSTs and recent BCG vaccination. Additionally, the study results put in question the clause found in the CDC’s “Technical Instructions For Tuberculosis Screening and Treatment” of not requiring immune response testing in patients with documented diagnostic test results from their respective country of origin. 222 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 198 Biomarkers & Vaccines 21/09/2016 Immunodominant protein MIP_05962 from Mycobacterium indicus pranii displays chaperone activity and induces Th1 mediated T cell response A. Sharma2, M.J. Equbal2, M. Saqib3, J.A. Sheikh4, S. Pandey1, N.Z. Ehtesham4, S. Bhaskar3, T.K. Chaudhuri2, S.E. Hasnain2-1 1 Dr Reddy’s Institute of Life Sciences, University of Hyderabad, Hyderabad 2Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas 3National Institute of Immunology 4National Institute of Pathology, Safdarjung, New Delhi, India Tuberculosis still remains a major threat to human beings. At present BCG is the only vaccine for TB, however protective efficacy varies from 80% to none to all and that too is only effective in preventing childhood TB and also fails to impart protection during reactivation of latent TB. Therefore, vigorous efforts are on for new TB vaccine development. Mycobacterium indicus pranii (MIP), a saprophytic, non-pathogen organism, is emerging as a promising intervention against a number of diseases by virtue of its strong immunomodulatory functions and continues to be extensively used as an intervention against leprosy. Comparative genome sequences analyses of MIP, BCG and M.leprae, revealed that MIP and M.leprae contain 29 novel genes having significant antigencity index revealed by in-silico analysis and absent in BCG (Saini V, et al. Nucleic Acids Research, 2012). Most of these gene products are potentially highly immunogenic proteins. One of these putatively immunogenic proteins, MIP_05962 belongs to Hsp20 protein family due to the presence of α-crystallin domain and also has a very high protein identity with antigen of M.tuberculosis and Hsp18 of M.leprae. Hsp18 of M.leprae, a homolog of MIP_05962, is a highly antigenic protein, carries a number of various common T cell epitopes with M.tuberculosis and elicits good CD4+T-cell response. With this background, the chaperone and immunological functions of MIP_05962 gene were investigated. Purified recombinant MIP_05962, expressed in E.coli, showed thermal aggregation prevention, thermal inactivation prevention and in-vitro refolding of substrate proteins, exhibited interaction with non-native protein, provided in-vivo viability of E.coli and also showed in-vivo assisted refolding of substrate protein. These properties of MIP_05962 protein lead us to conclude that the protein resembles molecular chaperone. Immunological properties of this protein were investigated in mice model. MIP_05962 showed significant proliferation of lymphocytes which confirmed that MIP_05962 is highly antigenic and induced significant Th1 type of T cell mediated response both in-vitro and intracellularly. It also evoked significant effector memory response in CD4+T cells. Th1-type cell mediated response is critical for controlling TB infection; Therefore MIP_05962 has a potential for use as a subunit vaccine or booster with BCG against tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 223 199 Biomarkers & Vaccines 21/09/2016 Th1-type T cell immunity driven by TLR2-mediated activation of dendritic cells and vaccine potential against tuberculosis: An example of Mycobacterium tuberculosis Rv3628 W.S. Kim, J.S. Kim, S.B. Cha, H. Kim, K.W. Kwon, S.J. Shin Department of Microbiology, Yonsei University College of Medicine, Seoul, South Korea Identification of vaccine target antigens (Ags) that induce Ag-specific Th1 immunity is the first step toward the development of a tuberculosis vaccine. Here, we evaluated the Mycobacterium tuberculosis (Mtb) protein Rv3628, a soluble inorganic pyrophosphatase, as a vaccine target and characterized the molecular details of its interaction with dendritic cells (DCs). Rv3628 activated DCs, increasing their expression of cell surface molecules and augmenting their production of TNFα, IL-1β, IL-6, and IL-12p70. Rv3628 mediated these effects by binding to TLR2 and activating downstream MyD88-, MAPK- and NF-κB-dependent signaling pathways. Rv3628-stimulated DCs induced the expansion of OVA-specific CD4+ and CD8+ T cells, which secreted IFN-γ and IL-2. Rv3628-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 Ag in samples of lung and spleen cells collected from Mtb-infected mice. Finally, an Rv3628 subunit vaccine adjuvanted with dimethyldioctadecylammonium liposomes containing monophosphoryl lipid-A caused significant reductions in bacterial counts and lung inflammation after challenge with the hyper-virulent Mtb K strain. Importantly, protective efficacy was correlated with the generation of Rv3628-specific CD4+ T cells co-producing IFN-γ, TNF-α and IL-2 and exhibiting an elevated IFN-γ recall response. Thus, Rv3628 polarizes DCs toward a Th1 phenotype and promotes protective immunity against Mtb infection. 224 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 200 Biomarkers & Vaccines 21/09/2016 Pulmonary immunity and durable protection induced by the ID93/GLA-SE vaccine candidate against the hyper-virulent Korean Beijing Mycobacterium tuberculosis strain K S.B. Cha1, W.S. Kim1, H. Kim1, K.W. Kwon1, S.N. Cho1, R.N. Coler2, S.G. Reed2, S.J. Shin1 1 Department of Microbiology, Yonsei University College of Medicine, Seoul, South Korea 2 Infectious Disease Research Institute, Seattle, United States The majority of tuberculosis (TB) vaccine candidates advanced to clinical trials have been evaluated preclinically using laboratory-adapted strains. However, it has been proposed that challenge with clinical isolates in preclinical vaccine testing could provide further and more practical validation. Here, we tested the ID93/GLA-SE TB vaccine candidate against the clinical Mycobacterium tuberculosis (Mtb) strain K (Mtb K) belonging to the Beijing family, the most prevalent Mtb strain in South Korea. Mice immunized with ID93/GLA-SE exhibited a significant reduction in bacteria and reduced lung inflammation against Mtb K when compared to non-immunized controls. In addition, we analyzed the immune responses in the lungs of ID93/GLA-SE-immunized mice, and showed that ID93/GLA-SE was able to elicit sustained Th1-biased immune responses including antigen-specific multifunctional CD4+ T cell co-producing IFN-γ, TNF-α, and IL-2 as well as a high magnitude of IFNγ response for up to 10 weeks post-challenge. Notably, further investigation of T cell subsets in the lung following challenge showed remarkable generation of CD8+ central memory T cells by ID93/GLA-SE-immunization. Our findings showed that ID93/GLA-SE vaccine confers a high level of robust protection against the hypervirulent Mtb Beijing infection which was characterized by pulmonary Th1-polarized T-cell immune responses. These findings may also provide relevant information for potential utility of this vaccine candidate in East-Asian countries where the Beijing genotype is highly prevalent. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 225 201 Biomarkers & Vaccines 21/09/2016 Impared peripheral blood CD8 T cell differentiation in HIV patients with pulmonary tuberculosis E. Vasileva1-4, G. Maximov2, I. Kudryavtsev3, M. Serebryakova3, V. Verbov4, A. Totolian4 1 N.F. Gamaleya FRCEM, Moscow 2City Tuberculosis Dispensary 3Institute of Experimental Medicine 4Saint-Petersburg Pasteur Institute, Saint-Petersburg, Russia Background: Tuberculosis (TB) is the leading cause of death among HIV-positive patients. Previously we have shown that there is no difference in the diagnostic value of QuantiFERON®-TB Gold In-Tube test (QFT) among patients with HIV infection, in comparison with patients without HIV. But it is known, that immune deficiency caused by HIV due primarily to a decrease in the number and the functions of CD4 T cells, that result in reduction of production and biological action of effector cytokines, especially IFNγ, that is measured in QFT. Methodology/Principal Findings: To answer the question which cells have produced IFNγ, we recruited 30 patients with HIV infection and pulmonary tuberculosis (TB+HIV+, n = 30) and patients without HIV infection with active tuberculosis (TB+HIV-, n = 29). In addition to the QFT we assess the degree of maturity of peripheral blood T cells using flow cytometry. Peripheral blood samples were stained with antibodies to CD3, CD4, CD8, CD27, CD28, CD45, CD45RA and CD62L. There was no significant difference (p=0,724) between the percentage of positive QFT results in TB+HIV+ (70%, median 1,36 IU/ml) and TB+HIV– (62 % median, 0,77 IU/ml). If to speak about CD4+ T cells, the percentages of naïve (N, CD27+CD28+CD45RA+CD62L+), central (CM, CD27+CD28+CD45RA+CD62L–), transitional (TM, CD27+CD28+CD45RA–CD62L–) and effector (EM, CD27–CD28+) memory were significantly higher in TB patients without HIV (p<0.001, p<0.001, p<0.001 and p=0.004, respectively). Whereas the percentages of TM, EM and terminally-differentiated CD45RA-positive effector (CD27–CD28–) CD8 T cells were significantly lower in TB patients without HIV (p=0.002, p<0.001 and p<0.001, respectively). Conclusions: These findings indicate that there is a significant difference in relative contents of CD8 T cell subsets in TB patients without HIV in comparison with TB+HIV+. Our data suggest that HIV-TB coinfection impairs CD8+ T-cell differentiation. Thus, it is possible, that especially CD8 cells produce IFNγ in patients with HIV in QFT. But in this case IFNγ has no protection against Mycobacterium tuberculosis. So, it is important to examine this trend in a group of HIV patients without TB. 226 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 202 Biomarkers & Vaccines 21/09/2016 Death and Detection: Development of a human challenge strain of Mycobacterium tuberculosis J. Wagner1, J. Fohtung1, A. Lotterman1, T. Mellors2, J. Hill2, B. Walker3, N. Cadieux3, S. Fortune1, E. Rubin1 1 Harvard T.H. Chan School of Public Health, Boston, Ma 2Thayer School of Engineering, Dartmouth College, Hanover 3Aeras, Rockville, United States Human challenge studies utilizing model strains are a powerful tool for the development of effective treatments and vaccinations for infectious disease. Due to the lengthy required treatment time, high rate of relapse, and difficulty in quantitative assessment of bacterial load, no human challenge model strain currently exists for the pathogen Mycobacterium tuberculosis. An ideal human challenge strain will require both a “kill switch” to completely and efficiently eliminate the infection in a host after a specified period of time, and a detection system designed to give real time quantitative measurement of bacterial load. Here we report the development of systems to accomplish these goals. Utilizing an orthogonal system for the incorporation of the non-canonical amino acid p- iodo-Lphenylalanine (pIF) into essential proteins, we have developed a strain of M. smegmatis that is dependent on the supplementation of pIF for survival with an escape rate of ~10-7-10-8 escapes/CFU. By placing strains grown in the presence of pIF into media lacking the supplement, we observe several generations of growth until the “protein reservoir” is depleted resulting in growth arrest of the bacterial population. In order to quantify the bacterial load in the lungs, we have genetically engineered a strain of M. tuberculosis to produce the volatile methyl salicylate, also known as the wintergreen scent. Volatile production through GC/MS can be quantified using this molecular marker to monitor bacterial loads. These two systems represent the first steps towards development of a human challenge strain of M. tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 227 203 Biochemistry & Chemical biology 21/09/2016 Using a novel hybrid method to target DnaG primase for the design of a new class of Mycobacterium tuberculosis antibiotics B. Akabayov Chemistry, Ben-Gurion University of the Negev, Be'er Sheva, Israel The DNA replication process in Mycobacterium tuberculosis (Mtb) is a promising but underexploited target for the development of novel antibiotics. We have developed an approach to identify inhibitors for Mtb DnaG primase, which is a key enzyme in the DNA replication machinery of Mtb. For the development process we have used DNA primase from bacteriophage T7. T7 primase has several structural features that are similar to bacterial (including Mtb) primases, making it an ideal model to study bacterial primases. Using NMR screening, fragment molecules that bind T7 primase were identified and then exploited in virtual filtration to select larger molecules from a virtual library. The molecules were docked to the primase active site using the available T7 primase crystal structure and ranked based on their binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Importantly, some of the molecules inhibit also the activity of DnaG primase of Mtb. Our studies yielded new class of antituberculous agents and provide new tools for fragment-based lead discovery. 228 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 204 Biochemistry & Chemical biology 21/09/2016 Lcp1 is a phosphotransferase responsible for ligating arabinogalactan to peptidoglycan in Mycobacterium tuberculosis J. Harrison2, G. Lloyd2, M. Joe1, T.L. Lowary1, E. Reynolds2, H. Walters-Morgan2, A. Bhatt2, A. Lovering2, G. Besra2, L.J. Alderwick2 1 Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Gunning-Lemieux Chemistry Centre, Edmonton, Canada 2Institute of Microbiology & Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom Mycobacterium tuberculosis, the etiological agent of tuberculosis, has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The main structural elements of the cell wall consist of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are covalently attached to a complex polysaccharide, arabinogalactan (AG) via a unique a-L-rhamnopyranose–(1®3)-a-D-GlcNAc-(1®P) linker unit. Whilst the molecular genetics associated with PG and AG biosynthetic pathways have been largely delineated, the mechanism by which these two major pathways converge has remained elusive. In Gram positive organisms, the LytR-CpsA-psr (LCP) family of proteins are responsible for ligating cell wall teichoic acids to peptidoglycan, through a linker unit that bears a striking resemblance to that found in mycobacterial arabinogalactan. In this study we have identified Rv3267 as a mycobacterial LCP homolog that encodes for a phosphotransferase which we have named Lcp1. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is capable of ligating AG to PG in a cell free radiolabeled assay. Importance Tuberculosis is an infectious disease caused by the bacterial organism Mycobacterium tuberculosis. Survival of M. tuberculosis rests critically on the integrity of its unique cell wall, therefore a better understanding of how the genes and enzymes involved in cell wall assembly work is fundamental for us to develop new drugs to treat this disease. In this study, we have identified Lcp1 as an essential phosphotransferase that ligates together arabinogalactan and peptidoglycan, two crucial cell wall macromolecules found within the mycobacterial cell wall. The discovery of Lcp1 sheds new light on the final stages of mycobacterial cell wall assembly and represents a key biosynthetic step that could be exploited for new anti-TB drug discovery. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 229 205 Biochemistry & Chemical biology 21/09/2016 Cycloserines inactivate the PLP-dependent branched-chain aminotransferase IlvE from Mycobacterium tuberculosis T. Amorim Franco, L. Favrot, S. Cameron, O. Vergnole, J. Blanchard Biochemistry, John S. Blanchard Laboratory, Albert Einstein College of Medicine, Bronx, United States D-cycloserine (DCS; or Seromycin) is a cyclic amino acid analog used in the treatment of multidrugresistant tuberculosis (MDR-TB). This natural product displays irreversible inhibitory activity against the pyridoxal 5’-phosphate (PLP)-dependent alanine racemase (AR) and, reversible inhibition of the D-alanine-D-alanine ligase (DAL). DCS has been shown to inactivate PLP-dependent enzymes from different organisms, raising the question of whether the effects of this drug in MDR-TB are only due to its inhibition of AR and DAL. In the present study, we have examined the ability of DCS- and L-cycloserine (LCS) to inhibit the essential PLP-dependent branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE). MtIlvE, is inhibited by DCS and LCS, both in a time- and concentration-dependent manner. In addition, we have crystallized the D-cycloserine-inhibited enzyme, and solved the structure to 1.7 Å. The bound, noncovalent PMP-DCS adduct reveals that the DCS ring is aromatic as evidenced by our 13C-NMR studies, and as proposed and observed in other enzyme systems. In addition, we have evidence of a disulfide bond formation that covalently links the two monomers, unlikely any other orthologue. MIC studies revealed that LCS is a much more potent inhibitor of M. tuberculosis than D-cycloserine. Together these results confirm that the effects of cycloserine are broader than previously thought. 230 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 206 Biochemistry & Chemical biology 21/09/2016 The active site architecture in peroxiredoxins: a case scenario for Mycobacterium tuberculosis AhpE B. Pedre, L. Van Bergen, A. Palló, V. Dufe, K. Wahni, I. Van Molle, L. Astolfi Rosado, H. Erdogan, M. Alonso, F. De Proft, J. Messens Structural Biology Brussel, Vrije Universiteit Brussel, Brussels, Belgium Peroxiredoxins catalyze the reduction of peroxides, a process of vital importance to survive oxidative stress. A nucleophilic cysteine, also known as peroxidatic cysteine, is responsible for this catalytic process. We used the alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE) as a model to investigate the role of the chemical environment on the specificity of the reaction. By an integrative structural (R116A - PDB 4XIH; F37G – PDB 5C04), kinetic and computational approach, we explain the mutational effects of key residues in its environment. This study shows that the active site residues are specifically oriented to create an environment which selectively favours a reaction with peroxides. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 231 207 Biochemistry & Chemical biology 21/09/2016 The pMy vector series: a versatile toolkit for the recombinant production of mycobacterial proteins K. Beckham1, S. Staack1, N. Hanna1, A. Geerlof2, M. Wilmanns1, A. Parret1 1 European Molecular Biology Laboratory, Hamburg 2Institute of Structural Biology, Helmholtz Zentrum München, Munich, Germany Structural characterisation of mycobacterial proteins is a key step in the development of novel therapeutic agents. Several studies have reported that the use of Escherichia coli as an expression host for mycobacterial targets can result in misfolded or insoluble proteins1. Switching to the related Mycobacterium smegmatis expression system2 offers several advantages, including the presence of mycobacterium-specific chaperones and post-translational modification pathways which can promote the correct folding of mycobacterial proteins. Here, we report a new series of modular vectors designed for mycobacterial protein production in M. smegmatis. The pMy vector series includes a range of promoters, resistance cassettes and affinity tag positions which can be used in combination for co-expression of protein complexes. In this study, we systematically investigated the expression of several difficult proteins including membrane proteins, toxic proteins and protein complexes using different inducible promoter systems. By exchanging the antibiotic resistance cassette, we have created vectors suitable for the co-expression of two genes which can be controlled by two independent promoters. This vector series expands the current toolkit for the overexpression of mycobacterial proteins in M. smegmatis. All the vectors generated in the study are available at Addgene. References 1. Bashiri and Baker (2015) Protein Science 24:1 2. Noens et al. (2011) BMC Biotechnology 11:27 232 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 208 Biochemistry & Chemical biology 21/09/2016 Deleting a unique polypeptide γ-loop of mycobacterial F-ATP synthase affects its ATP catalysis and H+-pumping A. Hotra3-2-1, M. Suter4, G. Biukovic4, P. Ragunathan2, S. Kundu4, T. Dick4, G. Grüber2 1 Nanyang Institute of Technology in Health and Medicine, Interdisciplinary Graduate School, Nanyang Technological University, Singapore 2School of Biological Sciences, Nanyang Technological University Singapore 3School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 4Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore The discovery of the drug bedaquiline, which specifically targets mycobacterial F-ATP synthase, proved that the enzyme is essential for both aerobically growing and dormant mycobacteria (1,2). In mycobacteria the enzyme catalyses ATP synthesis by the catalytic domain (α3/β3 subunits) at the expense of the torque energy that is generated by the rotating transmembrane H+-pump (a/c9 subunits) and the connecting central stalk γ/ε subunits. The reversible reaction, a weak or so called “latent” ATP hydrolysis was also observed in some of the fast growing mycobacteria (3,4). Previously, we described the solution structure of mycobacterial subunit γ (γ1-204) and its unique polypeptide loop γ166-179, the latter was assigned by a sequence analysis in the close proximity of rotating c-ring (5). Here we determined whether the loop γ166-179 affects ATP catalysis and subsequent H+-pumping (6). We performed functional studies of F-ATP synthase containing γ (wild type) or γ without the loop (Δγ166-179), using inverted membrane vesicles (IMV) of strain M. smegmatis mc2 155 and a set of different F-ATP synthase inhibitors. The overall reduction of ATP hydrolysis in the presence of different inhibitors, confirmed that the wild type (WT) F-ATP synthase has a low ATP hydrolytic activity. An increase of the ATP hydrolytic activity by 34% and the reduction of the ATP synthesis activity by 54% were observed in IMV of the mutant lacking the loop, as compared with WT F-ATP synthase. So far, ATP hydrolysis driven H+-pumping was not observed in mycobacteria (4). However, deletion of the γ-loop resulted in coupling of ATP hydrolysis and H+pumping (6). In summary, these results suggest that the γ-loop plays a distinctive role in regulating and coupling of ATP synthesis/hydrolysis with H+-pumping. 1. Andries, K. et al. (2005). Science, 307, (5707), 223-227. 2. Koul, A. et al. (2008). J. Biol. Chem., 283, (37), 25273-25280. 3. Higashi, T. et al. (1975). J Biol Chem, 250, (16), 6541-6548. 4. Haagsma, A.C. et al. (2010). FEMS Microbiol Lett, 313, (1), 68-74. 5. Priya, R. et al. (2013). J Bioenerg Biomembr, 45, (1-2), 121-129. 6. Hotra, A. et al. (2016). FEBS J, 1-16. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 233 209 Biochemistry & Chemical biology 21/09/2016 Functional, biochemical and structural characterization of MSMEG_6753, a novel FabG-like protein from Mycobacterium smegmatis M. Blaise2, N. Van Wyk2, F. Baneres Roquet2, M. Drancourt1, Y. Guerardel4, L. Kremer2-3 1 Université Aix-Marseille, URMITE, UMR63, CNRS 7278, IRD 198, INSERM 1095, Marseille 2Centre d’étude d’agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRSFRE3689, 1919 route de Mende, 34293 Montpellier 3INSERM-CPBS, Montpellier 4Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, IFR 147, Université des Sciences et Technologies de Lille, Villeneuve D'ascq, France Ketoacyl-acyl carrier protein (ACP) reductase (FabG) catalyse the reduction of thioesters linked to an ACP. FabG are ubiquitous in bacteria and found associated to the type II fatty acid synthase (FAS-II). Mining the Mycobacterium smegmatis genome database unravelled the existence of several putative FabG-like enzymes. Among them, MSMEG_6753 exhibits 30% primary sequence identity with FabG1/MabA, the essential ketoacyl-ACP reductase of FAS-II in Mycobacterium tuberculosis. Interestingly, MSMEG_6753 is adjacent to MSMEG_6754, encoding a recently characterized enoyl-ACP dehydratase and to MSMEG_6755, encoding a putative enoyl reductase. Thus, this genome organisation is evoking a fatty acid biosynthetic pathway functionally related to FAS-II. Recombinant MSMEG_6753 was purified and assayed for ketoacyl-reductase activity by monitoring the reduction of acetoacetyl-CoA in the presence of NADPH, albeit MSMEG_6753 is catalytically less efficient than MabA from M. smegmatis. In addition, MSMEG_6753 was able to functionally complement a FabG thermosensitive E. coli mutant, further confirming its activity in relation to fatty acid synthesis. A ΔMSMEG_6753 deletion mutant was constructed in M. smegmatis, indicating that this gene is dispensable for mycobacterial growth. However, the mutant exhibited differences in fatty acid composition as compared to the parental strain. Furthermore, we have solved the crystal structures of MSMEG_6753 in its apo form and as well as in complex with NADP+ at 1.9Å and 2.2Å resolution, respectively. The overall structures were highly similar to the one of M. tuberculosis MabA and to other bacterial FabG structures that are part of FAS-II. Unexpectedly, the binding of NADP+ to MSMEG_6753 induces an important structural rearrangement. Our structural analyses support also the capacity of MSMEG_6753 to process smaller acyl-chains than MabA. Overall, these data add new structural insights towards our understanding of the enzymatic mechanism of FabG proteins and demonstrate that MSMEG_6753 encodes a new FabG-like member acting on fatty acids. 234 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 210 Biochemistry & Chemical biology 21/09/2016 Investigating Tuberculosis Drug Metabolism Phenotype (DMP) using Metabolomics Approach M. Das2, R. Arya2, S. Debnath1, R. Debnath1, A. Lodh1, S. Bishwal2-3, A. Das1, R. Nanda2 1 Department of Tuberculosis and Respiratory Disease, Agartala Government Medical College,Tripura, Agartala 2Transnational Health Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi 3School of Life Sciences, Sambalpur University, Sambalpur, India Treatment of tuberculosis involve lengthy (at least 6 months) and intensive intervention with multiple antibiotics primarily Rifampicin (RMP), Isoniazid (INH), Pyrazinamide (PZA) and Ethambutol (ETB). Drug metabolism phenotype or how a patient metabolizes these drugs determines its efficacy, adverse effect vis a vis adherence and resistance development. Unbiased metabolomics is useful to identify endogenous and exogenous metabolites like drugs and its metabolites present in different circulatory and excretory biofluids. Untargeted metabolome analysis was carried out of urine collected from drug naïve active TB patients (n=20) at time points (2/6/12/24/36/48 hours) post dose, using a gas chromatography-time of flight-mass spectrometry (GC-Tof-MS). Broad objective of this activity was to carry out a rigorous analysis to extract important insights from the generated metabolome metadata (324x124). From this, we could successfully identify three parent drugs viz. INH, ETB, PZA and majority of their metabolites and their excretion pattern at different time points. We demonstrated drug metabolism phenotype (AcINH/INH, Pyrazinoic acid-POA/PZA) of the study population. Most importantly, using correlation analysis we identified a novel metabolite of ETB and few acute response metabolites of endogenous origin influenced by TB drugs. This novel metabolite may explain mechanism of neuropathic side effect of ETB administration in some patients. Our observations highlight the usefulness of untargeted metabolomics to understand drug metabolism phenotype of TB patients at population level. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 235 211 Biochemistry & Chemical biology 21/09/2016 Mur pathway enzymes as potential drug targets: Development of a one-pot assay for screening novel inhibitors targeting Mur enzymes K. Eniyan1, A. Kumar2, G. Rayasam2, A. Perdih3, U. Bajpai1 1 Ant-Mycobacterial Drug Discovery Lab, Department of Biomedical Science, Acharya Narendra Dev College 2Council of Scientific and Industrial Research, Open Source Drug Discovery, New Delhi, India 3National Institute of Chemistry, Ljubljana, Slovenia Background: The alarming rise in multi-drug resistance in Mycobacterium tuberculosis urges the need for discovering novel drug targets in the pathogen. Enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis in M. tuberculosis appear promising candidates for finding novel antituberculosis drugs.. Mur pathway enzymes (MurA–MurF) catalyze the synthesis of UDP-Nacetylmuramyl pentapeptide, a key precursor molecule required for the formation of the peptidoglycan monomeric building blocks. The enzymes are essential for bacterial viability, their homologues are reported to be absent in eukaryotic cells and have not yet been targeted by clinically approved antibacterial compounds Methods: In this study, the six enzymes (MurA-MurF) from Mycobacterium tuberculosis were cloned, expressed and purified by affinity chromatography. Through successive coupled enzyme assays, a one-pot assay designed to reconstitute the Mur pathway in vitro has been developed. The intermediates and the final product formed in the assays were identified by ESI-MS. To examine the sensitivity of the one-pot assay for screening of inhibitors, D-Cycloserine and a few furan-based benzene-derived compounds were tested. Results: A one-pot assay for the in vitro synthesis of UDP-MurNAc pentapeptide is established and validated as an effective screen for potential inhibitors. D-Cycloserine and two furan-based benzene-derived compounds have been identified as multi-target inhibitors of Mur pathway enzymes. Significance: Mur enzymes have not been tested for their potential as drug targets for antitubercular drug development. The one-pot assay developed in this study is amenable to high throughput screening (HTS) and provides a strategy to screen for novel inhibitors against multiple enzymes in Mur pathway of Mycobacterium tuberculosis. Also, it is a micro-titer based assay that requires a short period of incubation and does not mandate addition of expensive intermediates (sugar nucleotides) of the pathway, which further adds to its potential as a cost-effective HTS of drug candidates. 236 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 212 Biochemistry & Chemical biology 21/09/2016 Structural Characterization of Mycobacterium smegmatis RimJ, an N-acetyltransferase Protein L. Favrot, S. Hegde, J. Blanchard Biochemistry Department, Albert Einstein College of Medicine, Ny, United States The GCN5-related N-acetyltransferases (GNAT) family is a major family of enzymes found in both eukaryotes and prokaryotes. The members of the GNAT family are responsible for the acetylation of a wide array of substrates, from small molecules to macromolecules. RimJ is an Nacetyltransferase, thought to transfer an acetyl group from acetyl-CoA (AcCoA) to the N-terminus of the ribosomal protein S5 (Nα-acetylation). Other proposed functions for RimJ include the acetylation of thymosin α1, an immunostimulating peptide, and the Z-domain protein in E. coli, as well as a role in the ribosome assembly. Here we report the three-dimensional structure of Mycobacterium smegmatis RimJ (MsRimJ), an ortholog of MtbRimJ, which was solved using single wavelength anomalous dispersion. The enzyme was co-crystallized with AcCoA or CoA and the structures of both complexes were determined to 1.9 Å and 1.7 Å, respectively. Additionally, the structure of an apo form was solved to 1.4 Å. The MsRimJ structure displays a common GNAT fold. Based on gel filtration experiments, MsRimJ functions as a monomer in solution, which is unusual for Nacetyltransferases. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 237 213 Biochemistry & Chemical biology 21/09/2016 Characterisation of DprE1-mediated benzothiazinone resistance in Mycobacterium tuberculosis C. Foo, B. Lechartier, G. Kolly, J. Neres, S. Boy-Röttger, J. Rybniker, A. Lupien, C. Sala, J. Piton, S. Cole EPFL, Lausanne, France Benzothiazinones (BTZs) are a class of compounds found to be extremely potent against both drugsusceptible and drug-resistant Mycobacterium tuberculosis. The potency of BTZ is explained by their specificity to their target decaprenylphosphoryl-D-ribose oxidase (DprE1), in particular by covalent binding of the activated form of the compound to the critical C387 residue of the enzyme. To probe the role of C387, we used promiscuous site-directed mutagenesis to introduce other codons at this position in dprE1 of M. tuberculosis. The resultant viable BTZ-resistant mutants were characterised in vitro, ex vivo and biochemically to gain insight into the effects of these mutations on DprE1 function and on M. tuberculosis. Five different mutations at C387 (C387G, C387A, C387S, C387N, C387T) conferred various levels of resistance to BTZ and exhibited different phenotypes. C387G and N resulted in a slower growth rate of the mycobacterium on solid medium, which could be attributed to the significant decrease in catalytic efficiency of the DprE1 enzyme. All five mutations rendered the mycobacterium less cytotoxic for macrophages. Finally, differences in the potency of covalent and non-covalent DprE1 inhibitors in the presence of C387 mutations were revealed by enzymatic assays. As expected from the mechanism of action, the covalent inhibitor PBTZ169 only partially inhibited the mutant DprE1 enzymes as compared to near complete inhibition with a non-covalent DprE1 inhibitor Ty38c. This study emphasises the importance of the C387 residue for DprE1 activity and in the killing action of covalent inhibitors such as BTZs and other recently-identified nitro-aromatic inhibitors. 238 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 214 Biochemistry & Chemical biology 21/09/2016 Effect of doses for b6 and isoniazid under treatment of destructive tuberculosis L. Gaevaya, L. Hayova Department of Biological and Bioorganic chemistry, Bogomolets National Medical University, Kiev, Ukraine Purpose: Identify the optimal combination of drugs, the treatment of which reached at least the residual kidney damage, assessment is made based on the results of the morphological characteristics of the tissues of the internal organs in terms of treatment of different ratios of doses of isoniazid and vitamin B6. Materials and metod: Work the outcomes of destructive pulmonary tuberculosis different doses of isoniazid and B6. Results: Research on determination of protective action of isoniazid and B vitamins B6 respect to the internal organs of guinea pigs under the experimental tuberculosis are intended to help find a correlation between the dose of isoniazid and B6, which is essential to optimize treatment regimens TB and evaluating antitoxic action of vitamin B6 in the conditions of an overdose of isoniazid. Conclusions: The slightest manifestation of side effects on morphological characteristics, found in cases of (10 + 50) and (32 + 5) mg / kg, which is the best result of treatment compared with treatment doses (10 + 5) mg / kg, where side effects is more significant. Keywords: TB of the lungs, spleen, liver, kidneys, indices of infestation, isoniazid, vitamin B6, the optimal mathematical model Tuberculosis 2016 – September 19-23, 2016 – Paris, France 239 215 Biochemistry & Chemical biology 21/09/2016 Proteomic analysis of Mycobacterium tuberculosis pknG knockout mutant: insights into its role in biological processes relevant for pathogenicity A. Lima3, M. Gil3, A. Cascioferro1, J. Rossello3, B. Rivera3, M. Portela3-4, F. Le Chevalier1, W. Frigui1, N. Lisa5, M. Bellinzoni5, M. Alvarez2, C. Batthyány3-2, R. Brosch1, P. Alzari5, R. Durán3 1 Integrated Mycobacterial Pathogenomics Unit, Institut Pasteur, Paris, France 2Departamento de Bioquímica, Facultad de Medicina, UdelaR 3Analytical Biochemistry and Proteomics Unit, Institut Pasteur de Montevideo and IIBCE 4Facultad de Ciencias, Universidad de la República, Montevideo 5Structural Microbiology, Institut Pasteur, Paris, Uruguay The success of Mycobacterium tuberculosis as human pathogen resides mostly in its ability to maintain a latent infection in the host. It has been demonstrated that the Ser/Thr-kinase PknG from M. tuberculosis regulates critical processes in bacterial physiology and pathophysiology by still poorly understood mechanisms. To further characterize the role of PknG in mycobacteria, we carried out interactomic and quantitative proteomics approaches. Using a novel strategy for sequential elution of PknG partners we obtain a list of potential kinase substrates that participate mainly in the control of nitrogen metabolism and cell division. To validate these substrates as physiologically relevant, we performed quantitative comparative proteomic studies of M. tuberculosis pknG knockout mutant (KO) and H37Rv strain (WT) using 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) strategy. 2DDIGE approach showed differences in the relative abundance of spots of the same protein but with different pI, pattern consistent with potential changes in the phosphorylation status. We corroborate that some of the substrates identified by the interactomic approach were indeed physiological substrates of PknG. In addition we analyzed the global changes in the proteome of KO and WT strains of M. tuberculosis. We identified more than one-thousand proteins for each condition using a shotgun approach, and more than one-hundred proteins were differentially expressed between strains. Altogether, functional analyses of differential proteins identified using both proteomic approaches allowed us to distinguish diverse biological processes altered in the PknG deletion mutant, including nitrogen metabolism, cell wall synthesis and complex lipid biosynthesis, processes that are relevant for M. tuberculosis pathogenicity. 240 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 216 Biochemistry & Chemical biology 21/09/2016 Pyrazinamide resistance is caused by two distinct mechanisms: prevention of Coenzyme A depletion and loss of virulence factor synthesis P. Gopal1, M. Yee1, J. Sarathy1, J.L. Low1, J.P. Sarathy2, F. Kaya2, V. Dartois2, M. Gengenbacher1, T. Dick1 1 MIcrobiology, National University of Singapore, Singapore, Singapore 2Public Health Research Institute, New Jersey, United States Pyrazinamide (PZA) is a critical component of first and second line treatment of tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a genetic screen to isolate Mycobacterium bovis BCG mutants resistant to pyrazinoic acid (POA), the bioactive derivative of PZA, followed by whole genome sequencing of 26 POA resistant strains. Rather than finding mutations in fatty acid synthase FAS I and ribosomal protein S1 / RpsA, we found resistance conferring mutations in two pathways: missense mutations in aspartate decarboxylase panD, involved in synthesis of the essential acyl carrier coenzyme A (CoA), and frameshift mutations in the vitro non-essential polyketide synthase genes mas and ppsA-E, involved in the synthesis of the virulence factor phthiocerol dimycocerosate (PDIM). The POA-resistant strains fell into different phenotypic classes based on their resistance levels to POA, which corresponded with their respective genotypic classes. Probing for cross resistance to two structural analogs of POA, nicotinic acid and benzoic acid, showed that the analogs share the PDIM but not the CoA related mechanism of action with POA. Sequencing ten POA resistant Mycobacterium tuberculosis H37Rv isolates confirmed the presence of at least two distinct mechanisms of resistance to the drug. Emergence of resistance through the loss of a virulence factor in vitro may explain the lack of clear molecular patterns in PZA resistant clinical isolates, other than mutations in the prodrug converting enzyme. The apparent interference of POA with virulence pathways may contribute to the drug’s excellent in vivo efficacy compared to its modest in vitro potency. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 241 217 Biochemistry & Chemical biology 21/09/2016 Dare to be different - the ESX-1 protein secretion system in Staphylococcus aureus F. Jäger, M. Zoltner, H. Kneuper, W. Hunter, T. Palmer University of Dundee, Dundee, United Kingdom The cell membrane represents a permeability barrier between the cell and its environment and forms a permanently closed system. Therefore specialised transporters are necessary to allow direct exchange of substrates and proteins between the cell cytoplasm and the external medium. Transported proteins play essential roles in nutrient acquisition as well as in virulence. The Type VII/ESX-1 protein secretion system was first described in Mycobacterium tuberculosis where it was shown to secrete ESAT6 (early secreted antigen, 6kDa) and CFP10 (culture filtrate protein, 10 kDa). Both of these proteins are important T-cell targets and essential for the virulence of M. tuberculosis. A protein secretion system distantly related to the ESX-1 protein secretion system in M. tuberculosis was later identified and characterized in the human and animal pathogen Staphylococcus aureus, sharing ESAT6/CFP10-like and EssC-like components. EssC proteins are member of a family of the FtsK/SpoIIIE family of ATPases [1], with three interlocking ATPase domains at the C-terminus. It was recently shown that multimerisation of EccC (the EssC homolog in actinobacteria) depends upon the binding of EsxB to a pocket on the most C-terminal ATPase domain [2]. I have been studying the four membrane proteins EssA, EssB, EssC and EsaA, of the Type VII/Ess protein secretion system in S. aureus. While EssC is related to the actinobacterial EccC, EssA, EssB and EsaA are essential components of the secretion machinery that are unique to firmicutes. Crosslinking and blue native PAGE analysis have shown that the EssB, EssC and EsaA proteins individually form homomeric complexes, but do not appear to interact with one another, or with EssA. Surprisingly, I was able to show through crosslinking that the formation of higher molecular weight multimers of EssC in S. aureus is not reliant on EsxB or EsxA. Therefore it seems that oligomerisation of EssC may be controlled differently to that of EccC [3]. Bioinformatic analysis of EssC has indicated the presence of a conserved serine phosphorylation site in the N-terminal region of the protein. My current work is focused on identifying whether EssC is phosphorylated and if so whether this controls the activity of the type VII secretion system posttranslationally. My latest results will be presented. 1. M. J. Pallen, “The ESAT-6/WXG100 superfamily -- and a new Gram-positive secretion system?,” Trends Microbiol., vol. 10, no. 5, pp. 209–12, May 2002. 2. O. S. Rosenberg, D. Dovala, X. Li, L. Connolly, A. Bendebury, J. Finer-Moore, J. Holton, Y. Cheng, R. M. Stroud, and J. S. Cox, “Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion,” Cell, pp. 1–12, 2015. 3. F. Jäger, M. Zoltner, H. Kneuper, W. N. Hunter, and T. Palmer, “Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus,” FEBS Lett., vol. 590, no. 3, pp. 349–357, 2016. 242 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 218 Biochemistry & Chemical biology 21/09/2016 Fast and specific detection of mycobacteria with a novel fluorogenic derivative M. Kamariza2, C. Bertozzi1-3 1 Howard Hughes Medical Institute 2Biology Department 3Chemistry Department, Stanford University, Stanford, United States Tuberculosis (TB) is the leading cause of death from infectious disease worldwide. To reduce the time-to-diagnosis of TB, better tools for the fast, sensitive detection of the causative agent M. tuberculosis (Mtb) are urgently needed. Towards this end, we designed a fluorogenic derivative that shows a greater than 700-fold enhancement when transitioning from aqueous to hydrophobic environments and is biosynthetically incorporated into mycomembranes. Furthermore, the labeled mycobacteria are detected within minutes with specificity to live mycobacteria. This combination of properties facilitates the rapid no-wash visualization of a panel of mycobacterial and corynebacterial species and enables the detection of live Mtb within sputum samples. The potential impact of this work is vast and may lead to a complete modernization of the current state of TB diagnosis in high burden regions. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 243 219 Biochemistry & Chemical biology 21/09/2016 Structural and mechanistic insight into 1-Cys peroxiredoxin AhpE from Mycobacterium tuberculosis A. Kumar, A. Balakrishna, W. Nartey, S. Manimekalai, G. Grüber School of Biological Sciences, Nanyang technological University, Singapore, Singapore Mycobacterium tuberculosis (Mtb) has the ability to persist within the human host for a long time in a dormant stage and remerges when the immune system is compromised. One important condition, which is responsible for dormancy, is reduced availability of oxygen. Mtb is constantly exposed to numerous oxido-reductive stresses during its pathogenic cycle of transmission and infection. However the unique ability of Mtb, to not only survive the redox stress manifested by host but also to synchronize its metabolic pathway and expression of virulence factor accordingly, is central to its survival. Under such severe conditions, Mtb employs an elaborate anti-oxidant defence mechanism expressing a plethora of anti-oxidant proteins which includes peroxiredoxins, disulfide reductases, superoxide dismutase etc. Among the family of Peroxiredoxins, Mtb expresses 1-cysteine peroxiredoxin, known as alkylhydroperoxide reductase E (MtAhpE). The reduced MtAhpE (MtAhpESH) scavenges peroxides and peroxynitrite and is converted to MtAhpE-SOH. To provide continuous availability of MtAhpE-SH, MtAhpE-SOH has to become reduced. In the present study, we used NMR spectroscopy to delineate the reduced (MtAhpE-SH), sulphenic (MtAhpE-SOH) and sulphinic (MtAhpE-SO2H) states of MtAhpE through cysteinyl-labelling, and report for the first time evidence of a Mycoredoxin-independent mechanism of MtAhpE reduction. This is confirmed by crystallographic studies, where a mycothiol-reduced- and an oxidized conformation of MtAhpE was solved at 2.43 Å resolution. Combined with NMR-studies, the crystallographic structures reveal conformational changes of important residues during the catalytic cycle of MtAhpE (unpubl. data). Reference: 1. Hugo, M. et al. Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis. J. Biol. Chem. 289, 5228-5239 (2014). 244 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 220 Biochemistry & Chemical biology 21/09/2016 Mycobacterial cell wall catabolism A. Mery2, L. Shen2, A. Viljoen3, S. Villaume1, C. Mariller2, S. Vincent1, L. Kremer3, Y. Guerardel2 1 Université de Namur, Laboratoire de Chimie Bio-organique, Namur, Belgium 2Univ. Lille, CNRS, UMR8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, Lille 3Mycobacterial Pathogenesis and Novel Therapeutic Targets, CNRS-FRE3689, Montpellier, France Arabinogalactan (AG) is a crucial element of the mycobacterial cell wall representing approximately 35 % of its components. AG is also remarkable because its sugars, D-Ara and L-Gal, are all under furanose form but, unlike most bacterial polysaccharides, AG lacks repeating units and is instead composed of a few distinct structural motifs. This polysaccharide has an essential function by connecting the mycolic acid layer to the inner peptidoglycan layer to form the mycolylarabinogalactan-peptidoglycan (mAGP) complex. Because of its crucial importance in the complex life style of mycobacterias, the biosynthesis of mAGP has always been essential for the development of potential new drug targets. In contrast, earlier work on the existence of an endogenous endo-D-arabinase in mycobacteria has also shown that the mycobacterial cell wall catabolism and especially mAGP is an original and innovative route toward the discovery of new anti-tuberculosis drugs. In this context, this work is aimed to prove that mycobacteria can degrade their own mAGP focusing particularly on the enzymes capable of cleaving the arabinan and galactan portions of the AG. By using ion chromatography coupled to mass spectrometry analysis of enzymatic products, we developed a tool to investigate the glycosidase activities in mycobacteria. At the present moment this methodology is used to monitor the endo-D-arabinase activity (in M. smegmatis) in order to purify and identify it. We have also searched for putative glycoside hydrolase-coding genes by mining the genome database and focusing focused our search on the identification of glycosyl hydrolases that can use D-Araf and L-Galf as substrates. This second approach allowed us to identify and express in E. coli the Rv3096 protein from M. tuberculosis. Functional assay showed that this protein released exclusively free galactose when incubated with purified AG. Altogether, we have successfully developed a straightforward methodology to screen for glycosidase activities and identify enzymes. This enabled us to identify activities that degrade both D-arabinan and L-galactan moieties of mAGP. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 245 221 Biochemistry & Chemical biology 21/09/2016 Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids L. Blanc1, M. Gilleron1, J. Prandi1, P. Brodin3, M.S. Jang3, Y. Poquet1, O. Neyrolles1, D. Drocourt2, G. Tiraby2, A. Vercellone1, J. Nigou1 1 Tuberculosis & Infection Biology, CNRS - Université de Toulouse 2Research Department, Invivogen, Toulouse, France 3Institut Pasteur Korea, Seoul, South Korea M. tuberculosis detection by human innate immune system involves different receptors called "Pattern Recognition Receptors” (PRRs), including Toll-like Receptors (TLRs). The concerted action of PRRs results, on one hand, in the phagocytosis of mycobacteria and, on the other hand, in the activation of intracellular signaling pathways leading to nuclear translocation of transcription factors (such as NF-κB and AP-1) and the expression of genes coding for cytokines and chemokines. However, M. tuberculosis is able to inhibit innate immune responses, thus favoring its survival inside the infected host. To identify the inhibitory mechanisms used by M. tuberculosis, we have screened a transposition mutant library of M. tuberculosis on a human macrophage cell line THP-1, expressing a NF-κB/AP-1 reporter system. We identified several mutants inducing a NF-κB activation stronger than that of the wild-type strain. One of these mutants was found to be altered for the synthesis of cell envelope glycolipids, namely sulfoglycolipids, suggesting that the latters can interfere with innate immune responses. We further deciphered the molecular mechanisms involved and determined that sulfoglycolipids are competitive antagonists of TLR2, thereby inhibiting the recognition of M. tuberculosis by this receptor. Producing antagonists of PRRs is thus a strategy used by M. tuberculosis to undermine innate immunity. 246 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 222 Biochemistry & Chemical biology 21/09/2016 ATP hydrolysis and ATPase domain motion in Mycobacterium tuberculosis DNA gyrase G. Janvier3, C. Pissis4, L. Regad5, H. Munier-Lehmann2, S. Petrella3, C. Mayer3 1 INSERM 2Chimie Biocatalyse 3DBSC - Structural Microbiology Unit 4PF6, Institut Pasteur 5MTi, University Paris Diderot, Paris, France Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. This enzyme regulates DNA topology by creating a double-stranded break in one DNA duplex and transporting another DNA duplex through this break. The spread of FQ resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target DNA gyrase in novel ways. The DNA gyrase ATPase domain provides the energy required for catalysis by ATP hydrolysis. In the present work, we discuss several aspects of the Mtb DNA gyrase ATPase domain. On the basis of two crystal structures at 2.9 and 3.3 Å resolution and structural dynamics studies, we propose a mechanochemical scheme to explain how ATP hydrolysis is coupled to domain motion. In this study we also validate a novel assay for screening DNA gyrase inhibitors by the setup of an ATPase activity test. A library of 640 compounds representative of the chemical diversity of the “Chimiothèque Nationale” was screened against the Mtb DNA gyrase ATPase domain. In the first campaign, eleven novel inhibitors were identified but only one hit inhibits Mtb ATPase activity (> 90%). Finally, we developed chemoinformatic-based statistical approaches to characterize the ATP-binding site in type II topoisomerase to design a superligand. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 247 223 Biochemistry & Chemical biology 21/09/2016 Structural, functional and genetic studies of the BkaR regulon of Mycobacterium tuberculosis J. Pritchard1-2, N. Keep1, S. Kendall2 1 Institute of Structural Molecular Biology 2Royal Veterinary College, London, United Kingdom The tetracycline repressor (TetR) family regulators are abundant in the Mycobacterium tuberculosis genome and control the expression of genes involved in fatty acid metabolism, drug resistance and virulence. Many genes in TetR regulons have been implicated in the survival of the bacteria, making TetR regulators potential drug targets. TetR regulators bind to DNA to repress transcription and are only removed from DNA in the presence of a ligand. One member of the TetR family in M. tuberculosis is the branched-chain keto acid regulator (bkaR), which is thought to control the expression of 12 genes in a regulon. The ligand for bkaR is unknown, but the genes of the bkaR regulon have been shown to be involved in the catabolism of branchedchain amino acids (BCAA); leucine, isoleucine and valine. This regulon has been shown to be important for the survival of the bacteria in macrophages. The physiological relevance of BCAA catabolism in M. tuberculosis is unknown, but it is thought that utilisation may be conditional. The overall goal of this study is to determine the physiological role of the bkaR regulon in M. tuberculosis. We aim to use structural and biophysical approaches to characterise the regulation by bkaR and to determine the promoter binding properties. This project also aims to identify the ligand for the regulator and to determine the conditions under which the regulon is expressed. 248 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 224 Biochemistry & Chemical biology 21/09/2016 Siderophore monoxygenase: A potent drug target for Tuberculosis S. Rao, J. Rao2 1 Dept of Biotechnology, Dayananda sagar College Of Engg, Bengaluru, India 2Richland college, Dallas, United States Iron is an essential nutrient and is typically present at concentrations too low to support proliferation of microbial pathogens in mammalian hosts. To overcome this iron deficiency during infection many invasive human pathogens such as Mycobacterium tuberculosis, Aspergillus fumigates, Shewanella putrefaciens produce and secrete siderophores. The role of siderophores is to scavenge ferric iron from the host in order for the bacteria to proliferate. Biosynthesis of these siderophores involves the action of novel flavin –containing N-hydroxylating monoxygenases (NMO). Homologous NMOs are present in a number of siderophore-producing organisms and disruption of these monooxygenases in Burkholderia cepacia, and Aspergillus fumigates results in loss of effective persistence and colonization and has been reported to be absolutely essential for virulence. Specifically, we are targeting the enzymes that hydroxylate the siderophores to form a hydroxamate moiety, which is essential for iron binding and the lack of homologous enzymes in humans suggest that these enzymes are an attractive drug target for the treatment of tuberculosis and other bacterial diseases. Putrescine monoxygenase (PMO) is a flavin –dependent monoxygenase that catalyzes the NAD(P)H- and oxygen dependent hydroxylation of the putrescine to give N-hydroxyputrescine involved in the biosynthetic pathway of the siderophore putrebactin is targeted. Thus, enzymes involved in the biosynthesis of mycobactin/ putrebactin represent potential drug targets. With this objective the cloning of pubA gene of Shewanella putrefaciens whose genomic seq is available from Gen Bank is PCR synthesized based on the ORF sequence using genomic DNA of 4840251 bp as template. The PCR amplicon is confirmed by sequencing and nucleotide BLAST analysis. The amplicon is ligated wit pET Topo vector and the construct was propogated in TOP10 E coli cells and confirmed by PCR analysis, sequencing and restriction digestion. Later transformed into E.coli BL21 cells for full length expression. IPTG induced protein expression of recombinant entry proteins fused with 6-His and V5 epitope tags at their N termini and purified using metal affinity and ionic exchange chromatographies. Recombinant PMO represents the first member of this class of enzymes isolated in the active form, with a tightly bound FAD cofactor. The kcat value for formation of hydroxylated putrescine under steady-state conditions was 8.0 min–1, and Km values of 0.5 mM for putrescine 1.8 mM for NADH, and 2.46mM for NADPH were calculated. Crystallization and drug binding studies are in progress. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 249 225 Biochemistry & Chemical biology 21/09/2016 Metabolic labeling using azido pentoses: A novel and rapid tool to specifically target Mycobacterium tuberculosis bacteria K. Kolbe1-4, L. Möckl5, V. Sohst1, J. Brandenburg1, R. Engel3, S. Malm2, C. Bräuchle5, O. Holst3, T. Lindhorst4, N. Reiling1 1 Microbial Interface Biology 2Molecular and Experimental Mycobacteriology 3Structural Biochemistry, Research Center Borstel, Borstel 4Otto Diels Institute of Organic Chemistry, Christiana Albertina University, Kiel 5Department of Physical Chemistry, Ludwig Maximilian University, Munich, Germany Tuberculosis, caused by Mycobacterium tuberculosis (Mtb) is the single leading bacterial cause of death from infectious disease worldwide. The complex cell envelope of Mtb bacteria represents a major virulence factor and contributes to the intrinsic difficulties to eradicate the pathogen and treat the disease. However, the unique carbohydrate constituents of the mycobacterial cell envelope provide the possibility to specifically target Mtb. Synthesized derivatives of selected carbohydrates can be taken up, metabolized and subsequently be introduced into the cell envelope of Mtb bacteria. In this study three new azido pentoses were synthesized and successfully used for metabolic labeling of Mtb bacteria. The data strongly suggest the presence of arabinose and ribose transporters in the Mtb cell envelope as well as unknown biosynthetic pathways for arabinose derivatives. The investigated azido sugars exhibited no toxicity and high labeling efficiency also in Mtb clinical isolates. Thus the newly developed metabolic method allows rapid fluorescent labeling of different Mtb strains without time consuming genetic modifications. Metabolically labeled Mtb bacteria were further used in infection studies with human primary macrophages, showing comparable pathogen-specific virulence characteristics when compared to untreated bacteria. The new azido pentoses can now be employed to identify unknown uptake and metabolic mechanisms of Mtb bacteria and to study strain specific pathogenicity in host cells, which is strongly associated with the clinical outcome of tuberculosis. In addition, carbohydrate derivatives may form a new and unexpected way to specifically reach and target Mtb bacteria, which might facilitate the improvement of tuberculosis therapy in future. 250 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 226 Biochemistry & Chemical biology 21/09/2016 Dynamics of trehalose mycolates in live mycobacteria F. Rodriguez-Rivera2, C. Bertozzi2-1 1 Howard Hughes Medical Institute, Stanford University 2Chemistry, Stanford University, Stanford, United States Mycobacterium tuberculosis showcases a cell wall that serves as a formidable barrier against targeted drugs and biological stresses. The highly conserved cell wall of mycobacteria benefits from a mycomembrane, which has been correlated to intrinsic resistance to chemotherapeutics. Even though extensive characterization of individual species within the plethora of glycolipids found in the mycomembrane has been achieved, native organization and dynamics remain poorly understood. Scarcity of tools has hampered the study of real-time mycomembrane dynamics in live bacterial cells. Glycolipids with high degree of structural complexity and with significant roles in hostpathogen interactions remain elusive to biochemical and biophysical characterization in their native environment. Metabolic engineering allowed rational design of unnatural trehalose reporters that label trehalose mycolates selectively with a one-step procedure in bacterial cells. Our studies demonstrate that trehalose glycolipids can be visualized during the course of infection in cellulo and in the M. marinum/zebrafish model. This work illustrates the potential to identify relevant hostpathogen interactions in an in vivo tuberculosis pathogenesis model and enables visualization of the dynamics of trehalose glycolipids with high spatial and temporal resolution. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 251 227 Biochemistry & Chemical biology 21/09/2016 Pyrazinoic acid exerts anti-mycobacterial activity via reduction of intracellular coenzyme A level J. Sarathy1, P. Gopal1, J. Sarathy2, F. Kaya2, V. Dartois2, T. Dick1 1 Department of microbiology and immunology, Yong Loo Lin School of Medicine, National university of singapore, Singapore, Singapore 2Public Health Research Institute and New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, United States Pyrazinamide (PZA) is a key drug used in the treatment of Tuberculosis (TB). Despite being in use since the 1950s, its mode of action remains a mystery. In this study, a possible mechanism of action was investigated: the depletion of the essential cofactor Coenzyme A (CoA). Through the use of a fluorescence-based CoA assay and Liquid chromatography–mass spectrometry (LC-MS), Pyrazinoic acid (POA), the bioactive form of PZA, was shown to reduce the CoA level in wild type mycobacteria and this coincided with mycobacterial growth termination by the drug. However, POA resistance mutations in the panD gene, a proposed POA target which functions in the CoA biosynthesis pathway, was shown to prevent POA-induced CoA depletion. Wild type mycobacteria supplemented with exogenous pantothenate mimicked this phenotype and from this it is conceivable that the mechanistic basis of panD mutation-conferred resistance is that panD mutation prevents POA-induced inhibition of the CoA biosynthesis pathway. Interestingly, POA resistance mutations in the mas and ppsA-E genes, which are novel POA targets that function in the synthesis of virulence factor phthiocerol dimycocerosate (PDIM), were shown to not prevent POA-induced COA depletion. This provides further credence to POA modulating CoA levels through inhibition of the CoA biosynthesis pathway instead via a different mechanism and it also suggests a possible novel CoA-independent mode of action of POA where it targets the PDIM biosynthesis pathway. Taken together, this study has helped provide a better understanding how PZA functions and this will be crucial for the development of mechanistically-similar novel anti-TB drugs in the future. Acknowledgements This work was supported by the Singapore Ministry of Health’s National Medical Research Council under its Translational Clinical Research Flagship Grant (NMRC/TCR/011-NUHS/2014) and its Centre grant MINE / Research core no. 4 (NMRC/CG/013/2013) to A/Prof Thomas Dick, and is part of the Singapore Programme of Research Investigating New Approaches to Treatment of Tuberculosis (SPRINT-TB;www.sprinttb.org) led by Nick Paton. We would like to thank Sabai Phyu, School of Medicine BSL3 core facility for support; Martin Gengenbacher, TB research laboratory, for discussion and help, and Kristina Rutkute, SPRINT-TB, for management. 252 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 228 Biochemistry & Chemical biology 21/09/2016 Free trehalose is accumulated in dormant Mycobacterium smegmatis cells and is required for their early resuscitation phase M. Shleeva, G. Demina, K. Trutneva, D. Ostrovsky, A. Kaprelyants A.N. Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Moscow, Russia, Moscow, Russia The main aspect of the latent tuberculosis studies is elucidation of the molecular mechanisms underlying M.tuberculosis dormancy in host organism. Until now the role of trehalose in mycobacteria have been associated only with a cell wall glycolipid known as “cord factor”. It consists of a unit of trehalose esterified to two mycolic acid residues, which gives rise to α,α-trehalose 6,6′-dimycolates (bound form of trehalose). In this work we show that under external gradual acidification resulted in formation of dormant M.smegmatis cells, the content of accumulated free trehalose in dormant cells was greatly elevated. The composition of low molecular weight compounds in cell-free extracts of dormant Mycobacterium smegmatis cells was investigated with 1H-, 13C- and 31P-NMR spectroscopy. We found that up to 75% of total organic substances were represented by trehalose. The rest 25% contained unknown minor components, dinucleotides, phosphomonoethers and other phosphates. In water extract of active cells taken from early stationary phase it was detected only 15 % of trehalose. During transition to dormant state the expression of genes involved in OtsA- OtsB and TreY- TreZ pathways of trehalose synthesis (biosynthetic ways) was significantly increased. Gene expression of TreS pathway (degradatory way) was negligible. Trehalase activity was much higher in active cells compared to dormant forms although the amount of the enzyme was approximately equal in these states of mycobacteria according to electrophoresis data. In the process of dormant form resuscitation the intracellular cAMP level was increased followed by the decreasing of the free trehalose concentration during first 4-6 hours before RNA synthesis started. The presence of trehalase inhibitor validamicin A slowed down considerably the reactivation of dormant cells. Thus, for the first time, we have shown that free trehalose is accumulated in significant amount in dormant mycobacteria and participates in early molecular events leading to cell reactivation. Consequently, the inhibitores of trehalose metabolism should possibly help to prevent the dissemination of tuberculosis, lepra and other mycobacterial deseases by decreasing the resuscitation of dormant pathogenic mycobacteria or even the development of dormancy. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 253 229 Biochemistry & Chemical biology 21/09/2016 A simple TLC-based method to determine the enzyme activity of two mycobacterial Nacetyl-muramyl-L-alanine amidases N. Van Wyk3, T. Küssau3, K. Sørensen1, K. Jensen1, M. Thygesen1, M. Drancourt2, L. Kremer3-4, M. Blaise3 1 Department of Chemistry, Faculty of Science, Centre for Carbohydrate Recognition and Signalling, University of Copenhagen, Copenhagen, Denmark 2Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Centre National de la Recherche Scientifique (CNRS), Institut de Recherche pour le Développement, Faculté de Médecine, Aix-Marseille Université, Marseille 3Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), Centre National de la Recherche Scientifique FRE3689, Université de Montpellier 4INSERM, CPBS, Montpellier, France As each bacterial replication event requires the partial hydrolysis of the cell wall peptidoglycan (PG), screening for compounds that could inhibit the enzymes involved in the hydrolysis of PG could prove a fruitful exercise. One set of enzymes responsible for PG hydrolysis is known as PG amidases which includes N-acetyl-muramyl-L-alanine amidases that cleave the bond that exists between the amino sugar moiety (N-acetyl-muramic acid) and the peptide stem of a PG subunit. To our knowledge, the PG amidases are yet unexplored as a possible drug target. The caveat in examining PG amidases is the lack of a suitable direct enzyme assay without the need of expensive mass spectrometry equipment. Here, we describe a simple thin-layer chromatography method using acetyl-muramyl-dipeptide (MDP) as the substrate for testing the activity of two mycobacterial Nacetyl-muramyl-L-alanine amidases – Rv3717 of Mycobacterium tuberculosis and Mab_0318 of M. abscessus. Using a mobile phase consisting of ethanol, butanol and water at a ratio of 32:50:18 both the substrate and end products (acetyl-muramic acid and the dipeptide consisting of L-alanine and isoglutamine) could be easily separated and visualized once stained with a 2% ninhydrin solution and the TLC plate is charred. Due to the visual nature, cost-effectiveness, reproducibility and relative ease this TLC-based assay bodes well for the future screening of compounds that could inhibit these PG amidases. 254 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 230 Biochemistry & Chemical biology 21/09/2016 Mycobacterial Siderophore Biosynthesis Regulation through Post-Translational Acetylation O. Vergnolle, H. Xu, J. Tufariello, L. Favrot, W.R. Jacobs, J. Blanchard Albert Einstein College of Medicine, Bronx, United States Mycobacterial siderophores are critical components for bacterial virulence in the host. Mycobacterium tuberculosis (Mtb) synthesizes two related aryl-capped polyketide-polypeptide siderophores, named mycobactin and carboxymycobactin to scavenge intracellular macrophage iron. MbtA, the first enzyme of the mycobactin core biosynthesis catalyses two half reaction: 1) salicylic acid activation as an acyladenylate before subsequent 2) ligation to the pantothenyl group of the acyl carrier protein domain of MbtB. We demonstrated that MbtA can be reversibly posttranslationally acetylated by the mycobacterial protein lysine acetyltransferase (Pat, Rv0998) leading to enzyme inhibition. MbtA acetylation can be reverted by the mycobacterial deacetylase (DAc, Rv1151c) resulting in MbtA enzymatic reactivation. We also report the first crystal structure of Mycobacterium smegmatis MbtA solved to 2.3 Å. Furthermore, deletion of Pat and DAc genes in Mtb emphasize the significance of those specific two genes for normal mycobactin production during iron starvation. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 255 231 Biochemistry & Chemical biology 21/09/2016 Clinical and genotypic profile of TB Diabetes patients at a tertiary hospital in Ghana A. Wiredu, A. Baidoo, A. Forson Department of Chest Diseases, Korle-bu teaching Hospital, Accra, Ghana Background: We aimed to describe the prevalence of tuberculosis (TB) –Diabetes in Ghana and to investigate its association with MTBC lineages. Method: Seven hundred and sixty-seven sputum positive patients were recruited in this study The study subjects were screened for DM and diagnoses were made on the basis of the WHO criteria Species classification and sub lineage identification was by SNP-typing and Spoligotyping respectively. Associations between the different phylogenetic lineages of MTBC and epidemiological variables were assessed using Univariate and multivariate logistic regression. Results: Six hundred and seventy nine: 476 (70.1 %) males and 203 (29.9%) females indicated their diabetic status of which 85 (12.5%; 61 males and 24 females) were confirmed as diabetic with HBA1C levels above 6.5mmol/L, 589, (86.8%) as non-diabetic and the remaining 5 (0.7%) as prediabetic patient. With the exception of one, all the diabetic participants were diagnosed with pulmonary tuberculosis (TB). Seventy-seven (90.5%) patients were categorized as new cases with 6 relapse cases and 2 defaulted cases. Genotypic data showed 74 patients harboring M. tuberculosis sensu stricto, 10 harboring M. africanum with the remaining 1 M. caprae. Four of the 74 (5.4%) had elevated blood pressure with a long history of hypertension. Nine patients (1.3%) were co infected with HIV: 8 MTBss and 1 with Maf. All examined TB cases had abnormal chest X-ray with the exception one particular patient showing severe cases of infiltration and presence of macro nodules in all six zones of the chest coupled with cavitation at the right upper zone. Maf was significantly associated with Rt upper mid and lower zone opacification (odds ratio=22.03, 95% confidence interval (CI): 2.59-289, P<0.0014). Thirty-three patients (38.9%) presented with high bacteria load (3+). Conclusion: This study reports an association between diabetes and MTBC lineages in Ghana. Given the substantial burden of DM and TB comorbidity, we recommend that TB be screened routinely for DM to ensure the use of appropriate treatment regimen 256 Tuberculosis 2016 – September 19-23, 2016 – Paris, France POSTER SESSION 3 Thursday, September 22 SUMMARY Non-tuberculous & Emerging mycobacterial pathogens ............. 259 Host-responses to mycobacteria /interaction .............................. 265 Host-Pathogen interaction and beyond ....................................... 307 232 Non-tuberculous & Emerging mycobacterial pathogens 22/09/2016 Discovery of novel anti-mycobacterial drug therapy in biofilm of pathogenic nontuberculous mycobacterial keratitis T. Aung2, J. Chor2, J. Yam1, Y. Liang1, R. Beuerman2 1 Singapore Centre for Environmental Life Science Engineering 2Anti-infective group, Singapore Eye Research Institute, Singapore, Singapore Bacterial keratitis is a leading cause of corneal opacification and contributing to the global burden of blindness. Mycobacterial keratitis shows increasing incidence globally and is very difficult to eradicate compared to the other common gram-positive and gram-negative infections of the cornea. Mycobacteria easily forms biofilms in human tissues which may be a critical factor ifn sterilizing these infections. Treatment often requires two or three different antibiotics in prolonged combination treatment plan with accompanying surgical debridement. Thus, a new treatment strategy or antibiotic or both would be welcomed. Our results suggest using the strong synergism of Amikacin and Gatifloxacin for treating Mycobacterium fortuitum (ATCC 49404) while adding DNase to this combination yielded the best efficacy in eradicating mycobacteria biofilms in vitro. In this study, we reported the very first evidence of biofilm formation on the cornea by employing a novel neutropenic mouse keratitis model using M. fortuitum (ATCC 49404). Our results indicated that mycobacterial infections in keratitis were in mature biofilm modes with large amounts of extracellular DNA and micro colony formation as evaluated by Confocal Laser Scanning Microscopy. We suggest that the biofilm plays a critical role in this type of infection leading to the poor treatment outcomes in clinical practice. Our results indicated that combination of Amikacin and Gatifloxacin, was more effective for M. fortuitum (ATCC 49404) in murine keratitis model than the current standard treatment for atypical mycobacteria, Amikacin and fourth generation fluoroquinolone, Gatifloxacin. In an in vivo murine keratitis model, we successfully reported a novel treatment strategy, i.e., the destruction of biofilm matrix component, extracellular DNA, increasing the efficacy of antibiotics for mycobacterial keratitis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 259 233 Non-tuberculous & Emerging mycobacterial pathogens 22/09/2016 A new chemical entity active against Mycobacterium abscessus targets mycolic acid transport C. Dupont3-2, A. Viljoen3, F. Dubar1, M. Blaise3, A. Bernut3, A. Pawlik6, C. Bouchier5, R. Brosch6, Y. Guerardel1, J. Lelièvre7, L. Ballell7, J.L. Herrmann2, C. Biot1, L. Kremer4-3 1 UMR 8576 - UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, CNRS, Lille 2UMR 1173, INSERM, Université de Versailles St Quentin, Montigny Le Bretonneux 3 FRE 3689 Pathogénie mycobactérienne et nouvelles cibles thérapeutiques, CNRS, Université de Montpellier 4FRE 3689 Pathogénie mycobactérienne et nouvelles cibles thérapeutiques, INSERM CPBS, Montpellier 5PF1 Genopole, Institut Pasteur 6Unité de Pathogénomique Mycobacterienne Intégrée, Institut Pasteur, Paris, France 7Diseases of the Developing World, GlaxoSmithKline Tres Cantos, Madrid, Spain Mycobacterium abscessus complex is an emerging pathogen responsible for severe lung infections with parenchymal involvement especially in patients with cystic fibrosis. Highly resistant to most antibiotics, the presence of M. abscessus is deleterious to pulmonary transplantation. Therefore, there is an urgent need to identify new compounds active against this pathogen. M. abscessus shares many biochemical pathways with Mycobacterium tuberculosis suggesting than antitubercular compounds may also be active against M. abscessus. Starting from a library of compound previously validated for activity against M. tuberculosis, we identified a piperidinol-based molecule, designated PIPD1. This compound exhibited a potent activity against a wide panel of clinical M. abscessus strains with an MIC of 0.125 µg/ml. Moreover, PIPD1 was also found to be active in M. abscessus-infected macrophages. In addition, infected zebrafish embryos treated with PIPD1 displayed an increased survival rate that correlated with reduced bacterial loads. To gain insights into the mechanism of action of PIPD1, whole genome sequencing of spontaneous M. abscessus strains resistant to PIPD1 revealed several point mutations, all found in MAB_4508, encoding a transmembrane protein homologous to MmpL3, a known mycolic acid transporter in M. tuberculosis. Biochemical analyses confirmed that, while de novo biosynthesis of mycolic acids was unaffected, PIPD1 treatment strongly inhibited the transport of trehalose monomycolate. This led to impaired mycolylation of arabinogalactan, and to subsequent mycobacterial death. Thanks to a predictive MAB_4508 tridimensional homology model, the mutations conferring resistance to PIPD1 were found to be clustered, defining a putative PIPD1-binding pocket. PIPD1 represents a so far untapped class of chemical structures highly active against M. abscessus and opens the way to future translational development opportunities by targeting transport of mycolic acids. 260 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 234 Non-tuberculous & Emerging mycobacterial pathogens 22/09/2016 MgtC (MAB_3593), a host-induced factor and a vaccine candidate against Mycobacterium abscessus infection V. Le Moigne1, C. Belon2-4, C. Goulard1, G. Accard2-4, B. Pitard5, J.L. Gaillard1, L. Kremer3, J.L. Herrmann1, A.B. Blanc-Potard2-4 1 EPIM, Université Versailles St-Quentin-en-Yvelines -INSERM U1173, Montigny-LeBretonneux 2UMR5235, CNRS 3Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS FRE 3689 4Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier, Montpellier 5IN-CELL-ART, Nantes, France Mycobacterium abscessus is an emerging pathogenic mycobacterium involved in pulmonary and cutaneo-mucous infections, which constitutes a serious threat for cystic fibrosis (CF) patients. The lack of an efficient treatment regimen and the emergence of multi-drug resistance in clinical isolates require the development of new therapeutic strategies against this pathogen. Reverse genetics revealed genes present in M. abscessus but absent from saprophytic mycobacteria, and potentially involved in pathogenicity. Among them, MAB_3593 encodes MgtC, a known virulence factor involved in intramacrophage survival and adaptation to Mg2+ deprivation in several major bacterial pathogens. Herein, we first investigated the contribution of MgtC in M. abscessus virulence in infected macrophages. Despite a strong induction of M. abscessus MgtC at both transcriptional and translational levels when bacteria reside inside macrophages or upon Mg2+ deprivation, an M. abscessus knock-out mgtC mutant exhibited a slight, but non significant, replication defect within macrophages. However, our results indicated that inhibition of MgtC in vivo through immunization with M. abscessus mgtC DNA, formulated with a tetra functional amphiphilic block copolymer, exerted a protective effect against an aerosolized M. abscessus challenge in CF (ΔF508 FVB) mice. The formulated DNA immunization was associated with the production of MgtC specific antibodies, which may stimulate a protective effect by counteracting MgtC during M. abscessus infection. Our results, thus, emphasize the importance of M. abscessus MgtC in vivo, and provide a basis for the development of novel therapeutic tools against M. abscessus infections in CF patients. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 261 235 Non-tuberculous & Emerging mycobacterial pathogens 22/09/2016 The distinct fate of smooth and rough mycobacterium abscessus variants inside macrophages A. Roux3, A. Viljoen1, A. Bah6, R. Simeone5, A. Bernut4, T. Deramaudt2, M. Rottman3, J.L. Gaillard3, L. Majlessi5, R. Brosch5, I. Vergne6, C. De Chastellier1, L. Kremer4, J.L. Herrmann3 1 Centre d'Immunologie de Marseille-Luminy, Marseille 2UMR1179, Montigny Le Bretonneux 3UMR 1173, Montigny-Le-Bretonneux 4Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé, Montpellier 5Unité de Pathogénomique mycobactérienne, Paris 6IPBS, Toulouse, France Mycobacterium abscessus is a pathogenic rapid growing mycobacterium responsible for pulmonary and cutaneo-mucous infections in immunocompetent patients and in patients with cystic fibrosis. It transitions from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that the M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S variantsare usually found as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. Conversely, infection with the R variantleads to phagosomes harbouring more multiplebacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable to restrict the intraphagosomal acidification of macrophages and limits apoptosis and autophagy. Unexpectedly, the phagosomal membrane enclosing the S forms showed signs of alteration such as breaks or partial lysis. Although such events were not frequently encountered, they suggest that the S form is capable to maintain phagosome-cytosol communications. This view support M. abscessus S as sharing several traits relevant to slowgrowing mycobacterial species with respect to pathogenicity and virulence. 262 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 236 Non-tuberculous & Emerging mycobacterial pathogens 22/09/2016 Association of genetic markers with the pattern of antibiotic resistance of Mycobacterium avium subsp. hominissuis clinical isolates in South Korea S.Y. Kim1, W.J. Koh1, S.J. Shin2 1 Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine 2 Department of Microbiology, Yonsei University College of Medicine, Seoul, South Korea The aim of this study was to genetically characterize clinical isolates from patients diagnosed with Mycobacterium avium lung disease and to investigate the clinical significance. Multi-locus sequencing analysis (MLSA) and pattern of insertion sequence analysis of M. avium isolates from 92 Korean patients revealed that all isolates were M. avium subspecies hominissuis. In hsp65 sequevar analysis, codes 2, 15, and 16 were most frequently found (88/92) with similar proportions among cases additionally two isolates belonging to code N2 and an unreported code were identified, respectively. In insertion element analysis, all isolates were IS1311 positive and IS900 negative. Four of the M. avium subsp. hominissuis isolates did not harbor IS1245 and 1 of the M. avium isolates intriguingly harbored DT1, which is thought to be a M. intracellulare-specific element. M. avium subsp. hominissuis harboring ISMav6 are prevalent in Korea. No significant association between clinical manifestation and treatment response in patients with hsp65 code type and ISMav6 has been found, indicating that any specific strain/genotype among M. avium subsp. hominissuis organisms was not a major source causing M. avium lung disease. Interestingly, the presence of ISMav6 was significantly related to trends of more resistance to moxifloxacin. Conclusively, the genotype of Korean M. avium subsp. hominissuis isolates is not a disease determinant responsible for lung disease and further investigation of specific virulent factors of M. avium subsp. hominissuis needs to be investigated further. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 263 237 Non-tuberculous & Emerging mycobacterial pathogens 22/09/2016 MAB_3551c encodes the primary triacylglycerol synthase involved in lipid accumulation in Mycobacterium abscessus A. Viljoen2-1, M. Blaise2, C. De Chastellier1, L. Kremer2-3 1 Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, Inserm, U1104, CNRS UMR7280, Marseille 2Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé 3INSERM, CNRS FRE3689, Montpellier, France Mycobacterium abscessus (Mabs) is a fast-growing mycobacterial species and an emerging infective agent. The presence of seven genes encoding putative triacylglycerol (TAG) synthases (Tgs) in Mabs, suggests that during infection this mycobacterial species may accumulate TAG as a source of carbon and energy in the form of intracellular lipid inclusions (ILI), which could be used for long term survival within patients suffering from a chronic Mabs infection. Targeted gene deletion and gene-complementation studies were used to investigate the importance of the seven putative Tgs-encoding genes in TAG production in Mabs. Analysis of the TAG content in the different strains was carried out by lipid extraction followed by thin layer chromatography analysis. The most important TAG-producing Tgs’s identified by the complementation studies were purified, allowing characterization of their substrate preference. Residues critical to Tgs activity were identified by site-directed mutagenesis. Subsequently, a well-defined foamy macrophage (FM) model was used for quantitative electron microscopy to monitor and quantify ILI formation in Mabs. Deletion of MAB_3551c (tgs1) resulted in a pronounced decrease of TAG production in Mabs. However, complementation studies of this mutant with tgs1 to tgs7 revealed that Tgs4 also expressed considerable Tgs activity. Recombinant Tgs1 and Tgs4 were purified and among the two, Tgs1 exhibited the greatest TGS activity in the presence of diacylglycerol and acyl-CoA. Subsequently, H144 and Q145 of the putative acyltransferase domain were identified to be critical to the enzymatic activity. Importantly, when macrophages were infected with Mabs and stimulated to become foamy, Mabs accumulated TAG in the form of ILI. In addition, the tgs1 deletion mutant produced less and smaller ILI within FM than the parental strain, while over-expression of Tgs1 resulted in increased ILI size. We demonstrate, for the first time, ILI accumulation in Mabs inside FM. We propose Tgs1 as the major factor in this phenotype and provide novel biochemical features of this family of enzymes in mycobacteria, including substrate preferences for long acyl-CoA chains and residues critical to its activity. Future developments of in vivo animal models are required to address the importance of tgs1 in survival and persistence of Mabs during latent infection. 264 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 238 Host-responses to mycobacteria /interaction 22/09/2016 Profiling the early immune response of macrophages to Mycobacterium tuberculosis infection using RNA-seq N. Andreu2, J. Phelan2, P.F. De Sessions1, T.G. Clark2, M.L. Hibberd2-1 1 Genome Institute of Singapore, A*STAR, Singapore, Singapore 2London School of Hygiene and Tropical Medicine, London, United Kingdom Tuberculosis disease kills 1.5 million people every year. Latent Mycobacterium tuberculosis infection persists within an estimated third of the world’s population, and 10% lead to active tuberculosis. Understanding how the host immune system is able to contain or even eradicate the infection in the remaining 90% of the infected cases is crucial to the development of new therapies and vaccines aimed at boosting these defence mechanisms. Macrophages play an essential role in the early immune response against M. tuberculosis and are the cell type preferentially infected in vivo. In vitro, naïve macrophages are adept at killing at least some bacilli during the first two days of infection, although this ability wanes with time. To explore the early response of macrophages to M. tuberculosis infection we have characterised their transcriptomes using RNA-seq. In particular, we define the transcriptomes of macrophages infected with live and dead M. tuberculosis at 4 and 24 hours post-infection. Using this approach, we have detected many differentially expressed genes and pathways previously described as important during M. tuberculosis infection, confirming the ability of this approach to detect relevant processes. In addition, we have also identified novel genes whose function in resistance to M. tuberculosis infection has not been investigated. The results presented here detail the immune response of macrophages to M. tuberculosis infection at greater depth and confidence than previously achieved. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 265 239 Host-responses to mycobacteria /interaction 22/09/2016 Impact of helminth infection on antimycobacterial immune responses in UK migrants S. Anwar1, H. Fletcher1, M. Brown2, H.M. Dockrell1, F. Toulza1 1 Infection and Immunity, London School of Hygiene and Tropical Medicine 2University College of London, London, United Kingdom Latent tuberculosis and helminth infections are co-endemic in many parts of the world. Chronic helminth infections can modulate the immune responses against Mycobacterium tuberculosis (Mtb). The consequent immunomodulation may affect the ability of the host to control the growth or killing of Mtb. This study aims at investigating the modulations of the immune response profile of latent tuberculosis (LTBI) and helminth co-infected patients and whether these modulations are associated with a decrease in mycobacterial growth inhibition using a mycobacterial growth inhibition assay (MGIA). UK migrants attending University College Hospital London, UK with or without diagnosed helminth infection (Strongyloides spp and Schistosoma spp) and/or latent TB were bled at recruitment and 4 months after completing anthelmintic treatment. PPD specific IFN-γ responses in PBMC are tested by ex-vivo ELISpot assay and flow cytometry. We have previously shown the frequency of CD4+IFNγ+T cells following PPD stimulation is lower in helminth and latent TB coinfected UK migrants, compared to LTBI only, while antihelminth treatment resulted in a reduction in the frequency of CD4+FoxP3+T cells and increased Mtb specific CD4+IFNγ+ T cell responses (Toulza et al, Eur J Imm 2016). Helminth infected patients showed reduced mycobacterial growth inhibition compared to healthy controls which improved after anthelmintic treatment. Modulations of cytokine profile were also observed in these patients following treatment. Mycobacterial growth inhibition and multiple immune parameters simultaneously may provide immune signatures indicative of protective immunity against Mycobacterium tuberculosis, as well as modulation following treatment of helminth co-infected patients. Acknowledgement: Funded by EU consortia; TBVAC2020; # 643381, IDEA; # 241642 and Commonwealth Scholarship Commission, UK. 266 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 240 Host-responses to mycobacteria /interaction 22/09/2016 Complement activation by Mycobacterium tuberculosis is phenotype-dependent T. Keating3-1, S. Thomas2, L. Alderwick1, S. Taylor2, J. Bacon3 1 School of Biosciences, University of Birmingham, Birmingham 2Pathogen Immunology Group 3TB Research, Public Health England, National Infection Service, Salisbury, United Kingdom Pathogenesis of pulmonary TB is highly complex, with the measurement of cellular responses often used to assess protection in human and animal models. Compared to other major bacterial pathogens, relatively little is known about the nature of Mycobacterium tuberculosis-Complement interactions. The deposition of C3b on bacterial surfaces is thought to be beneficial to the pathogen by enhancing uptake by alveolar macrophages, an environment within which M. tuberculosis is adapted to survive. The M. tuberculosis cell wall is rich in mannose-containing structures and these have been shown to be important for complement activation with MBL levels and have been linked with susceptibility to infection in humans. We have investigated how bacterial phenotype, specifically biofilm and planktonic growth, alters complement pathway activation. Using IgG-depleted human plasma as the complement source, significantly enhanced deposition of C3b and C5b-9 was detected on the surface of planktonic M. tuberculosis, compared to cells from pellicle biofilms or planktonic cells from the non-pathogenic Mycobacterium smegmatis. This was seen at 10% complement levels and at levels physiologically relevant to the lung where the alternative pathway is less active (2%). Use of a blocking monoclonal to C1q resulted in reductions in C3b deposition on both phenotypes, showing direct activation of the classical pathway, and this was more reduced in biofilms. At 2% complement, C1q blocking completely abrogated C3b deposition on M. smegmatis. Direct measurement of C1q and MBL deposition on bacterial surfaces showed higher levels on planktonic compared to biofilm M. tuberculosis cells, with no MBL detectable on M. smegmatis. Biochemical analyses of planktonic and biofilm cells showed altered proportions of glucose in cell wall associated alpha-glucans of the biofilm cells. Carbohydrate extracts are being used in a complement C3b activation ELISA determine whether these alpha-glucan changes are leading to reduced C3b generation on the biofilm-derived polysaccharide. Currently IFN-y is used as an indicator of vaccine protection in TB. However, this has failed to predict protection of a vaccine in recent clinical trials. A greater understanding of the interplay between antibody, complement, and T cell responses could lead to additional correlates that span both the humoral and cell-mediated responses. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 267 241 Host-responses to mycobacteria /interaction 22/09/2016 Mycobacterium abscessus-induced granuloma formation is strictly dependent on TNF signaling and neutrophil trafficking A. Bernut2, J.L. Herrmann1, G. Lutfalla3, L. Kremer2-4 1 UMR1173, Université de Versailles St Quentin, INSERM, Montigny Le Bretonneux 2CPBS, FR3689 3DIMNP, UMR5235, CNRS 4CPBS, FR3689, INSERM, Montpellier, France Mycobacterium abscessus (Mabs) is considered the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. Infections with Mabs are increasingly found in chronic lung diseases, especially with cystic fibrosis, and they are often refractory to antibiotic therapy. Mabs has two morphotypes with distinct effects on host cells and biological responses. The smooth (S) variant is recognized as the initial airway colonizer while the rough (R) is known to be a potent inflammatory inducer associated with invasive disease, but the underlying immunopathological mechanisms of the infection remain unsolved. We conducted a comparative stepwise dissection of the inflammatory response in S and R pathogenesis by monitoring transparent infected zebrafish embryos. Quantitative RT-PCR, reporter zebrafish lines and antisense technology (morpholinos) were used to determine the contribution of the innate immune system and the role of inflammation in Mabs infection. An intense TNFα and IL8 pro-inflammatory response was measured by qRT-PCR. Through the use of mpeg1:mCherry or mpx:eGFP transgenic zebrafish lines harboring fluorescent macrophages and neutrophils, we found that, like macrophages, neutrophils interacted with Mabs at the initial site of infection. Loss of IL8 function resulted in increased mortality with both variants and was associated with impaired of neutrophils mobilization to the infection foci. Next, the role of TNFα in the control of Mabs infection was subsequently investigated using TNFR1 morpholinos. TNFR1 morphants were found to be extremely susceptible to both Mabs infections. Impaired TNF signaling disrupted the IL8-dependent neutrophil mobilization and the defect in neutrophil trafficking led to the formation of aberrant granulomas, extensive mycobacterial cording, unrestricted extracellular bacterial growth and subsequent larval death. The role of neutrophils for the granuloma formation was confirmed in IL8- or in neutrophil-depleted embryos. These results indicate that the TNF/IL8 inflammatory axis provides a protective sanctuary for Mabs. Our unanticipated findings indicate that neutrophils are critical for the establishment and maintenance of Mabs granulomas and play an important protective role against Mabs. These observations are particularly relevant to infections in cystic fibrosis patients, which are characterized by severe inflammation dominated by neutrophil influx. 268 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 242 Host-responses to mycobacteria /interaction 22/09/2016 Human Xenobiotic Nuclear Receptor PXR Augments Mycobacterium tuberculosis Survival. E. Bhagyaraj, R. Nanduri1, A. Saini1, H. Kitdorlang Dkhar1, N. Ahuja1, V. Chandra1, S. Mahajan1, R. Kalra1, D. Tiwari1, C. Sharma1, A. Janmeja2, P. Gupta1 1 IMTECH, CSIR 2Pulmonary medicine, Government Medical College and Hospital, Chandigarh, India The current challenge in tuberculosis (TB) treatment is the rapid increase in the emergence of drug resistant TB and this necessitates alternative therapeutic strategies that are less likely to induce drug resistance. Identification and targeting of host factors that could modulate the outcome of M. tuberculosis infection is a promising strategy. Human nuclear receptor Pregnane X receptor (hPXR) plays a crucial role in drug metabolism, drug resistance, energy homeostasis and immune response. So far its role in M. tuberculosis infection has not been explored. In this study, we investigated hPXR role in M. tuberculosis infection in human monocyte derived macrophages (hMDMs) generated from human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. Here, we demonstrate that PXR augments M. tuberculosis survival inside the host macrophages by promoting the foamy macrophage (FM) formation and abrogating phagolysosomal fusion, inflammation and apoptosis. Additionally, M. tuberculosis cell wall lipids, particularly mycolic acids (MAs) crosstalk with hPXR by interacting with its promiscuous ligand binding domain. To confirm our in vitro findings and to avoid the reported species barrier in PXR function, we adopted an in vivo mouse model expressing hPXR wherein expression of hPXR in mice promotes M. tuberculosis survival. Therefore, pharmacological intervention and designing antagonists to hPXR may prove to be a promising adjunct therapy for TB. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 269 243 Host-responses to mycobacteria /interaction 22/09/2016 The Wnt ligand Wnt6 promotes the survival of Mycobacterium tuberculosis in macrophages by modulating host cell metabolism J. Brandenburg1, D. Schwudke1, T. Goldmann1, S. Marwitz1, M. Leitges3, A. Kispert2, N. Reiling1 1 Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel 2Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany 3The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway The Wnt signaling pathway, an ancient and highly conserved signaling network, controls embryonic development and tissue homeostasis and has also been shown to exert immuno-regulatory functions during inflammatory and infectious diseases including tuberculosis. We recently showed that the Wnt ligand Wnt6 is expressed in the lung of Mycobacterium tuberculosis (Mtb)-infected mice and demonstrated that Mtb-induced Wnt6 shifts macrophage polarization towards a M2-like phenotype. Here, the functional role of Wnt6 and downstream factors during Mtb infection was analyzed in primary murine and human macrophages by use of a variety of methods, ranging from gene expression to mass spectrometry-based lipidome and CFU analyses. We were able to show that Wnt6 expression promotes the survival of Mtb within its main host cell by targeting cellular metabolism. 270 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 244 Host-responses to mycobacteria /interaction 22/09/2016 Th22 cells in mycobacterial immunity and the effect of HIV co-infection R. Bunjun2, C. Riou2, F. Omondi2, R. Wilkinson1-4-3, W. Burgers2 1 Clinical Infectious Diseases Research Initiative 2Division of Virology, Institute of Infectious Disease and Molecular Medicine, Cape Town, South Africa 3Department of Medicine, Imperial College London 4Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom HIV-1 infected individuals are at greater risk of developing tuberculosis (TB), irrespective of CD4 count. We investigated mycobacterial CD4+ T cell responses during early HIV infection, to identify defects present prior to profound CD4 depletion. T cell immunity was measured in the blood of healthy persons with evidence of immune sensitisation to M. tuberculosis (Mtb) who were either HIV-1-infected (median CD4 count 619 cells/mm3, ART naïve) or uninfected (n=24 in each group). Whole blood was stimulated with BCG and Mtb PPD and analysed for cytokine production using multiparameter flow cytometry. Characterisation of CD4+ T cell responses in HIV-1-uninfected persons after BCG stimulation revealed a high frequency of IL-22-producing CD4+ cells, that was significantly greater than the magnitude of the IFN-g response (median 0.91% versus 0.55% of CD4+ T cells, respectively; p=0.024). By comparison IL-17 responses were of low frequency (median 0.11%). There was a positive correlation between the IFN-γ and IL-22 response to BCG (r=0.83, p<0.0001), suggesting co-expression of these cytokines. However, the majority (80%) of IL-22+ cells did not co-express either IFN-γ or IL-17, suggesting rather that these cells are a distinct ‘Th22’ subset. Further characterisation of these Th22 cells revealed a memory differentiation phenotype similar to Mtbspecific Th1 cells, with 80% of cells displaying an early-differentiated phenotype (CD45RO+CD27+) and 20% of cells being late-differentiated (CD45RO+CD27-). Like Th17 cells, mycobacteriaresponsive Th22 cells expressed the chemokine receptor CCR6 (median 96% of cells producing IL22 alone), and had an intermediate phenotype between Th1 and Th17 cells for CXCR3 (28%), CCR4 (26%) and CCR10 (3%) expression. Interestingly, in HIV-1 infected individuals the IL-22+ response was 3-fold lower than in the uninfected group (p=0.0007), despite well preserved CD4 counts. This decrease was similar to the depletion of IFN-g+ CD4+ T cells in the same individuals during HIV-1 infection (p=0.0006). Thus, Th22 T cells make up a substantial and under-recognised part of the mycobacterial response, that together with the Th1 response is significantly decreased even in early HIV-1 infection. These findings highlight the effect of HIV on multiple immune subsets that may be important for control of Mtb. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 271 245 Host-responses to mycobacteria /interaction 22/09/2016 Acid sphingomyelinase is required for immune control of Mycobacterium tuberculosis infection M. Burmeister1, N. Reiling2, D. Adam3, U. Schaible1 1 Cellular Microbiology 2Microbial Interface Biology, Research Center Borstel, Borstel 3Institute for Immunology, Christian-Albrechts-University, Kiel, Germany Dysfunctional lysosomes interfering with phagocyte functions can alter host defense, antigen processing and downstream immune responses. The etiological agent of tuberculosis (Tb), Mycobacterium tuberculosis (Mtb), manipulates the macrophage's phagosomal system to escape elimination and modulate inflammation. The lysosomal acid sphingomyelinase (aSMase) generates biologically active ceramide species, which modulate autophagy, vesicle formation and apoptosis, thereby linking lipid mediator signalling, inflammation and membrane trafficking. Here, we analyze the functions of aSMase in lipid signalling and vesicle trafficking during Mtb infection. The murine Tb model of aerosol infection revealed an increased mycobacterial burden in lung, spleen and liver of aSMase KO vs. wild type (WT) mice. Furthermore, Mtb-infected aSMase KO mice showed enhanced systemic as well as pulmonary production of the proinflammatory cytokines MCP-1 and TNF-α and an exacerbated histopathology characterized by lipid storing foamy macrophages and multinucleated giant cells. We also observed alterations in immune cell recruitment with less CD4+ T cells, DCs and NK T cells in lungs of KO mice, while there was an increase in macrophages and CD8+ T cells. Currently, we analyze whether increased susceptibility to Mtb is due to a loss of macrophage function associated with intracellular lipid accumulation, which promotes Mtb growth or either hampers TH1 and NK T cell recruitment or antigen presentation / T cell activation. Understanding the function of aSMase in Mtb infection may identify signalling pathways and target structures for host directed therapeutic (HDT) strategies, which can accompany classical antimycobacterial treatment. 272 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 246 Host-responses to mycobacteria /interaction 22/09/2016 Mycobacterium tuberculosis RipA induces a prominent production of IL-10 in dendritic cells though activating TLR4-ROS-MAPK signaling H. Choi, J.S. Kim, H. Kim, W.S. Kim, S.J. Shin Department of Microbiology, Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Currently the biggest problem for tuberculosis (TB) control worldwide is preventing reactivation of latent TB. In this context, we hypothesized that some Mycobacterium tuberculosis (Mtb) antigens could be involved in the reactivation process of TB by altering the immune status, especially downregulating immune signaling and immune cell function towards a pathogen-favoring environment. RipA, a key enzyme in Mtb cell division, may be actively involved in this process by interacting with various immune cells playing an important role in the regulation of T cell immunity. In this study, we investigated how RipA manipulates the host immune system toward pathogen-favored conditions by interacting with dendritic cells (DCs). RipA-treated DCs significantly up-regulated the expression of cell surface molecules (CD80, CD86, MHC class I and II) along with remarkable secretions of proinflammatory cytokines, IL-6, IL-1β, and TNF-α, in a dose-dependent manner. Interestingly, unlike LPS, RipA significantly produced IL-10 in DCs through TLR4 binding and subsequent activation of the p38 MAPK and NF-κB signaling pathways. Furthermore, we found that intracellular reactive oxygen species (ROS) is required for the p38 MAPK activation and for RipA-induced IL-10 generation in DCs. Collectively, our results suggest that RipA induces a prominent production of IL10 through TRL4-dependent ROS and p38 MAPK signaling pathways and may contribute to the reactivation process by regulating host immune response. This study was supported by grants from the Basic Science Research Program through the Ministry of Science, ICT, and Future Planning (NRF-2014R1A2A1A11053330). Tuberculosis 2016 – September 19-23, 2016 – Paris, France 273 247 Host-responses to mycobacteria /interaction 22/09/2016 RNA-seq transcriptional profiling of PPD-b-stimulated peripheral blood from cattle infected with Mycobacterium bovis C. Correia3, K. Mcloughlin3, N. Nalpas3-1, D. Magee3, J.A. Browne3, K. Killick3, H. Vordermeier5, B. Villarreal-Ramos5, S. Gordon4-2, D. Machugh3-2 1 Proteome Center Tübingen, University Of Tübingen, Tübingen, Germany 2UCD Conway Institute of Biomolecular and Biomedical Research 3UCD School of Agriculture and Food Science, Animal Genomics Laboratory 4UCD School of Veterinary Medicine, University College Dublin, Dublin, Ireland 5Animal and Plant Health Agency, Weybridge, United Kingdom Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), costs an estimated $3 billion to global agriculture annually. The impacts of M. bovis infection are manifold, including risks to animal and public health, disruptions to trade, and reduced agricultural productivity. During the last decade, the maturation of high-throughput sequencing technologies coupled with well-annotated genome resources, has provided an unprecedented opportunity to gain a deeper understanding of host-pathogen interactions for many infectious diseases. Within this context, transcriptional profiling of the host immune response to M. bovis infection is a powerful approach for identifying host genes and cellular pathways important to disease pathology and for biomarker development. For the present study, ten age-matched male Holstein-Friesian calves were infected endobronchially with M. bovis (~2,000 CFU). Peripheral blood samples were collected in duplicate at four time points (-1 wk pre-infection, +1 wk, +2 wk, and +10 wk post-infection) and used for a) an overnight stimulation with purified protein derivative of bovine tuberculin (PPD-b) at 37°C or b) a control overnight incubation at 37°C without PPD-b stimulation. After isolation of total RNA, poly(A)+ purified RNA was used to generate strand-specific RNA-seq libraries for high-throughput sequencing (HTS). Sequencing reads were quality checked, adapter and quality filtered, and then aligned to the Bos taurus reference genome UMD3.1.1. Following summarisation of gene counts, lowly expressed transcripts were removed prior to subsequent gene annotation and differential expression analyses. This computational workflow revealed 929 differentially expressed (DE) genes at -1 wk pre-infection, 1,619 DE genes +1 wk post-infection, 1,170 DE genes at +2 wk, and 5,535 DE genes at +10 wk (when compared to the non-PPDb-stimulated group at each time point; FDR correction threshold ≤ 0.05). DE gene lists were used for functional enrichment with Ingenuity® Systems Pathway Analysis (IPA) to identify canonical cellular pathways perturbed across the infection time course in peripheral blood due to PPD-b stimulation. 274 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 248 Host-responses to mycobacteria /interaction 22/09/2016 Mycobacterium tuberculosis induces a quiescent bioenergetic phenotype in infected macrophages through irreversible suppression of oxidative phosphorylation B. Cumming1, K. Addicott1, A.J.C. Steyn1-2 1 KwaZulu-Natal Research Institute for Tuberculosis and HIV, Durban, South Africa 2Department of Microbiology, University of Alabama at Birmingham, Birmingham, United States Immunometabolism has become increasingly important in the development of new host-directed therapies against human diseases such as atherosclerosis, diabetes and some cancers. About one third of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb) and infection with Mtb does not confer immunity against future infections demonstrating Mtb has definitive evasion strategies to manipulate the immune response and prevent complete eradication. To explore the use of immunometabolism as a potential target for adjunctive host-directed therapy in the treatment of tuberculosis, we investigated the effects of pathogenic Mtb and non-pathogenic M. bovis BCG on the energy metabolism of the infected macrophage. Using extracellular flux analysis, Mtb infection was shown to decrease the rates of both ATP-generating pathways, oxidative phosphorylation (OXPHOS) and glycolysis, in macrophages, shifting the bioenergetic phenotype of the host cell to one synonymous with a state of quiescence. In contrast, macrophages infected with M. bovis BCG maintained OXPHOS and glycolysis at levels similar to those in uninfected macrophages. Heat-killed Mtb decreased OXPHOS rates of macrophages comparable to that of viable Mtb infections, but maintained the glycolytic rate present in uninfected macrophages. Pharmacological killing of intracellular Mtb by treatment with combinations of rifampicin and isoniazid, or bedaquiline and clofazimine, increased the rates of glycolysis to levels observed in uninfected macrophages, however it did not improve host OXPHOS. Thus, the presence of dead Mtb in the macrophages still suppresses OXPHOS, which suggests that the infected cells have a greater dependency on glycolysis for their ATP requirements. This was confirmed by treatment of Mtb infected macrophages with 2-deoxyglucose, which inhibits the enzyme catalysing the first step in glycolysis, namely hexokinase. Unlike the therapeutic effects of 2-deoxyglucose in cancers using aerobic glycolysis for ATP generation, 2-deoxyglucose did not reverse the bioenergetic phenotype of Mtb infected macrophages. Metformin, an AMPK-agonist that has been shown to inhibit gluconeogenesis and promote OXPHOS in diabetics, did not restore OXPHOS in Mtb infected cells. Going forward, we plan to discover the mechanisms whereby Mtb subdues host OXPHOS; if this has detrimental effects on macrophage mitochondria and whether or not this suppression can be pharmacologically reversed or inhibited. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 275 249 Host-responses to mycobacteria /interaction 22/09/2016 Calcimycin inhibits intracellular mycobacterial growth by regulating autophagy S. Mawatwal3, S. Kidwai2, A. Kapuria3, A. Singh1, S. Agarwal2, R. Singh2, R. Dhiman3 1 Structural Biology Laboratory, G. N. Ramachandran Protein Centre, CSIR-Institute of Microbial Technology, Chandigarh 2Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Faridabad, Haryana 3Department of Life Science, National Institute of Technology, Rourkela, India Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis resides and multiplies in macrophages by inhibiting various host defensive pathways like autophagy. Increased autophagy by different inducers like adenosine triphosphate (ATP) has been shown to be antimycobacterial through calcium-dependent mechanism that decreased M. bovis BCG viability. Our preliminary screening experiment using whole cell based approach has identified Calcimycin, which increases intracellular calcium as a potent inhibitor of M. tb growth. We observed that Calcimycin was bactericidal in action and inhibited M. tb growth in vitro by 99% at 1.25 M. So, present study was undertaken to decipher the role of Calcimycin on intracellular mycobacteria in THP-1 cells. We found by MTT assay that 0.4 µM of calcimycin is non-toxic even after 72 h of treatment. Viability findings were further corroborated by Trypan blue dye exclusion assay where calcimycin treatment for 72 h reduced cell viability to 86.4 ± 1.8%, 73.0 ± 11.9% and 62.2 ± 10.2% at 0.6 µM, 0.8 µM and 1µM respectively. Time kinetic experiment was performed with 0.4 µM of calcimycin to study its effect on the autophagy in THP-1 cells. We found optimum up-regulation of different autophagy markers like Beclin-1, Atg 7 and Atg 3 after 12 h of treatment through western blotting. Increased conversion of LC3-I to LC3-II, widely used marker of autophagy induction was also observed in treated cells suggesting increased autophagy. Addition of 3-methyadenine (3-MA), an autophagy inhibitor, abrogated the effect of calcimycin on autophagy. We found down-regulation of Beclin-1, Atg 7, Atg 3 and LC3-II expression in calcimycin treated cells in the presence of 3-MA. Increased autophagy in calcimycin treated cells led to decrease in intracellular M. bovis BCG viability and addition of 3-MA reversed this effect. Studies are currently underway to determine the mechanism of how calcimycin induced autophagy exerts its antimycobacterial effect. 276 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 250 Host-responses to mycobacteria /interaction 22/09/2016 Insights into modulatory roles of vitamin C on human monocytic cell line, THP-1: An ex vivo model for studying host-pathogen interaction S. Dhingra, M. Nandi, K. Sikri, J.S. Tyagi Biotechnology, All India Institute of Medical Sciences, Delhi, India Macrophages are considered to be the first line of defence in response to any pathogen. The THP-1 cell line is widely used for studying macrophage functions and for analyzing host cell-pathogen interactions. Vitamin C (vit C) is established as a free radical scavenger, an essential cofactor of many enzymes and is required for immune effector cell function of macrophages. We performed a genome-wide temporal gene expression and functional enrichment analysis of THP-1 cells treated with 100 μM of vit C, a physiologically relevant concentration of the vitamin. Extensive modulation of gene expression was observed from 8 hours to 96 hours as reflected by an increase in the number of differential regulated genes. Further, functional enrichment analysis based on statistically stringent criteria revealed a gamut of functional responses, namely, ‘Regulation of gene expression’, ‘Signal transduction’, ‘Cell cycle’, ‘Immune system process’, ‘Regulation of cAMP metabolic process’, ‘Cholesterol transport’ and ‘Ion homeostasis’ were enriched. A comparative analysis of vit C-mediated modulation of gene expression data in THP-1 cells and human skin fibroblasts disclosed an overlap in certain functional processes such as ‘Regulation of transcription’, ‘Cell cycle’ and ‘Extracellular matrix organization’ and THP-1 specific responses, namely, ‘Regulation of gene expression’ and ‘Ion homeostasis’. It was noteworthy that vit C modulated the ‘Immune system’ process throughout the time-course. We have reported earlier that tubercle bacilli treated with vit C develop an isoniazid-tolerant phenotype that is widely considered to be an indicator of bacterial dormancy. This vit C-induced drug tolerant response occurred in vitro as well as in infected THP-1 cells. In view of the ability of vit C to induce a ‘dormant’ phenotype in Mtb, this temporal transcriptome profiling study of baseline gene expression to assess the response of THP-1 cells to vit C would pave the way for utilizing the vit C-based THP-1 infection model to study biochemical and cellular responses of host cells to ‘dormant’ Mtb infection and other intracellular pathogens. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 277 251 Host-responses to mycobacteria /interaction 22/09/2016 Mycobacterial N-Glycolyl Muramyl Dipeptide Drives Cell-Mediated Immunity from Complete Freund’s Adjuvant J. Dubé2-4-1, D. Montamat-Sicotte4-1, F. Mcintosh4-1, M. Behr3-1-4-2 1 McGill International TB Centre 2Dept. of Microbiology & Immunology, McGill University 3Dept. of Medicine 4Research Institute, McGill University Health Centre, Montreal, Quebec, Canada The capacity for the mycobacterial cell to act as an adjuvant has long been recognized, notably in the form of complete Freund’s adjuvant (CFA, killed M. tuberculosis in mineral oil). However, work remains to define the molecular entities unique to mycobacteria that contribute to cell-mediated immunity (CMI), as was first demonstrated by Chase and Landsteiner circa 1940. Subsequent work by Lederer and colleagues suggested the peptidoglycan fragment N-acetyl muramyl dipeptide (MDP) was the minimal component for CFA adjuvancy. MDP was later shown to elicit innate immune responses via the pattern recognition receptor NOD2. Together, this has led to two related hypotheses: 1) the CMI adjuvant effect is in part mediated by NOD2, and 2) mycobacteria, instead of other more conventional bacteria, were used by Freund because of their unique N-glycolylated MDP. To determine the contribution of N-glycolyl MDP, acting via NOD2, to the CMI response elicited by CFA, we have investigated these two hypotheses at two levels: the whole animal, using an in vivo murine model of immunization, and the cell, by contrasting the effect of N-glycolyl MDP with Nacetyl MDP in different ex vivo models of innate and adaptive immunity. Ultimately, we aim to ‘complete’ Freund’s incomplete adjuvant (mineral oil without mycobacteria) by adding specific mycobacterial pathogen-associated molecular patterns (PAMPs). Data indicate that the majority of CMI elicited by CFA is dependent on the pattern recognition molecule NOD2, strongly implicating MDP in CFA adjuvancy. At the cellular level, N-glycolyl MDP augments dendritic cell (DC) functions as compared to N-acetyl MDP, when measuring cytokine secretion, upregulation of costimulatory molecules and T-cell activation. Complementing the PAMP data, we observe that these DC read-outs are diminished with either of two genetic manipulations: 1) using Nod2-/- DCs, or 2) infecting DCs with mutated mycobacteria that are unable to produce Nglycolyl MDP. This improved understanding of mycobacterial cell adjuvancy will inform our perspective on the immunomodulatory capacity of mycobacterial pathogens and may provide insights about BCG as a vaccine and an immunotherapeutic agent. 278 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 252 Host-responses to mycobacteria /interaction 22/09/2016 Insights into the role of a Mycobacterium abscessus specific MmpL transporter involved in the macrophage-mycobacterium interaction V. Dubois3, L. Laencina3, A. Viljoen1, V. Le Moigne3, G. Sapriel3, E. Dumas2, E. J Rubin4, M. Blaise1, L. Kremer1, J.L. Herrmann3, F. Girard-Misguich3 1 FRE3689, Montpellier 2UMR8079, CNRS, Paris Saclay 3U1173, UVSQ, Saint-Quentin-EnYvelines, France 4Department of Immunology and Infectious Diseases, Massachusetts, United States Mycobacterium abscessus (Mabs) is a non-tuberculous rapidly growing mycobacterium mainly responsible for lung infections. Prevalence of Mabs infections has been increasing worldwide, and specifically among Cystic Fibrosis patients. Mabs has been shown to grow and survive in eukaryotic cells like macrophages. Benefiting of a transposon mutant library obtained in a Mabs subsp massiliense smooth clinical Korean isolate, we aimed at deciphering the genes allowing Mabs to survive intracellularly. 6000 Tn mutants were screened individually for their survival impairment in macrophages. One specific mutant was identified with a Tn insertion within MAB_0855 (MAB_0855::Tn), encoding a mycobacterial membrane protein Large (MmpL) protein. MmpL protein members are mycobacterial transporters involved in the maturation and export of cell wall lipids and can also act as drug efflux proteins. These proteins are well-described in Mycobacterium tuberculosis: some of them play a role in M. tuberculosis virulence such as MmpL8 and MmpL10. Although no orthologs of MAB_0855 are found in M. tuberculosis by Bidirectional Best Hit (BBH), a Blast analysis and phylogenetic studies revealed the presence of MAB_0855 within the M. tuberculosis mmpL8, 10 and 12 gene cluster. A Thin Layer Chromatography of the extractible lipids unraveled subtle changes in its parietal lipids composition. One consequence of this cell wall defect results in impaired adhesion to macrophages and a subsequent diminution of MAB_0855::Tn internalization by the cells. In addition, the intramacrophage survival of MAB_0855::Tn is highly reduced in comparison to the parental strain. To further confirm the phenotype of MAB_0855::Tn, the MAB_0855 gene has been inactivated in both the smooth and rough reference strains of Mabs. Functional complementations are currently being carried out in these two gene deletion mutants to confirm and validate the contribution of MAB_0855 in the observed phenotypes. The impact of the gene deletion will also be studied in whole infected zebrafish embryos. Here, we identified and characterized a new MmpL protein implicated in shaping the cell-wall and in the Mabs/macrophage interactions, further supporting the importance of these transporters in Mabs virulence. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 279 253 Host-responses to mycobacteria /interaction 22/09/2016 Ancient and modern M. tuberculosis lineages differently modulate the innate immune response and autophagy in human macrophages E. Petruccioli3, A. Romagnoli3, I. Palucci2, E. Carata1, L. Petrone3, S. Camassa2, L. Dini1, E. Girardi3, G. Delogu2, D. Goletti3, G. Fimia3-1 1 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, University of Salento, Lecce 2Department of Microbiology, Catholic University 3Epidemiology and preclinical research, Inmi l. Spallanzani irccs, Rome, Italy Introduction: Recent studies highlighted the impact of M. tuberculosis (Mtb) genetic diversity on relevant pathogenetic properties, with Mtb-modern lineages associating with reduced immune responses that consequently led to higher Mtb intracellular replication and, as such, potentially linked to rapid progression from infection to disease. Moreover, laboratory strain H37Rv reduces macrophage autophagy flux, which can be fully restored by the host specific immune response, leading to a reduction of the Mtb intracellular survival. Aim: To in vitro evaluate the impact of the Mtb genetic diversity on the immune response, Mtb replication and modulation of the autophagic flux in human primary macrophages. Methods: Different Mtb clinical isolates belonging to strains from different lineages (1-5), in addition to H37Rv, were used to infect human macrophages. We overtime evaluated Mtb replication (CFU); cytokines (cytometric bead array) in cell supernatants; autophagy flux (immunoblot). Results: Mtb strains belonging to modern lineage 4 show high rate of replication, associated to a significant production of inflammatory cytokines (IL1, IL6, TNFa), but not of IL-8. Moreover, high autophagy flux was observed in macrophages infected with lineage 4 Mtb, which is stimulated via an IL1-dependent signaling. Instead, Mtb strains belonging to the ancient lineage 1 and 5, were characterized by a significant lower cytokine production, reduced bacterial replication and decreased autophagy flux. Conclusion: Mtb belonging to modern lineages show an unpredicted high capability to stimulate the innate immune responses and the autophagy flux of in vitro infected macrophages. However, activation of both processes is not effective in limiting the intracellular replication of these MTB strains. Conversely, these patterns may be correlated to their increased pathogenic properties with respect to ancient ones. 280 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 254 Host-responses to mycobacteria /interaction 22/09/2016 Investigating the role of rv2660c and rv2661c during mycobacterial stress repsonses and non-replicating persistence N. Hafneh, G. Mukamolova Dept. Of infection, immunity & inflammation, University of Leicester, Leicester, United Kingdom One key aspect of Mycobacterium tuberculosis (Mtb) pathogenesis is its ability to survive stress conditions encountered during macrophage infection and transit into a state of non-replicating persistence (NRP) which is characterised by low metabolic activity and alteration of gene expression. This forms the basis of global risk of latent tuberculosis and justify efforts to identify key latency-specific antigens as component of vaccines for prevention of TB reactivation. rv2660c encoding a conserved hypothetical protein was shown to be significantly up-regulated in several NRP models and in late stages of both murine and macaque infections. The multistage vaccine H56 which included a recombinant Rv2660c protected cynomolgus macaques against active TB and reactivation of latent TB (1), while Rv2660c peptides induced specific T-cell responses in TB patients (2). Although the existence of Rv2660c has been recently challenged by identification of a small non-coding RNA, ncrv12659 overlapping with a coding sequence of rv2660c (3), the importance of the genomic region annotating rv2660c, rv2661c and ncRv12659 in Mtb pathogenesis and stress response has not been investigated. To address this question, we have generated Mtb H37Rv strains with deleted or over-expressing rv2660c and rv2661c and characterise their biological phenotypes. ∆rv2661c, ∆rv2660c, ∆rv2661c+rv2660c mutants strains showed no growth defect in either 7H9 Middlebrook or Sauton’s media. In addition, these strains survived similarly to the wild type Mtb during starvation and under hypoxic conditions. However, survival of ∆rv2661c+rv2660c mutant was significantly impaired during oxidative stress, while over-expression of rv2661c+rv2660c from a strong hsp60 promoter improved Mtb survival in the same condition. Our findings suggest that Rv2660c and Rv2661c may facilitate Mtb response to reactive oxygen and nitrogen species. Further experiments will elucidate the role of Rv2660c and Rv2661 in Mtb infection and persistence in vivo. References: 1. Lin et al, J Clin Invest. 2012,122: 303-14. 2. Govender et al, Vaccine. 2010;29: 51-7 3. Houghton et al, PLoS One. 2013, 8(12): e80047. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 281 255 Host-responses to mycobacteria /interaction 22/09/2016 Mycobacterium tuberculosis peptidyl-prolyl isomerases modulates host immune response and aids in intraphagosomal survival D. Tripathi1, S. Pandey2-3, M. Khubaib3-2, N.Z. Ehtesham2, S.E. Hasnain1 1 Kusuma School of Biological Sciences, Indian Institute of Technology 2Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, Delhi 3School of Life Sciences, University of Hyderabad, Hyderabad, India Tuberculosis caused by the intracellular pathogen Mycobacterium tuberculosis (M.tb), remains a potential threat regardless of strong efforts to alleviate its toll on humanity. Intracellular/intraphagosomal survival plays a critical role in the infection cycle of the pathogen, a process which majorly relies on array of virulence factors to colonize and replicate within the host macrophages. Studies involving immunological characterization of these effector molecules can bridge huge gaps in our understanding of M.tb biology and facilitate better therapeutic and diagnostic interventions. M.tb is known to possess two Ppiases (cyclophilins), PpiA and PpiB. M.tb PpiA is a part of the secretome and is known to interact with host proteins involved in immune defense mechanism and signal transduction, while PpiB has been reported in membrane fraction and mannosylation enriched culture filtrate. In the present study, we describe the potential role of Ppiases in intraphagosomal survival of the pathogen. We present evidence that M.tb Ppiases play role in modulating host immune responses. ELISA results revealed presence of antibodies to M.tb Ppiases in the patient sera as compared to the sera of healthy individuals. Treatment of THP-1 cells with increasing dose of rPpiA induced secretion of pro-inflammatory cytokines. Alternatively, treatment with rPpiB inhibited secretion of TNFα and induced secretion of IL-10. Furthermore, heterologous expression of M.tb PpiA and PpiB in Mycobacterium smegmatis increased its survival in THP-1 cells as compared to the vector control. Our results demonstrate that M.tb Ppiases are immunogenic proteins that can possibly modulate host immune response and enhance persistence of the pathogen within the host by subverting host cell generated stresses. 282 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 256 Host-responses to mycobacteria /interaction 22/09/2016 Pulmonary Microbiota: Impact on Lung Morphology, Respiratory Diseases and Immune Responses M. Hauptmann, Y. Yun, U. Schaible Cellular Microbiology, Research Center Borstel, Borstel, Germany With the advent of high throughput sequencing tools, it became evident that the healthy lung is not sterile as considered so far, but contains a diverse repertoire of aerobic and anaerobic bacterial species. In contrast, standard microbiological techniques only encompassed a small percentage of total microbiota in healthy lungs. These lung microbiota isolates however can now be used for murine lung colonization experiments. Colonizing germ-free mice with Lactobacillus spec. isolated from lungs of wild mice, had profound influence on lung morphology. We observed higher numbers of alveoli and increased mucus production. As research on the impact of pulmonary microbiota is still in its beginning, it is still an open question how pulmonary microbiota influence immune responses and the outcome of respiratory diseases. To address these questions, we have established an in vivo model that allows manipulations of pulmonary microbiota in adolescent mice and subsequent infection with Mycobacterium tuberculosis (M. tb). Our results revealed that the course of M. tb infection was similar between specific pathogen free (SPF), antibiotic-treated, and antibiotic-treated mice that were recolonized with bacterial isolates from wild mice lungs. However, a tendency towards higher percentage of IFN-γ+ CD4+ T cells was observed in recolonized mice. At present, we do not know whether this response was due to non-physiologically high burden of commensal bacteria during M. tb infection, or whether immune responses in the lung can be trained by microbial exposure, as has been suggested from models of asthma and allergies. Therefore, we currently investigate the effect of microbiota-alterations during early life on M. tb infection by the use of germ-free mice. The emerging field of lung microbiota research will provide new insights on how environmental factors shape the disposition for respiratory disorders like infections, allergies, COPD and acute respiratory distress syndrome. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 283 257 Host-responses to mycobacteria /interaction 22/09/2016 Characterization of IgG responses and isolation of B cells from healthy individuals and TB patients S. Hwa2-1, D. Mahamed2, A. Sigal2-1 1 Systems Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany 2Sigal Lab, K-RITH, Durban, South Africa Isolation of human monoclonal antibodies is a powerful tool for understanding the humoral response against pathogens. In both humans and nonhuman primates with active TB, anti-Mycobacterium tuberculosis (Mtb) serum titers increase and the reactivity toward specific antigens shifts. Transcriptomic data also suggest a B cell role in health versus TB disease states. We hypothesize that immunoglobulins do play a role in protection against TB disease and aim to recover antibodies with functional activity. We developed an automated image analysis method to screen immunoglobulin samples for effects on phagocytosis and growth of Mtb in primary human monocyte-derived macrophages. Polyclonal serum IgG from healthy South African volunteers restricted the growth of Mtb in macrophages compared to IgG derived from TB patients or US-origin normal serum. We also showed proof of concept that B cells can be sorted by binding fluorescently labelled whole bacteria and/or purified antigens, and constructed a feeder cell line which supports proliferation and IgG secretion from primary B cells without further cytokine supplementation. Single cells sorted into microtiter plates containing feeder cells yielded secreted IgG up to several hundred ng/mL, as well as cDNA sequences of heavy and light chain mRNA. Monoclonal antibodies recovered from the single-cell cultures will be screened for effects on phagocytosis as above. We are currently enrolling volunteers undergoing lung resection for TB or non-TB lung disease controls which will allow us to characterize the antigen specificities and functional activities of antibodies from B cells in the draining lymph nodes. 284 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 258 Host-responses to mycobacteria /interaction 22/09/2016 Interferon gamma production upon mycobacterial stimulations is controlled by major loci on chromosome 8q and 3q in various populations exposed to tuberculosis F. Jabot-Hanin3-6, A. Cobat3-6, J. Feinberg3-6, J. Bustamante3-6, S. Boisson-Dupuis9-3-6, J.L. Casanova8-9-3-4-6, E.G. Hoal7, E. Schurr1-2, A. Alcaîs9-3-6, C. Delacourt5, L. Abel9-3-6 1 McGill International TB Centre 2Department of Human Genetics and Department of Medicine, McGill University, Montreal, Canada 3Laboratory of Human Genetics of Infectious Diseases, Necker Branch,U1163, INSERM 4Pediatric Hematology-Immunology Unit 5Pediatric Pneumology Unit, Necker Hospital for Sick Children, AP-HP 6Imagine Institute, Paris Descartes University, Sorbonne Paris Cité, Paris, France 7Molecular Biology and Human Genetics, MRC Centre for Molecular and Cellular Biology, DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, Stellenbosch University, Tygerberg, South Africa 8Howard Hughes Medical Institute 9St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, Ny, United States More recent and more specific than Tuberculin Skin Test (TST), Interferon Gamma (IFN-γ) release assays (IGRAs) provide an in vitro measurement of antimycobacterial immunity that is widely used as a test for tuberculosis (TB) infection. IGRA outcomes are highly heritable in various populations, but the nature of the involved genetic factors remains unknown. We conducted a genome-wide linkage analysis of IGRA phenotypes in 143 families from a TB household contact study in France with the new Maximal Likelihood Binomial method which considers the sibship as a whole and makes no assumptions about the distribution of the phenotype. A replication study in 135 families from South Africa was also conducted to confirm the loci identified. We identified a major locus on chromosome 8q controlling IFN-γ production in response to stimulation with live BCG (LOD score=3.81, p=1.40x10-5). We also detected a second locus on chromosome 3q that controlled IFN-γ levels in response to stimulation with ESAT-6 antigen when accounting for the IFN-γ production shared with that induced by BCG (LOD score=3.72, p=1.8x10-5). Both loci were replicated in South African families, where TB is hyperendemic, and do not overlap with the TST1 and the TST2 loci controlling TST positivity per se and the intensity of TST reactivity, respectively. Refined identification of the new identified loci is ongoing by ultra-fine association studies of each of the two phenotypes within the corresponding linked regions, with interesting preliminary results. The identification of these two new linkage signals in populations of various ethnic origins living in different M. tuberculosis exposure settings provides new clues to the genetic control of human antimycobacterial immunity. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 285 259 Host-responses to mycobacteria /interaction 22/09/2016 Cord-forming Mycobacterium tuberculosis causes the formation of macrophage extracellular traps S. Kalsum, C. Braian, M. Lindroth, M. Lerm Dept. of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden In 1882, Robert Koch observed microscopic cords as one of the first phenotypic characteristics in Mycobacterium tuberculosis, the causative agent of tuberculosis. When grown in a liquid medium without detergent, they form pellicle-like structures, or cords, in which the orientation of the long axis of each cell is parallel to the long axis of the cord. The formation of cords has been shown to correlate with increased virulence. Like neutrophils, the formation of extracellular traps has been recently recognized in macrophages and referred as macrophage extracellular traps (METs). METs, like neutrophil extracellular traps, are composed of DNA and histones. We found that cord-forming mycobacteria (H37Rv) can induce MET formation in human monocytes-derived macrophages (hMDMs). Formation of METs in hMDMs is independent of ROS production but dependent on M. tuberculosis virulent factor ESAT-6. Now, we want to evaluate the response of cord-forming mycobacteria in a more complex system such as our recently developed human lung tissue model. Knowledge on how different phenotypes of M. tuberculosis interacts with human cells and tissues is fundamental for the understanding of the devastating disease that this pathogen is causing. 286 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 260 Host-responses to mycobacteria /interaction 22/09/2016 Mycobacterium smegmatis Δespg3 mutant a potential live bacterial vaccine vector for antigen delivery N. Kannan, M. Haug, T. Flo, M. Steigedal CEMIR,, NTNU, Trondheim, Norway Background and Objective: Mycobacteria possess a family of protein secretion systems, ESX-1 to ESX-5, which has been demonstrated to aid in altering host pathways and promote bacterial survival. One of them, ESX-3, is ubiquitously present in all mycobacterial species and is regulated by iron and zinc; also it has been shown that ESX-3 is involved in siderophore mycobactin based iron uptake. Recently, a mutant strain of the non-pathogenic M. smegmatis lacking ESX-3, complemented with Mycobacterium tuberculosis esx-3 has shown promise as a vaccine candidate. We wanted to investigate how the vaccine effect was created so efficiently. Methods: To study the host response to mutant M.smegmatis, we investigated the survival of the mutant within in macrophages and dendritic cells. In addition we measured host response as ability to produce various cytokines and other immune modulators. Finally, we also investigated the antigen presenting ability of the M. smegmatis strains using a model ovalbumin system. Results and conclusion: Preliminary data indicate that mutant M. smegmatis has reduced survival when compared to the wild type when added to macrophages and dendritic cells. As a consequence of this we observe a significant change in expression profile of a panel of immune modulators. Further, we observed improved antigen presentation upon exposure to the mutant vector when compared to the wild type. Currently, we are conducting mouse experiments, to check if the mutant strain may provide protective immunity when compared to BCG. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 287 261 Host-responses to mycobacteria /interaction 22/09/2016 Rv0753c, a novel dendritic cell-activating antigen of Mycobacterium tuberculosis, drives Th1-type T-cell immunity J.S. Kim, W.S. Kim, H.H. Choi, H.M. Kim, K.W. Kwon, S.B. Cha, S.N. Cho, S.J. Shin Department of Microbiology and Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is an outstanding pathogen that modulates the host immune response. This inconvenient truth drives the continual identification of antigens that generate protective immunity, including Th1-type T cell immunity. Here, the contribution of methylmalonate semialdehyde dehydrogenase (MmsA, Rv0753c) of Mtb to immune responses was examined in the context of dendritic cell (DC) activation and T cell immunity both in vitro and in vivo. The results showed that MmsA induced DC activation by activating the MAPK and NF-κB signaling pathways. Additionally, MmsA-treated DCs activated naïve T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and IL-2, and induced T cell proliferation. These results indicate that MmsA is a novel DC maturation-inducing antigen that drives the Th1 immune response. Thus, MmsA was found to potentially regulate immune responses via DC activation toward Th1-type T cell immunity, enhancing our understanding of Mtb pathogenesis. 288 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 262 Host-responses to mycobacteria /interaction 22/09/2016 Epigenetic reprogramming of peripheral blood mononuclear cells in BCG-vaccinated individuals M. Lerm Dept of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden Bacille Calmette-Guerin (BCG) was developed in the 1920s as a vaccine against tuberculosis (TB) and albeit having variable efficacy against TB, it is one of the world’s most widely used vaccines. The mechanisms responsible for the protective effects of BCG are not well understood. Here, we isolated peripheral blood mononuclear cells (PBMCs) from BCG-vaccinated subjects and performed global DNA methylation analysis in combination with functional assays to address epigenetic changes and functional responses induced through BCG vaccination. Enhanced containment of Mycobacterium tuberculosis replication could be observed in monocyte-derived macrophages from a sub-group of BCG-vaccinated individuals (classified as responders). A stable and robust differential DNA methylation pattern in response to BCG could be observed in PBMCs isolated from the responders but not from the non-responders. Gene ontology analysis revealed that promoters with altered DNA methylation pattern were strongly enriched among genes belonging to immune pathways in responders but no enrichment could be observed in the non-responders. Our findings suggest that BCG-induced epigenetic reprogramming of immune cell function can enhance antimycobacterial immunity. Understanding why BCG induces this response in responders but not in non-responders could provide clues to improvement of TB vaccine efficacy. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 289 263 Host-responses to mycobacteria /interaction 22/09/2016 B-lymphocytes forming follicle-like structures in the lung tissue of TB-infected mice: phenotypic and functional features I. Linge, E. Kondratieva, V. Avdienko, A. Dyatloff, E. Petrova, A. Apt, T. Kondratieva Laboratory for Immunogenetics, Central Institute for Tuberculosis, Moscow, Russia In the course of tuberculosis (TB) infection, B cells form follicle-like aggregates in close vicinity of TB lung granuloma. Such aggregates are known in TB patients, as well as in mycobacteria-infected mice and monkeys; however, their role in TB immunity yet remains obscure. To shed more light on this problem, we assessed the surface phenotype and function of lung B cells during TB in genetically TB-susceptible I/St mice in considerable detail. Our results demonstrate that the majority of lung B cells closely resemble classical B2 (surface phenotype CD19+IgMloIgDhiCD21/35intCD5CD11b-CD43-) but differ from the latter by the absence of B2-cell marker CD23. Although it is speculated that the CD23 expression may drop after B-cell activation, in our system the overall proportion of activated B cells, as measured by CD80 and CD69 expression and the numbers of the CD138+ plasma cells, was less than 10 percent, suggesting that CD23 expression does not reflect the activation status. Thus, we expected that lung B-cells’ functions should not differ much from B cells populating lymphoid organs. Indeed, lung B-cells constitutively expressed MHC II molecules, with a slight elevated after infection. Accordingly, lung B-cells presented mycobacterial antigens to immune CD4+ T-cells as effectively as their splenic counterparts. We also assessed the secretion level of key pro- and anti-inflammatory cytokines by lung B-cells and observed production of pro-inflammatory IL-6 (very high) and IL-11 (moderate), whereas marginal to no production of classical type 1 (TNF-α, IFN-γ), or anti-inflammatory IL-10 and TGF-β. Assessment of antibodies evidenced that a very large proportion of the total antibody response in tuberculous lung shows no specificity to mycobacteria. A major proportion of immunoglobulins produced by lung B cells from infected mice did not react with mycobacterial antigens, despite a high reactivity of sera from the same mice. A panel of monoclonal antibodies obtained from lung B cells contained only few clones demonstrating anti-mycobacterial specificity or reactivity with the infected lung tissue extract, whereas the majority of clones’ specificity remained unidentified. Taken together, our results suggest that B cell response against TB infection in the lung has a clear pathological and a doubtful protective role. This work was financially supported by the Russian Scientific Foundation (grant 15-15- 30020). 290 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 264 Host-responses to mycobacteria /interaction 22/09/2016 Human and bovine tubercle bacilli elicit distinct innate responses during early stage infection of bovine alveolar macrophages K.M. Malone6, K. Rue-Albrecht1, D. Magee1, K. Conlon6, N. Nalpas1, J.A. Browne1, A. Smyth6, E. Gormley5, D.E. Mc-Hugh1-2, S. Gordon6-4-3-2 1 Animal Genomics Laboratory, School of Agriculture and Food Science 2Conway Institute of Biomolecular and Biomedical Research 3School of Biomolecular and Biomedical Science 4School of Medicine 5Tuberculosis Diagnostics and Immunology Research Centre, School of Veterinary Medicine 6Tuberculosis Molecular Microbiology group, School of Veterinary Medicine, University College Dublin, Dublin, Ireland Members of the Mycobacterium tuberculosis complex (MTBC) exhibit a high degree of genetic identity and yet show distinct host preference; for example Mycobacterium bovis is highly virulent for cattle while the human pathogen M. tuberculosis is attenuated in the bovine host. Differential immunopathology exhibited by MTBC members may have a basis in host innate immune mechanisms; hence the initial interaction between pathogen and host may be key for the host preference observed within the MTBC. As a route towards defining the differential innate immune response to host-adapted tubercle bacilli, we have performed the first in-depth comparative transcriptomic analysis of the bovine alveolar macrophage during infection with M. bovis AF2122/97 and M. tuberculosis H37Rv. In addition, both the transcriptomic and proteomic profiles of M. bovis AF2122/97 and M. tuberculosis H37Rv were evaluated during in vitro growth to identify differential expressed effectors between the pathogens that could drive divergent interactions with the host cell. The bovine alveolar macrophage response to infection with M. bovis and M. tuberculosis was found to be highly similar over the first 24 hours of infection, with substantial differentially expressed genes between the M. bovis- and M. tuberculosis-infected macrophages only observed 48 hours post infection. Differences observed in cell death and autophagy responses, eicosanoid metabolism and type I interferon signalling suggest a distinct engagement by M. bovis with the bovine innate immune system, and suggest increased cytosolic access during the first 48 hours compared to M. tuberculosis. In addition, profiling of both pathogens revealed increased expression of the ESX-1 secretion system in M. bovis in contrast to M. tuberculosis; this latter observation presents a potential mechanism to explain the increased cytosolic access of the bovine bacillus during the initial stages of macrophage infection. The work presented here therefore provides insight into factors that may underlie host preference between the bovine and human tubercle bacilli during the early stages of infection. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 291 265 Host-responses to mycobacteria /interaction 22/09/2016 Characterization of the repertoire of lipid-derived epitopes presented by CD1 proteins during mycobacterial infection J. Mazurek2-3, C. Carrat2, G. Gaibelet1, E. Fabre2, E. Layre2, M. Gilleron2 1 Institut de Génomique Fonctionnelle, Montpellier 2Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, Toulouse, France 3Karolinska Institutet, University Hospital Huddinge, Stockholm, Sweden Lipid-containing antigens are presented to T cells in the complex with CD1 molecules. Several glycolipids derived from mycobacterial cell wall have been found among CD1 ligands. Moreover, CD1-restricted T cells recognizing mycobacterial lipid antigens have been isolated from the blood of Mycobacterium tuberculosis (Mtb)-infected patients. Such cells secrete the Th1 cytokines and are cytotoxic, which indicates that the lipid antigen-mounted immune response plays a role in controlling mycobacterial infections. Particularly, Mtb-derived mycolic acid, glycerol and glucose monomycolates, phosphatidyl-myoinositol mannosides (PIM) and diacylated sulfoglycolipids (Ac2SGL) have been found to be associated with CD1b molecules. Whilst some lipid antigens, e.g. Ac2SGL, are presented to T cells without being processed, others, like PIM6, require prior processing to activate T cells. Up to now, identification of a lipid antigen depended on the availability of the specific T cell clone(s) recognizing the lipid epitope which severely limits the identification of the new CD ligands. In this project we aim at characterizing the entire repertoire of Mtb-derived lipid antigens bound by CD1b. We employ a novel strategy to define such antigens that is non-biased by the availability of T cell clones. Furthermore, our methodology allows for isolation of CD1b-bound lipids without need for using detergents, which severely impact the readout by dissociating lipids from the CD1b binding groove. We constructed antigen presenting cells (APCs) expressing protease-cleavable CD1b molecules (pCD1b). Such pCD1b molecules could be proteolitically cleaved and purified on an affinity chromatography column without destabilization of their antigen binding capacity. Next, lipids separated from CD1b proteins by the organic solvent extraction are identified by liquid chromatography followed by mass spectrometry analysis. Using this strategy we will identify the lipid epitopes associated with CD1b molecules upon exposure of the APCs to the Mtb cell wall-derived lipid mixture, and also upon infection with Mtb. Identified lipid epitopes will next be tested for their antigenicity on the blood obtained from the Mtb-infected individuals and the immune response triggered by such epitopes will be characterized. 292 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 266 Host-responses to mycobacteria /interaction 22/09/2016 Establishment of a cell culture method to functionally study the TB-helminth co-infection M. Méndez1, N. Angulo1, R. Gilman3-1, M. Verástegui1, O. Stendahl2, M. Lerm2, R. Blomgran2 1 Laboratorio de Investigación en Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, Lima, Peru 2Department of Clinical and Experimental Medicine, Division of Medical Microbiology, Linköping University, Linköping, Sweden 3Department of International Health, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States Helminth infections are among the most common infections worldwide and are associated with an increased host susceptibility to develop tuberculosis (TB) disease. By inducing a strong Th2 immune response, helminths impair Th1 responses that are necessary for protection against TB. This reduction in Th1 response has been mostly studied at the systemic level by assessing the cytokine profiles and in adaptive immunity by analyzing T-cells. However, M. tuberculosis can replicate within innate immune cells, especially the macrophage, and manipulate the fate of these cells contributing to the severity of the disease. Within this context, functional studies on macrophages from TB-helminth coinfected individuals are lacking. Our aim is to investigate the immunological effect of helminth infection on the macrophages of TB patients. For this, we start by optimizing a cell-culture system in which we can obtain monocyte-derived macrophages from cryopreserved PBMCs and differentiated with autologous serum. In this phase we work with healthy donors to later, once established the method, carry out an observational study on a Peruvian Amazon region that is endemic for both helminthiasis and TB. By using autologous serum to differentiate macrophages ex-vivo, we intend to provide conditions closer to their original environment and determine macrophage polarization, ability to control M. tuberculosis infection and cytokine secretion pattern. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 293 267 Host-responses to mycobacteria /interaction 22/09/2016 Factors Affecting TB transmission from adult to children within households in the Gambia A. Mendy1, L. Tientcheu1, U. Egere1, E. Coker1, A. Ayorinde1, B. Kampmann1-2, D. Leopold1 1 Vaccines and Immunity Theme, TB Immunology Lab, Medical Research Council (MRC) Gambia unit, Banjul, Gambia 2Faculty of Medicine, Imperial College London, London, United Kingdom Background: Childhood tuberculosis (TB) has significant impact on public health worldwide and it is believed that most children acquire TB from an adult smear-positive index case within their household. To further examine this hypothesis and to investigate transmissibility of strains within the household setting, we compared strain-types in adults and their child contacts. We also examined the influence of bacillary burden and strain-type on clinical outcome of contacts. Materials and Methods: Stored isolates from smear positive adult TB cases (n =136) were selected according to clinical outcomes of their household contacts (children ˂ 15 years old). Mycobacteria were isolated from both adult and –where available- children samples via culture, and typed using spoligotyping to enable strain classification. Results: The AFB grade of adult index cases correlated with clinical outcome of the children with microbiologically confirmed TB, clinically diagnosed probable TB, asymptomatic but TST positive and asymptomatic, TST negative children showed 60%, 35%, 34% and 33% highest AFB grade (3+) levels respectively. Strain-type determination by spoligotyping showed that 93% of children had acquired EuroAmerican lineages, while 7% had M. Africanum lineage. Combining results for adult contacts of children with confirmed TB and probable TB, 76% showed Mtb-Euro-American, 17% M. africanum and 7% Mtb-Indo-Oceanic. Contacts of TST positive children showed 59% Mtb-Euro-American, 32% M. africanum, 8% Mtb-Indo-Oceanic and 2% Mtb-Beijing. Those of TST negative children showed 63% Mtb-Euro-American, 26% M. africanum, 9% Mtb-Indo-Oceanic and 2% Mtb-Beijing. Conclusion: The data so far support other published data, which show that a higher bacillary burden in the index case increases the likelihood of TB transmission to child contacts. Adult patients appear to be more likely to transmit TB if they were carrying Euro-American lineages rather than West African strains. 294 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 268 Host-responses to mycobacteria /interaction 22/09/2016 Antigen HLA-DR at patients with different clinical manifestations of tuberculosis T. Morozova, T. Salina Department of Tuberculosis, Saratov State Medical University, Saratov, Russia HLA-DR - antigen MHC (major histocompatibility complex) class II needed to generate an adequate immune response. Aim: Study of expression HLA-DR on immune cells of patients with different clinical manifestations of tuberculosis. Materials: Examined 54 patients newly diagnosed pulmonary tuberculosis (TB) and 22 healthy. Depending on the clinical manifestations of TB patients divided into 2 groups) Groupa 1 included 18 patients with severe and complicated forms of tuberculosis. Groupa 2 included 36 patients with limited and favorable flowing TB. Assessed the relative number of cells HLA-DR, in the peripheral blood using immunophenotyping method with monoclonal antibodies (anti-HLA-DR). Results: All patients with TB prior to antibiotic treatment were found an increase number of cells HLA-DR - (M ± δ) (medium-arithmetic and standard deviation) - 16 ± 41,8% Me (median) - 40.6 Mo (moda) - 17.4 interval 17,4-75) compared with healthy (M - 32,5,0 ± 12%, Me - 33.3 Mo - 33 3, the range 14,4-50,3), p = 0.0017. The expression level of marker HLA-DR significantly higher in Group 1 (M - 17 ± 49,3% Me - 45 Mo - 65,4, 24,4-75 interval) compared with Group 2 (M - 38,2 ± 15%, Me - 36.4 Mo - 17.4, range 17,4-64,2, p1-2 = 0,0122) and healthy (M - 32,5 ± 12%, Me - 33.3 Mo - 33.3, interval - 14,4-50,3, p1-3 = 0,0086, p2-3 = 0,0495). All patients at baseline and after 2 months of TB treatment found decrease in the number of cells of HLA-ДР after treatment of M - 41,8 ± 16% vs 30,4 ± 9%,respectively, p = 0.0009. Conclusions: 1. In patients TB in acute phase of disease is observed elevated levels of cells HLA-DR in blood compared with healthy. 2. The level of expression HLA-DR depends on the severity TB and is most pronounced in patients with severe forms of disease. 3. In course of antibacterial therapy HLA-DR cells is reduced, which may indicate the relationship of its expression with the level of the bacterial population M. tuberculosis. These results open prospects for a targeted use of immune preparations Tuberculosis 2016 – September 19-23, 2016 – Paris, France 295 269 Host-responses to mycobacteria /interaction 22/09/2016 Human plasma lipidome variations during Mycobacterium tuberculosis infection H. Neo3-2-1, S. Ji3, F. Torta3, A. Bendt3, C. Chee4, Y. Wang4, A. Singhal5, G. De Libero6, M. Wenk3 1 Agilent Technologies Singapore Pte Ltd 2Department of Biological Sciences, Faculty of Science 3Singapore Lipidomics Incubator (SLING), National University of Singapore 4Tuberculosis Unit, Tan Tock Seng Hospital, Singapore 5Singapore Immunology Network (SigN), A*STAR, Singpaore, Singapore 6Department of Biomedicine, University Hospital Basel, Basel, Switzerland Eradication of tuberculosis (TB) remains challenging with lengthy treatment durations and the existence of drug resistant Mycobacterium tuberculosis (Mtb). As human pathogens continue to infect and acquire resistance to current anti-microbial drugs, recent focus has turned to potential host-directed therapeutics in search for novel treatment strategies. Pathogens such as Mtb make use of host lipids as building blocks and influence the host cell physiology to enable their survival and replication. Hence, detection of changes in the host lipidome during TB progression may enable novel and precise diagnostic tools as well as potentially providing insights into host-pathogen interactions. To test this hypothesis, we aimed to identify changes in the human plasma lipidome of active TB patients (n=88) in comparison with the lipidome of healthy controls (n=62) and latent individuals (n=36). After lipid extraction, analyses were performed by liquid chromatography mass spectrometry (LC/MS) using targeted analysis for the major classes of lipids. The lipid classes that showed the most significant differences (p < 0.05) were molecular species of ceramides, sphingomyelins, plasmalogen and ether phosphatidylethanolamines. These lipids were significantly lower in the plasma of active TB patients. Validation of these potential disease biomarkers in longitudinal studies with human plasma samples from active TB patients (n=19) who were on established TB drug treatment over a period of six months was carried out and will be discussed. 296 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 270 Host-responses to mycobacteria /interaction 22/09/2016 An interferon-γ-independent antimycobacterial mechanism M. Resende1-2-4, M. Cardoso4, M. Borges3, A. Gil Castro1-2, R. Appelberg4 1 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga 2ICVS/3B’s – PT Government Associate Laboratory, Braga/guimarães 3Faculty of Pharmacy, University of Porto 4Institute for Molecular and Cell Biology (IBMC)/ I3S – Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal Granuloma formation is a hallmark of several infectious diseases, including those caused by Mycobacterium sp. These structures are composed of accumulations of inflammatory cells and it has been shown that cytokines such as IFNγ and TNFα are required for granuloma assembly during M. avium infections in mice. MIIG mice have macrophages, monocytes and dendritic cells that are unresponsive to IFNγ. We observed that although IFNγ-ko mice present an exacerbated infection, the same is not true for MIIG animals, where the same levels of protection as the wild type (WT) animals were observed in the liver and partial protection in the spleen. Moreover MIIG mice still develop well defined granulomas (as opposed to IFNγ-ko mice), suggesting that IFNγ-mediated macrophage activation is not required for granuloma assembly. This work also shows that MIIG animals exhibit increased iron accumulations within the granulomas and increased cell recruitment with higher CD4+ T cells numbers as well as increased IFNγ and TNFα expression, suggesting that TNFα may have a role on protection and may compensate the lack of macrophage response to IFNγ in the MIIG model. Indeed blocking TNFα with monoclonal antibodies in M. avium infected MIIG animals induced an increased bacterial burden accompanied by a reduction of iron accumulation in the granulomas. How the axis TNFα expression/iron accumulation overcomes the lack of IFNγ signalization controlling the bacteria proliferation is being studied. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 297 271 Host-responses to mycobacteria /interaction 22/09/2016 Selective reduction of IFN-γ single positive mycobacteria-specific CD4+ T cells in HIV-1 infected individuals with latent tuberculosis infection C. Riou2, R. Bunjun2, T.L. Muller2, A. Kiravu2, Z. Ginbot2, T. Oni3-1-4, R. Goliath3-1, R. Wilkinson3-1-6-5, W. Burgers3-2 1 Clinical Infectious Diseases Research Initiative 2Department of Pathology, Division of Medical virology 3Institute of Infectious Disease and Molecular Medicine 4School of Public Health and Family Medicine, Division of Public Health Medicine, University of Cape Town, Cape Town, South Africa 5Department of Medicine, Imperial College London 6Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom While in HIV-uninfected persons the risk of progression from latent to active tuberculosis (TB) is 210% in a lifetime, it increases to a 5-10% annual risk in HIV-infected individuals. Moreover, HIV-1 is recognized to increase the risk for TB even before CD4+ T cell deficiency is profound. In order to understand the mechanisms involved in the maintenance and/or impairment of TB latency, it is of interest to define in detail the extent to which HIV affects TB immune responses. Using flow cytometry, we compared the frequency, polyfunctional (IFN-γ, TNF-α and IL-2) and memory profile (CD45RO, CD27) of ESAT-6/CFP-10-, PPD- and BCG-responding CD4+ T cells in Mycobacterium tuberculosis-sensitized HIV-uninfected individuals (n=25) and HIV-infected subjects (n=24) with well-preserved CD4 counts (median: 625 cells/mm3). Our data revealed a significant and selective reduction of IFN-γ single positive CD4+ T cells, for all mycobacterial antigens tested in HIV-infected participants, compared to HIV-uninfected individuals (median fold reduction of 3.2, 10 and 5.6 for ESAT-6/CFP-10, PPD and BCG, respectively). The proportion of IFN-γ single positive cells correlated inversely with viral replication (p=0.007, r=-0.66 for BCG responses and p=0.03, r=-0.4 for PPD and ESAT-6/CFP-10 responses). Interestingly, no changes were observed in the magnitude of polyfunctional cells (producing IFN-γ+IL-2+TNF-α+) between the two groups. Moreover, IFN-γ single positive cells were significantly enriched in a late differentiated phenotype (CD45RO+CD27-, median: 22%, IQR: 17-39), compared to polyfunctional cells (median: 12%, IQR: 12-18). As differentiated CD4+ T cells are more susceptible to HIV infection; this could, in part, contribute to the preferential depletion of this subset. Overall, we found that HIV-1 infection impairs TB immunity not only by numerically reducing mycobacteria-specific CD4+ T cells, but also alters the balance of their functional profile by selectively depleting IFN-γ single positive cells. Our results provide new insights into potential mechanisms that perturb immune control of latent TB infection during HIV infection. 298 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 272 Host-responses to mycobacteria /interaction 22/09/2016 Antimycobacterial activity in whole blood from type I diabetes patients S. Raposo-García3, S. García-García5, J. Guerra-Laso3, J. Juan-García5, C. Diez-Tascón2, T. Nebreda-Mayoral4, E. López-Fidalgo1, R. López-Medrano6, O. Rivero-Lezcano1-7 1 Unidad de Investigación, Leon 2Anatomía Patológica 3Medicina Interna 4Microbiología 5Neumología, Complejo Asistencial Universitario de León, León 6Microbiología, Hospital El Bierzo, Ponferrada 7Instituto de Estudios de Ciencias de la Salud de Castilla y León, Soria, Spain In contrast with isolated phagocytes (usually monocytes and neutrophils), which are difficult to activate against pathogenic mycobacteria, whole blood has shown to exhibit a strong mycobactericidal activity. Diabetic patients are recognized in epidemiological studies as a population susceptible to tuberculosis. We have used the whole blood model for in vitro infection to analyze the susceptibility to tuberculosis in type I diabetes patients. We have applied a factorial design to analyze the effect of four cytokines (IFNγ, TNFα, GM-CSF and IL-15) and their interactions on the mycobactericidal activity of whole blood. Samples were obtained from 18 volunteers: 9 type I diabetes patients and 9 non-diabetic controls, matched by age and gender, and 18 in vitro infections with different cytokine combinations were performed with each sample. Upon infection with Mycobacterium tuberculosis and incubation for two days in a rotary shaker, antimicrobial activity was estimated by colony counting. Additionally, we studied the phagocytic activity of neutrophils by flow cytometry, using a Mycobacterium bovis BCG transformed with the gene of the fluorescent protein DsRed. Mycobactericidal activity was significantly lower in blood from diabetic patients (P*=0.005). When patients and controls were analyzed independently, it was observed that IL-15 stimulated the antimicrobial activity in patients, but not in controls. In addition, the phagocytic activity of neutrophils from diabetic patients was significantly lower (P*=0.006). Monocytes from patients showed also a lower phagocytic activity, although it did not reach statistical significance (P*=0.066). These results prompt further research to assess whether the whole blood model may be suitable for testing the tuberculosis susceptibility of different groups as exemplified by type I diabetic patients that present a lower capacity to kill M. tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 299 273 Host-responses to mycobacteria /interaction 22/09/2016 The impact of influenza coinfection on the control and progression of tuberculosis S. Ring1, L. Eggers1, G. Gabriel2, B. Schneider1 1 Coinfection Unit, Priority Area Infections, Research Center Borstel, Borstel 2Viral Zoonosis and Adaptation, Heinrich Pette Institute, Hamburg, Germany Mycobacterium tuberculosis (Mtb) can establish long-term infection in the host even in the presence of an otherwise intact immune system. The problem with persistent infections like tuberculosis (Tb) is that they usually have to be controlled in the host for decades. Coinfections acquired throughout life may impact on the balance of the immune response and skew it into one direction that is disadvantageous for the host. Influenza virus establishes pulmonary infection and is known to impair host responses to secondary bacterial infections. Epidemiological data raise concerns about a possible impact of influenza on patients with Tb. In an experimental coinfection model we study the impact of an early influenza A virus (IAV) coinfection on mycobacterial control and the initiation of the Mtb-specific adaptive immune response. We found that control of Mtb replication is impaired shortly after IAV challenge whereas viral clearance is not hampered. Importantly, the long-term control of Mtb after IAV coinfection differs between males and females, indicating a role for sexrelated factors. While female mice regain control over Mtb infection after 7 weeks, males are unable to restrict mycobacterial replication and show severely increased lung pathology after IAV coinfection. Our goal is to elucidate the influenza-induced changes in the lung and to identify sexdependent factors that render males more susceptible to coinfection than females. 300 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 274 Host-responses to mycobacteria /interaction 22/09/2016 Mycobacterium tuberculosis PE27 activates dendritic cells and contributes to Th1-polarized memory immune responses during in vivo infection W.S. Kim, J.S. Kim, H. Kim, K.W. Kwon, S.J. Shin Department of Microbiology, Yonsei University College of Medicine, Seoul, South Korea A gradual understanding of the proline-glutamate (PE) and proline-proline-glutamate (PPE) families, which compromise 10% of the coding regions in the Mycobacterium tuberculosis (Mtb) genome, has uncovered unique roles in host–pathogen interactions. However, the immunological function of PE27 (Rv2769c), the largest PE member, remains unclear. Here, we explored the functional roles and related signaling mechanisms of PE27 in the interaction with dendritic cells (DCs) to shape the T cell response. PE27 phenotypically and functionally induces DC maturation by up-regulating CD80, CD86, MHC class I and MHC class II expression on the DC surface to promote the production of TNF-a, IL-1B, IL-6, and IL-12p70 but not IL-10. Additionally, we found that PE27mediated DC activation requires the participation of mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) signaling pathways. Interestingly, PE27-treated DCs directed naïve CD4+ T cells to secrete IFN-g and activate T-bet but not GATA-3. These DC also induced PE27-specific IFN-g-producing memory T cells in Mtb-infected mice indicating that PE27 contributes to Th1polarization. Taken together, these findings suggest that PE27 possesses Th1-polarizing potential through DC maturation and could be useful in the design of TB vaccines. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 301 275 Host-responses to mycobacteria /interaction 22/09/2016 Blockade of the Kv1.3 K+ channel enhances BCG vaccine efficacy by expanding central memory T lymphocytes D.K. Singh1-2, V.P. Dwivedi2, A. Ranganathan2-1, G. Das2 1 Recombinant Gene Products Group, ICGEB 2Center for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India Tuberculosis (TB) is the oldest known infectious disease, yet there is no effective vaccine against adult pulmonary TB. Emerging evidence indicates that T helper (Th)1 and Th17 cells play important roles in host protection against TB. However, TB vaccine efficacy in mice is critically dependent on the balance between antigen-specific central memory T (Tcm) to effector memory T (Tem) cells. Vaccine efficacy is determined on the generation of Tcm cells, which are a continuous source of Tem cells. BCG induces strong Th1 effector cell responses and these cells function effectively in neonates. However, over time these cells become gradually exhausted by recurrent exposure to the pathogen, rendering the host again susceptible to infection. Therefore, to provide the host with a continuous supply of Tem cells, a large pool of Tcm cells is needed. The generation of Tem cells increases with the severity of bacterial pathogenesis and these cells rapidly produce copious amount of IFN-γ upon antigenic challenge. Tem cells, however, are terminally differentiated effector cells with low or no proliferative capacity. Therefore, maintenance of long-term protective memory responses is thought to rely on Tcm cells with high proliferative capacity. Thus, increasing the pool of Tcm (CD4+CD62LhiCD44hi) cells by concomitant regulation of Tem (CD4+CD62LloCD44hi) cells during vaccination may be an effective strategy to develop a vaccine that induces long-lasting and robust recall responses. In our study we found that pharmacological inhibition of Kv1.3, a potassium channel preferentially expressed by Tem cells, with clofazimine selectively expands Tcm cells during vaccination with Bacillus Calmette Guerin (BCG) which, therefore, have the potential to continuously replace effector T cells at the site of infection. Specifically, a high Tcm to Tem cell ratio is essential for optimal vaccine efficacy. Further, animals that received clofazimine following BCG vaccination exhibited significantly enhanced resistance against TB as well as robust Th1 and Th17 responses. This superior activity could be transferred to naïve syngeneic animals by CD4+ T cells. Thus, Kv1.3 blockade via inclusion of clofazimine during BCG vaccination is a promising approach for enhancing the efficacy of the BCG vaccine in humans. 302 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 276 Host-responses to mycobacteria /interaction 22/09/2016 Host and Mycobacterium tuberculosis determinants of immune cells activation J. Sousa7-8, H. Novais Bastos1-2-5, N. S. Osório1-2, M. Isabel Veiga1-2, H. Machado1-2, F. Cardoso1-2, J. Gaifem1-2, A. Ramos4-6, T. Carvalho4-6, J. Vieira7-8, C. Vieira7-8, F. Rodrigues1-2, J. Tiago Guimarães4-3-6, A. Gil Castro1-2, M. Saraiva7-8 1 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga 2ICVS/3B’s-PT Government Associate Laboratory, Braga/guimarães 3Department of Biochemistry, Faculty of Medicine, University of Porto 4Department of Clinical Pathology, São João Hospital Center, Porto 5Department of Pneumology, São João Hospital Center, Porto 6EPIUnit Institute of Public Health, University of Porto 7i3S - Instituto de Investigação e Inovação em Saúde 8IBMC, University of Porto, Porto, Portugal A striking feature of tuberculosis (TB) is the variability of disease outcomes, ranging from pathogen clearance or latency establishment to active disease. Immune imbalances clearly contribute to this heterogeneity, assuming a rising importance as therapeutic targets. Several studies show a central role of the cross-talk between host and bacteria in defining the immune response and the outcome of disease. To underpin the molecular mechanisms regulating this cross-talk, we are studying a cohort of 192 TB patients, showing different severity of disease at presentation. Thirty Mycobacterium tuberculosis (Mtb) clinical isolates associated with different clinical severity of TB were selected and PBMCs obtained from healthy IGRA+ donors (HD) or TB patients infected. Cytokine production by the infected cells was then measured. Independently of the host genetics, we identified two distinct groups of Mtb isolates: high vs low inflammatory triggers. Although a straight relation of the cytokine profile with TB severity was not found, PBMCs from patients responded differently than those from HD. These results show that although the profile of PBMC activation is Mtb-dependent, host factors can also contribute to different disease outcomes. Resorting to whole-genome and functional analysis, we are now investigating the host and pathogen determinants underlying the observed differences. Our findings highlight the importance of considering both host- and bacteria-associated characteristics when designing immunemodulatory interventions for TB. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 303 277 Host-responses to mycobacteria /interaction 22/09/2016 Therapeutic targeting of PPM1A to eliminate macrophages persistently infected with Mycobacterium tuberculosis K. Schaaf1, S.R. Smith1, O. Kutsch1, J. Sun2 1 Medicine 2Microbiology, University of Alabama at Birmingham, Birmingham, United States The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to replicate intracellularly and evade the cell-mediated immune response. Thus, reversal of the anti-apoptotic response during Mtb infections would have therapeutic benefits. However, a complete understanding of the host factors responsible for the signaling pathways that control apoptosis during Mtb infection remains elusive. We recently described that Mtb infection upregulates expression of the host phosphatase PPM1A, which reduces the antibacterial and antiviral response of macrophages1. Here we established a central role for PPM1A in the control of macrophage apoptosis during Mtb infection. Overproduction of PPM1A protected macrophages from etoposide (intrinsic) and tumor necrosis factor-α (extrinsic) induced apoptosis, and more importantly suppressed macrophage apoptosis during Mtb-infection. Conversely, targeted depletion of PPM1A by shRNA restored the ability of Mtb-infected macrophages to undergo apoptosis. Kinome analysis and subsequent studies revealed that PPM1A-controlled inhibition of apoptosis is mediated by a mechanism that involves abrogation of the c-Jun N-terminal kinase (JNK) activity. Accordingly, activation of JNK by subtoxic concentrations of anisomycin restored the ability of macrophages to undergo apoptosis, and most importantly, induced selective killing of primary human macrophages infected with Mtb. Our data indicate that therapeutic targeting of PPM1A or its downstream signaling network could provide novel host directed therapies for the elimination of persistent bacterial infections such as Mtb. This strategy could be particularly beneficial for Mtb/HIV co-infected patients since we also describe that HIV-infected macrophages display upregulated PPM1A expression leading to arrest of the cellular innate antiviral response and apoptosis pathways. Reference: 1. 304 Sun J, Schaaf K, Duverger A, Wolschendorf F, Speer A, Wagner F, Niederweis M, Kutsch O. Protein Phosphatase, Mg2+/Mn2+-dependent 1A controls the innate antiviral and antibacterial response of macrophages during HIV-1 and Mycobacterium tuberculosis infection. Oncotarget 2016. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 278 Host-responses to mycobacteria /interaction 22/09/2016 Immature granulocytes are increased in early clearance of Mycobacterium tuberculosis infection A. Verrall5, L. Apriani1, K. Sharples2, J. Ussher3, A. Indrati1, A. Nurani1, E. Diandini1, B. Alisjahbana1, R. Van Crevel6, P. Hill4 1 TB-HIV Working Group, Universitas Padjadjaran, Bandung, Indonesia 2Department of Medicine 3Department of Microbiology and Immunology 4Department of Preventive and Social Medicine, Dunedin 5Department of Pathology and Molecular Medicine, University of Otago, Wellington South, New Zealand 6Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboudumc, Nijmegen, The Netherlands Background: Early clearance (EC) is the eradication of inhaled Mycobacterium tuberculosis (Mtb) by innate mechanisms before an adaptive immune response develops. Understanding EC could lead to novel Mtb vaccines or host directed therapies. Neutrophils may have a protective role in the early response to Mtb infection. Hence, we tested the hypotheses that peripheral blood total neutrophil and immature granulocyte (IG) counts are associated with remaining uninfected after household exposure to a smear positive tuberculosis (TB) case. Methods: A study of household contacts of TB cases in Indonesia recruits contacts within 2 weeks of the case starting treatment. Mtb infection is determined by Interferon Gamma Release Assay (IGRA) at baseline (and if negative, again at 14 weeks). At baseline the duration and intensity of exposure to the index case is recorded, a full blood count is performed using a Sysmex XE 5000 analyser, and other specimens are archived. We compared neutrophils and IG in contacts who remained persistently IGRA negative, a group that contains a high proportion of ECs, to susceptible contacts who converted from negative at baseline to positive at 14 weeks. Adjusted risk ratios (ARR) for IGRA conversion were estimated using generalised estimating equations that accounted for confounders and the clustered nature of household data. Results: Of 941 contacts, 545 (57.9%) were IGRA positive at baseline, 222 (23.6%) were persistently IGRA negative, and 75 (8.0%) converted. Immature granulocytes were detected in 39% of persistently IGRA negative contacts compared to 31% of converters. The median neutrophil count was 3.95 (IQR 3.3 - 5.0) for persistently IGRA negative contacts and 4.05 (Inter Quartile Range (IQR): 2.9 - 5.1) for converters. IGRA conversion was independently associated with lower IG (P=0.003) but the relationship of lower neutrophils with conversion was not statistically significant (P=0.06). The relative risk of conversion decreased sharply as IG and neutrophils increased (figure). Conclusion: Immature granulocytes are associated with EC in this cohort of Mtb exposed case contacts. We will elaborate key elements of this signal through RNA sequencing and flow cytometry sub-studies that are currently underway. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 305 279 Host-responses to mycobacteria /interaction 22/09/2016 Identification of genes required for BCG survival in bovine lymph nodes. K. Williams3, B. Villarreal-Ramos4, H. Wu3, S. Manjari2, A. Prasad2, M. Veerasami1, G. Stewart3, M. Vordermeier4, J. Mcfadden3 1 Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai 2Centre for Development of Advanced Computing (C-DAC), Pune, India 3Faculty of Medical and Health Sciences, University of Surrey, Guildford 4Animal and Plant Health Agency (APHA), Weybridge, United Kingdom Although bovine tuberculosis is a major global problem, BCG vaccination is not used to control bovine tuberculosis, despite its proven efficacy in cattle, as vaccination interferes with the current diagnostic PPD test. This project aims to generate a new recombinant BCG strain and complementary diagnostic test, which would allow the vaccination of cattle to greatly reduce bovine tuberculosis prevalence. Our approach: 1. Identify genes required for in vivo survival of BCG by Tn-seq 2. Remove several antigens from BCG not required for in vivo survival (new vaccine) 3. Construct protein cocktail comprising these antigens (new skin test) A saturated BCG Danish Tn library was constructed and injected into the prescapular lymph nodes of 3 cows. After 3 weeks the 6 lymph nodes were harvested and Tn mutants recovered. Tn-seq data from input and output libraries was analysed by TRANSIT. Known skin test antigens (such as MPB70 & MPB83) were found not to be essential for in vivo survival, validating the removal of these antigens from BCG for our new vaccine. Conversely, BCG genes found to be essential or attenuated in the bovine lymph node model could represent genes important for host survival. 306 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 280 Host-Pathogen interaction and beyond 22/09/2016 The expired microbiome sampled by use of face masks M. Abdulwhhab, M. Barer, C. Williams, K. Haldar, M. Ramsheh, P. Haldar, N. Garton Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom We have been evaluating the use of modified face masks as a means of sampling the microbial content of exhaled air particles. We are using this approach to study respiratory diseases such as tuberculosis, pneumonia and chronic obstructive pulmonary disease. We have shown that the approach is effective in detecting tubercle bacilli in TB patients (PLoS One 9:e104921) and are now assessing its value in providing samples from the lower airways in other conditions. We report here our preliminary results from a study of over 100 samples from healthy volunteers and consenting patients with respiratory disease. Subjects were invited to wear adapted face masks for periods of up to 1 hour while undertaking specified respiratory efforts including coughing, normal breathing, instructed tidal breathing and reading out loud. As with our TB study, samples were acquired on gelatine filters from which we then obtained extracts for DNA and protein analysis. Bacterial 16SrDNA-directed analyses indicate contamination rates of between 104 and 106 copies per hour of phyla including Firmicutes, Gamma- and Beta-proteobacteria, Actinobacteria and Bacteroidetes. Evidence of mask contamination with particles from the lower and upper respiratory tracts has been obtained from these phylogenetic assignments and from quantifying surfactant protein A and albumin. Mask sampling provides important new opportunities to characterise the microbial content of human expired air in tuberculosis and other lung infections. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 307 281 Host-Pathogen interaction and beyond 22/09/2016 Deciphering the role of shape regulators in growth and survival of mycobacteria D. Arora2, Y. Chawla2, B. Malakar2, A. Singh1, V. Nandicoori2 1 CSIR-IGIB 2Signal Transduction Laboratory-I, National Institute of Immunology, New Delhi, India Establishment of successful bacterial infection in host requires undeterred cell growth and division. Mycobacterial cell wall, the prime barrier protecting the cell from immune assault undergoes substantial remodeling events during its survival in host. How mycobacteria regulate cell shape maintenance during this process is an unexplored facet. Continuous cell integrity and shape maintenance will require coordination of biosynthetic, structural and regulatory elements of cell division. Studies on genes involved in maintaining bacterial cell shape during mycobacterial division are scant. Here, we report the role of mycobacterial shape regulators genes in the growth and survival of the pathogen in vitro and in host. Overexpression and deletion studies suggests critical role for these genes in maintaining normal cell morphologies. Furthermore, our studies delineate the significance of the previously uncharacterized active domains of these proteins and suggest a phosphorylation-mediated regulation in conserving their normal activity. Animal infection studies further suggests critical role for these genes in survival of the pathogen in host. Together, our data indicate that these shape regulators are required for mycobacterial cell fitness during host-induced stress. 308 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 282 Host-Pathogen interaction and beyond 22/09/2016 Breaking fat! How mycobacteria become obese in Dictyostelium mutants lacking lipid droplets C. Barisch, T. Soldati University of Geneva, Geneva, Switzerland Lipid droplets (LDs) store energy in form of neutral lipids and exist in virtually every cell type. Induction of foamy, LD-filled macrophages is a general host response to mycobacterial infection and develops shortly after granuloma formation. Intracellular mycobacteria have been seen in the vicinity or even “plunging” inside LDs, possibly corroborating that the bacteria are able to exploit host lipids for their own metabolism. The mechanism by which the bacteria get access to LDs is still unknown. Using the Dictyostelium/Mycobacterium marinum infection system as a model for foamy macrophages, we have found that M. marinum exploits lipids from host LDs to build up its own lipid inclusions (ILIs). Strikingly, the Dictyostelium homologue of perilipin and the murine perilipin 2 surround bacteria that had escaped to the cytosol of Dictyostelium or microglial BV-2 cells, respectively. In addition, bacterial growth was inhibited in perilipin mutants. To test which lipids are preferentially transported into the mycobacterium-containing compartment, we interfere with host enzymes involved in triacylglycerol (TAG) synthesis. Interestingly, the Long Chain Fatty Acid CoA Synthase 1, that activates fatty acids (FAs), is recruited to the mycobacterium niche. In addition, LDs decorated with diacylglycerol acyltransferase (Dgat) 2 were seen clustering around cytosolic bacteria at late infection stages. Dictyostelium Dgat mutants are unable to generate TAGs and LDs. Instead, the exogenous FAs are esterified into phospholipids, inducing uncontrolled proliferations of the ER-membrane. Strikingly, M. marinum is able to exploit this source of host phospholipids resulting in rapid reversal of ERproliferations. Moreover, the bacteria build up many more ILIs compared to the wild type. This excessive “fattening” is not accompanied by a change in intracellular growth and metabolic activity, and thus provides evidence that the storage of neutral lipids does not necessarily induce dormancy. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 309 283 Host-Pathogen interaction and beyond 22/09/2016 Cloning, expression and characterization of Mycobacterium tuberculosis antigen, HBHA, produced in the yeast Pichia pastoris C. Benabdessalem2, R.Y.M. Ouni2, C. Rouanet1, F. Haj Aissa2, M. Loyens1, D. Raze1, C. Locht1, R. Barbouche2 1 INSERM U1019 - CNRS UMR 8204, Univ Lille Nord de France, CIIL - Center for Infection and Immunity of Lille, Institut Pasteur de Lille, Lille, France 2Laboratoire de Transmission, Contrôle et Immunobiologie des Infections, Institut Pasteur de Tunis, Tunis, Tunisia Identification and preventive treatment of latently infected cases (LTBI) with Mycobacterium tuberculosis (Mtb) are currently essential components of the global Post-2015 tuberculosis (TB) strategy. Since commercially available Interferon Gamma Release Assays (IGRAs) cannot, alone, discriminate active TB from LTBI individuals it will be crucial to identify alternative antigens to use for IGRA. The test HBHA-IGRA is a novel test that shows a high specificity for LTBI. This assay is based on the HBHA (Heparin Binding Haemagglutinin Antigen) a latency associated antigen of Mtb which induces a strong IFNg response during latent but not active TB. It has been shown that the methylation on its C-terminal domain is crucial for an effective T cell response to this antigen. The current HBHA-IGRA is based on the use of the native antigen purified from BCG strain since the conventional expression system, E.coli, is not able to carry out methylation. The development of a recombinant methylated form of this antigen would be crucial for the commercialization of HBHA-IGRA. Herein we used the yeast Pichia (P.) pastoris to express a recombinant form of the HBHA. Indeed, P. pastoris similar to Mtb has a GC rich codon usage and also retains protein post-translational events, which may better mimic native antigens. In previous studies we showed that P. pastoris could be a good alternative expression system for mycobacterial genes. Three, already developed monoclonal antibodies (MoAbs), two of which are specific for the methylation of the native form of HBHA were used to recognize the produced recombinant form as well. Using shaker flasks, we generated a P. pastoris clone expressing a recombinant form of HBHA (rHBHA). Interestingly, we showed that the rHBHA is methylated since it is well recognized by both methylation specific MoAbs. We are studying by mass spectrometry whether the methylation performed by the yeast is similar to mycobacterial one. 310 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 284 Host-Pathogen interaction and beyond 22/09/2016 The initial encounter of pathogenic mycobacteria with tissue resident macrophages determines infection outcome C. Cambier2, L. Ramakrishnan1 1 University of Cambridge, Cambridge, United Kingdom 2Chemistry, Bertozzi, Stanford, Stanford, United States Despite the notoriety of tuberculosis as a human killer, epidemiological studies suggest that some exposed individuals can clear tuberculosis independently of adaptive immune responses. Using the zebrafish, we provide direct mechanistic evidence for early clearance of infection, and show that it is mediated by the innate microbicidal activity of tissue-resident macrophages. We find that resident macrophages are first-responders to bacterial pathogens, including mycobacteria. However, virulent mycobacteria deploy an escape strategy after phagocytosis by these microbicidal cells. This escape requires mycobacterial phenolic glycolipid (PGL), which programs the infected resident macrophages to recruit permissive monocytes, into which the mycobacteria transfer. Thus infection outcome - clearance versus progression - depends on the relative strengths of two competing programs in the resident macrophage - intrinsic microbicidal activity versus PGL-induced recruitment of permissive monocytes. Our findings suggest immunological or pharmacological PGLblocking strategies as interventions to prevent tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 311 285 Host-Pathogen interaction and beyond 22/09/2016 Mycobacterium tuberculosis LppM displays an original structure and domain composition linked to a dual localisation M. Cohen-Gonsaud Centre de Biochimie Structurale, Montpellier, France Mycobacterium tuberculosis (Mtb) encodes several bacterial effectors impacting the colonization of phagocytes. LppM (Rv2171) is both implied in phagocytosis and able to efficiently block phagosomal acidification in the macrophage, two key features contributing to Mtb persistence. We showed that LppM is anchored to the mycobacterial cell wall by a C-terminal membrane domain. But the protein also exists as a truncated protein secreted into the culture medium. The LppM solution structure we solved here displays no similarity with other Mtb lipoproteins also involved in phagosomal maturation (ie LprG). In addition, we demonstrated the protein is able to bind scarce phosphatidylinositol mannosides derivatives, molecules involved in the host processes manipulation. Therefore, our data demonstrate a dual localization and open new perspective on Mtb secretion and protein localization regulation. 312 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 286 Host-Pathogen interaction and beyond 22/09/2016 Developing a persistent zebrafish embryo infection model S. Commandeur1, N. Iakobachvili2, M. Nur2, G. Mukamolova2, A. Van Der Sar1, W. Bitter1 1 Department of Medical Microbiology and Infection Control, VUmc, Amsterdam, The Netherlands 2Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, United Kingdom Mycobacterium tuberculosis (Mtb) is able to reside in the host without inducing any clinical symptoms of the disease. A so-called latent tuberculosis (TB) infection can be present for decades. Mtb is able to adapt to the harsh environmental conditions it encounters in the host, resulting in a persistent phase of Mtb. Such persisters are phenotypically drug resistant and require long-term treatment of a combination of antibiotics. The rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb is fueled by this long-term therapy and thus dramatically shortening of the length of TB treatment is urgently required. New drugs are needed that directly target the persister population to reach this goal. In this work we develop a model that can be used to identify compounds that efficiently kill such persistent mycobacteria. We use Mycobacterium marinum (Mm) and the zebrafish embryo host as model system because (i) Mm infection of zebrafish embryos induces similar pathology as observed for Mtb infection in humans and (ii) it is useful for high throughput screens in search for novel compounds. Because latent infections cannot be reached in the zebrafish embryo model due to time constraints, we have to establish the bacterial persistent phase in vitro prior infection. To create persistent Mm we analyzed whether Mm is able to adapt to in vitro stress conditions and identified that carbon-rich starvation conditions leads to a persister phase in Mm. However, the persister phenotype was not maintained in vivo. Mtb expresses resuscitation promoting factors (Rpfs) which are described to aid in resuscitation of persister mycobacteria. We hypothesized that Rpf knock-outs will prevent outgrowth in the zebrafish embryos. Single and double Rpf knock-outs were generated and analyzed for their growth abilities after persister formation both in vitro and in vivo.We conclude that Mm is able to adapt to stress conditions and that the absence of Rpfs seem to play a role in maintaining the persister phenotype in the zebrafish embryo model. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 313 287 Host-Pathogen interaction and beyond 22/09/2016 Mycobacterium tuberculosis survival upon efferocytosis of infected neutrophils by macrophages requires ESAT-6 T. Dallenga1-2, U. Repnik4, J. Eich2, G. Griffiths4, R. Reimer3, U.E. Schaible1-2 1 German Centre for Infection Research 2Cellular Microbiology, Research Center Borstel, Borstel 3Core Facility Microscopy & Image Analysis, Heinrich-Pette-Institute, Hamburg, Germany 4Department of Biosciences, University of Oslo, Oslo, Norway Extensively and multidrug-resistant isolates of Mycobacterium tuberculosis are on the rise worldwide. Novel host-directed therapies are promising adjunct measures to antibiotics treatment. Neutrophils represent the main infected cell population in sputum and bronchoalveolar lavages from patients with active tuberculosis, but fail to kill M. tuberculosis. Instead, virulent but not RD1deficient mycobacteria induce necrotic cell death in a reactive oxygen species-dependent manner, facilitating mycobacterial escape from killing. Dead neutrophils are removed by macrophages in a process termed efferocytosis. We studied the role of efferocytic removal of mycobacteria-infected apoptotic vs. necrotic neutrophils in macrophage defense against M. tuberculosis. We found that efferocytosis of necrotic neutrophils infected with wild type M. tuberculosis promoted survival and growth of the mycobacteria, whereas mutants lacking either RD1 or the RD1-encoded ESAT-6 alone failed to grow in efferocytes. In contrast, mycobacteria directly entering macrophages required other RD1-encoded genes for intracellular growth, additionally to ESAT-6. The decision whether efferocytosed M. tuberculosis survived in macrophages was determined by differential intracellular trafficking and cell death pathways upon infection by either wild type or attenuated mutant strains. By combined time-lapse live cell imaging, correlative confocal-based 3D-reconstruction and electron microscopy we monitored infection, cell death and efferocytosis. Data suggests differential phagosomal sorting of dead PMN material from Mtb within macrophages early after uptake. Taken together, efferocytosis of neutrophils infected with virulent M. tuberculosis promotes infection in an ESAT-6-depending manner. Our data further emphasize the detrimental role of neutrophils for tuberculosis pathology and disease outcome, making them a predestined candidate for both, point of care testing as well as target for host-directed therapies. 314 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 288 Host-Pathogen interaction and beyond 22/09/2016 Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection C. Péan2, M. Schiebler1, S. Tan2, K. Brown1, M. Maserumule1, S. Menezes3, M. Pilátová3, K. Bronda3, P. Guermonprez3, B. Stramer3, A. Floto1, M. Dionne2 1 University of Cambridge, Cambridge 2Dept of Life Sciences and MRC CMBI, Imperial College London 3King's College London, London, United Kingdom Macrophages are essential to the host defense against mycobacteria; however, macrophages are often inefficient at killing virulent mycobacteria and constitute a niche for mycobacterial persistence and proliferation. We use a Drosophila model to identify host mechanisms that regulate mycobacterial growth within phagocytes. We show that, in this model, mycobacterium-induced cytokine-STAT signaling is detrimental to the host, reducing resistance to Mycobacterium marinum partly by reducing Atg2 expression and thus facilitating pathogen-induced deregulation of lipid droplets. Flies with cytokine-STAT signaling inhibited live longer when infected and exhibit markedly lower mycobacterial burden and improved macrophage survival; this is accompanied by significant and specific overexpression of Atg2. Atg2 overexpression is sufficient to reduce proliferation of mycobacteria in Drosophila phagocytes. Increased Atg2 appears not to affect bulk autophagy; instead, Atg2 overexpression partially normalizes lipid deposition in infected phagocytes. Direct inhibition of macrophage triglyceride biosynthesis likewise reduces intracellular lipid droplets and mycobacterial numbers. Similarly, in human macrophages, IL-6 reduces ATG2A levels and contributes to mycobacterial growth by deregulating macrophage lipid metabolism. Our results reveal a new mechanism by which cytokines control lipid droplet homeostasis and consequently resistance to mycobacterial infection. That inhibition of macrophage triglyceride synthesis reduces mycobacterial growth and the identification of ATG2 as a key cytokine effector highlight potential host-directed therapies in tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 315 289 Host-Pathogen interaction and beyond 22/09/2016 Peptidyl prolyl isomerase family of Mycobacterium tuberculosis mediates the modulation of host macrophages aiding the infection process N. Dubey2, M. Gupta1, A.K. Sharma1, Y. Singh2 1 Institute of genomics and integrative biology 2Department of Zoology, University of Delhi, Delhi, India Mycobacterium tuberculosis (Mtb), the etiological agent of the disease tuberculosis can evade the host immune system to either develop an active infection or to stay in the latent form for years. This, in part, is achieved by the secretion of virulence factors which not only assist in initial invasion but also in the progression of the disease towards the granulomatous form. The focus of our study is mycobacterial secretory proteins Peptidyl prolyl isomerases (PPiases) and we are trying to determine their role in mycobacterial pathogenesis. PPiases mediate the inter-conversion of cisand trans- conformers of protein, conferring the stability and activity to the cellular proteome. Mtb encodes two PPIases, PPiA and PPiB. Though the family executes many housekeeping functions, recent literature suggests a probable role of PPiases as potent virulence factors in many pathogens such as Listeria, Salmonella and Brucella. Interestingly, Mtb PPiA is a secretory isomerase and its secretion is restricted to the pathogenic species of Mycobacteria genus. Also, the transcriptional upregulation of Mtb PPiA is observed in intra-phagosomal bacteria. Our studies with the gene deletion strain of PPiA in H37Rv strain indicate towards its role during the late time point of mice infection. To explore the mechanism of action of PPiA, we have utilized in-silico tools to identify the novel interacting partners of Mtb PPiA in the host. These targets were further confirmed by co-immuno pull down and ELISA based interaction assays. Two interacting partners that we chose to study are cell surface receptor protein Integrin and an innate immune response protein, Lipocalin. While Integrins mediates the extracellular matrix modulations via matrix metallo-proteases, Lipocalins are host siderocalins, which are known to inhibit the iron translocation required for bacterial survival by competing with the Mtb carboxymycobactins. Our study, (i) elaborates the role of Mtb PPiA in employing Integrin as a strategy for host invasion (ii) the implication of PPiA & lipocalin interaction on intracellular bacterial iron requirements.Mtb PPiB is a membrane bound protein which undergoes mannosylation. Its role during bacterial phagocytosis by host is also being studied. This study, for the first time, delineates the functional significance of the PPiase family during tuberculosis pathology. Our research on Mtb PPIases will provide us better understanding of the cues utilized by the bacteria to evolve over its host. 316 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 290 Host-Pathogen interaction and beyond 22/09/2016 Pst/SenX3-RegX3 directly regulates ESX-5 secretion in Mycobacterium tuberculosis S. Elliott, A. Tischler University of Minnesota, Minneapolis, United States The Type VII secretion system ESX-5 has been implicated in Mycobacterium tuberculosis (Mtb) virulence, though both the regulatory mechanism and the signal inducing ESX-5 activity were unknown. We recently discovered that ESX-5 is activated during phosphate (Pi) limitation, an environmental signal Mtb likely experiences during infection. ESX-5 activity is directly regulated at the transcriptional level by the Pst/SenX3-RegX3 system, which controls gene expression in response to Pi availability. Pi limitation resulted in RegX3-dependent overexpression of esx-5 transcripts, as well as overproduction and hyper-secretion of ESX-5 proteins. We propose that regulation of ESX-5 activity in response to Pi limitation is a critical function of the Pst/SenX3-RegX3 system that promotes Mtb virulence. Deletion of pstA1, which encodes a Pst component, causes constitutive activation of RegX3, hyper-secretion of ESX-5 substrates, and attenuation in the mouse aerosol infection model. We hypothesize that disruption of ESX-5 regulation is responsible for attenuation of the ΔpstA1 mutant. Since EsxN is one of the hyper-secreted ESX-5 substrates and is highly antigenic, we tested whether deletion of esxN could reverse the ΔpstA1 virulence defect. We infected C57BL/6 and NOS2-/- mice with a ΔpstA1ΔesxN mutant, and evaluated bacterial replication and persistence in the lungs. The ΔpstA1ΔesxN mutant did not reverse the virulence defect, suggesting that hyper-secretion of all, or some combination of, ESX-5 substrates causes attenuation. To further investigate this possibility, we first determined the precise regulatory mechanism governing ESX-5 secretion. We previously demonstrated that RegX3 directly binds to a region within the esx-5 locus. Using electrophoretic mobility shift assays (EMSA), we screened a series of unlabeled competitor probes and identified a direct repeat sequence that is essential for RegX3 binding. Subsequent mutation of this region to evaluate the importance of RegX3-dependent regulation of ESX-5 secretion for Mtb virulence is currently in progress. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 317 291 Host-Pathogen interaction and beyond 22/09/2016 Mycobacterium tuberculosis subverts autophagosome formation by inducing miR-155 expression in human dendritic cells M.P. Etna3, A. Sinigaglia2, A. Grassi2, E. Giacomini3, M. Pardini3, M. Severa3, M. Cruciani3, A. Romagnoli4, L. Barzon2, B. Di Camillo2, G.M. Fimia4-1, R. Manganelli2, E.M Coccia3 1 Department of Biological and Environmental Science and Technology, University of Salento, Lecce 2Department of Molecular Medicine, University of Padua, Padua 3Department of Infectious, Parasitic and Immune-mediated diseases, Istituto Superiore di Sanità 4National Institute for Infectious Diseases "L. Spallanzani”, Rome, Italy Given the re-emergence of tuberculosis (TB), increasing efforts are devoted to understand the events occurring along Mycobacterium tuberculosis (Mtb)-host interaction, w ith a partic ular interest for the strategies engaged by Mtb to escape immune response. In this study, we sought to characterize the interaction of Mtb w ith human dendritic cells (DC), a key cell type involved in the regulation of innate and adaptive immune responses against pathogens. In particular, we analyzed autophagy, as an immune process exploited by Mtb to evade the host antimicrobial response and, in this context, we focused on microRNAs (miRNAs), whose role in controlling the Mtb-DC interplay remains unclear. Microarray analysis w as conducted at different time points after the Mtb infection thus unveiling the modulation of a specific pattern of cellular miRNAs. Among cellular miRNAs de-regulated in Mtbinfected DC, w e analy zed the function of miR-155 for which target predic tion analysis identified as a putative target Atg3, the E2 ubiquitin-like-conjugating enzyme involved in LC3-lipidation. Interestingly, we observed that at late time points of infection Mtb impairs both ATG3 protein levels and the number of LC3 puncta per cell, thus supporting the hypothesis of a miR-155-dependent mechanism for Atg3 regulation. Accordingly, dual luciferase 3’UTR reporter assay demonstrated that miR-155 dow n-regulated the 3’UTR of Atg3. All these observations are in line w ith the reduction of ATG3 protein level detected in miR-155-transfected DC and, as a consequence, w ith a poor conversion of LC3 in its autophagosome-associated form. These data further underlined the importance of autophagy in controlling the fate of Mtb infection, indeed in addition to the well-characterized block of the autophagic flux mediated by Mtb, here we illustrated how this pathogen takes advantage from cellular miRNA regulation to impair autophagosome formation thus escaping host immune response. These evidences provided new intriguingly information on host pathogen interaction involved in Mtb-driven control of autophagy flux to favor its own intracellular survival and persistence in the host. This w ork was supported by grant RF-2010235199 from the Italian Ministry of Health (to EMC). 318 Tubercul osis 2016 – September 19-23, 2016 – Paris, Franc e 292 Host-Pathogen interaction and beyond 22/09/2016 Mycobacterium avium interfering with phagosome maturation evades an antibacterial program activated by phagolysosomal TLR7/8/MyD88 in human primary macrophages A. Gidon, S. Åsberg, C. Louet, L. Ryan, T. Flo Centre of Molecular Inflammation Research, and Dept of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway In developed countries the prevalence of non-tuberculous mycobacterial infections caused by M. avium (Mav) is increasing in individuals who are immunocompromised due to underlying disease or use of immunosuppressant drugs. Mav lacks several of the key virulence factors of Mtb, but nevertheless can establish chronic infections. Pathogenic mycobacteria reside in macrophages and are thought to avoid lysosomal targeting and degradation through poorly understood mechanisms involving secreted phosphatases and cell wall lipids. We have previously demonstrated that Mav is associated with Rab11-related compartments accessible to transferrin four days post infection. However, how trafficking of mycobacteria relates to inflammatory signalling is poorly characterized, especially for Mav. Using high-resolution confocal imaging of human primary macrophages infected with Mav we show evidence that mycobacterial phagosomes are not arrested at an early endosomal stage but rather progress to a late endosomal/lysosomal stage where inflammatory signalling is initiated through TLR7/8 recruitment of MyD88 and nuclear translocation of NF-kB and IRF-1, inflammatory cytokine production and Mav growth restriction. A fraction of the mycobacteria escapes and re-establish in a less hostile "stealth" compartment, evading destruction, TLR detection and growth restriction. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 319 293 Host-Pathogen interaction and beyond 22/09/2016 Role of VirS in acid induced responses of Mycobacterium tuberculosis G. Khare, S. Singh, A.K. Tyagi Department of Biochemistry, University of Delhi South Campus, Delhi, India More than five decades of tuberculosis (TB) control programmes using potentially efficacious drugs have not succeeded in reducing the burden of this disease in most parts of the world. Mycobacterium tuberculosis is one of the most successful pathogens that effectively counteracts the host antimicrobial responses and is able to reside in macrophages, the very cells involved in the killing of the incoming pathogens. Phagosomal acidification is considered as the major host defense mechanism against the microbial pathogens in addition to the production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). However, M.tuberculosis is endowed with various properties that help its survival in the macrophages by subverting the phagosomal maturation pathways. The ability of mycobacterium to resist the killing in these harsher acidified conditions highlights the importance of maintaining its pH homeostasis for its long term persistence in macrophages during infection, however, the underlying mechanisms for this are poorly understood. Till to date, the identity of a specific sensor of acidic pH in M.tuberculosis is not known. In this context, our laboratory and others have identified the specific induction of an AraC/XylS-type transcriptional regulator VirS (Rv 3082c) in response to acidic pH. The fact that acidic conditions led to its up-regulation suggested that the genes regulated by VirS (VirS-regulon) might be associated with the maintenance of pH homeostasis in the cell. Further, the infection of guinea pigs with the M. tuberculosis virS mutant was shown to result in an ~800 fold reduction in the splenic bacillary load. The present study describes the role of VirS in the acid induced responses of M.tuberculosis by employing the gene knockouts in in vitro broth cultures and macrophages. Purification of VirS was carried out and the DNA binding region for VirS was determined by using EMSA and footprinting studies. Moreover, microarray studies were carried out to identify the genes regulated by VirS in response to acidic stress. The study further focused on understanding the gene regulation under acidic conditions faced by the pathogen and the role of acid induced transcriptional regulator VirS in this process. 320 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 294 Host-Pathogen interaction and beyond 22/09/2016 Imaging NADH-NAD+ redox homeostasis of Mycobacterium tuberculosis during infection S. Bhatt, I. Iqbal, A. Kumar CSIR-Institute of MIcrobial Technology, Chandigarh, India The NADH:NAD+ ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. In this study, we have generated reporter strains of fast and slow growing mycobacterium species for measurement of the NADH:NAD+ levels in vitro and in vivo. Genetically encoded fluorescent biosensors of the NADH:NAD+ ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, and treatment with antimycobacterial drugs lead to the accumulation of NADH in mycobacterial cells. Interestingly, we observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD+ levels in resident Mtb cells. Furthermore, we observed that the sub-vacuolar localization dictates the metabolic state of Mtb; Mtb cells residing in phagolysosomes have a higher NADH:NAD+ ratio and Mtb cells colocalizing with the early endosome have lower NADH:NAD+. Thus in this study we report novel engineered reporter strains of mycobacteria capable of measuring NADH:NAD+ redox homeostasis in response to antimycobacterial drug and other physiologically relevant stresses. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 321 295 Host-Pathogen interaction and beyond 22/09/2016 Genome Wide Mutagenesis strategies in Dictyostelium discoideum and Mycobacterium marinum to decipher the conserved genetic basis of mycobacteria intracellular infections L. Lefrançois1, T. Mendum2, G. Stewart2, T. Soldati1 1 Dept of Biochemistry, University of Geneva, Geneva, Switzerland 2FHMS, University of Surrey, Guildford, United Kingdom This study aims at understanding how Mycobacterium bovis and Mycobacterium marinum manipulate the fundamental processes of innate immunity, in particular the phagosome environment. Because genetic analysis of host factors is difficult in diploid mammalian cells, we propose to use Dictyostelium discoideum as a model phagocyte. This social amoeba exhibits evolutionarily conserved mechanisms of cell-intrinsic defence and has a haploid genome opened to forward genetics. Our plan is to develop the host-pathogen system D. discoideum-M. marinum as a powerful genetically tractable model for mycobacterial infection. To that purpose, we will apply genome-wide mutagenesis and high throughput sequencing. Transposon mutagenesis (Tn-Seq) in M. marinum and M. bovis will be performed using the MycoMar transposon. Restriction Enzyme-Mediated Insertional Mutagenesis (REMI-Seq) will be performed in D. discoideum using a blasticidin resistance cassette. The complexity of the libraries generated is about 100,000 mutants, with an insertion every 100bp in M. marinum and in 92% of protein coding genes in D. discoideum. The precise identification and relative abundance of insertions before and after a given selection allows us to quantitatively compare the compositions of pools with a high dynamic range. As a proof of principle, we are validating the approach by applying the Tn-Seq method for the first time to M. marinum. Then, we want to test whether M. marinum can use similar carbon sources as Mycobacterium tuberculosis, namely fatty acids and sterols, by performing selections on Tn-seq pools in different media. In addition, we will also investigate whether accumulation of propionate produced by cholesterol catabolism is toxic for M. marinum and can be alleviated by adding acetate. Moreover, we will infect D. discoideum and macrophages with both M. marium and M. bovis Tn-seq pools to identify genes required for survival in these two phagocytes. Finally, infection of D. discoideum REMI-seq pools with M. marinum will reveal host genes implicated in resistance and susceptibility to infection. These promissing and innovative approaches will allow a comprehensive definition of the host and pathogen genes important for the intracellular host-pathogen interactions and an overview of mycobacterial virulence mechanims conserved during macrophages and Dictyostelium infection. 322 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 296 Host-Pathogen interaction and beyond 22/09/2016 Beyond macrophages: the role of lymphatic endothelial cells in tuberculosis T. Lerner7, C. De Souza Carvalho-Wodarz2, U. Repnik3, M. Russell7, S. Borel7, C. Diedrich5, M. Rohde1, H. Wainwright4, L. Collinson7, R. Wilkinson7-5-6, G. Griffiths3, M. Gutierrez7 1 Helmholtz Institute for Infection Research, Braunschweig 2Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrucken, Germany 3University of Oslo, Oslo, Norway 4UCT Faculty of Health Sciences and NHLS Laboratories 5University of Cape Town, Cape Town, South Africa 6Imperial College 7The Francis Crick Institute, London, United Kingdom Lymphatic tuberculosis (TB) is the second most common manifestation of TB in humans but the role of lymphatics in TB is poorly characterised. By analysing the localization of Mycobacterium tuberculosis (Mtb) in lymph node samples from human TB patients, we identified lymphatic endothelial cells (LECs) lining the lymphatic vessels as a niche for Mtb. In cultured primary human LECs (hLECs), the uptake of mycobacteria via phagocytosis was mediated through the mannose receptor. Moreover, Mtb formed distinctive intracellular cords within the cytosol, a process that required the type VII secretion system ESX-1. Interestingly, these M. tuberculosis cords were not recognised by the autophagy machinery. Surprisingly, by using correlative light electron microscopy we demonstrated that Mtb also replicated in autophagosomes. However, upon activating hLECs with interferon gamma (IFN-γ), the growth of the bacteria was restricted. This restriction of bacterial growth was via the induction of autophagy and also via nitric oxide production. Thus, Mtb localises in distinct sub-populations in LECs that differentially interact with the autophagy pathway resulting in either Mtb growth or restriction. These findings highlight the importance of LECs in the pathogenesis of tuberculosis, and implicate LECs as a potential reservoir for M. tuberculosis in humans. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 323 297 Host-Pathogen interaction and beyond 22/09/2016 ESX-1 exploits type I IFN-signaling to inhibit IFNg-mediated autophagy and growth restriction of intracellular mycobacteria J. Lienard1, E. Movert1, C. Valfridsson1, E. Sturegård2, F. Carlsson1 1 Experimental Medical Science, Immunology section, Host-pathogen interactions, Lund 2Laboratory Medicine, Medical Microbiology section, Lund University, Malmö, Sweden The ability of macrophages to eradicate intracellular pathogens is normally greatly enhanced by IFNg, a cytokine produced mainly after onset of adaptive immunity. However, adaptive immunity is unable to provide sterilizing immunity against mycobacteria, suggesting that mycobacteria have evolved virulence strategies to inhibit the bactericidal effect of IFNg-signaling in macrophages. Still, the host-pathogen interactions and cellular mechanisms responsible for this feature have remained elusive. Our data suggest that the ESX-1 type VII secretion systems of Mycobacterium tuberculosis and Mycobacterium marinum exploit type I IFN-signaling to promote an IL-12low/IL-10high regulatory macrophage phenotype characterized by secretion of IL-10, IL-27 and IL-6. This mechanism has no impact on intracellular growth in the absence of IFNg, but suppresses IFNg-mediated autophagy and growth restriction, indicating that the regulatory phenotype extends to function. The IFNgrefractory phenotype is partly mediated by IL-27-signaling, establishing functional relevance for this downstream cytokine. These findings identify a novel macrophage-modulating function for the ESX1 secretion system that might contribute to suppress the efficacy of adaptive immunity, and provide mechanistic insight into the antagonistic cross talk between type I IFNs and IFNg in mycobacterial infection. 324 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 298 Host-Pathogen interaction and beyond 22/09/2016 The anti-inflammatory role of C-type lectin DC-SIGN in human alternatively activated macrophages and the response against Mycobacterium tuberculosis A. Troegeler2, L. Balboa1, C. Lastrucci4, C. Duval2, I. Mercier2, A. Bénard2, T. Al Saati3, R. Poincloux2, I. Kondova5, F. Verreck5, I. Maridonneau-Parini2, M.C. Sasiain1, C. Cougoule2, O. Neyrolles2, G. Lugo-Villarino2 1 IMEX-CONICET, Academia Nacional de Medicina, Buenos Aires, Argentina 2Tuberculosis and Infection Biology, CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), UPS (Université Paul Sabatier) 3INSERM/UPS/US006 CREFRE, CHU Purpan, Toulouse, France 4 EMBL/CRG, Barcelona Institute of Science and Technology, Barcelona, Spain 5Biomedical Primate Research Centre, Rijswijk, The Netherlands DC-SIGN is a C-type lectin receptor (CLR) that serves as a biological marker for human alternatively activated macrophages, M(IL-4). Although DC-SIGN plays important roles in M. tuberculosis (Mtb) interactions with dendritic cells, its contribution to the mycobacteria-macrophage interaction remains poorly understood. Here, we investigate the pertinence of DC-SIGN in the control of Mtb intracellular growth and inflammatory response by M(IL-4). We found that siRNAmediated inactivation of DC-SIGN renders M(IL-4) less permissible to Mtb intracellular growth compared to DC-SIGN-expressing cells, despite the equal levels of the initial Mtb uptake. Transcriptomic analysis in DC-SIGN-deficient M(IL-4) revealed a pro-inflammatory gene signature characterized by up-regulation of IL-6, TNF and CXCL1, which was confirmed by RT-qPCR and ELISA analyses. Conditioning of human monocytes with supernatants from Mtb-infected DC-SIGNdeficient M(IL-4) resulted in a shift towards a pro-inflammatory phenotype, supporting the role of DC-SIGN in controlling macrophage inflammation. At the molecular level, we show that DC-SIGN antagonizes the pro-inflammatory response mediated by Dectin-1. Treatment of M(IL-4) with mannosylated lipoarabinomannan (an Mtb-derived glycolipid and ligand for DC-SIGN) induces STAT3 activation in a DC-SIGN-dependent manner, suggesting a novel signaling pathway in the DC-SIGN-Dectin-1 crosstalk. Finally, we demonstrate that DC-SIGN expression can be accentuated in macrophages under different contexts using samples from TB patients, and become abundant in TB pulmonary lesions of non-human primates, arguing for the pertinence DC-SIGN+ macrophages in TB pathology. Collectively, this study highlights a dual role for DC-SIGN as, on the one hand, being a host factor granting advantage for Mtb to parasitize macrophages and, on the other hand, representing a molecular switch to turn off the pro-inflammatory response in these cells to prevent potential immunopathology. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 325 299 Host-Pathogen interaction and beyond 22/09/2016 Growth of Mycobacterium tuberculosis in dead macrophages drives a host cell death cascade D. Mahamed2-3, M. Boulle2-3, S. Skroch2-3, L. Oom2-3, G. Sreejit2-3, K. Pillay2-3, O. Catinas2, M. Naicker2-3, S. Rampersad2-3, C. Mathonsi2-3, J. Hunter2-3, A. Pym2-3, G. Lustig2-3, A. Sigal2-1 1 Max Planck Institute for Infection Biology, Berlin, Germany 2KwaZulu Natal Research Institute for TB-HIV 3University of KwaZulu-Natal, Durban, South Africa Active Mycobacterium tuberculosis (Mtb) infection leads to extensive host cell death and bacterial growth, releasing Mtb into the airways to be transmitted to another host. Here we set out to determine the factors contributing to bacterial growth and host cell death at the level of individual macrophages, the primary Mtb host cells. Primary human macrophages, differentiated from peripheral blood monocyes, were infected with a fluorescently labelled strain of Mtb H37Rv and tracked using live cell time lapse microcopy. To facilitate quantitative analysis of complex macrophage-Mtb interactions, custom written image analysis software was developed to track cell fate. We quantified the number of bacteria in each pickup of Mtb by a macrophage, whether the bacilli were cell-free or inside a dead infected cell, and the fate of the internalizing macrophage after pickup. Macrophages internalizing clumps of Mtb died with a frequency which increased with total Mtb uptake and number of Mtb in a single clump. Interestingly, macrophage death did not eliminate intracellular bacteria. Instead, it led to rapid Mtb multiplication inside dead host cells, with a median doubling time of 25±10 hours. Growth in dead infected cells was significantly faster than extracellular Mtb growth (doubling time 38±11 hours), while live macrophages controlled Mtb replication efficiently. Bacteria grew as a clump inside dead macrophages, creating a locally high Mtb concentration. Internalization of these dead infected cells resulted in nearly 90% macrophage death, indicating that once an infected cell dies, additional cycles of cell death will occur. Such a cascade of host cell death provides a mechanism for the propagation of TB infection at the cellular level which, once initiated, would be difficult to contain. 326 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 300 Host-Pathogen interaction and beyond 22/09/2016 Inhibition of matrix metalloproteinases interfere with the formation of tuberculosis granuloma in a human lung tissue model V.R. Parasa1, J. Muvva2, J. Jeronimo Frias Rose1, S. Brighenti2, M. Lerm1 1 Division of Medical Microbiology and Molecular Medicine, Linköping University, Linköping 2Center for Infectious Medicne, Karolinksa University Hospital, Karolinska Institute, Stockholm, Sweden Granulomas, organized cluster of immune and non-immune cells, are the hallmarks of pulmonary tuberculosis (TB). Matrix metalloproteinases (MMPs), a family of proteases involved in extracellular matrix destruction and tissue remodeling, have been implicated in the pulmonary pathology of TB, specifically during extensive tissue damage and disseminated TB. However, the exact stage of the disease at which the specific MMPs act in human TB is uncertain. We hypothesize that MMPs are required for early granuloma formation and that by blocking the activity of specific MMPs, the process of granuloma formation could be altered, thus perturbing the initial growth of M. tuberculosis. Studying TB at its initial stages, like granuloma formation, requires the assessment of early events during infection in a three-dimensional system. Therefore, we developed an in vitro lung tissue model [1,2] to investigate the role of MMPs during TB granuloma formation. Confocal microscopy was used for the visualization of granulomas. Analysis of MMPs 1,2,3,7,8,9,11,12 and13 is done in the different cell types extracted from the M. tuberculosis-infected tissue models using quantitative PCR and immunohistochemistry. Validation of the MMPs by comparison with tissues from human TB patients are under investigation. Our results show that the granuloma formation observed in the in vitro model upon infection with virulent M. tuberculosis was inhibited in the presence of a global MMP inhibitor. Identification of the specific MMPs involved in the process of TB granuloma formation is underway. We further plan to investigate the effect of MMP inhibitors on the inflammatory response and bacterial load in the tissue model. Our project will give insight into how MMP inhibitors may help managing TB. 1. Parasa VR et al. Dis. Model. Mech. 2014;7:281-8. 2. Braian C et al. J. Vis. Exp. 2015;104 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 327 301 Host-Pathogen interaction and beyond 22/09/2016 Enhanced presence of proteins related to type I interferon signalling at the Mycobacterium tuberculosis phagosome N. Reiling1, M. Hiller1, C. Steinhäuser1, D. Linke3, V. Tchikov2, A. Tholey3, S. Schütze2, J. Brandenburg1 1 Microbial Interface Biology, Research Center Borstel, Borstel 2Institute of Immunology 3Systematic Proteomics and Bioanalytics, Christiane Albrechts University, Kiel, Germany This project explores the molecular mechanisms operating at the interface of Mycobacterium tuberculosis strains of clinical relevance and their first intracellular habitat, the phagosome. This compartment determines intracellular survival and growth of the pathogen, host cell destruction and, ultimately, impacts on the outcome of tuberculosis (TB). Recently we identified clade-specific virulence pattern of M. tuberculosis complex (MTBC) strains: Exclusively human-adapted M. tuberculosis lineages, also termed clade I, comprising modern lineages showed a significantly enhanced capability to replicate inside in macrophages, when compared to clade II strains, which include ancient lineages. This project now focusses on the sequelae differentially induced by MTBC clade I and II strains at the level of the phagosome. A recently developed immunomagnetic isolation procedure to isolate pathogen-containing phagosomes was used to characterize live and heat inactivated MTBC clade I strain-containing phagosomes from macrophages to comparatively evaluate their protein content by mass spectrometry. These data show that viable M. tuberculosis induces the presence of proteins related to type I interferon signalling to its phagosome. Experiments, which focus on the induction and the presence of these factors in M. tuberculosisinfected macrophages will be discussed. 328 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 302 Host-Pathogen interaction and beyond 22/09/2016 The pro-apoptotic factor Bax regulates macrophage necrosis during Mycobacterial infection F. Roca Soler, S. Redmond, L. Ramakrishnan Department of Medicine, University of Cambridge, Cambridge, United Kingdom In the zebrafish-Mycobacterium marinum model for tuberculosis infection, excess TNF triggers necrosis of infected macrophages which leads to exuberant extracellular bacterial growth and host susceptibility (Tobin et al.,2012; Roca and Ramakrishnan, 2013). It has been shown that in infected macrophages, excess TNF induces production of mitochondrial reactive oxygen species (ROS) which activate two death pathways that converge in macrophage necrosis: cyclophilin D-dependent mitochondrial transition pore and acid sphingomyelinase-dependent ceramide production that leads to lysosomal permeabilization. In exploring the events downstream of lysosomal permeabilization, we found that ceramide acts in a Cathepsin D-dependent manner to induce cell death. In the cytosol Cathepsin D initiates the activation of pro-apoptotic factors including Bid and Bax. We engineered Bax mutants that lack its different functional domains and studied the ability of each mutant protein to regulate apoptosis and necrosis. Then we infected Bax-deficient zebrafish larvae expressing the Bax mutants that were unable to induce apoptosis. We found the BH3 domain, which is required for Bax oligomerization during apoptosis, not to be required for necrosis under excess TNF conditions. Rather, an Nterminal transmembrane helix suggested to be the first portion of Bax in contact with the mitochondrial membrane was found to be required for necrosis. Current studies are focused on understanding post-translational modifications of Bax that determine whether activation of the same protein promotes apoptosis or necrosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 329 303 Host-Pathogen interaction and beyond 22/09/2016 A secreted Mycobacterium tuberculosis protein promotes disseminated disease: insights from an outbreak J. Saelens1, D. Sisk1, S. Lee3, J. Stout2, D. Tobin1 1 Molecular Genetics and Microbiology, Tobin lab, Duke University 2Department of Medicine, Division of Infectious Diseases and International Health 3Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, United States Extrapulmonary tuberculosis causes significant morbidity and mortality worldwide. Here, we report an unusual outbreak of tuberculosis with an exceptionally high rate of disseminated disease in immunocompetent adults. We developed a zebrafish model of mycobacterial dissemination to examine specific genetic variants from the outbreak strain. We identify one variant impacting a secreted protein, and demonstrate a role for this variant in altering macrophage motility and the rate of disseminated infections. 330 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 304 Host-Pathogen interaction and beyond 22/09/2016 CD4+ T Cells Recognizing PE/PPE Antigens Directly or via Cross Reactivity are Protective against Pulmonary Mycobacterium tuberculosis Infection F. Sayes2, A. Pawlik2, W. Frigui2, M.I. Gröschel2, S. Crommelynck2, C. Fayolle3-1, F. Cia6, G.J. Bancroft6, D. Bottai5-4, C. Leclerc3-1, R. Brosch2, L. Majlessi2 1 INSERM U1041 2Unité de Pathogénomique Mycobactérienne Intégrée 3Unité de Régulation Immunitaire et Vaccinologie, Institut Pasteur, Paris, France 4Department of Biology 5Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, University of Pisa, Pisa, Italy 6London School of Hygiene and Tropical Medicine, London, United Kingdom Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 331 305 Host-Pathogen interaction and beyond 22/09/2016 Single-cell FRET cytometry assay improves knowledge on the cytosolic access of Mycobacterium tuberculosis R. Simeone2, F. Sayes2, O. Song1, M. Gröschel2, P. Brodin1, R. Brosch2, L. Majlessi2 1 INSERM U1019, CNRS UMR 8204, Lille 2Pathogénomique Mycobactérienne Intégrée, Institut Pasteur, Paris, France The capacity of Mtb to block phagosome-lysosome fusion and allow intraphagosomal replication has long been considered as a characteristic feature of Mtb which is mainly an intraphagosomal pathogen. However, the pathogenic potential of Mtb is intimately linked to the interplay between the host immune response and the persistence of the bacteria and notably the access of Mtb to the host cytosol has numerous consequences on the activation of different axes of innate immunity. To get novel insights into this important cell biological process, we had previously adapted to Mtb studies, a single-cell Fluorescence Resonance Energy Transfer (FRET)-based assay, used in an automated fluorescent microscopy. This approach allowed us to confirm the implicative role of the ESX-1/type VII secretion system of Mtb and the secretion/export of two key virulent protein factors, the 6 kDa Early Secreted Antigenic Target (ESAT-6/EsxA) and the 10 kDa Culture Filtrate Protein10 (CFP-10/EsxB), in the induction of phagosomal rupture. This event, initially evidenced at 3 days post-infection, is then followed by host cell death (Simeone et al., PloS Pathogens 2012). We recently adapted this FRET-based single cell method to multi-color cytoometry. Using this more sensitive and quantitative approach, we studied the ESX-1-dependent phagosomal rupture in several phagocyte types and under different physiological conditions. One of our findings is that the phagosomal rupture occurs as early as 3h post-infection and evolves in a proportional manner with the activation of inflammasome and IFN-b production. By tracking rare infected phagocytes within the lungs and spleens of infected mice, we provided the first and unique evidence that Mtb-induced phagosomal rupture occurs in vivo in different subsets of phagocytes Furthermore, by use of mouse macrophages with a functional or non-functional allele of the Natural Resistance-Associated Macrophage Protein-1 (Nramp-1), we established a link between the inhibition of phagosomal acidification and phagosomal membrane rupture (Simeone et al., PloS Pathogens 2015). Our results propose that Mtb is more than a passive pathogen and that its life cycle in host cells of Mtb is highly complex. Our work open up new research pathways and contribute to a better understanding of the host-pathogen interaction of Mtb. 332 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 306 Host-Pathogen interaction and beyond 22/09/2016 Deciphering the mechanisms of HIV-1 exacerbation induced by Mycobacterium tuberculosis in monocytes/macrophages S. Souriant3, L. Balboa2, K. Pingris3, D. Kviatcowsky2, B. Raynaud-Messina3, C. Cougoule3, I. Mercier3, M. Kuroda4, P. González-Montaner1, S. Inwentarz1, E.J. Moraña1, M.C. Sasiain2, O. Neyrolles3, I. Maridonneau-Parini3, G. Lugo-Villarino3, C. Vérollet3 1 Instituto Prof. Dr. Raúl Vaccarezza, Hospital de Infecciosas Dr. F.J. Muñiz 2Inmunologia de Enfermedades Respiratorias, Instituto de Medicina Experimental (IMEX)-CONICET, Buenos Aires, Argentina 3IPBS, CNRS UMR 5089, Toulouse, France 4Division of Immunology, Tulane National Primate Research Center, Covington, United States Tuberculosis (TB) and the Acquired ImmunoDeficiency Syndrome (AIDS) are two of the deadliest diseases due to a single infectious agent, namely Mycobacterium tuberculosis (Mtb) and the Human Immunodeficiency Virus (HIV-1), respectively. Worsening these public health issues is the fact that HIV-1 is frequently associated with Mtb. The well-known synergy between the two pathogens places an immense burden in the healthcare systems, particularly in resource-limited countries where coinfection with these pathogens are highly prevalent. Therefore, the development of new therapeutic strategies requires a better understanding of the synergistic relationship between Mtb and HIV-1. Both pathogens are able to impair the host immune response and share monocytes/macrophages as common host target cells. Our work aims at deciphering the mechanisms by which Mtb exacerbates HIV-1 infection in monocytes/macrophages. Here, we demonstrate that treatment of human primary monocytes with pleural effusions from TB patients (PE-TB) exacerbates HIV-1 infection (i.e. number of infected cells, virus entry and replication, and formation of multinucleated giant cells), in comparison to treatment with PE obtained from patients with other pulmonary infections (PE-nonTB). This increased HIV-1 infection is associated with a deregulator (M2c) activation program of monocytes/macrophages, characterized by the CD16+CD163+MerTK+CD169+ cell-surface marker signature, also observed on monocytes from the pleural cavity of tuberculosis patients. The establishment of an in vitro culture system, based on the utilization of supernatants from Mtb-infected primary human macrophages, allowed us to differentiate freshly isolated monocytes from healthy donor towards both the M2c activation program and the exacerbation of the HIV-1 infection of these cells, thus mimicking the effects enacted by PE-TB. Using this approach, we identified specific molecular mechanisms responsible for this HIV-1 infection amplification induced by Mtb in monocytes/macrophages, including the increased expression of HIV-1 entry co-receptors CCR5 and CXCR4, and the IL10/STAT3 signaling pathway. Collectively, this study improves our understanding of how Mtb modulates the differentiation process of human monocytes towards a macrophage activation program that increases susceptibility to viral infection and formation of potential cell reservoirs for HIV-1. Finally, this work will provide novel target candidates with diagnostic and therapeutic potential against the comorbidity established between AIDS and TB. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 333 307 Host-Pathogen interaction and beyond 22/09/2016 Modulating activity of the inflammasome during infection with Mycobacterium tuberculosis - a therapeutic adjunct? S. Subbarao, N. Krishnan, B. Robertson MRC Centre for Molecular Bacteriology, Imperial College, London, United Kingdom Introduction: Mycobacterium tuberculosis (Mtb) remains a considerable cause of morbidity and mortality.Whilst our host immunity is pivotal in containing Mtb infection, it has long been recognised as also contributing to tissue damage, cavitatory breakdown, paradoxically facilitating pathogen spread.There is an urgent need for alternative treatments such as immunomodulating agents, as an adjunct to anti-microbial chemotherapy. This would reduce TB-associated tissue destruction, increase anti-microbial access and reduce treatment length. The inflammasome is a cytosolic complex that facilitates pro-inflammatory and microbicidal responses through induction of IL-1b. This is highly regulated at the translational level, but uncontrolled activation is associated with hyper-inflammatory diseases.The induction of the inflammasome by H37Rv varies markedly within the literature. Method: We are examining how clinical isolates interact with the inflammasome and promote activation of IL-1b. We have also investigated whether other immunomodulatory drugs show potential as adjuncts to anti-tuberculosis chemotherapy. Results: Infection of immortalised BMDMs (wild-type, caspase 1/11-/-, NLRP3-/-, AIM2/NLRP3-/-) with Mtb clinical isolates elicits differential inflammasome induction and variable caspase-1 activation. Strains varied in induction of p20 IL-1b, which regulates conversion of pro-IL-1b into IL-1b, and is activated under acidic conditions by cathepsin D. In vitro, therapeutic blockade of the inflammasome (glibenclamide, CRID3, anakinra) demonstrates a dose dependent reduction in IL-1b release and synergism with rifampicin. Conclusion: We demonstrate the importance of using clinical isolates in vitro; the strain dependent inflammasome induction suggests a mycobacterial protease may cleave pro-IL-1b. Differential induction of p20 IL-1b, suggests varied regulation of IL-1b activation through cathepsin D activation. Blockade of the inflammasome in ‘high-risk’ individuals offers a tailored approach to TB therapy. In summary, Mtb has evolved to differentially manipulate the host innate response; compilation of how multiple strains manipulate the host together with the clinical and radiological data could identify individuals who would benefit from immunomodulatory therapy. 334 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 308 Host-Pathogen interaction and beyond 22/09/2016 Mortality of adult tuberculous meningitis is associated with Increased tryptophan metabolism and genetic variation in IDO1 in Indonesia R. Van Crevel3, A. Van Laarhoven3, C. Clish, Clary4, S. Dian1, R. Notebaart3, C. Ruesen3, J. Annisa1, L. Chaidir1, M. Netea3, B. Alisjahbana1, V. Kumar2, A. Ganiem1 1 Health Research Unit, TB HIV working group, Universitas Padjadjaran, Bandung, Indonesia 2 Department of Genetics, University of Groningen, University Medical Centre Groningen, Groningen 3Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboudumc, Nijmegen, The Netherlands 4The Broad Institute of MIT and Harvard, Boston, United States Background: Damaging or ineffective host immune responses may contribute to the high mortality of tuberculous meningitis (TBM), but the underlying mechanisms remain largely unknown. We examined cerebrospinal fluid (CSF) inflammatory markers, metabolomics and host genetics in a cohort of adult TBM patients in Indonesia. Material/methods: 307 HIV-negative TBM patients were included based on typical CSF leukocyte count and CSF:blood glucose ratio, and positive M. tuberculosis culture. Metabolomics analysis using liquid chromatography mass spectrometry was performed on CSF and serum of 33 culture positive TBM patients and 22 controls with CSF cell count < 5 cells/μl and negative mycobacterial culture. 358 metabolites were identified in CSF and 397 in serum of cases and controls. Data were visualized using principal component analysis (PCA), classified with partial least square (PLS) analysis, and correlating to diagnosis and survival status through pathway enrichment analysis. Polymorphisms in genes coding for enzymes in identified pathways were genotyped using an Illumina HumanCoreExome-24 BeadChip, and correlated to survival. Results: PCA showed clear visual separation of TBM patients and controls based on metabolite concentrations in CSF but not in serum. In CSF this corresponded to a 94% accuracy for separating patients from controls and 75% for TBM survivors versus non-survivors. Pathway analysis showed thiamine metabolism, cyanoamino acid metabolism, aminoacyl-tRNA-biosynthesis and tryptophan metabolism as top upregulated pathways in TBM (false discovery rates p< 10-20). CSF tryptophan was 9.8-fold lower in patients versus controls, while its metabolites (kynurenine, quinolinate) were all higher in patients, reflecting strongly increased Indoleamine 2,3-dioxygenase 1 (IDO1) activity. Interestingly, CSF tryptophan levels were lower especially in patients who survived (31-fold compared to controls) and much less affected (4-fold compared to controls) in those who died. Polmorphisms in IDO1, which codes for the rate-limiting enzyme in conversion of tryptophan to kynunrenine were associated with survival. Conclusions: This first metabolomics study of TBM shows strongly increased tryptophan metabolism in TBM. Tryptophan is essential for growth of M. tuberculosis, and tryptophan metabolism skews the balance between regulatory and effector T-cells. Our results suggest that induction of tryptophan metabolism could limit M. tuberculosis growth and damaging immunopathology in TBM. these findings may help development of new immunomodulatory strategies for TBM and could also aid in its diagnosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 335 POSTER SESSION 4 Friday, September 23 SUMMARY Drug design-new developments .................................................. 339 From animal models to man ........................................................ 380 309 Drug design-new developments 23/09/2016 Bacteriocin AS-48 is active against Mycobacterium tuberculosis C. Aguilar Pérez5-6, B. Gracia5-6, A. Vitoria5-4-6, R. Cebrián1, M. Maqueda1, J.A. Ainsa2-3-5-6 1 Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Granada 2CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid 3Instituto de Biocomputacion y Fisica de Sistemas Complejos, BIFI, Universidad de Zaragoza 4Servicio de Microbiología, Hospital Clínico Universitario Lozano Blesa 5Instituto de Investigacion Sanitaria de Aragon (IIS-Aragon) 6Genética de Micobacterias, University of Zaragoza, Zaragoza, Spain The increasing incidence of multi-drug resistant strains of Mycobacterium tuberculosis and the very few drugs available for treatment is promoting the development of a new line of drugs that try to solve these problems. Following this idea, we explored the antibacterial peptide AS-48, produced by E. faecalis, which is targeting the bacterial membrane, and it is active against several gram-positive bacteria. Targeting the membrane is an advantage considering that there are a lot biosynthetic pathways involved, so it is less likely to select a resistance mutation. This study has assessed the bactericidal effect of AS-48 in M. tuberculosis strains, including H37Rv and other clinical and reference strains, and also against some non-tuberculous clinical mycobacterial species. We demonstrated that AS-48 has a bactericidal action against M. tuberculosis and other mycobacterial species. The combination of AS-48 with either lysozyme or first line drugs (commonly used in the treatment of tuberculosis) increases bactericidal action of AS48, showing a synergic interaction. Under these conditions, AS-48 kills M. tuberculosis at a lower dose, and exhibits a MIC (Minimal Inhibitory Concentration) close to some of the first line anti-TB agents. In addition, we assayed cytotoxicity of AS-48 against THP-1 and MHS macrophage cell lines, and found that at concentrations close to the MIC of AS-48 we could not detect any cytotoxic effect. In summary, we consider that bacteriocin AS-48 has an interesting potential in antituberculosis therapy because of its activity against mycobacterial species and its low cytotoxicity against cell lines. KeyWords: AS-48, antimicrobial peptide, antituberculosis activity, synergy. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 339 310 Drug design-new developments 23/09/2016 Pathway-based whole cell screening of compounds against mycobacteria: targeting genome integrity W. Aragaw2, U. Lakshmanan1, C. Ling1, A. Poulsen1, J. Hill1, Y. Yamada2, T. Dick2 1 Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR) 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Tuberculosis (TB) continues to pose heavy toll on global health. The increasing trends of drug resistance along with other etiologic factors made the TB disease more difficult to control. A most serious aspect of the problem of drug resistance is the emergence of strains resistant to many of the standard anti-TB drugs. To counter the spread of drug resistance and to effectively control the disease epidemic as a whole, new medicines are demanded. A major challenge to developing new TB drugs is identifying whole cell active compounds with their putative targets. In this study, we employ a cell-based high throughput screening approach coupled to pathway reporter system to find novel target-lead couples. We broadly target genome integrity with a premise that inhibition of the indispensable protein components involved in mycobacterial genome stability would result in desirable therapeutics. To identify chemotypes that interfere with genome integrity, we use a recombinant Mycobacterium bovis BCG harbouring an integrative plasmid with mCherry reporter gene transcriptionally fused to a DNA damage inducible promoter (BCG::pMV306-PrecA-mCherry). In the primary screening, compounds with growth inhibition activity against BCG are identified from a commercial library (Enamine) of 70,000 synthetic compounds. The screen yielded 18 hit compounds which upon re-test showed dose dependent activity. All confirmed hits were then subjected to the subsequent pathway specific screening. Two whole cell actives (compounds E04 and E08) elicited a dose dependent induction of mCherry expression in the DNA damage reporter assay signifying their interference with mycobacterial genome integrity. In vitro potency studies in BCG showed a pattern of concentration dependent growth inhibition (MIC50 = 6.25 µM). With respect to cidal activity, both compounds exhibited similar kill pattern with a 3 log reduction in CFU/ml at 25 µM. Cytotoxicity profiling revealed no apparent in vitro toxicity and haemolytic activity up to 10X the MIC concentration. To provide further evidence for the possible target(s) involved, spontaneous resistant mutants are being isolated and characterized. 340 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 311 Drug design-new developments 23/09/2016 Identification of novel drug target pathways and scaffolds to combat tuberculosis G. Arora VIDRC, TB LAB, Translational health science and technology institute, Delhi-Ncr, India Due to emergence of drug resistant TB strains, there is an urgent need to validate newer drug target pathways and scaffolds with novel mechanism of action. In my lab we are validating amino acid and carbon metabolism pathways as drug targets to combat M. tuberculosis. L-serine biosynthetic pathway represents one such pathway, in which phosphoserine phosphatase (PSP), a key essential enzyme involved in conversion of O-phospho-L-serine to L-serine, acts at the final step. We have done detailed biochemical characterization of PSP homolog in M. tuberculosis, SerB2 and developed a malachite green based high throughput assay system to identify novel SerB2 specific inhibitors. We have identified 10 novel scaffolds that were structurally different from known PSP inhibitors and few of these were highly specific in their ability to inhibit SerB2 enzyme in comparison to human PSP enzyme, non-cytotoxic against mammalian cell lines and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline were bactericidal in activity and killed intracellular bacteria in a dose dependent manner. We are also focusing on carbon metabolic pathways such as Tricarboxylic acid pathway, which is a converging point of many catabolic pathways of central carbon metabolism, to understand how they play a key role in physiology and virulence of M. tuberculosis and to identify novel scaffolds that target these pathways. In addition, using whole-cell screening approach, we identified 28 compounds out of 5000 screened as novel inhibitors of M. tuberculosis from small molecule library. Future experiments would include cytotoxicity evaluation of the hits against mammalian cell lines and their mechanistic studies. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 341 312 Drug design-new developments 23/09/2016 Boromycin kills mycobacterial persisters without detectable resistance W. Moreira, D. Aziz, T. Dick Department of Microbiology and Immunology, National University of Singapore, Singapore, Singapore Boromycin is a boron-containing polyether macrolide antibiotic isolated from Streptomyces antibioticus. It was shown to be active against Gram positive bacteria and to act as an ionophore for potassium ions. The antibiotic is ineffective against Gram negative bacteria where the outer membrane appears to block access of the molecule to the cytoplasmic membrane. Here we asked whether Boromycin is active against Mycobacterium tuberculosis which, similar to Gram negative bacteria, possesses an outer membrane. The results show that Boromycin is a potent inhibitor of mycobacterial growth (MIC50 = 80 nM) with strong bactericidal activity against growing and nongrowing drug tolerant persister bacilli. Exposure to Boromycin resulted in a rapid loss of membrane potential, reduction of the intracellular ATP level and leakage of cytoplasmic protein. Consistent with Boromycin acting as a potassium ionophore, addition of KCl to the medium blocked its antimycobacterial activity. In contrast to the potent antimycobacterial activities of the polyether macrolide, its cytotoxicity and haemolytic activity were low (CC50 = 30 uM, HC50 = 40 uM) with a selectivity index of more than 300. Spontaneous resistant mutants could not be isolated suggesting a mutation frequency of less than 10-9 / CFU. Taken together, the results suggests that targeting the mycobacterial membrane / ion gradient may be an attractive chemotherapeutic intervention to kill otherwise drug tolerant persister bacilli, and to slow down the development of genetic antibiotic resistance. 342 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 313 Drug design-new developments 23/09/2016 Development and validation of low-cost dna amplification-based assays for rapid diagnosis of tuberculosis and detection of mutations associated with rifampicin resistance in moroccan patients E. Bentaleb3-2, M. Abid4, M.D. El Messaoudi1, S. Amzazi2, H. Sefrioui3, H. Ait Benhassou3 1 Laboratoire de Tuberculose, Institut Pasteur du Maroc, Casablanca 2Department of Biology, Laboratory of Biochemistry and Immunology, Mohammed V University of Rabat 3Medical Biotechnology Center, Moroccan Foundation for Advanced Science, Innovation and Research ( MAScIR), Rabat 4Laboratoire de Génétique Mycobactérienne, Institut Pasteur du Maroc, Tangier, Morocco Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. Each year, about 9 million people develop the disease and approximately 1.5 million of them die. In Morocco as in most developing countries where the timely and accurate diagnosis of TB remains a great challenge, TB constitutes a major public health threat with 30,000 new cases each year. Furthermore, control of TB has been complicated by the emergence of multidrug-resistant (MDR) M. tuberculosis strains. Currently, the conventional methods such as light microscopy and classic culture used to detect Mycobacterium tuberculosis in Moroccan patients, especially in less equipped regions are complex, unreliable, labor-intensive, technically challenging and time-consuming. In that context, we are actually developing at MAScIR (Moroccan foundation for Advanced Science, Innovation and Research) and for the first time in Morocco a new TB detection test based on loopmediated isothermal amplification technology (LAMP). Loop-mediated isothermal amplification (LAMP) has emerged as a powerful tool for rapid diagnosis of Tuberculosis in endemic settings of resource-poor countries, due to its simplicity, rapidity and cost-effectiveness. Our TB-LAMP assay is a single step LAMP (SS-LAMP) based on the amplification of IS6110 specific to Mycobacterium tuberculosis complex (MTBC). It has been successfully tested on DNA extracted from culture and also directly on liquefied sputum samples without any prior DNA extraction or denaturation,and also without the final enzymatic inactivation step. On the other hand, to encounter the alarming increase in the global incidence of MDR-TB infection, a need for methods that can rapidly and effectively identify drug-resistant cases becomes an emergency. Thus, we are currently developing a highly sensitive, accurate, and rapid and an affordable assay, based on qPCR-HRM technology, able to detect and identifiy mutations in rpoB gene responsible of rifampicin resistance to Mycobacterium tuberculosis, by using five recombinant plasmids as reference. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 343 314 Drug design-new developments 23/09/2016 In vitro and in vivo profiling of a new anti-Tuberculosis serie discovered by phenotypic screening I. Blanc, L. Goullieux, C. Lair, L. Somody, C. Briot, C. Cantalloube, X. Boulenc, E. Fontaine, M. Magnon, M. Doubovetzky, A. Milla, A. Oualim, A. Pellet, E. Bacqué, S. Lagrange, L. Fraisse R&D, SANOFI, Marcy L'etoile, France Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb). It is the first leading infectious cause of death worldwide. In order to address this unmet medical need, Sanofi has entered into collaboration with TB Alliance, a key player in the TB drug discovery field, with the objective to develop new combinations with the potential to shorten treatment of TB. This poster will report the discovery and properties of a novel anti-TB series that stems from a growth inhibition phenotypic screening. We will present, in particular, the in vitro and in vivo profile of RA01, an optimized analogue of the series. in vitro, RA01 is very potent against replicating Mtb, extra- or intra-macrophages as well as against representative clinical and mono-resistant Mtb strains. RA01 also exhibits a significant serum-shift though retaining submicromolar potency and is weakly active on non-replicating Mtb. In vivo, RA01 showed good exposure and satisfactory PK parameters in mice, at pharmacologically active doses. It is efficacious in an Highly Acute TB mouse model (MED at 100 mg/kg). It has also shown similar efficacy in an Acute TB model along with a weak effect in a Chronic TB model that features Non Replicating Mtb. Overall, RA01 is a potent inhibitor of Mtb growth. Combined to other antibiotics, it has the potential to improve current TB treatments. 344 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 315 Drug design-new developments 23/09/2016 iniBAC induction is vitamin B12 and MutAB-dependent in Mycobacterium marinum M. Boot, M. Sparrius, K. Jim, S. Commandeur, R. Van De Weerd, A. Speer, W. Bitter Medical Microbiology and Infection control, VU medical center, Amsterdam, The Netherlands Although the drug targets of most first-line antibiotics for Mycobacterium tuberculosis have been identified, less research has focused on the intra-bacterial stress response that follows upon treatment with antibiotics. Studying the roles of drug-induced stress genes may lead to the identification of crucial stress-coping mechanisms that can provide additional drug targets to increase treatment efficacy. The three-gene operon iniBAC has no known function and is strongly up-regulated upon treatment with isoniazid and ethambutol. We have confirmed upregulation of the iniBAC genes in Mycobacterium marinum and found that the operon is also induced during cell infection experiments, albeit that the response is more heterogenic as compared to bacterial culture. Next, we set out to elucidate the genetic network that results in iniBAC induction in M. marinum. By means of transposon mutagenesis, we identified that the operon is highly induced upon mutations in genes encoding enzymes of the vitamin B12 biosynthesis pathway and the vitamin B12-dependent methylmalonyl–CoA-mutase (MutAB). Subsequent lipid analysis showed that a mutA::tn mutant has decreased PDIM levels, suggesting a link between iniBAC induction and the production of methylbranched lipids. Moreover, a similar screen in M. bovis BCG identified PDIM mutations to cause the up-regulation of iniBAC genes. We have attempted to determine the metabolic cue that causes iniBAC upregulation and conclude that there is a strong correlation between the utilization of fatty acids and the induction of the iniBAC operon. Based on these data, we propose that iniBAC is induced in response to mutations that cause defects in the biosynthesis of methyl-branched lipids. The resulting metabolic stress caused by these mutations, or caused by ethambutol or isoniazid treatment, may be relieved by iniBAC to increase of bacterial survival. We are currently dissecting the role of IniBAC proteins in this process. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 345 316 Drug design-new developments 23/09/2016 Developement of new synthetic inhibitors of Mycobacterium tuberculosis DprE2 G. Chiodarelli1-3, G. Besra2, L.R. Cox3, J. Lelievre1, M. Cacho-Izquierdo1 1 GlaxoSmithKline, Tres Cantos, Spain 2School of Biosciences 3School of Chemistry, University of Birmingham, Birmingham, United Kingdom Mycobacterium tuberculosis is the pathogen responsible for human tuberculosis (TB). TB is still one of the leading killers accounting about 9 million cases and 1.7 million deaths every year. The current TB treatment is a six-month regimen with the forty years old drugs rifampicin, isoniazid, ethambutol and pyrazinamide. The length of the treatment makes compliance difficult thereby favouring the emergence of the resistant strains multi-drug resistant TB (MDR-TB) and extensively-drug resistant TB (XDR-TB), for this reason is important to find more potent drugs and shorter treatment. DprE1 and DprE2 are the two subunits of the epimerase enzyme involved in the biosynthesis of decaprenyl phosphor-D-arabinofuranose (DPA). DPA is the only furan sugar donor used by the bacterium to synthesise the arabinogalctan (AG) and lipoarabinomannan (LAM), two of the major constituents of the bacteria cell wall. Because of the essentiality and broad range of inhibitors targeting DprE1, this enzyme is considered one of the most interesting targets for the development of new anti-tubercular agents. In contrast with DprE1, DprE2 has received far less attention even if it has been demonstrated that DprE2 is essential as well. The goal of this project is to develop a lead compound from the DprE2 hit GSK426032A discovered by high throughtput screening by GSK in Tres Cantos (Spain). At the moment the SAR investigation around our hit is limited by the lack of isolated and purified DprE2, this does not allow us to have access to crystal structures of the protein and enzymatic assays. Our approach to investigate the SAR of the hit is to synthesise and test against M. tuberculosis whole cells several series of derivatives generated by modifications in different sites in the original structure of the hit. The information derived from the SAR studies can help us to obtain derivatives with improved physical chemical properties and safety profile. 346 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 317 Drug design-new developments 23/09/2016 Relational Sequencing TB Data Platform (ReSeqTB): a global collaborative effort to build and deploy a centralized relational data warehouse for investigating the correlations between M. tuberculosis genotypes and phenotypic drug resistance P. Miotto2, E. Tagliani2, B. Tessema6, I. Comas4-3, N. Hillery8, L. Chindelevitch1, M. Ezewudo7, J. Posey7, T. Rodwell8, D. Dolinger6, M. Schito9, C. Reseqtb Consortium5 1 Simon Fraser University, Vancouver, Canada 2Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan, Italy 3CIBER on Epidemiology and Public Health, Madrid 4Tuberculosis Genomics Unit, Institute of Biomedicine of Valencia, Valencia, Spain 5ReSeqTB, Consortium 6Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland 7Centers for Disease Control and Prevention, Atlanta 8University of California, San Diego 9Critical Path institute, Tucson, United States A global consortium has been established to develop the Relational Sequencing TB Data Platform (ReSeqTB, https://platform.reseqtb.org/), aimed at understanding the genetic basis of drug resistance (DR) in Mycobacterium tuberculosis (MTB) by correlating genotypic data with phenotypic drug susceptibility testing (DST) and patient metadata (e.g. treatment outcomes). ReSeqTB is a standardized, validated analysis pipeline, relational database and curated archive, designed to be a plug-and-play and cloud-based bioinformatics solution for identifying canonical and novel drug-resistance related genetic variants from raw whole genome sequencing files. The platform has been developed to process and share global genotypic, phenotypic and related metadata on MTB with transparent, regulatory compliant methodology and clear documentation, while ensuring contributor-specific intellectual property and data privacy is preserved. The variant analysis pipeline works with raw Illumina® fastq data and implements a series of quality control thresholds. In parallel we have developed a statistical framework based on a combination of likelihood ratios (LR) and p-values. Mutations are categorized as having “high”, “moderate” or “minimal” confidence for association with DR, “not associated” or “indeterminate”. Both the variant pipeline and the statistical drug resistance association framework have been validated using 90 MTB strains drawn from a well-characterized drug resistant strain collection. The results were compared with other well-documented analysis and drug resistance databases like the PhyResSE web tool and the Knowledge Synthesis TBDR DB (www.TBDR.org). Once our statistical approach was validated we aimed at expanding the range of mutations significantly associated to resistance. We applied our approach to data obtained from a comprehensive systematic literature review for mutations and associated liquid/solid phenotypic DST for rifampicin, isoniazid, fluoroquinolones second-line injectable drugs, and pyrazinamide. The total number of MTB isolates used for evaluation ranged from 571 to 9537. Forty-four mutations with moderate to high confidence associations with DR were identified for the testing data set. The data set is being utilized by ReSeqTB as a baseline for drug resistance prediction and will be dynamically validated and updated as retrospective and prospective NGS data fills the ReSeqTB data platform. The final aim is to provide a tool for diagnostics development and for clinical drug resistance prediction. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 347 318 Drug design-new developments 23/09/2016 Pulmonary co-administration of the synergic drugs Ethionamide and Booster using aerosolized nanoparticles: a promising strategy to treat tuberculosis J. Costa-Gouveia1, E. Pancani3, S. Jouny1, V. Delorme1, G. Salzano3, N. Willand2, A. Baulard1, R. Gref3, P. Brodin1 1 U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, Univ. Lille, CNRS, Inserm, CHU Lille, Pasteur Institute of Lille 2U1177 - Drugs and Molecules for living Systems, Univ. Lille, Inserm, Pasteur Institute of Lille, Lille 3UMR 8214 - ISMO, Univ. Paris Sud, Univ. Paris-Saclay, CNRS, Orsay, France Tuberculosis (TB) is a leading cause of death worldwide. The use of ethionamide (ETH), an important second line anti-TB drug, is hampered by low oral bioavailability and the onset of severe side effects. Recently discovered “booster”, molecules strongly increasing the efficacy of ETH, could restore the use of this drug and thus improve the current clinical outcome of drug-resistant TB. To investigate the simultaneously delivery of ETH and its booster BDM41906 in the lungs, we coencapsulated the synergic compounds in biodegradable polymeric nanoparticles (NPs), overcoming the bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of the Booster. The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform, showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, “green” β-cyclodextrin (pCD) NPs displayed the best physiochemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mice lungs using a Microsprayer®, led to a significant decrease of the pulmonary bacterial load as compared to untreated mice. This study paves the way for a future use of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair. Acknowledgments: Financial support for this work was provided by the European Community (CycloN Hit Grant n° 608407), the Feder (12001407 (D-AL) Equipex Imaginex BioMed) and the Region Nord Pas de Calais (convention n° 12000080). 348 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 319 Drug design-new developments 23/09/2016 Identification of novel mutations that confer resistance to pyrazinamide in virulent Mycobacterium tuberculosis N. Dillon, Y. Minato, A. Baughn Microbiology, University of Minnesota, Minneapoils, United States Pyrazinamide is often characterized as an irreplaceable component of the current short course therapy, yet its efficacy is threatened by the emergence of drug resistance. One third of pyrazinamide resistant clinical isolates have no identified mechanism of drug resistance. The elucidation of novel pyrazinamide resistance mutations from clinical isolates is complicated by the absence of parental strains for sequence verification. We therefore sought to generate pyrazinamide resistant mutants in the virulent laboratory strain H37Rv through transposon mutagenesis. Anticipating a preponderance of pncA mutations during pyrazinamide exposure due to its characterized role in resistance, we instead selected isolates on pyrazinoic acid, the bioactivated form of pyrazinamide, negating the selective advantage of pncA mutations. Prior to this work, only a single mutation, in panD, had been found to lead to pyrazinoic acid resistance. Using our mutagenesis screen we selected sixteen random colonies from the transposon library for further phenotypic characterization. Phenotypic characterization of these strains demonstrated that each isolate was resistant to both pyrazinoic acid and pyrazinamide under various culture conditions. These isolates did not have increased resistance to isoniazid, indicating resistance is specific to pyrazinamide. Of the sixteen colonies we identified nine unique insertions not previously linked to pyrazinamide resistance. We therefore employed a tn-seq based approach to analyze the remainder of the library. In all, we identified a little over one hundred different genes whose mutagenesis led to pyrazinoic acid resistance. The identified insertions clustered to genes responsible for central metabolism, proteosome function, and cellular stress response. Comparison of our data with published genomes for clinical isolates with no mutations linked to pyrazinamide resistance now allows us to identify the polymorphisms responsible for the loss of drug activity in vivo. Through isolation and identification of the mutations responsible for one third of resistant clinical isolates we can begin understanding and combating the emergence of pyrazinamide resistance in Mycobacterium tuberculosis infections. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 349 320 Drug design-new developments 23/09/2016 Does the side-chain of delamanid and Q203 convey special affinity for Mycobacteria? tested on proven and possible anti-Mtb alkaloid derivatives R. Feilcke, P. Imming Institute of Pharmacy, Pharmaceutical Chemistry, Martin-Luther-University Halle-Wittenberg, Germany, Halle (saale), Germany In 2014 (EMA, 2014), delamanid met the need for new antitubercular drugs. This was an immense progress against the rising number of cases with XDR and MDR-tuberculosis, but the possibilities of therapy still lack highly active substances that will lead to a short treatment regimen. The side chain of this molecule attracted our attention. It is almost similar to the side chain of Q203 that is currently in phase I clinical study (WGND, 2016). We investigated if this side chain would confer antimycobacterial activity to other scaffolds. We therefore attached 4-(4-(trifluoromethoxy)phenoxy)piperidine to different heterocycles and submitted the derivatives to whole cell antimycobacterial assays. Figure 1: Delamanid and Q203 with a similar side chain. Core structures were chosen among established antibiotics e.g. metronidazol as well as among alkaloids e.g hippadine. This pyrrolophenanthrinone is derived from Amaryllidaceae alkaloids and shows some antibacterial activity. Not much attention was paid to this class of compounds as antibacterials yet, even though the extract of the leaves of Crinum purpurascens showed good activity in some tests (Nkanwen et al., 2009). In order to facilitate access to this alkaloid class, a short synthetic route was investigated that led to one of the core structures of the tested molecules. Figure 2: Core structure derived from pyrrolophenanthrinone alkaloids. Results of the whole cell assays as well as insights into synthetic details will be presented on the poster in September 2016. References: • European Medicines Agency (EMA), Summary of the European Public Assessment Report (EPAR) for Deltyba, [http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/002552/h uman_med_001699.jsp&mid=WC0b01ac058001d124], Accessed on 25th of May 2016 • Working Group on New TB Drugs (WGND), Drug Pipeline, [http://www.newtbdrugs.org/pipeline.php], Accessed on 25th of May 2016 • Nkanwen, E., et al. (2009), Antibacterial agents from the leaves of Crinum purpurascens herb (Amaryllidaceae), African health sciences 9, (264-269) 350 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 321 Drug design-new developments 23/09/2016 Covalent Modification of the Mycobacterium tuberculosis FAS-II Dehydratase by Isoxyl and Thiacetazone A. Grzegorzewicz4, N. Eynard1-2, A. Quémard1-2, E.J. North5, A. Margolis4, J.J. Lindenberger6, J. Korduláková3, V. Jones4, P.J. Brennan4, R.E. Lee5, D.R. Ronning6, M.R. Mcneil4, M. Jackson4 1 Département Tuberculose et Biologie des Infections, CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale) 2Université de Toulouse, Toulouse, France 3Department of Biochemistry, Comenius University, Bratislava, Slovakia 4Microbiology Immunology and Pathology, Colorado State University, Fort Collins 5Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis 6Department of Chemistry and Biochemistry, University of Toledo, Toledo, United States Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation, and the nature of their activated forms remained unknown. Our data demonstrates that both ISO and TAC specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase, thereby inhibiting HadAB activity. The results unveil for the first time the nature of the active forms of ISO and TAC and explain the basis for the structure−activity relationship of and resistance to these thiourea prodrugs. The results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 351 322 Drug design-new developments 23/09/2016 Whole blood Bactericidal Activity of faropenem with and without rifampicin against Mycobacterium tuberculosis M. Gurumurthy1, R. Verma1, C. Naftalin1, D. Hee1, Q. Lu3, K.Y. Seng2, L. Lee1, N. Paton1 1 Department of Medicine 2Department of Pharmacology, National University of Singapore 3Singapore Clinical Research Institute, Singapore, Singapore Background: In vitro studies indicate that faropenem (an oral penem) has activity against TB and synergy with rifampicin and may have potential for repurposing as a TB drug. We investigated this further in a whole-blood bactericidal activity (WBA) trial. Methods: Healthy volunteers were randomised to receive a single dose of faropenem (600mg; with amoxicillin/clavulanic acid 500mg/125mg; FAR-AMC group, n=8); rifampicin (10mg/kg; RIF group; n=14); or the combination (RIF-FAR-AMC group; n=14). Blood was drawn pre-dose and at 0.5, 1, 2, 3, 4, 5, 6 and 8 hours post-dose. Drug levels were measured using liquid chromatography-tandem mass spectrometry. WBA was measured by inoculating blood samples with Mycobacterium tuberculosis (Mtb) H37Rv and estimating the change in bacterial colony forming units (CFU) at the end of 72 hours’ incubation. The primary outcome measure was cumulative WBA at 8 hours following drug ingestion. Results: We found no evidence of activity in the FAR-AMC group (cumulative WBA 0.06ΔlogCFU; p=0.99 vs zero change). Cumulative WBA showed a trend in favour of the RIF-FAR-AMC group at 8 hours (-0.57±0.09 and -0.60±0.10ΔlogCFU in RIF and RIF-FAR-AMC groups respectively; p=0.113; p=0.023 for comparison over the first 1 hour post-dose). The Cmax and AUC of faropenem were 5.4mg/L and 16.2h*mg/L respectively, and MIC against Mtb H37Rv was 5-10 mg/L. Conclusions: Faropenem does not appear to be useful as TB drug when used alone, possibly due to low plasma levels relative to the MIC. However, we found limited evidence of modest synergy with rifampicin that may merit further testing in clinical trials. This study is registered in Clintrials.gov (NCT02393586). 352 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 323 Drug design-new developments 23/09/2016 3,5-Dinitrobenzylsulfanyl heterocycles as novel antitubercular agents: structure – activity relationship study G. Karabanovich1, L. Valášková1, J. Němeček1, J. Roh1, J. Stolaříková2, K. Mikušová3, R. Székely4, P. Pávek1, V. Klimešová1, A. Hrabalek1 1 Department of Inorganic and Organic Chemistry, Charles University, Faculty of Pharmacy in Hradec Kralove, Heyrovského, Hradec Králové 2Department of Bacteriology and Mycology, Regional Institute of Public Health, Partyzánské Náměstí, Ostrava, Czech Republic 3Department of Biochemistry, Comenius University, Faculty of Natural Sciences, Mlynská Dolina, Bratislava, Slovakia 4Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland In this work we deal with the synthesis and structure-activity relationships of 1-alkyl/aryl-5-[(3,5dinitrobenzyl)sulfanyl]-1H-tetrazoles, 5-substituted-2-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 4,5-disubstituted-3-[(3,5-dinitrobenzyl)sulfanyl]-4H-1,2,4-triazoles as novel antitubercular agents.[1, 2] The influence of the position of nitro groups, introduction of additional group to 3,5dinitrobenzylsulfanyl moiety, reduction of one nitro group or its replacement with other electronwithdrawing group on antimycobacterial activity were intensively studied. The role of heterocycle and the linker between heterocycle and 3,5-dinitrophenyl moiety was also widely explored. Compounds with the most promising activities were further evaluated against multi-drug resistant tuberculosis strains, other bacteria or fungi. The effects on viability of mammalian cell lines, e.g. isolated human hepatocytes, were also studied. All derivatives bearing 3,5-dinitrobenzyl moiety exhibited high antimycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains and showed no activity against other bacteria or fungi. Moreover, these compounds exhibited low in vitro toxicity in mammalian cell lines. The highest efficacy was displayed by 5-substituted-2-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles and 1,3,4-thiadiazoles with minimum inhibitory concentrations as low as 0.03 µM (0.011-0.026 µg/mL). Metabolic radiolabeling experiments indicated that 3,5-dinitrobenzylsulfanyl-1,3,4-oxadiazoles inhibit the synthesis of mycobacterial nucleic acids. However, the determination of specific target in mycobacterium is under evaluation. The study was supported by the Czech Science Foundation project (14-08423S) and Charles University in Prague (SVV 260 291). 1. Karabanovich G. Eur. J. Med. Chem. 2014, 82, 324-340. 2. Karabanovich G. J. Med. Chem. 2016, 59 (6), 2362–2380. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 353 324 Drug design-new developments 23/09/2016 rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary tuberculosis A. Khosravi Boroujeni2, A. Ghadiri1, H. Meghdadi2, A.H. Sina3, M. Mirsaeidi4 1 Immunology and Cell & Molecular Research Center 2Microbiology and Infectious & Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences 3Danesh Medical Laboratory, Ahvaz, Iran 4Department of Medicine, University of Miami, Miller School of Medicine, Miami, United States Background: The current study was aimed to analyze the mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB) using the nested rpoB-PCR technique combined with cloning. Methods: Seventy formalin-fixed, paraffin-embedded samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Results: In total, 67 (95.7%) were positive for the presence of MTB. Sequence analysis of the 81bp region of rpoB gene, demonstrated mutations in 41 (61.2%) out of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln à His, at codon 513, presented in 30 (75.6%) isolates. Conclusions: This study confirmed that 75.6% of RIF-resistant strains have sequence alterations in codon 513 of the 81bp region of rpoB gene. In addition, the simultaneous mutation at the codons 512 and 513, were demonstrated in 34.3% of the isolates. 354 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 325 Drug design-new developments 23/09/2016 Genetic diversity of multidrug resistant Mycobacterium tuberculosis strains isolated from tuberculosis patients in Iran, using MIRU-VNTR technique A. Khosravi Boroujeni, S. Khandan Dezfuli1, A. Hashemi Shahraki2, M. Hashemzadeh1 1 Infectious & tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz 2Epidemiology, Institute Pasteur of Iran, Tehran, Iran Introduction: Tuberculosis (TB) is considered as one of the most important infectious diseases in the world, and recent rise and spread of multi-drug resistant (MDR) Mycobacterium tuberculosis (MTB) strains, have made the matter worsened. Therefore, there is an urgent need to understand and estimate the incidence of tuberculosis in large and small biographical ranges by aid of molecular biology methods. Due to the importance of TB prevalence in Iran, this study was designed to investigate the genetic diversity among MDR strains of MTB by MIRU-VNTR typing scheme. Materials and Methods: A total of 88 drug resistant M. tuberculosis isolates belong to pulmonary TB cases were collected from several TB reference centers of Iran. Drug susceptibility testing for Isoniazid and Rifampin was performed using the agar proportion method and MDR isolates were underwent genotyping by using 12-locus- based MIRU-VNTR typing. Results: On performing proportion method, 22 isolates were identified as MDR. By typing of MDR isolates using 12-loci MIRU-VNTR technique, high diversity were demonstrated in MDR strains and these were classified into 20 distinct MIRU-VNTR genotypes, including 2 clusters (2 strains/cluster) and 18 unique patterns. MIRU loci 10 and 26, were the most discriminatory loci with 8 and 7 alleles respectively.; while MIRU loci 2, 20, 24 and 39 were found to be the least discriminatory with 1-2 alleles each. We noticed a mixed infection in isolate 53, as this isolate comprised simultaneous 2 alleles in MIRU loci 40, 10, 16 and 39. The discriminatory power of MIRU-VNTR was high (HGDI=0.991%) in present study with clustering rate of 0.086%. Conclusions: The results show that MIRU-VNTR typing is a useful tool for studying genetic diversity of MTB in regional settings. Besides, this genotypic analysis will help the health sectors to construct an effective control program for the disease. Additionally, it can detect mixed infection which can facilitate management of treatment. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 355 326 Drug design-new developments 23/09/2016 Oligotuftsin-based Peptide Carriers for Novel Antimycobacterial Active Agents: Synthesis and In vitro Evaluation M. Krátký1, J. Vinšová1, Z. Baranyai3, S. Bösze3, N. Szabó2 1 Department of Inorganic and Organic Chemistry, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Hradec Králové, Czech Republic 2Laboratory of Bacteriology, Korányi National Institute for Tuberculosis and Respiratory Medicine 3MTA-ELTE Research Group of Peptide Chemistry, Budapest, Hungary The global tuberculosis epidemic and increasing emergence of drug-resistant Mycobacterium tuberculosis strains as well as non-tuberculous mycobacteria (NTM) call for intensive research on new therapeutic interventions including novel drugs and drug delivery systems (DDS) for them.1 DDS have been used largely for the modification of inconvenient properties of bioactive molecules. Illustratively, they are able to increase solubility, bioavailability or decrease toxicity. Additionally, they are useful in targeted drug delivery.2 We selected tuftsin-based oligopeptides as potential carriers for our small antimycobacterial active molecules. Tuftsin derivatives are non-toxic, non-immunogenic and biodegradable. They also stimulate immune response and target macrophages specifically, thus enhancing cellular uptake and selectivity index. A pilot study with isoniazid indicates that this concept seems to be viable.3 Salicylanilides (2-hydroxy-N-phenylbenzamides) have exhibited a significant in vitro activity against both tuberculous and NTM including drug-resistant strains at low micromolar concentrations. However, they share a significant cytotoxicity and poor solubility.4 These obstacles can be overcome, i.a., by employment of DDS. Peptide carriers based on repeated oligotuftsin sequence [TKPKG]n were obtained using solidphase synthesis (Fmoc/tBu strategy, rink amide MBHA resin, diisopropylcarbodiimide/HOBt in Nmethylpyrrolidone). N-Terminus and/or lysine side chain amino group(s) were modified by various substituents (carboxylic acids, fluorescent labels, short peptides spacers, aminooxyacetic acid etc.). Carriers were cleaved from resin, purified and then coupled with salicylanilide derivatives bearing a carbonyl group via oxime bond. Obtained conjugates were purified and characterized (MS, RPHPLC). The conjugates were evaluated for their in vitro extracellular antimycobacterial activity (two strains of M. tuberculosis, M. abscessus), intracellular activity in infected macrophages, cytotoxic and cytostatic properties and cellular uptake. In general, salicylanilide-oligotuftsin conjugates showed improved activity against both extracellular and intracellular mycobacteria, enhanced cellular uptake together with decreased toxicity. They were effective also against multidrug-resistant M. tuberculosis and “chemoresistant” M. abscessus. These promising in vitro results could stimulate further research on this field. References 1. Vinšová J., Krátký M.: Nova Science Publishers 2010, ISBN 978-1-61668-233-0. 2. Tiwari G. et al.: Int. J. Pharm. Invest. 2, 2 (2012). 3. Horvati K. et al.: J. Pept. Sci. 15, 385 (2009). 4. Krátký M., Vinšová J.: Curr. Pharm. Des. 17, 3494 (2011). 356 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 327 Drug design-new developments 23/09/2016 Pharmacomodulation on the piperidinol skeleton: Design, synthesis, and biological evaluation of novel PIPD1 derivatives as Mycobacterium abscessus agents F. Dubar4, C. Dupont2-3, V. Le Moigne3, J.L. Herrmann3, Y. Guerardel4, L. Kremer2-1, C. Biot4 1 CPBS, INSERM 2Centre National de la Recherche Scientifique FRE 3689, Centre d’études d’agents Pathogènes et Biotechnologies pour la Sante, Université de Montpellier, Montpellier 3 3UMR1173, INSERM, Université de Versailles St Quentin, Versailles St Quentin 4UGSF - France, Université Lille 1, Villeneuve D'ascq, France Mycobacterium abscessus (M. abscessus) is a nontuberculous mycobacteria (NTM) that is responsible for a wide range of disease, particularly pulmonary diseases. M. abscessus complex is the most drug resistant of the mycobacterial pathogens.1 Even classical antitubercular drugs (Clarithromycn, amikacin and Cefoxitin) are used in the treatment to M. abscessus, the long treatment period are often unsuccessful. Moreover, no vaccine is currently avaiblable against this pathogen.2 In this context, increased effort to develop new drugs for NTM diseases are really needed. Recently, we identified a new piperidinol-based molecule, PIPD1.3 This promising compound exhibit potent activity against clinical M. abscesssus strains in vitro and in infected macrophages. In this study, novel derivatives of PIPD1 were designed, synthesized and tested for their in vitro antimycobacterial effects on M. abscessus strains. The range of CMI is from 2 µg/ml to 0.25 µg/ml. In vitro cytotoxicity effects of these new compounds were also evaluated in vitro against murine J774 cells line in order to determine the therapeutic index of these series. The structure-activity relationships (SARs) were discussed. 1. Henkle E., Hedberg K., Schafer S., Novosad S., Winthrop K.L. (2015) Population-based incidence of Pulmonary Nontuberculous Mycobacterial Disease in Oregon 2007 to 2012. Ann Am Thorac Soc 12 : 642-647. 2. Le Moigne V., Gaillard J.L., Herrmann J.L. (2016) Vaccine strategies against bacterial pathogens in cystic fibrosis patients. Médecine et Maladies Infectieuses 46: 1, 4–9. 3. Dupont C., Viljoen A., Dubar F., Blaise M., Bernut A., Pawlik A., Bouchier C., Brosch R., Guérardel Y., Lelièvre J., Ballell L., Herrmann J.L., Biot C., Kremer L. (2016) A new piperidinol derivative targeting mycolic acid transport in Mycobacterium abscessus. Mol Microbiol. doi: 10.1111/mmi.13406. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 357 328 Drug design-new developments 23/09/2016 GTPase-Obg mRNA expression analysis in Mycobacterium under stress conditions V. Kumar2, H.N. Singh1, A.K. Tomar2, S. Yadav2 1 School of Sciences, Noida International University, Gautam Budh Nagar, UP, India, Gautam Budh Nagar 2Department of Biophysics, All India Institute Of Medical Sciences, New Delhi, India To counter Multi Drug Resistance (MDR) and Extensively Drug Resistance (XDR) strains of Mycobacterium tuberculosis, there is an urgent need of identification and characterization of new drug targets. GTPase-Obg is a GTP binding protein that belongs to a highly conserved protein family in both prokaryotes and eukaryotes. It plays many roles in bacteria, including ribosome biogenesis, protein translation regulation, stress response, cell growth regulation, etc. Past studies have reported that GTPase-Obg from Mycobacterium tuberculosis interacts with ribosome, but its role in the bacterial defence mechanism is unclear to date. Here, our study reports expression, purification, biophysical characterization and expression analysis under stress conditions of GTPase-Obg. Circular dichroism experiments revealed that GTPase-Obg underwent conformational changes in presence of GTP. mRNA expression analysis was performed using real time PCR in various stress conditions, including heat and cold shock and antibiotics. Significant increase in GTPase-Obg mRNA was observed during different stress conditions, highlighting a possible role of GTPase-Obg in Mycobacterium stress response. Further, three-dimensional structure of GTPaseObg was modelled and its interaction with some known GTPase inhibitors was studies in silico. This study concludes that GTPase-Obg acquires functional stability in presence of GTP through helix destabilization, may have a crucial role in Mycobacterium stress response and might be explored as a new target to counter drug resistance. 358 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 329 Drug design-new developments 23/09/2016 Genetic evidence for a new binding site of Bedaquiline (TMC207) in the ε subunit of the mycobacterial F-ATP synthase S. Kundu2, G. Biukovic2, G. Grüber1, T. Dick2 1 School of Biological Science, Nanyang Technological University 2Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore The F-ATP synthase binding and inhibition mechanism of Bedaquiline (BDQ; former name TMC207), a newly approved TB drug, is the focus of this project. Previous studies ([1][2][3]) suggest that the c-subunit of the mycobacterial F-ATP synthase contains the binding site of the drug. However, we showed recently - employing biochemical, biophysical and modeling methods - that TMC207 may in addition bind to the interface of subunits ε, γ and c [4,5]. Here we confirm the involvement of subunit ε in this new binding mode in intact bacteria using genetics. Recombineering [6] was employed to introduce targeted missense mutations into the ε subunit, encoding gene at sites of ε close to the postulated BDQ-binding site. Exchanging these amino acids was expected to result in shifts of the minimal inhibitory concentration (MIC50) indication increased or decreased binding of the drug to its ε target. The amino acid changes introduced were W16A and R37G [4]. To determine whether the amino acid exchange mutations affect MIC50s of M. smegmatis atpCW16A and M. smegmatis atpCR37G, growth inhibition dose response curves were generated with the drug. The results showed no significant difference between wildtype bacteria and M. smegmatis atpCR37G suggesting that the interaction of residue R37 of ε with BDQ may not be essential for BDQ-binding. In contrast, M. smegmatis atpCW16A showed a 5-fold decrease in its MIC50 form 10 +/- 2 nM in the wildtype strain to 2 +/- 1 nM in the mutant strain. This result suggests that W16 of subunit ε is close to the BDQ-binding site as predicted previously by in vitro tryptophan fluorescence- and NMR spectroscopy [4]. It furthermore suggests that the substitution of the bulky sidechain of W16 with a smaller side chain of alanine allows for a tighter binding of BDQ to ε. In conclusion, we provide genetic in vivo evidence for a new binding and inhibition mechanism of BDQ involving subunit ε of the mycobacterial F-ATP synthase. This work is supported by the Ministry of Health (MOH), Singapore (NMRC, CBRG12nov049; G. Grüber and T. Dick). Subhashri Kundu receives NUS Research Scholarship from Yong Loo Lin School of Medicine. References: 1. de Jonge, M.R., Koymans, L.H., Guillemont, J.E., Koul, A. and Andries, K., 2007. A computational model of the inhibition of Mycobacterium tuberculosis ATPase by a new drug candidate R207910. PROTEINS: Structure, Function, and Bioinformatics, 67(4), pp.971-980. 2. Segala, E., Sougakoff, W., Nevejans-Chauffour, A., Jarlier, V. and Petrella, S., 2012. New mutations in the mycobacterial ATP synthase: new insights into the binding of the diarylquinoline TMC207 to the ATP synthase C-ring structure. Antimicrobial agents and chemotherapy, 56(5), pp.2326-2334. 3. Huitric, E., Verhasselt, P., Koul, A., Andries, K., Hoffner, S. and Andersson, D.I., 2010. Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor. Antimicrobial agents and chemotherapy, 54(3), pp.1022-1028. 4. Biuković, G., Basak, S., Manimekalai, M.S.S., Rishikesan, S., Roessle, M., Dick, T., Rao, S.P., Hunke, C. and Grüber, G., 2013. Variations of Subunit ε of the Mycobacterium tuberculosis F1Fo ATP Synthase and a Novel Model for Mechanism of Action of the Tuberculosis Drug TMC207. Antimicrobial agents and chemotherapy, 57(1), pp.168-176. 5. Hotra, A., Suter, M., Biuković, G., Ragunathan, P., Kundu, S., Dick, T. and Grüber, G. (2016) Deletion of a unique loop in the mycobacterial F-ATP synthase γ subunit sheds light in its inhibitory role in ATP hydrolysis driven H+-pumping. FEBS J. in press (doi: 10.1111/febs.13715). 6. Van Kessel, Julia C., and Graham F. Hatfull. "Efficient point mutagenesis in mycobacteria using single‐stranded DNA recombineering: characterization of antimycobacterial drug targets." Molecular microbiology 67.5 (2008): 1094-1107. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 359 330 Drug design-new developments 23/09/2016 Efficient nanoencapsulation of Bedaquiline L. De Matteis6, D. Jary1-2, A. Lucía8-4-3, I. Serrano7, S. García6, F. Navarro1-2, J. M. De La Fuente7, J.A. Ainsa8-4-3-5 1 Technologies for Healthcare and Biology division, CEA, LETI, MINATEC Campus 2Univ. Grenoble Alpes, Grenoble, France 3Instituto de Investigacion Sanitaria de Aragon (IIS-Aragon) 4CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III 5Instituto de Biocomputacion y Fisica de Sistemas Complejos, BIFI 6Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza 7Instituto de Ciencia de Materiales de Aragon (ICMA), Universidad de ZaragozaCSIC 8Dpto. Microbiología, medicina preventiva y salud publica. Grupo Genética de Micobacterias., University of Zaragoza, Zaragoza, Spain The problem of antibiotic resistance in Mycobacterium tuberculosis has been declared a global health emergence by the WHO. Rising cases of MDR-TB, and XDR-TB are making increasingly difficult to treat tuberculosis nowadays (1). After 50 years without any new drug for TB, in 2012 the FDA approved the use of bedaquiline, the first drug designed specifically to treat MDR-TB. It is very effective but shows serious side effects. Consequently, this antibiotic should be prescribed only when no other treatment options are available (2). Besides classical drug discovery strategies, new approaches are urgently needed to get a faster, more efficient and less harmful treatment. Novel drug delivery systems based on nanocarriers are a promising strategy to overcome current therapeutic challenges due to their unique physicochemical properties (3, 4), which include small size or high surface to volume ratio. Nanocarriers improve the aqueous solubility of poorly soluble drugs, protects the drugs, and allow a controlled release of the medication to decrease the frequency of administration. Additionally they can be modified to control their biodistribution allowing selective transport to the sites of infection. The development of effective and safe nanotherapy methods is particularly relevant in the treatment of MDR-TB, as very long treatments with highly toxic second-line drugs are required for those cases. In this sense the encapsulation of bedaquiline in novel nanocarriers is of special interest (5). In this work, chitosan based nanocapsules and Lipid NanoParticles, based on the Lipidots® technology, have been synthesized for the encapsulation of bedaquiline. The encapsulation of this antibiotic has been optimized for both nanocarriers, upon quantification of drug loading efficiency, improvement of the drug payload, nanoparticles stability amelioration in storage condition. For the best candidates, drug release has been determined in biological media for in vitro assays. The antimycobacterial activity has finally been evaluated to confirm that the drug is still active after encapsulation. Also, their cytotoxicity has been assayed in different cell lines. 1. WHO, Drug resistant TB: surveillance and response. Supplement to global tuberculosis report 2014. Available in http://www.who.int/tb/publications/global_report/gtbr14_supplement_web.pdf 2. Mingote LR, Namutamba D, Apina F, Barnabas N, Contreras C, Elnour T, Frick MW, Lee C, Seaworth B, Shelly D, Skipper N, Tavora dos Santos Filho E. Lancet. 2015, 385: 477-479. 3. Mishra B, Patel BB, Tiwari S. Nanomedicine: NBM. 2010, 6: 9-24. 4. Onoue S, Yamada S, Chan HK. Int J Nanomed. 2014; 9: 1025-1037. 5. Mustafa S, Pai RS, Singh G, Devi K. J Drug Targeting. 2015, 23: 287-304. 360 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 331 Drug design-new developments 23/09/2016 An optimized background regimen with the next generation benzothiazinone PBTZ169 for active tuberculosis A. Lupien3, A. Vocat3, B. Lechartier2, V. Makarov1, S. Cole3 1 Bakh Institute Of Biochemistry, Russian Academy of Science, Moscow, Russia 2Service de pneumologie, CHUV 3Global Health Institute, École polytechnique fédérale de Lausanne (EPFL), Lausanne, Switzerland Tuberculosis (TB) mainstay treatment relies on a four-drug regimen including isoniazid, rifampicin, pyrazinamide (PZA) and ethambutol for six months. The emergence of multidrug (MDR) and extensively drug-resistant (XDR) TB has now challenged the efficiency of this treatment to cure TB. Intensive efforts have been made to develop new anti-TB drugs. Several of these novel or repurposed drugs are in clinical trials to evaluate their efficacy at curing TB. We are focusing on the promising piperazine-containing benzothiazinone PBTZ169 that inhibits the essential flavo‐protein enzyme DprE1. PBTZ169 is active against MDR‐ and XDR‐clinical isolates of Mycobacterium tuberculosis. When combined with other TB drugs, PBTZ169 acts synergistically with bedaquiline (BDQ) and clofazimine (CFM). A new regimen comprising PBTZ169, BDQ and PZA was found to be more efficacious than the standard three-drug treatment in a murine model of chronic disease. PBTZ169 is therefore an attractive drug candidate to treat TB in humans. The aim of this project is to determine the activity in vitro of PBTZ169 in combination with anti-TB drugs, to elaborate a potential OBR (optimized background regimen) against active TB. To do so, PBTZ169 was tested against active M. tuberculosis using two-drug checkerboard and viability assays. PBTZ169 has additive activity when it is combined with first- and second-line anti-TB drugs. At the MIC, PBTZ169 combined with BDQ, CFM and delamanid produce a two log reduction in the bacterial burden (synergism) compared to the drugs alone. For all the other drugs tested in combination with PBTZ169 (meropenem, clarithromycin, sutezolid, linezolid and lansoprazole sulfide), we observed additive activity. Neither antagonism nor increased cytotoxicity were found suggesting that PBTZ169 could be added to most drug combinations without any adverse effects. Interestingly, several two-drug combinations that do not include PBTZ169 showed concentrationdependent synergism. Further studies on these two-drug combinations using ex vivo models and three-drug regimens are ongoing. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 361 332 Drug design-new developments 23/09/2016 Killing and sensitizing of Mycobacterium tuberculosis to antibiotics and TB drugs using a protein complex from human milk V. Meikle2, A.P. Håkansson1, M. Niederweis2 1 Experimental Infection Medicine, Malmö, Lund University, Lund, Sweden 2Microbiology, University of Alabama at Birmingham, Birmingham, United States Mycobacterium tuberculosis (Mtb) is intrinsically resistant to many antibiotics which are effective against other bacteria. Increasingly infections with Mtb occur with drug resistant strains that cause TB with very poor prognosis. The development of new antibiotics is hugely expensive and return of investment is low. An alternative strategy is to sensitize bacteria to current antibiotics e.g. by drug efflux pump inhibitors. We used HAMLET (human alpha-lactalbumin made lethal to tumor cells), a complex of an altered form of human milk alpha-lactalbumin with fatty acids, that uses a novel death pathway to kill bacterial pathogens such as Streptococcus pneumoniae and examined whether HAMLET is active against Mtb. Using the microplate Alamar blue assay we showed that HAMLET completely inhibited growth of Mtb H37Rv at a concentration of 200 µg/mL. Non-toxic amounts of HAMLET potentiated the efficacy of antibiotics and TB drugs by 12 to 130-fold. HAMLET also killed Mtb in infected THP-1 macrophages with a minimal inhibitory concentration of 200 µg/ml, while concentrations up to 400 µg/ml did not affect macrophage viability. This result demonstrates that HAMLET is active against intracellular Mtb. The inhibitory activity of HAMLET against Mtb and its large potentiating effect on antibiotics and TB drugs, open an exciting avenue for developing safe, alternative and more efficient treatment regimens which do not require the development of new drugs, may overcome the resistance of current MDR-, XDR- and TDR-Mtb strains and may include antibiotics which, by themselves, are not effective against Mtb. 362 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 333 Drug design-new developments 23/09/2016 Design, Syntheses and Studies of New Potent and Selective Anti-tuberculosis Agents Based on Imidazopyridine and Imidazothiazole Cores G. Moraski Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, United States Multidrug (MDR) and extensive drug resistant (XDR) forms of TB are becoming common. New treatments are desperately needed. Our early studies focused on syntheses of mycobactins (essential virulence factors for Mtb, that facilitate iron uptake), analogs and drug conjugates. Simultaneous fragment based screening of all synthetic intermediates led to the discovery of new small molecule anti-TB agents including appropriately functionalized imidazopyridines and imidazothiazoles that have remarkably potent anti-TB activity. In order to fully explore the potential of this new class of anti-TB agents, collaborations were formed with the Lilly TB initiative, the Infectious Disease Research Institute (IDRI) and the NIH, including the laboratories of Dr. Clif Barry, and Prof. Jeffrey Schorey’s group at Notre Dame. The early chemistry developed by Garrett Moraski at Notre Dame (now Montana State University) was readily adapted to the Automated Synthesis and Purification Laboratory (ASPL) at Eli Lilly and Co and allowed further expansion of SAR. Additional collaborative efforts, including high throughput screening (University of Illinois, Chicago), development and utilization of separate sensitive assays (IDRI) and determination of in vivo pharmacokinetics at Lilly and the NIH and elucidation of the mode of action (inhibition of QcrB) have accelerated studies that have advanced the studies to determine in vivo efficacy in rodents and primates. The combined results that will be described indicate that imidazo[1,2-a]pyridine-3-carboxamides and the corresponding imidazothiazoles are exciting, potent, selective anti-TB agents that merit additional development opportunities. While chemistry is the driver, substantial navigation of the project has been due to productive interdisciplinary collaborations. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 363 334 Drug design-new developments 23/09/2016 A tail tale: what does it take to get under TB skin W. Moreira1, D. Aziz1, C. Huan2, T. Yang2, S. Nyantakyi2, M.L. Go2, T. Dick1 1 Microbiology 2Pharmacy, National University of Singapore, Singapore, Singapore Resistance and persistence represent two major challenges faced by current anti-TB regimens. TB drugs, like most antibiotics, target specific macromolecules. Genetically acquired antibiotic resistance arises mainly from mutations in target genes and occurs readily in patients due to noncompliance to lengthy multi-drug regimens and spatio-temporal pockets of monotherapy. Persistence of infection is thought to be due to various types of metabolically quiescent non-growing drug tolerant bacteria. Ideally new TB drugs should kill persister bacilli and show a low propensity for the development of genetic drug resistance. The bacterial membrane integrity and its associated homeostatic functions (e.g. selective permeability, energy metabolism) remains an essential and vulnerable target regardless of the metabolic status of the cell. Additionally, resistance development to membrane-targeting antibiotics is expected to be slow as the target is the product of complex biosynthetic pathways, as opposed to a single macromolecule. The large number of antimicrobial peptides made by the host validates the membrane as an antibacterial target site. Furthermore, the successful clinical use of a few membrane-targeting antibiotics (e.g. colistin, polymixin, daptomycin) supports this notion. Remarkably, these molecules are inactive against mycobacteria. These large compounds (MW >3000) are possibly blocked by the outer membrane and cannot reach the cytoplasmic membrane of mycobacteria. It is interesting to note that most of these large molecules possess a lipophilic side chain that favours their anchoring in the membrane. We hypothesized that a lipophilic side chain could suffice to target a small molecule to the cytoplasmic membrane. Such small molecule would not be blocked by the outer membrane. We made use of the widely use indole scaffold to design a small molecule that interferes and disrupt mycobacterial membrane integrity and functions. We provide chemical and biochemical evidences suggesting that these compounds compromise the membrane integrity causing depolarization and disruption of energy metabolism leading to cell death. Induction of promoter-reporter associated with envelope stresssensing pathway further support these findings. These compounds were active against non-growing persister bacilli and spontaneous resistant mutants could not be isolated (mutation frequency <109). Taken together these results suggest that targeting mycobacterial membrane integrity may be an attractive chemotherapeutic intervention level to kill otherwise drug tolerant persister bacilli, and to slow down the development of genetic antibiotic resistance. 364 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 335 Drug design-new developments 23/09/2016 Membrane targeting drugs against mycobacteria - a road less travelled D. Mukherjee2, H. Zou4, S. Liu1-4, R. Beuerman1-3-4, T. Dick2 1 SRP Neuroscience and Behavioural Disorders, Duke-NUS Graduate Medical School 2Department of Microbiology & Immunology 3Department of Ophthalmology, National University of Singapore 4Singapore Eye Research Institute, Singapore, Singapore Aims: It is well known that the major issues with TB treatment are resistance and persistence. Drugs with novel mechanisms of action are needed to help address these issues. We test the hypothesis that targeting the cytoplasmic membrane may be an effective way to kill persister mycobacteria as well as delay the emergence of resistance. Methods: In vitro activity of AM-0016, a novel semi-synthetic xanthone-based antibacterial, was assessed against growing and persister mycobacteria. Resistance mutation frequencies were also determined. Membrane potential was assessed biochemically and electron microscopic analyses were carried out. Results: AM-0016 rapidly sterilized growing tubercle bacillus cultures and displayed strong bactericidal activity against hypoxic and drug induced persister bacteria. Spontaneous resistance mutation frequency was lower than 10-8. Exposure to AM-0016 resulted in rapid collapse of the membrane potential. Imaging revealed deformation of the cell envelope. Conclusions: Targeting the cytoplasmic membrane may be an attractive approach to eliminate persister mycobacteria and slow down the emergence of genetic drug resistance. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 365 336 Drug design-new developments 23/09/2016 Fragment-based Mycobacterium tuberculosis whole cell screen delivers hits for drug discovery D. Negatu2-3, J. Liu1, C. Aldrich4, M. Gengenbacher1-2, T. Dick1-3 1 Yong Loo Lin School of Medicine, Department of Medicine, BSL-3 Core Facility 2Yong Loo Lin School of Medicine, Department of Medicine, Tuberculosis Research Laboratory 3Yong Loo Lin School of Medicine, Department of Microbiology and Immunology, Antibacterial Drug Discovery Laboratory, National University of Singapore, Singapore, Singapore 4Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States Mycobacterium tuberculosis (Mtb) is now the leading cause of infectious disease mortality by a single pathogen. New drugs, ideally with novel mechanism (s) of action are needed to treat drugresistant strains and simplify the current lengthy treatment regimen. Many TB drugs (isoniazid, pyrazinamide, para-aminosalicylic acid, ethionamide) are small molecular weight compounds that are bioactivated to reactive metabolites that inhibit multiple metabolic pathways. By todays standards, these drugs would be considered fragments owing to their low molecular weight (MW<170), a property which would generally exclude them from most compound collections used in high-throughput screening (HTS). We hypothesize that fragment libraries represent a rich source of untapped chemical matter for the identification of new antitubercular agents, which may act through multi-targeting and potentially by bioactivation. We screened a library of 1000 chemically diverse fragment compounds. At a concentration of 1 mM, the growth inhibition of M. tuberculosis H37Rv measured by turbidity delivered 84 (8.4%) primary hits at 70% cut off. Of the hits, 33 compounds showed at least 50% growth inhibition at 500 µM in dose response assays, which is comparable with the MIC50 of pyrazinamide, a first line TB drug. Fifteen compounds also displayed activity against the clinically relevant non-tuberculosis mycobacteria species, Mycobacterium avium and Mycobacterium abscessus, exhibiting at least 50% growth inhibition at 500 µM. Further profiling of the hits for membrane toxicity (hemolysis assay) and cytotoxicity (HepG2, HEK, A549) identified 19 compounds with acceptable hemolytic activity and selectivity (Selectivity Index > 10) in tested cell lines. Our findings suggest that fragment libraries may be valuable resources for Mtb drug discovery delivering low molecular weight hits with attractive physicochemical properties. 366 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 337 Drug design-new developments 23/09/2016 Evaluating indazole sulfonamides targeting IMPDH as potential leads against Mycobacterium tuberculosis Y. Park4, K. Rhee6, T. Ioerger5, J. Sacchettini5, V. Dartois7, T. Blundell2, M. Rizzi1, S. Donini1, K. Arora4, L. Via4, P. Ray3, S. Green3, C. Barry4, H. Boshoff4 1 Dipartimento di Scienze del Farmaco, University of Piemonte Orientale, Via Bovio, Italy 2Department of Biochemistry, University of Cambridge, Cambridge 3Drug Discovery Unit, University of Dundee, Dundee, United Kingdom 4Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, Bethesda 5Department of Computer Science and Engineering, Texas A&M University, College Station 6Division of Infectious Diseases, Department of Medicine, Weill Cornell Medical College, New York 7Public Health Research Institute, New Jersey Medical School, Newark, United States A potent indazole sulfonamide was identified through high-throughput screening of Mycobacterium tuberculosis (Mtb). This non-cytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular GFP. Isolation of resistant mutants followed by whole-genome sequencing showed in two cases an unusual gene amplification of a 40 gene region spanning Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition and growth inhibition could be potentiated by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Further development of SAR allowed good correlation of IMPDH IC50 values and minimum inhibitory concentration (MIC) against Mtb and allowed active derivatives to be synthesized with a chemical linkage permitting bead attachment. Beads containing these immobilized derivatives specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above MIC for 24 hours were required for a cidal effect, levels that have been difficult to achieve in either mice or marmosets. It also requires rapid growth of Mtb for cidal activity in vitro growth rate dependent killing assay. In addition direct measurement of guanine levels in resected lung tissue from tuberculosis patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 367 338 Drug design-new developments 23/09/2016 Structure-activity relationships and biological profile of novel diarylthiazoles as antitubercular drugs G. Prosser2, N. Kalia2, S. Green1, P. Ray1, H. Boshoff2, C. Barry2 1 Drug Discovery Unit, University of Dundee, Dundee, United Kingdom 2Tuberculosis Research Section, NIH, Bethesda, United States Novel anti-tubercular drugs that can reduce treatment duration and are active against drug resistant strains are urgently needed to combat disease spread and progression. Using high-throughput screening of synthetic compound libraries we have identified a novel diarylthiazole scaffold as having potent anti-tubercular activity, under both aerobic and anaerobic conditions and within infected J774 macrophages. Structure-Activity Relationship studies have identified key aspects of the core scaffold that can be modified to increase potency and solubility, and a lead candidate has demonstrated promising oral exposure in a murine model. Spontaneous resistant mutants raised against a series lead compound contained mutations within the prrB gene, coding for the sensorhistidine kinase partner of the PrrAB two-component system. Over-expression of the operon in H37Rv via an integrative vector failed to induce significant alterations in drug MICs. Lack of knowledge on the physiological role(s) of this TCS stimulated us to perform genetic and biochemical studies on the operon and its individual components. We present data on drug-protein interactions in vitro between select diarylthiazoles and purified recombinant PrrB and each of its distinct domains, and also identify other potential native mycobacterial protein binding partners using a drug-immobilized affinity-based pull-down assay. The physiological role of the operon has been investigated through elucidation of the consensus DNA binding motif of the PrrA response regulator and subsequent genome-wide in silico analysis of putative PrrA-promoter binding sites. Genetic essentiality of the operon and each of its individual components in both M. tuberculosis and M. smegmatis was demonstrated through an inability to delete the corresponding gene(s) in the absence of a second copy integrated elsewhere in the genome. We are currently generating regulated knock-down strains for each gene and plan to identify regulon members using microarray technology. Together, our results identify a promising novel anti-tubercular drug-target pair and provide insight into the physiological role(s) of the enigmatic and essential PrrAB two-component system in Mycobacteria. 368 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 339 Drug design-new developments 23/09/2016 Repurposing clinically approved cephalosporins for tuberculosis therapy S. Ramon Garcia1-2, R. González Del Río2, A. Santos Villarejo2, G. Sweet1, F. Cunningham2, D. Barros2, L. Ballell2, A. Mendoza-Losana2, S. Ferrer-Bazaga2, C. Thompson1 1 Microbiology & Immunology, The University of British Columbia, Vancouver, Canada 2Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Spain While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we explored their potential repositioning as new anti-TB drugs and identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. We previously demonstrated that combinatorial drug therapy, traditionally designed to avoid emergence of drug resistant Mycobacterium tuberculosis (Mtb) strains, might also be employed to increase the efficacies of available antibiotics, allowing them to be repurposed for TB therapy within synergistic combinations1. Rifampicin, the cornerstone drug for TB therapy, is not administered at its optimal clinical dose due to long-established toxicity concerns. If the anti-mycobacterial activity of rifampicin could be increased, TB therapy could be shortened, thus reducing the rate of transmission and the emergence of drug resistance2. In pursuit of this vision, we screened an in-house library of ca. 600 commercially available antibiotics, and found that the cephalosporins had strong synergies with rifampicin and ethambutol, a first-line anti-TB drug. In addition, common chemical patterns required for single drug activity against Mtb were identified using structure-activity relationships (SAR) studies. Synergy was also observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (cefadroxil; or other beta-lactams) and clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Uptake experiments demonstrated that synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. The fact that these cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies. References: 1. Ramon-Garcia, S. et al. Synergistic drug combinations for tuberculosis therapy identified by a novel high-throughput screen. Antimicrob Agents Chemother 55, 3861-3869 (2011). 2. van Ingen, J. et al. Why Do We Use 600 mg of Rifampicin in Tuberculosis Treatment? Clin Infect Dis 52, e194-199 (2011). Tuberculosis 2016 – September 19-23, 2016 – Paris, France 369 340 Drug design-new developments 23/09/2016 Identification and prioritisation of hits from whole cell phenotypic screening of mycobacterium tuberculosis in the context of treatment shortening, lessons and future directions P. Ray2, H. Boshoff3, K. Arora3, P. Tsang3, T. Bayliss2, J. Harrison2, D. Murugesan2, K. Buchanan2, S. Green2, F. Zuccotto2, K. Read2, P. Scullion2, R. Epemolu2, C. Mackenzie2, V. Mizrahi1, D. Warner1, C. Barry3, P. Wyatt2 1 Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa 2Drug Discovery Unit, University of Dundee, Dundee, United Kingdom 3Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, United States Results of a phenotypic M. tuberculosis H37Rv screening campaign against two independently compiled commercial libraries of over five hundred thousand compounds and a proprietary agrochemical library of over fifty five thousand compounds is disclosed. In addition to conventional HTS triage processes, which include selecting starting points based on physicochemical properties and cytotoxicity, we introduce early H37Rv biology, ADME-Tox and physicochemical filters to aid prioritisation of novel starting points which are envisaged more likely to lead to treatment shortening drug discovery projects. A robust process for evaluating and prioritising >100 confirmed hit starting points for synthetic lead-generation and mechanism of action activities is described. Future directions include the need for screening untapped areas of chemical space beneficial for lesion and TB pentration. 370 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 341 Drug design-new developments 23/09/2016 Synthesis and evaluation of new benzothiazinones with piperazinylamide side chain A. Richter1, T. Wetzlar1, K. Voigt2, U. Möllmann2, S. Franzblau3, P. Imming1 1 Martin Luther Universität Halle Wittenberg, Halle 2Leibniz Institute for Natural Product Research and Infection Biology Hans-Knöll-Institute, Jena, Germany 3Institute for Tuberculosis Research, University of Illinois, Chicago, United States Benzothiazinones (BTZs) display very strong antimycobacterial activity in vitro [1]. Some derivatives of this compound class, for example BTZ043 [2] and PBTZ169 [3], are very active against Mycobacterium tuberculosis with MICs < 1 nM. The outstanding in vitro activity has not yet been fully translated into in vivo effectivity, thus requiring relatively high doses of 50 – 200 mg/kg daily in order to reduce CFUs in mice considerably [1, 4]. BTZs inhibit the mycobacterial enzyme decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE1), interfering with the construction of a functioning cell wall. Important for the activity is a nitro group in the molecule, which is reduced by FADH2/DprE1 to a nitroso BTZ that binds covalently to the thiol group of Cys-387 near the catalytic center [5,6]. In Fig. 1 we depict the complex process of this twostep mechanism-based inhibition: Figure 1. Steps of DprE1 inhibition by BTZs We synthesized a panel of new benzothiazinones based on the scaffold shown in Fig. 2 with the purpose of improving aqueous solubility while maintaining high activity. The synthesis was possible by an efficient one step pathway using thioureas. Figure 2. Scaffold of BTZs synthesised and studied (X = c or S, n = 1 or 2) The set contains different amide as well as sulfonamide side chains at the R1 position. Activities covered a wide range from 1000 nM to 0.2 nM (MICs against M. vaccae). Some of the new BTZs reached or even surpassed the activity of the lead substance BTZ043 in this assay. Solubility data of these compounds were generated by CLND (chemiluminescence nitrogen detection) measurements. Although we focused on hydrophilic side chains, the solubility was equal or even slightly lower compared to BTZ043. Furthermore, the nitro group was replaced with other groups that have less potential of toxicity while maintaining the covalent mode of inhibition. 1. Makarov, V., et al.: Science 2009, 324, 801-804 2. Möllmann, U., Makarov, V., Cole, S. T.: WO2009010163A1 2009 3. Makarov, V., Cole, S. T.: WO2012/066518A1 2012 4. Makarov, V., et al.: EMBO Mol. Med. 2014, 6 (3), 372-383 5. K. Mikusová et al., Curr. Pharm. Design 2014, 20, 4249-4273 6. Trefzer, C., et al.: J. Am. Chem. Soc. 2012, 134 (2), 912-915 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 371 342 Drug design-new developments 23/09/2016 Structure-activity relationships of 3,5-dinitrophenyltetrazoles, oxadiazoles and triazoles as selective antitubercular agents J. Roh1, J. Nemecek1, G. Karabanovich1, L. Valaskova1, J. Stolaříková2, V. Klimešová1, A. Hrabalek1 1 Charles University in Prague, Faculty of Pharmacy, Hradec Králové 2Regional Institute of Public Health, Ostrava, Czech Republic In our previous work we found that 1-substituted and 2-substituted-5-(3,5dinitrobenzylsulfanyl)tetrazoles possessed highly selective antitubercular activity with minimal inhibitory concentration (MIC) values as low as 1 µM against drug susceptible strains of M. tuberculosis and 0.25 - 1 µM against six clinically isolated MDR M. tuberculosis strains. The further studies of these compounds and their analogues led to the development of 5-substituted 2(3,5-dinitrobenzylsulfanyl)oxadiazoles and thiadiazoles as novel antitubercular agents with outstanding activities against replicating and also nonreplicating strains of M. tuberculosis. Furthermore, this class of compounds possessed highly selective antimycobacterial effect, because they showed no antibacterial or antifungal activity, low cytotoxicity in mammalian cell lines and low genotoxicity. Despite the presence of 3,5-dinitrophenyl fragment, as in the potent inhibitors of mycobacterial decaprenylphosphoryl-β-D-ribofuranose 2´-oxidase (DprE1), these compounds did not inhibit DprE1 and more likely affect the synthesis of mycobacterial nucleic acids.[1] In the continuation of our work we further studied the structure-activity relationships of these lead compounds. In particular, we decided to remove the methylsulfanyl linker between the 3,5dinitrophenyl moiety and the heterocycle. Hence, the series of 3,5-dinitrophenyl-substituted tetrazoles, oxadiazoles and triazoles, with 3,5-dinitrophenyl group attached directly to heterocycle, were prepared. The change of the position of 3,5-dinitrophenyl fragment had no effect on the level of antimycobacterial activity - the majority of prepared compounds possessed excellent in vitro activities against M. tuberculosis H37Rv and several MDR stains of M. tuberculosis, with MIC values as low as 0.03 µM. These compounds again showed highly selective antimycobacterial effect, because they were not active against other bacteria or fungi and possessed low in vitro cytotoxicity. The study was supported by the Czech Science Foundation project (14-08423S) and Charles University in Prague (SVV 260 291). 1. G. Karabanovich et al. J. Med. Chem. 2016, 59, 2362−2380. 372 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 343 Drug design-new developments 23/09/2016 Combining the shotgun and sniper approach to target the mycobacterial cell wall A. Shetty2, U. Lakshmanan1, C. Ling1, A. Poulsen1, J. Hill1, Y. Yamada2, T. Dick2 1 Experimental and Therapeutics Centre, A* STAR 2Microbiology and Immunology, National University of Singapore, Singapore, Singapore Discovering new drugs for tuberculosis (TB) is like playing darts with spaghetti: promising lead molecules that are potent enzyme inhibitors but cannot penetrate the mycobacterial cell. Our goal is to identify attractive targets with their associated whole-cell active lead compounds for development of new clinical candidates. To achieve this, we have employed a pathway-based whole-cell screen approach, which allows screening against a defined pathway inside the bacterial cell using the double-membrane barrier as a filter. Of particular interest is the cell wall biosynthesis pathway because it is well validated as a drug target. Our study design employs a two-tier screening strategy for hit identification. First, a high-throughput screen of over 70,000 chemically diverse scaffolds for growth inhibitors of Mycobacterium bovis BCG (M. bovis BCG), which led to the selection of 18 compounds. Second, a pathway-based screen of the primary hits in a M. bovis BCG reporter strain carrying a cell wall stress inducible promoter, iniBAC fused to the mCherry reporter gene. Differential expression of the reporter gene allowed us to identify a new class of cell wall targeting compounds, E10 and E11. Both compounds are non-cytotoxic and have a minimum inhibitory concentration (MIC50) of about 10µM against M. bovis BCG and M. tuberculosis. Currently, we are carrying out efficacy studies against persisters and target deconvolution by chemical genetics (resistance generation coupled with whole genome sequencing). Tuberculosis 2016 – September 19-23, 2016 – Paris, France 373 344 Drug design-new developments 23/09/2016 Discovery and development of inhibitors of Mycobacterium tuberculosis primase and inorganic pyrophosphatase O. Tsodikov Department of Pharmaceutical Sciences, University of Kentucky, Lexington, United States Innovative strategies are needed to discover novel anti-tuberculosis therapeutics, as alternatives are needed to existing antibiotics that suffer from resistance, toxicity and ineffective mechanisms of action. Recently we developed a novel coupled primase-pyrophosphatase assay for discovery of inhibitors of two essential bacterial enzymes, primase (DnaG) and inorganic pyrophosphatase (PPiase). DnaG is responsible for synthesis of RNA primers for chromosomal DNA replication, and PPiase breaks down inorganic pyrophosphate (PPi) to drive all DNA and RNA synthesis reactions in the cell. We applied this assay to discovery of inhibitors of DnaG and PPiase from Mycobacterium tuberculosis. The high-throughput assay of a library of small molecules yielded low-microM inhibitors of primase and inorganic pyrophpsphatase. We obtained crystal structures of the RNA polymerase domain of M. tuberculosis DnaG and inorganic pyrophosphatase alone and in complex with inhibitors, which clarify the mechanisms of catalysis and inhibition and drive medicinal chemistry efforts to develop these compounds into therapeutic candidates. References 1. Biswas T, Resto-Roldan E, Sawyer SK, Artsimovitch I and Tsodikov OV. (2013) A novel nonradioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG. Nucleic Acids Res., 41, e56. 2. Gajadeera C#, Willby M#, Green KD, Shaul P, Fridman M, Garneau-Tsodikova S*, Posey JE* and Tsodikov OV*. (2015) Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG. J. Antibiot., 68, 153-157. (# first co-authors, * cocorresponding authors) 3. Pratt AC, Dewage SW, Pang AH, Biswas T, Barnard-Britson S, Cisneros GA* and Tsodikov OV*. (2015) Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis. J. Struct Biol., 192, 76-87. (* co-corresponding authors) 374 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 345 Drug design-new developments 23/09/2016 Pyrrolo[3,4-c]pyridine-1,3(2H)-diones: A novel anti-mycobacterial class targeting mycobacterial respiration R. Van Der Westhuyzen1, A. Moosa3, D. Warner3-2, L. Street1, K. Chibale1-2 1 Drug Discovery and Development Centre (H3-D) 2Institute of Infectious Disease and Molecular Medicine 3MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Cape Town, South Africa High throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound, which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (∆cydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of anti-mycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 375 346 Drug design-new developments 23/09/2016 Unexpected high prevalence of resistance-associated Rv0678 variants in MDR TB patients without documented prior use of clofazimine or bedaquiline C. Villellas2, N. Coeck1, C. Meehan1, N. Lounis2, S. Niemann3, B. De Jong1, L. Rigouts1, K. Andries2 1 Institute of Tropical Medicine, Antwerp 2Janssen Research and Development, Beerse, Belgium 3 National Reference Centre for Mycobacteria, Borstel, Germany Objectives: Resistance-associated variants (RAVs) in Rv0678, a gene regulating the expression of the MmpS5-MmpL5 efflux pump, have been shown to lead to 2- to 8- fold increases in bedaquiline (BDQ) MIC, and 2- to 4-fold increases in clofazimine (CFZ) MIC. The prevalence of these Rv0678 RAVs in clinical isolates and their impact on treatment outcomes are important factors to take into account in BDQ treatment guidelines. After discovering an Rv0678 RAV in a baseline isolate from a patient without documented prior use of CFZ or BDQ, the prevalence of these mutations in clinical isolates of TB patients was assessed. The potential effect of rifampicin in selecting Rv0678 mutants was investigated. Methods: Baseline isolates from two BDQ multidrug-resistant (MDR)-TB clinical trials were assessed for Rv0678 RAVs by Sanger sequencing and corresponding BDQ MICs were determined on 7H11 agar. Rv0678 RAVs were also investigated in non-MDR-TB sequences of the populationbased Hamburg cohort, using the SNP-calling pipeline Snippy. The comparative fitness of 2 fluorescent-marked strains (rpoB-Rv0678 double mutant and rpoB single mutant) was tested by flow cytometry in presence of a range of rifampicin concentrations. Results: Rv0678 RAVs were identified in 21/345 (6.1%) of MDR-TB baseline isolates. BDQ MICs of these isolates were either normal (0.03 to 0.24 µg/ml, n=10), high (0.48 to 1, n=8), or low (0.004 to 0.015, n=4). A variant at position -11 in the intergenic region between Rv0678 and Rv0677c in 11% of the baseline isolates appeared to increase the sensitivity for BDQ (median MIC = 0.015). In nonMDR-TB isolates, the frequency of Rv0678 RAVs was lower (4/817 or 0.5%). No difference in fitness between the double rpoB-Rv0678 mutant and the single rpoB mutant was found upon exposure to rifampicin. Conclusions: RAVs in Rv0678 mutations occur more frequently in MDR-TB patients than previously anticipated, and are not associated with prior use of BDQ or CFZ. Rifampicin is unlikely the selective pressure leading to this phenomenon. In the majority of cases, Rv0678 RAVs do not lead to BDQ MICs above the breakpoint. Given this unpredictable impact on MICs, phenotypic drugsusceptibility methods are preferred over molecular methods to assess BDQ susceptibility. 376 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 347 Drug design-new developments 23/09/2016 Derivatives of Oxyphosphorus Acids with Antimycobacterial Activity J. Vinsova1, M. Krátký1, J. Stolaříková2 1 Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Hradec Kralove 2Regional Institute of Public Health in Ostrava, Laboratory for Mycobacterial Diagnostics and Tuberculosis, Ostrava, Czech Republic The rise of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant (XDR-TB) cases and co-infection with HIV is alarming. These facts stimulate the development of new effective anti-tuberculosis drugs acting by new or different mechanism of action, attacking on the latent (dormant) forms which may become active and when are mutated, cause incurable pandemic. Based on our previous results of salicylanilide diethyl (thio)phosphates showing an increased antimycobacterial activity against both tuberculous and non-tuberculous mycobacteria, including drug-resistant strains, a decreased cytotoxicity and thus improved selectivity in comparison with the parent salicylanilides1,2, we have synthesized and evaluated other new substituted 2(phenylcarbamoyl)phenyl phosphenite derivatives. Parent compounds, i.e., the most active salicylanilides from previous studies1,2,3, prepared by microwave assistance from substituted salicylic acids and anilines in the presence of PCl33, were esterified via chlorides of appropriate oxyphosphorus acids in the presence of triethylamine2. Using dichlorides of phosphorus-based acids, substituted 3-phenyl-3-hydrobenzo[e] [1,3,2]oxazaphosphinin-4-one 2-oxides were synthesized. Some derivatives were obtained by a reaction of substituted salicylanilides with phosphite in the presence of tertiary base, 4(dimethylamino)pyridine and tetrachloromethane (Atherton-Todd reaction). All the prepared esters and heterocycles were investigated for their in vitro activity in Šula’s semisynthetic medium to determine their minimum inhibitory concentrations (MICs). The antimycobacterial activity was evaluated against Mycobacterium tuberculosis CNCTC (Czech National Collection of Type Cultures) 331/88 (H37Rv), CNCTC nontuberculous mycobacteria: Mycobacterium avium 330/88 and Mycobacterium kansasii 235/80 and a clinically isolated strain of M. kansasii 6509/96. The most active derivatives having MIC ≤ 1 µmol.L-1 underwent additional evaluation against MDR-TB strains clinically isolated from patients. Both synthesis and biological evaluation results have been patented.4 Their mechanism of action is currently under investigation. References: 1. Vinšová J. et al.: Molecules 19, 7152 (2014). 2. Vinšová J. et al.: Bioorg. Med. Chem. 22, 728 (2014). 3. Krátký M. et al.: Eur. J. Med. Chem. 45, 6106 (2010). 4. PCT/CZ2015/000129, Czech patent PV-2014-915. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 377 348 Drug design-new developments 23/09/2016 Copper-related innate immunity inspired drug discovery targeting Mycobacterium tuberculosis S. Shah, A. Dalecki, C. Crawford, F. Wolschendorf Medicine, University of Alabama at Birmingham, Birmingham, Al, United States Mycobacterium tuberculosis (Mtb) is becoming a drug-resistant superbug. In an effort to identify much needed new classes of compounds that act by novel mechanisms, we took inspiration from the innate immune system’s use of copper ions to defend against bacterial intrusion. A pilot screen uniquely tailored to identify compounds with copper-related anti-mycobacterial activities yielded 89 hit molecules with copper-dependent activity. For comparison, the same library gave only 16 molecules that inhibited growth of Mtb in a copper independent fashion, indicating that compounds with copper-related activities comprise a major, yet unrecognized, reservoir of potentially exploitable antibacterial activities. Chemoinformatic analysis revealed 7 distinguished hit clusters with the cluster of oxines comprising one of the largest. The minimal inhibitory concentration (MIC) of oxine itself was 0.16 μM against replicating and non-replicating Mtb. The presence of extracellular copper ions was absolutely essential for its activity on whole cells, suggesting a mode of action unrelated to its ability to extract catalytic metal ions from crucial metalloenzymes. Instead, oxine’s activity appears to be related to an increase in cell-associated copper, consistent with action as copper ionophore. Furthermore, we found that only copper ions confer antibacterial properties to the resulting oxine/metal-complex. The addition of Zn, Fe, or Mn, which also coordinate with oxine, neither enhanced nor ablated copper-specific effects, which is remarkable considering that the respective metal complexes are of nearly identical structure and geometry. Finally, in a macrophage infection model, oxine treatment significantly reduced intracellular bacterial burden by more than 50% within 48h. Given the stark copper-dependent nature of its activity, this has provided evidence for the first time that copper ions within Mtb-infected host cells directly enable the antibacterial properties of a copper-dependent inhibitor of Mtb. In summation, our findings highlight metal binding abilities of drugs as a potential focus for future medicinal chemistry and demonstrate the potential of innate immunity-inspired screening platforms to uncover molecules with novel modes of action against Mtb. 378 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 349 Drug design-new developments 23/09/2016 Evaluation of Genotype MTBDRsl version 2 for rapid detection of second-line antituberculosis drug resistance in Ghana identifies the first pre-XDR TB case D. Yeboah-Manu2, S. Osei-Wusu2-3, A. Asante-Poku2, I.D. Otchere2, S. Omari1, A. Forson1 1 Department of Chest Diseases, Korle-Bu Teaching Hospital 2Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana 3West Africa Centre for Cell Biology of Infectious Pathogens, University of Ghana, Legon, Accra, Ghana Background: Early profiling of clinical MTBC strains for drug resistance is essential for proper control of TB. Whereas the genotype MTBDRplus for first line drugs is globally accepted for its specificity and sensitivity, the MTBDRsl for second line v1 received conflicting responses leading to the development of v2. We evaluated the genotype MTBDRsl v2 for rapid screening of MTBC strains against second-line drugs in Ghana. Method: We screened 117 (76 MDR, 14 RIF mono-resistant and 27 INH mono-resistant) resistant TB isolates against moxifloxacin (MOX), streptomycin (STR) and amikacin (AMK) using the Epsilometer test (Etest). In addition, all the 76 MDR, 14 RIFr and 3 INHr were screened with the MTBDRsl. Genes (gyrA, gyrB, eis, rrs, tap, whiB7 and tlyA) of strains resistant to MOX or AMK were PCR amplified and sequenced for mutation analysis. Results: The isolates were obtained from 81.2% males and 28.8% females with an average age of 37.1 years. New cases, relapsed and treatment failures were respectively 69(59.0%), 47(40.2%) and 1(0.8%). Isolates phenotypically resistant to STR, AMK and MOX were 38 (32.5%), 13 (11.1%) and 1 (0.9%) respectively. Among the isolates, only 1 MDR isolate was diagnosed resistant to both MOX (MIC=3µg/mL) and AMK (MIC =16µg/mL) by both the phenotypic assay and MTBDRsl LPA. Gene sequencing analysis led to the identification of gyrA (G87C) and rrs (A514C and A1401G) in this isolate. Whereas none of the remaining isolates were resistant to MOX (MIC<0.125µg/mL), 9/76 (11.8%) MDR, 1/14 (7.1%) RIFr and 3/27 (11.1%) INHr were resistant to AMK with MICs ranging from 1 to 8µg/mL. However, none of these were identified as resistant to AMK by the MTBDRsl LPA. Except one MDR isolate harbouring a tlyA mutation (N236K), no other mutation was detected. The agreement between the phenotypic and the LPA was 87.1% and 100% whereas the sensitivity/specificity was 7.7%/100% and 100%/100% for AMK and MOX respectively. Conclusion: We report the first instance of a pre-XDR-TB case in Ghana. Our findings support the use of the MTBDRsl v2 in Ghana. However, its inability to detect low level resistance to AMK is a major concern. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 379 350 From animal models to man 23/09/2016 Discovery of potent and specific M. tuberculosis leucyl-tRNA synthetase inhibitors (GSK070) X. Li3, V. Hernandez3, F.L. Rock3, Y.S.L. Mak3, M. Mohan3, W. Mao3, Y. Zhou3, E.E. Easom3, J.J. Plattner3, W. Zou1, C. Alemparte2, E. Perez-Herran2, I. Giordano2, A. Mendoza2, D. Barros2, M.R.K. Alley3 1 BioDuro, Beijing, China 2Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Spain 3Anacor Pharmaceuticals, Palo Alto, United States Background: Leucyl-tRNA synthetase (LeuRS) is an essential enzyme for protein synthesis. LeuRS has two catalytic sites, an aminoacylation site, which charges tRNALeu with leucine and an editing or proof-reading site that hydrolyses incorrectly charged tRNALeu. Application of the oxaborole tRNA trapping (OBORT) mechanism (Rock & Mao et al. Science, 2007, 316: 1759) to M. tuberculosis (Mtb) LeuRS lead to the discovery of GSK070, which is in development for tuberculosis treatment. Material/methods: Enzyme and cell-based structure-activity relationships (SAR) were used to optimize potency. The LeuRS IC50 values were measured using the aminoacylation assay. The nosocomial pathogen MIC values were determined by CLSI M7-A10 and the M. tuberculosis H37Rv MIC values were determined using resazurin. Inhibition of human cell protein synthesis was determined in HepG2 cells. Results: We designed and synthesized a series of LeuRS inhibitors by introducing various substituents onto the aromatic ring of the 3-aminomethyl benzoxaborole scaffold. A number of compounds were identified, which showed good antitubercular activity with high selectivity over human cytoplasmic protein synthesis inhibition. Further evaluation of these Mtb LeuRS inhibitors led to the discovery of GSK070, which exhibits potent inhibition of Mtb LeuRS (IC50 = 0.216 µM) and antitubercular activity (Mtb H37Rv MIC = 0.08 µM). Additionally, it is highly specific for the Mtb LeuRS enzyme, as the IC50 for human cytoplasmic LeuRS is 140 µM. Conclusions: GSK070, selected as preclinical candidate, is a new antitubercular agent that targets Mtb LeuRS, a novel protein synthesis target for tuberculosis. 380 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 351 From animal models to man 23/09/2016 Early safety profile of gsk070 a new antitubercular M. Cacho1, E. Jiménez-Navarro1, A. Mendoza1, S. Gresham2, S. Turner2, R. Lincoln2, P. Bushdid4, L. Xi3, M.D. Allen3, D. Barros-Aguirre1 1 TB DPU, Tres Cantos, Spain 2R&D PTS Safety Assessment, GlaxoSmithKline, Ware, United Kingdom 3Anacor Pharmaceuticals, Inc, Palo Alto 4R&D PTS Safety Assessment Reproductive toxicology, GlaxoSmithKline, Upper Merion, United States Background: Oxaborols series are inhibitors of Leucyl t-RNA synthetase and selective antitubercular agents with a remarkable activity against a selection of DS-TB, MDR-TB and XDR-TB clinical isolates. This series exhibits excellent physicochemical properties and has demonstrated in vivo antitubercuular activity. Material/methods: GSK070 has been evaluated to discharge the following toxicology risks: general, genetic, reproductive, cardiovascular, phototoxicicty, and safety pharmacology. It has been tested through eXP cross functional panel of human receptors, enzymes and transporters. Ames test and MLA in the presence and absence of S9 fraction was performed to evaluate genetic toxicology. Inhibition of cardiac ion channels, hERG, Cav1.2 and Nav1.2 were tested by Patch clamp platforms and ex vivo rabbit ventricular wedge was assessed to evaluate cardiovascular liability and a 7 day repeat dose toxicity study in male rats. Results: GSK070 inhibited hERG and CaV1.2 receptors with IC50 of 10uM for both channels. In a subsequent rabbit ventricular wedge study there were no changes in any of the parameters measured, and from the cardiovascular rat study there is a >60-fold cover to the estimated free Cmax at a clinical dose of 33mg/day from the 10mg/kg/day dose at which no effects were observed. GSK070 was tested in a bacterial mutation screening assay (Ames test) +/- S9 fraction and was not mutagenic in any of the strains investigated and it was also negative in the MLA in the presence and absence of S9. GSK070 was tested in the rat whole embryo culture (WEC) in vitro test system and there is a minimum of an 18-fold margin from the NOEL at 10uM in the WEC and a 151-fold margin at 100mg/kg/day in a rat 7 day study. Conclusions: Based on an integrated assessment of all the safety findings (general tox, safety pharmacology, genotoxicity, reproductive toxicity) and taking into consideration that there are biomarkers and/or monitoring available for the toxicities observed in vivo. The preliminary toxicological profile (in vitro and in vivo) indicates an acceptable therapeutic window and the riskbenefit profile is such that there is no data that would preclude the progresion of this molecule. All animal studies were ethically reviewed and carried out in accordance with European Directive 86/609/EEC and the GSK Policy on the Care, Welfare and Treatment of Animals Tuberculosis 2016 – September 19-23, 2016 – Paris, France 381 352 From animal models to man 23/09/2016 In vitro grown human lung organoids as a model of tuberculosis N. Iakobachvili1, C. Lopez-Iglesias1, N. Sachs2, H. Clevers2, P. Peters1 1 M4I Nanoscopy Division, Maastricht University, Maastricht 2Hubrecht Institute, Utrecht, The Netherlands Tuberculosis remains a major global health threat; as a result, a huge number and variety of animals are being used for fundamental research into mycobacterial pathogenesis, virulence mechanisms and the development of novel treatment regimens and vaccines. Animal suffering is thought to be extensive and the validity of these models remains highly questionable as the causative agent of the disease, Mycobacterium tuberculosis, is an obligate human pathogen. Models of tuberculosis based on human cells currently do not extend beyond infection of immortalized cell lines, isolated and differentiated monocytes from human blood, and simple cocultures of the above. We are developing an in vitro model of tuberculosis using stem cells from healthy human lung tissue that have been cultured to expand, differentiate and naturally self-organize into organoids closely resembling human bronchi. The lab-grown “mini lungs” display important features of native lung epithelia and are composed of naturally differentiated cells of respiratory lineage (including ciliated, basal, secretory and epithelial cells) and their products. Together, the microenvironment of the lung is mimicked although some immune components are missing which we aim to separately introduce into organoid cultures. Mycobacteria are introduced to cultures by intraluminal injection and are monitored by 2-photon excitation microscopy and confocal microscopy when using live fluorescent stains; and by electron microscopy following high pressure freezing and freeze substitution. Whilst still in its preliminary stage, we believe that the use of a more physiologically relevant model such as the mini-lung organoids can produce more predictive data for human tuberculosis than animal models. The lung organoids can be further developed as a novel platform for screening of anti-mycobacterial compounds or adapted to model other respiratory pathogens. 382 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 353 From animal models to man 23/09/2016 Tuberculosis Preclinical and Clinical Resources at the National Institute of Allergy and Infectious Diseases. A. Kraigsley, J.P. Boyce, K. Lacourcière, B. Laughon, F.J. Leyva, M. Makhene, A. Mcbride, C.F. Sizemore NIH/NIAID/DMID/RDB, United States The Division of Microbiology and Infectious Diseases (DMID), part of the National Institute of Allergy and Infectious Disease (NIAID), part of the U.S. National Institutes of Health (NIH) is committed to finding new ways to better understand, diagnose, treat, and ultimately prevent tuberculosis (TB). The continued high burden of TB in many countries and the emergence of MDR TB and XDR TB has intensified the need for new interventions. In addition to supporting a comprehensive research and product development grants portfolio, NIAID/DMID also offers preclinical and clinical services to help researchers identify and develop products, technologies and targets relevant to treatment of tuberculosis. This presentation focuses on a summary of resources for preclinical and clinical biomedical research in TB that are issued through NIAID’s Biodefense and Emerging Infectious Diseases Programs. These services include in vitro evaluation of new chemical entities, in vivo animal model testing as well as early stage evaluation of new diagnostic candidates, vaccines, research reagents, genome sequencing and bioinformatics support. Many of these services are available at very low cost to U.S. and international investigators in academia, not-for-profit organizations, industry and governments through an application process. Requests are evaluated based on standard criteria including preliminary data and development plans. Institutional agreements will be executed to ensure confidentiality and protect intellectual property rights. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 383 354 From animal models to man 23/09/2016 Predicting the Human Dose for a novel LeuRS inhibitor, GSK070, for the Treatment of Tuberculosis M. Remuinan, F. Ortega, S. Crouch, A. Mendoza, R. Gomez, L. Alameda, S. Ferrer, D. Barros, X. Li1, M.R.K. Alley2 1 GSK, Tres Cantos, Spain 2Anacor Pharmaceuticals, Palo Alto, United States Background: GSK070 is a selective inhibitor of mycobacterial leucyl t-RNA synthetase (LeuRS) with a novel MoA for the treatment of tuberculosis (TB). After achieving positive results in the murine chronic assay for TB, preliminary human dose projections were performed to aid in clinical progression. Material/methods: Following in-vivo intravenous dosing in the preclincal species (mouse, rat and dog), the human pharmacokientic parameters of clearance and volume of distribution were derived using in-vitro/in-vivo extrapolation (IVIVE), allometry and simple PBPK modeling (CloePK Software, Cyprotex). A target efficacious exposure in human was determined using the dose (1.3mg/kg) at the maximum effect (EDmax) level observed in the therapeutic efficacy murine chronic assay. Human dose estimations were calculated using a target efficacious exposure of 3481ng*hr/ml which corresponds to the efficacious AUC0-24h at steady state measured in whole blood. Results: With the PK parameters from allometry and PBPK multiple scenarios were designed for the human dose projection in order to achieve the efficacious target exposure (whole blood and unbound). Predicted Vdss was moderate in all cases, while clearance was variable (low to moderate) depending on the modelling strategy used. All methodologies projected a low human dose (<200mg) to achieve the therapeutic target in whole blood (3481ng*h/mL). Conclusions: GSK070, a novel preclinical candidate for the treatment of tuberculosis, provides favorable predicted human pharmacokinetic parameters with a low projected human dose. All animal studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and the GSK Policy on the Care, Welfare and Treatment of Animals. 384 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 355 From animal models to man 23/09/2016 In vivo antitubercular activity of GSK070, a Mycobacterium tuberculosis leucyl-tRNA synthetase inhibitor J. Rullas1, L. Guijarro-Lopez1, A. Mendoza1, X. Li2, M.R.K. Alley2, D. Barros1 1 Biology TB, GlaxoSmithKline, Tres Cantos, Spain 2Anacor Pharmaceuticals Inc, Palo Alto, United States Background: Protein synthesis inhibitors play an important role in the treatment of tuberculosis. Parenteral aminoglycosides form a key part of the treatment of multidrug-resistant (MDR) TB and currently the oxazolidinones are the only class of protein synthesis inhibitors that are orally bioavailable and are effective against TB. The rise of resistance including extensively (XR) and totally resistant (TR) TB demands the need for new oral frontline drugs. Here, we show the excellent in vivo profile of GSK070, a new Mycobacterium tuberculosis leucyl-tRNA synthetase inhibitor (LeuRS). Material/methods: The experimental designs for the acute and combination studies have been previously described by Rullas et al. (AAC 2010; 54:2262) for the acute assay and Nuermberger et al. (AAC 2008; 52:1522) for the model used for the combinations, respectively. For the chronic assay, mice were infected intratracheally with 100 CFU, the treatment was administered once a day 7/7 for 8 weeks starting on week 6 after infection. Mice were sacrificed 24 hours after the last administrations, and lung CFU were counted by plating in OADC-7H11 media supplemented with 0.4% activated charcoal. All animal studies were ethically reviewed and carried out in accordance with European Directive 86/609/CEE and the GSK Policy on the Care, Welfare and Treatment of Animals. Results: For the acute and chronic assays, GSK070 administered orally once a day to infected C57BL/6 mice induced the killing in vivo of M. tuberculosis H37Rv. The maximum reduction of lung M. tuberculosis burden in the acute model (about 4 log10 units compared to untreated mice) was obtained upon daily oral administration of a dose of 1.1 mg/kg for 8 days. The maximum killing in the chronic model (about 2 log10 units) was obtained upon daily oral administration of a dose of 1.3 mg/kg for two months. In both models there was quantifiable remaining bacterial burden after the end of treatment, although when the treatment was prolonged, we could observe a further decline in the lung CFU counts in the chronic model. When administered in combination, the combo Pa824+Bedaquiline+Linezolid+GSK070 was more active than the standard of care (Rif+Iso+Pyr+Eta) or Pa-824+Bedaquiline+Linezolid. Conclusions: GSK070 has excellent in vivo antitubercular activity with demonstrated efficacy in both the standard acute and chronic murine assays, and when it was administered combination with new antitubercular drugs is more active than standard of care. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 385 356 From animal models to man 23/09/2016 Exploring the role of the RskA-SigK regulon in the virulence of Mycobacterium bovis A. Smyth4, F. Veyrier1, M. Behr2, E. Lavelle3, S. Gordon4 1 Institut Armand Frappier, Laval 2Department of Microbiology and Immunology, McGill University, Montreal, Canada 3Adjuvant Research Group, School of Biochemistry and Immunology, Trinity College Dublin 4School of Veterinary Medicine, University College Dublin, Dublin, Ireland Mycobacterium bovis and Mycobacterium tuberculosis (M. tb) share 99.95% nucleotide sequence identity, but while M. tb is almost exclusively a human pathogen M. bovis affects a wide range of domesticated and wild animal species and can cause zoonotic infections in humans. The precise molecular mechanisms that allow M. bovis to infect, cause disease and transmit across a wide variety of animal hosts has yet to be explained. A potential virulence mechanism that may play a role is the SigK regulon that is differentially regulated across the MTBC. The SigK regulon is under the control of the regulator RskA; two SNPs in rskA alter the gene in M. bovis, and as a result the SigK regulon is constitutively expressed in M. bovis. In comparison, M. tb shows minimal expression of genes in the SigK regulon under in vitro growth, but induces expression of the regulon during intracellular infection. No specific role in infection has been discovered for any of the SigK regulated proteins. A previous experimental infection of bovine alveolar macrophages with M. bovis 2122/97 and M. tb H37Rv has indicated differential cellular responses to the two species, most significantly an increase in the Type I interferon response to M. bovis. We aim to investigate what role the SigK regulon may have in causing this differential cellular response. The effects of constitutive SigK activation on mycobacterial interaction with innate immune cells will be analysed using a recombinant strain of M. tb containing rskA and sigK alleles of M. bovis and hence showing upregulation of the SigK regulon as observed in M. bovis. The cellular response to the recombinant M. tb strain is being explored using primary bovine macrophages and the murine cell line RAW 264.7, and analysed via transcriptomics and complementary targeted approaches. 386 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 357 From animal models to man 23/09/2016 Mycobacterial imaging with fluorescent benzothiazinone derivatives for in vitro and in vivo applications R. Sommer, S. Cole Global Health Institute, EPFL, Lausanne, Switzerland Tuberculosis (TB), a bacterial disease caused by Mycobacterium tuberculosis, remains a major global health problem and new antibacterial agents are required to fight the increasing number of drug-resistant strains. Benzothiazinones (BTZs), a new class of anti-TB molecules, are highly potent covalent inhibitors of DprE1, an essential enzyme involved in the synthesis of the mycobacterial cell wall. The drug candidate PBTZ169 is currently in phase I trials. We converted BTZ molecules into fluorescent probes by coupling the benzothiazinone core with different fluorophores, such as carboxytetramethylrhodamine (TAMRA) or the near-infrared fluorophore Dy750. These modified drugs still retained modest antimicrobial activity despite the addition of a bulky fluorescent moiety and were used to show by microscopic examination that DprE1 is situated at the poles of the bacilli in the periplasmic space of several mycobacterial species (M. marinum, M. smegmatis and M. tuberculosis). These probes are useful to track the target of BTZs and to investigate the mechanisms of drug action and the organization of the cell wall synthesis machinery. Given the high specificity of BTZs for mycobacteria, we also seek to use near-infrared fluorescent BTZ derivatives to evaluate the bacterial load in a mouse model of chronic TB by non-invasive in vivo optical imaging, thus allowing longitudinal studies, reduction of the number of animals and gain of time compared to the standard viability assays (CFU count) currently used to evaluate the efficacy of new drug regimens or vaccine candidates. M. tuberculosis reporter strains expressing near-infrared fluorescent proteins and/or luciferase will be used in parallel to optimize the sensitivity, reduce the limit of detection and increase the precision of the localization of the optical signal. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 387 358 From animal models to man 23/09/2016 The mycobacterial ESX-1 secretion system substrate ESAT-6 is not sufficient for membranolytic activity W. Conrad, M. Osman, J. Shanahan, F. Chu, K. Takaki, J. Cameron, R. Brosch, L. Ramakrishnan University of Cambridge, Cambridge, United Kingdom The mycobacterial ESX-1 secretion system is required for virulence. The ESX-1 substrate ESAT-6 is an immunodominant antigen and has been broadly implicated in ESX-1 virulence attributes. ESX1 deficient mycobacteria are defective for macrophage phagosome permeabilization, activation of cytosolic DNA sensing pathways, intramacrophage growth and macrophage death. These phenotypes are thought to be mediated via host membrane disruption which has been attributed to ESAT-6 because multiple studies have shown recombinant ESAT-6 to lyse eukaryotic membranes. We show that the membranolytic activity previously attributed to recombinant ESAT-6 is due to residual nonionic detergent ASB-14 detergent in the preparations. Our findings suggest reconsideration of all prior findings attributing ESX-1 lytic function to ESAT-6. Rather they demonstrate that ESX-1 mediated membranolytic activity is either mediated by ESAT-6 acting in conjunction with other ESX-1 substrates. Alternatively, it may simply be a marker of ESX-1 secretion, and cytolytic activity may be mediated by different ESX-1 substrates. These findings also warrant reconsideration of the role ESAT-6 plays in virulence. * Equal contributors 388 Tuberculosis 2016 – September 19-23, 2016 – Paris, France 359 From animal models to man 23/09/2016 The cytochrome bc1-aa3: an absurd target for tuberculosis? Kevin Pethe Nanyang Technological University, 59 Nanyang Drive, 636921 Singapore The clinical approval of bedaquiline (Sirturo) validated oxidative phosphorylation as a novel drug target space for tuberculosis. More recently, the discovery of small-molecules targeting the cytochrome bc1-aa3 (a terminal oxidase of the electron transport chain) has triggered interest on the respiratory cytochrome c pathway. A series of imidazopyridine amide compounds interfering with the function of the cytochrome bc1 were identified independently by several teams. Q203, the most advanced IPA derivative, is in clinical development phase I under a US FDA investigational new drug application. The metabolic redundancy of the electron transport chain, especially at the level of the terminal electron acceptor, raises some questions about the chemical vulnerability of the respiratory cytochrome c pathway. Our data demonstrate that the potency of Q203 is greatly influenced by many external factors, including nutrient availability and growth conditions. In addition, functional redundancy with alternate terminal electron acceptors modulates the essentiality of the cytochrome bc1-aa3. The possible consequences for clinical development of Q203 and for treatment of other mycobacteria infections will be discussed. Tuberculosis 2016 – September 19-23, 2016 – Paris, France 389 360 From animal models to man 23/09/2016 Methionine Aminopeptidase B (mapB), but not mapA, is essential for M. Tuberculosis viability Daniel. Barkan Koret School of Veterinary Medicine, Hebrew University, Rehovot, Israel Introduction: In all living cells, the initiating methionine in most newly synthesized proteins is removed by a Methionine Aminopeptidase enzyme (MAP). For reasons not completely clear, the genes coding for these enzymes are essential in all living cells. In cells that have two copies, there is either one essential and one redundant, or they are synthetically lethal. Mycobacterium tuberculosis has two genes – mapA and mapB. Their respective essentiality is currently unknown. A study using chemical inhibitors concluded mapA was essential, and mapB was not. In contrast, analysis of a transposon library concluded that mapB was the essential one. We decided to individually delete each of these genes to resolve which one is essential, and what is the phenotype of deletion of the non-essential one. Methods: For each gene, we pre-complemented M. bovis bacteria with an extra copy of the gene at the attB site, performed a deletion of the native gene with a transducing phage, and then attempted to remove the complementing genes using another integrating cassette. In one case, we also use conditional expression. Results: We successfully deleted each gene, when the bacteria was pre-complemented with that same gene. When we attempted to remove the complementing gene of mapA, we could easily do it, getting a full mapA deletion mutant. This mutant had a very mild growth retardation phenotype invitro. In contrast, removal of the complementing mapB from a ΔmapB mutant was not possible, thus proving mapB was essential for the survival of the bacteria in-vitro. Conclusions: Contrary to result previously obtained by chemical inhibition and by ASRNA, mapB, and not mapA, is the essential MAP gene in M. bovis (and Mtb). Future attempts at an inhibitor development should therefore focus on it, rather than mapA. The reason why this pathway is essential remains unclear, for the mare function of methionine removal appears, to us, not sufficient for rendering the genes essential. 390 Tuberculosis 2016 – September 19-23, 2016 – Paris, France AUTHORS & CO-AUTHORS INDEX A Abdallah A. Abdelaal H. Abdin M.Z. Abdulwhhab M. Abel L. Abid M. Abrahams K. Accard G. Adam D. Adamson J. Addicott K. Aebersold R. Afanasyev P. Afzali Behbahani M. Agarwal N. Agarwal S. Aguilar Pérez C. Aguilo N. Ahmed W. Ahuja N. Ainsa J.A. Ait Benhassou H. Ajuh P. Akabayov B. Akopian T. Al Saati T. Alameda L. Alavi S.M. Alcaîs A. Alderwick L.J. Aldrich C. Alemparte C. Alisjahbana B. Allen M.D. Alley M.R.K. Alonso M. Al-Saati T. Alvarez M. Alvarez S. Alzari P. Amallraja A. Amorim Franco T. Amzazi S. Andersen P.L. Andreas N. Andreu N. Andrew P. Andries K. Angala S. Angulo N. Anil Kumar V. Annisa J. Antonio M. Anton-Leberre V. Anwar S. Appelberg R. Apriani L. Author/Co-Author 13 90P 162P 280P 258P 95P, 313P 109P, 133P 234P 245P 131P 248P 67P 172P 65P 162P 152P, 249P 309P 30 138P 242P 309P, 330P 95P, 313P 136P 203P 126P 298P 354P 65P 258P 122P, 204P, 240P 336P 133P, 350P 89P, 278P, 308P 351P 55, 350P, 354P, 355P 206P 46 215P 30 215P 91P 205P 313P 26 177P 117P, 238P 116P 346P 10 266P 148P 308P 141P 46 239P 270P 278P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Apt A. Aragaw W. Arnold F. Arnvig K. Arora D. Arora G. Arora K. Arora N. Arries J. Arya R. Asante-Poku A. Åsberg S. Ascher D. Astolfi Rosado L. Ates L. Aubry A. Aung T. Avdienko V. Av-Gay Y. Ayorinde A. Aziz D. 263P 310P 167P 96P 150P, 281P 311P 337P, 340P 164P 97P 210P 66P, 86P, 142P, 349P 292P 19 206P 13, 163P 72P 232P 263P 98P 267P 312P, 334P B Author/Co-Author Bach H. 98P Bachy V.S. 181P Bacon J. 240P Bacqué E. 129P, 147P, 314P Baek S. 144P Baer C. 107P Bah A. 235P Bahrmand A. 183P Baidoo A. 231P Bailo Vergara R. 99P Bajpai U. 211P Balakrishna A. 219P Balboa L. 298P, 306P Balhana R. 100P, 102P, 134P Baliga N. 99P Ballell L. 109P, 233P, 339P Ballell-Pages L. 54 Ballinger E. 147P Banaei-Esfahani A. 67P Bancroft G.J. 304P Bandyopadhyay S. 170P Baneres Roquet F. 209P Banerjee N. 114P, 118P Bansal R. 148P Baranyai Z. 326P Barbouche R. 283P Barer M. 280P Barisch C. 282P Barros D. 109P, 339P, 350P, 354P, 355P Barros-Aguirre D. 55, 351P Barry C. 31, 60, 337P, 338P, 340P Barton A. 20 Barzon L. 291P Bassano L. 132P 393 Basu P.S. 100P, 102P Basu Roy R. 177P Batra S. 151P Battaglia S. 68P Batthyány C. 215P Bauer A. 129P Baughn A. 319P Baulard A. 53, 318P Bayliss T. 340P Beckert P. 84P Beckham K. 15, 165P, 207P Behar S. 47 Behr M. 41, 78P, 79P, 251P, 356P Bellinzoni M. 54, 215P Belon C. 234P Benabdessalem C. 283P Benard A. 46, 125P, 298P Bendt A. 269P Benedetti Y. 130P Bentaleb E. 313P Berger C. 172P, 173P Berná L. 4 Bernard C. 72P Berney M. 101P Berney-Meyer L. 101P Bernut A. 233P, 235P, 241P Bertozzi C. 218P, 226P Besra G. 109P, 122P, 133P, 204P, 316P Best K. 196P Beste D. 100P, 102P Bestebroer J. 163P Beuerman R. 232P, 335P Bhagyaraj E. 242P Bhaskar S. 198P Bhatnagar R. 114P, 118P Bhatt A. 99P, 123P, 204P Bhatt S. 294P Bhuwan M. 164P Biot C. 233P, 327P Birhanu A. 103P Bishwal S. 210P Biswas P. 104P Biswas R. 186P Bitter W. 13, 15, 163P, 165P, 168P, 286P, 315P Biukovic G. 208P, 329P Blaise M. 209P, 229P, 233P, 237P, 252P Blanc I. 129P, 130P, 147P, 314P Blanc L. 221P Blanchard J. 205P, 212P, 230P Blanco-Ruano D. 133P Blanc-Potard A.B. 234P Blazquez J. 69P Blomgran R. 266P Blundell T. 19, 337P Boisson-Dupuis S. 62, 258P Boom H. 71P Boot M. 315P Borel S. 296P 394 Borgers K. Borges M. Boritsch E. Borrás R. Borrell S. Borroni E. Boshoff H. Bosmans R. Bösze S. Botella H. Bottai D. Boucher N. Bouchier C. Boudinot P. Boulenc X. Boulle M. Boutte C. Boyce J.P. Boy-Röttger S. Braian C. Brandenburg J. Bräuchle C. Braverman J. Brennan P.J. Bresciani N. Brighenti S. Briot C. Brites D. Brodin P. Bronda K. Brosch R. Broset E. Brossier F. Brown K. Brown M. Browne J.A. Bryant J. Buchanan K. Buechler J. Bull T. Bunduc C. Bunjun R. Burgers W. Burke M. Burmeister M. Burns-Huang K. Bushdid P. Busso S.L. Bustamante J. Butcher P. Buttari N. C Cabibbe A.M. Caccamo N. Cacho M. 105P, 180P 270P 106P 182P 67P, 70P 68P 60, 147P, 337P, 338P, 340P 172P, 173P 326P 9, 17, 154P 304P 14 106P, 233P 46 129P, 130P, 314P 299P 107P 353P 213P 259P 225P, 243P, 301P 225P 44 321P 108P 300P 130P, 314P 71P, 81P 1, 221P, 305P, 318P 288P 3, 29, 106P, 175P, 215P, 233P, 235P, 304P, 305P 30, 178P 72P 39, 288P 239P 247P, 264P 39 340P 19 137P 15, 165P 244P, 271P 244P, 271P 51 245P 147P 351P 66P, 86P 258P 23 54 Author/Co-Author 68P 22 351P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Cacho-Izquierdo M. Cadieux N. Callewaert N. Camassa S. Cambier C. Cameron S. Cantalloube C. Carata E. Cardoso F. Cardoso M. Carlsson F. Carrat C. Carroll P. Carvalho T. Casanova J.L. Cascioferro A. Castañeda-García A. Castro R. Castro-Ramos M. Catanzaro A. Catanzaro D. Catinas O. Cebrián R. Cha H. Cha S.B. Chae H. Chaidir L. Chain B. Chan C. Chandra V. Chase M. Chaudhari T.K. Chaudhuri T.K. Chavez A. Chawla Y. Chee C. Chen C. Chen D. Chen S.L. Chernyaeva E. Chiarelli L.R. Chibale K. Chindelevitch L. Chiner-Oms Á. Chiodarelli G. Chng S. Cho S.N. Choi H. Chor J. Christensen D. Chung J.C.S. Church G. Cia F. Cianci M. Ciccarelli L. Cichonska A. Cirillo D.M. Clark R. 316P 202P 105P, 180P 253P 284P 205P 129P, 314P 253P 276P 270P 297P 265P 194P 276P 258P 215P 69P 70P 4 91P 91P 299P 309P 184P 185P, 199P, 200P, 261P 179P 89P, 308P 196P 93P, 157P 242P 7 164P 111P, 198P 7 281P 269P 60 172P, 173P 157P 73P, 74P 54 345P 317P 75P 316P 32, 176P 200P, 261P 246P, 261P 232P 194P 157P 7 304P 113P 15, 165P 23 68P, 108P 14 Tuberculosis 2016 – September 19-23, 2016 – Paris, France Clark T.G. 238P Clevers H. 352P Clish, Clary C. 308P Cobat A. 258P Coeck N. 346P Cohen-Gonsaud M. 285P Coker E. 267P Cole S. 19, 56, 64, 85P, 138P, 174P, 213P, 331P, 357P Coler R.N. 200P Collière L. 129P Collins B. 67P Collinson L. 296P Comas I. 75P, 182P, 317P Commandeur S. 286P, 315P Conlon K. 264P Cook G. 155P Cooper A.M. 45 Correia C. 247P Cortes T. 51, 117P Cortesía C. 175P Coscolla M. 67P, 141P Costa-Gouveia J. 318P Cougoule C. 298P, 306P Couturier C. 147P Cox J. 48, 109P Cox L.R. 316P Crawford C. 348P Crommelynck S. 304P Crouch S. 354P Cruciani M. 291P Cumming B. 131P, 248P Cunningham F. 339P Cusack S. 55 D Author/Co-Author Dahl J. 110P Dalecki A. 348P Dallenga T. 287P Danilchanka O. 166P Dartois V. 60, 216P, 227P, 337P Daryan A.K. 181P Das A. 186P, 210P Das G. 275P Das M. 210P Datta P. 20 Davis L. 141P De Carvalho L. 132P De Chastellier C. 235P, 237P De Jong B. 141P, 346P De Libero G. 269P De Matteis L. 330P De Proft F. 206P De Sessions P.F. 238P De Souza Carvalho-Wodarz C. 296P Debnath R. 210P Debnath S. 210P 395 Degiacomi G. 54 Del Carmen Sasiain M. 46 Delacourt C. 258P Delogu G. 253P Delorme V. 318P Demina G. 228P Den Hertog A. 25 Deramaudt T. 235P Derbyshire K. 14 Dhar N. 19, 85P, 156P Dhiman R. 249P Dhingra S. 250P Di Camillo B. 291P Diallo M. 7 Dian S. 89P, 308P Diandini E. 278P Dick T. 140P, 157P, 159P, 160P, 208P, 216P, 227P, 310P, 312P, 329P, 334P, 335P, 336P, 343P Diedrich C. 296P Dieli F. 22 Diels M. 141P Diez-Tascón C. 272P Dillon N. 319P Dini L. 253P Dionne M. 288P Divangahi M. 41 Dockrell H.M. 239P Dodd C.E. 49 Doherty A. 69P Doherty T.M. 193P Dolganov G. 141P Dolinger D. 317P Domenech P. 79P Donini S. 19, 337P Doornbos K. 166P Doubovetzky M. 129P, 314P Dougan G. 125P Drancourt M. 94P, 209P, 229P Drewes G. 109P Driessen M. 141P Drijkoningen J. 39 D. Robertson B. 143P Drocourt D. 221P Dubar F. 233P, 327P Dubé J. 251P Dubey N. 289P Dubois V. 169P, 252P Dufe V. 206P Dumas E. 252P Dupont C. 169P, 233P, 327P Durán R. 215P Dutilh B. 89P Dutta A. 186P Duval C. 298P Dwivedi V.P. 275P Dyatloff A. 263P 396 E E. Schaible U. Easom E.E. Egere U. Eggers L. Ehrt S. Ehtesham N.Z. Eich J. Eisenach K. Ekins S. El Messaoudi M.D. Elayati K. Elghraoui A. Elharar Y. Elliott S. Engel R. Eniyan K. Epemolu R. Equbal M.J. Erdogan H. Eskandarian H.A. Esposito M. Etna M.P. Evangelopoulos D. Evans J. Everall I. Eyangoh S. Eynard N. Ezewudo M. F Fabre E. Farrow-Johnson J. Fateh A. Favrot L. Fayolle C. Feilcke R. Feinberg J. Feldmann J. Fernandez-Reyes D. Ferrer S. Ferrer-Bazaga S. Festjens N. Fillatreau S. Fimia G. Finin P. Fink L. Fissette K. Fletcher H. Flo T. Floto A. Floto R.A. Flynn J. Fohtung J. Fontaine E. Foo C. Forson A. Author/Co-Author 194P 350P 267P 273P 9, 17, 154P 111P, 164P, 198P, 255P 287P 71P 54 313P 112P 76P, 77P, 83P 119P 290P 225P 211P 340P 198P 206P 18 54 291P 23 81P 39 40 321P 317P Author/Co-Author 265P 145P 183P 205P, 212P, 230P 304P 320P 258P 70P 23 354P 339P 105P, 180P 46 253P, 291P 131P 91P, 92P 141P 239P 170P, 260P, 292P 288P 39 60 202P 129P, 314P 213P 142P, 231P, 349P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Forti F. Fortune S. Fraisse L. Frank M. Franzblau S. Freire D. Frigui W. Fuerst J. Furusho H. Futterer K. 136P 7, 33, 115P, 126P, 202P 129P, 130P, 147P, 314P 84P 341P 113P 215P, 304P 3 172P, 173P 109P G Author/Co-Author 273P 214P 5, 66P, 67P, 70P, 71P, 81P, 86P, 141P Gaibelet G. 265P Gaifem J. 276P Gaillard J.L. 234P, 235P Ganiem A. 308P Gao Q. 6 Garcia B. 141P Garcia J. 137P García S. 330P García-García S. 272P Garcia-Pelayo C. 181P Garcia-Vallejo J. 163P Garg R. 114P Garton N. 280P Garza-Garcia A. 51 Geerlof A. 207P Gehre F. 141P Genevaux P. 113P Gengenbacher M. 157P, 160P, 216P, 336P Gennaro M. 20 Gerrick E. 7, 115P Geslin M. 129P Ghadiri A. 324P Ghanei M. 183P Ghanem M. 78P, 79P Ghisotti D. 136P Ghoma Linguissi L.S. 84P Giacomini E. 291P Gicquel B. 30, 46, 193P Gideon H.P. 195P Gidon A. 292P Gijsbers A. 173P Gil E. 196P Gil M. 215P Gil Castro A. 270P, 276P Gil-Brusola A. 182P Gilleron M. 10, 127P, 221P, 265P Gillespie S. 23, 158P Gilman R. 266P Ginbot Z. 271P Ginda K. 121P Giordano I. 350P Gabriel G. Gaevaya L. Gagneux S. Tuberculosis 2016 – September 19-23, 2016 – Paris, France Girardi E. 253P Girard-Misguich F. 169P, 252P Gisbers A. 172P Glenn S. 116P Glickman M.S. 9 Go M.L. 334P Gobe I. 100P, 102P Goig Serrano G. 182P Gold B. 37, 146P, 147P Goldberg A. 126P Goldmann T. 243P Goletti D. 253P Goliath R. 195P, 271P Gomez R. 354P Gomez-Romero M. 177P González Del Río R. 339P González-Candelas F. 75P González-Montaner P. 306P Gonzalo-Asensio J. 30, 178P, 190P Goodarzi H. 65P Goodmanson A. 91P Gopal P. 160P, 216P, 227P Gordon S. 59, 247P, 264P, 356P Gormley E. 264P Gosetti F. 54 Gouilleux L. 147P Goulard C. 234P Goullieux L. 314P Goyal R. 148P Grabowska A. 113P, 117P Gracia B. 309P Grassi A. 291P Gray T. 14 Green S. 337P, 338P, 340P Gref R. 318P Greif G. 4 Gresham S. 351P Griffiths G. 287P, 296P Grode L. 27 Grogono D. 39 Gröschel M. 304P, 305P Grüber G. 208P, 219P, 329P Grzegorzewicz A. 321P Guerardel Y. 209P, 220P, 233P, 327P Guerin M. 34 Guermonprez P. 288P Guerra-Laso J. 272P Guijarro-Lopez L. 133P, 355P Guilhot C. 8 Gupta A. 158P Gupta M. 118P, 289P Gupta P. 242P Gupta V. 114P Gur E. 119P Gurumurthy M. 322P Gutierrez C. 113P Gutierrez M. 132P, 296P Gygli S. 67P 397 H Author/Co-Author Hadizadeh Tasbiti A. 183P Hafneh N. 254P Hagens K. 194P Haj Aissa F. 283P Håkansson A.P. 332P Haks M.C. 22 Haldar K. 280P Haldar P. 280P Hammond R. 158P Han S.J. 179P, 185P Hanna N. 207P Harris S. 39, 66P, 86P Harrison J. 204P, 340P Hartkoorn R. 19 Harvey N. 177P Hasenoehrl E. 101P Hashemi Shahraki A. 325P Hashemzadeh M. 325P Hasnain S.E. 111P, 164P, 198P, 255P Hattendorff J. 71P Haug M. 260P Hauptmann M. 256P Haworth C. 39 Hayova L. 214P Hee D. 322P Hegde S. 138P, 212P Henrissat B. 94P Hernandez J.E. 120P Hernandez V. 350P Hernández-Pando R. 13 Herrmann J.L. 38, 169P, 233P, 234P, 235P, 241P, 252P, 327P Herrmann T. 85P Hibberd M.L. 238P Hill E. 39 Hill J. 202P, 310P, 343P Hill P. 278P Hiller M. 301P Hillery N. 317P Hilpert K. 137P Hingley-Wilson S. 120P Hmama Z. 187P Hoal E.G. 258P Hoffmann H. 68P Hoffmann-Thiel S. 68P Hoffner S. 83P, 93P Hofmann S. 137P Hogarth P.J. 181P Hohl Michael. 167P Holmes E. 177P Holst O. 225P Honeyborne I. 23 Honoré N. 106P Hopkins F. 7 Hotra A. 208P Houben E. 13, 15, 165P, 168P Houle D. 41 398 Houthuys E. Hrabalek A. Huan C. Hudrisier D. Huijnen M. Hunt D. Hunter J. Hunter W. Hurwitz R. Hwa S. Hwang M.H. I Iakobachvili N. Imming P. Indrati A. Inwentarz S. Ioerger T. Iqbal I. Isabel Veiga M. Izumi H. 180P 323P, 342P 334P 43, 46 89P 132P 299P 217P 128P 257P 80P Author/Co-Author 172P, 173P, 286P, 352P 320P, 341P 278P 306P 107P, 337P 294P 276P 3 J Author/Co-Author J Rubin E. 252P Jabot-Hanin F. 258P Jackson L. 91P Jackson M. 10, 127P, 321P Jacobs W.R. 2, 101P, 125P, 155P, 230P Jäger F. 217P Jakimowicz D. 121P Jang M.S. 221P Jang Y.H. 80P Jankute M. 122P Janmeja A. 242P Janvier G. 222P Jarlier V. 72P Jary D. 330P Javid A. 123P Javid B. 124P, 161P Jensen K. 229P Jeong H. 184P Jeronimo Frias Rose J. 300P Ji S. 269P Jiang X. 146P Jim K. 315P Jiménez B. 177P Jiménez C. 13 Jiménez-Navarro E. 351P Joe M. 204P Joloba M. 71P Jones V. 321P Jonnala S. 131P Joosten S.A. 22 Jouneau L. 46 Jouny S. 318P Juambeltz A. 4 Juan-García J. 272P Tuberculosis 2016 – September 19-23, 2016 – Paris, France K Author/Co-Author Kaiser P. 128P Kalia N. 338P Kalra R. 242P Kalscheuer R. 123P Kalsum S. 259P Kamariza M. 218P Kaminski L. 125P Kampmann B. 177P, 267P Kana B. 116P Kandror O. 126P Kaneko T. 130P Kannan N. 260P Kanno A.I. 178P Kant S. 114P Kaprelyants A. 228P Kapuria A. 249P Karabanovich G. 323P, 342P Kaufmann S.H. 46, 128P, 157P Kaya F. 216P, 227P Keane J. 112P Keating T. 240P Keep N. 223P Kendall S. 87P, 223P Keri G. 19 Kester J. 126P Khan R. 20 Khandan Dezfuli S. 325P Khanna V. 106P Khare G. 293P Khosravi A.D. 65P Khosravi Boroujeni A. 324P, 325P Khubaib M. 164P, 255P Kidwai S. 152P, 249P Killick K. 247P Kim H. 185P, 199P, 200P, 246P, 274P Kim H.M. 261P Kim J.M. 80P Kim J.S. 185P, 199P, 246P, 261P, 274P Kim N.R. 80P Kim S.Y. 236P Kim W.S. 185P, 199P, 200P, 246P, 261P, 274P Kim Y. 77P Kiravu A. 271P Kispert A. 243P Kissane K. 147P Kitdorlang Dkhar H. 242P Klieber S. 129P Klimešová V. 323P, 342P Kling A. 129P Kneuper H. 217P Koch A. 81P, 149P Koeken V. 308P Koh W.J. 236P Kohl T.A. 84P Kolbe K. 225P Kolly G. 213P Kondova I. 298P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Kondratieva E. 263P Kondratieva T. 263P Koram K. 66P, 86P Korduláková J. 127P, 321P Korman M. 119P Korotkov K. 168P Korotkova N. 168P Kraigsley A. 353P Krátký M. 326P, 347P Kreitmann L. 41 Kremer L. 42, 94P, 169P, 209P, 220P, 229P, 233P, 234P, 235P, 237P, 241P, 252P, 327P Kreutzfeldt K. 17 Krishnamoorthy G. 128P Krishnan N. 307P Krügel A. 167P Kudryavtsev I. 201P Kuijl C. 163P Kuittinen I. 23 Kumar A. 148P, 211P, 219P, 294P Kumar V. 308P, 328P Kundu P. 186P Kundu S. 208P, 329P Kuroda M. 306P Küssau T. 229P Kutsch O. 277P Kviatcowsky D. 306P Kwedi S. 84P Kwon K.W. 185P, 199P, 200P, 261P, 274P L Author/Co-Author L. Andersen P. 194P Laboudie P. 129P Lacourcière K. 353P Laencina L. 169P, 252P Lagrange S. 19, 57, 129P, 130P, 147P, 314P Lai R. 51 Lair C. 129P, 130P, 314P Lakshmanan U. 310P, 343P Lakssir B. 95P Lalande J.D. 41 Lamers M. 33 Lamprecht D. 131P Lang D. 156P Lang R. 46 Lang U. 33 Lapidus A. 73P Lapierre P. 14 Lardizabal A. 197P Larrouy-Maumus G. 10, 104P, 132P Lasserre M. 4 Lastrucci C. 298P Lau A. 187P Laughon B. 353P Lavelle E. 356P Layre E. 265P 399 Le Chevalier F. 215P Le Moigne V. 169P, 234P, 252P, 327P Lechartier B. 213P, 331P Leclerc C. 304P Lee H.S. 80P Lee L. 322P Lee M.H. 9 Lee R.E. 321P Lee S. 184P, 303P Lefrançois L. 295P Leite L.C. 178P Leitges M. 243P Lelievre J. 233P, 316P Lelli M. 85P Leopold D. 267P Lerm M. 171P, 259P, 262P, 266P, 300P Lerner T. 296P Leung L. 155P Leyva F.J. 353P Li X. 55, 350P, 354P, 355P Liang Y. 232P Lienard J. 297P Lima A. 215P Lin G. 37 Lincoln R. 351P Lindenberger J.J. 321P Lindhorst T. 225P Lindroth M. 259P Ling C. 310P, 343P Ling Y. 147P Linge I. 263P Linke D. 301P Lisa N. 215P Liu J. 336P Liu S. 335P Lloyd G. 204P Locht C. 283P Lodh A. 210P Loitto V. 171P López G. 175P Lopez Quezada L. 147P López-Fidalgo E. 272P Lopez-Iglesias C. 172P, 173P, 352P López-Medrano R. 272P Lopez-Perez P.M. 137P Lotterman A. 202P Louet C. 292P Lounis N. 346P Lovering A. 204P Low J.L. 216P Lowary T.L. 204P Loyens M. 283P Lu Q. 322P Lucía A. 330P Lugo-Villarino G. 298P, 306P Lupien A. 19, 213P, 331P Lupoli T. 37 Lustig G. 299P 400 Lutfalla G. Luukinen H. M Ma L. Ma N. Małecki T. Machado H. Machugh D. Mackenzie C. Madduri A. Madzou-Laboum I.K. Maerk M. Magee D. Magnon M. Mahajan S. Mahamed D. Mahmoudi S. Majlessi L. Mak Y.S.L. Makarov V. Makhene M. Makinoshima H. Malakar B. Maletta M. Malm S. Malone K.M. Mamishi S. Manfredi M. Manganelli R. Mangas K. Manimekalai S. Manina G. Manjari S. Mao W. Maqueda M. Marais S. Marengo E. Margolis A. Maridonneau-Parini I. Mariller C. Marino L. Marinova D. Marlovits T. Marriner G. Martin C. Martin D. Martin Z. Martín C. Martineau A. Marwitz S. Maserumule M. Matange N. Mathonsi C. Mawatwal S. Maximov G. Mayer C. 241P 189P Author/Co-Author 106P 120P 121P 276P 247P 340P 132P 84P 170P 247P, 264P 129P, 130P, 314P 242P 257P, 299P 188P 235P, 304P, 305P 350P 56, 116P, 331P 353P 9 281P 163P 84P, 225P 264P 188P 54 20, 54, 291P 3 219P 12 279P 350P 309P 51 54 321P 298P, 306P 220P 132P 30 15, 165P 60 30, 178P 81P 156P 190P 196P 243P 288P 104P 299P 249P 201P 222P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Mazurek J. Mc Neil M. Mcbride A. Mcfadden J. Mchugh T. Mc-Hugh D.E. Mcintosh F. Mckinney J. Mcloughlin K. Mcneil M.R. M. De La Fuente J. Meehan C. Meghdadi H. Meijgaarden K.E. Meikle V. Meiler E. Mellors T. Méndez M. Mendoza-Losana A. 265P 10 353P 102P, 120P, 134P, 279P 23 264P 41, 78P, 79P, 251P 19, 156P 247P 321P 330P 141P, 346P 324P 22 332P 133P 202P 266P 55, 133P, 339P, 350P, 351P, 354P, 355P Mendum T. 102P, 134P, 295P Mendy A. 267P Menezes S. 288P Mercier I. 46, 298P, 306P Merker M. 68P Mery A. 220P Messens J. 206P Mi J. 146P Mikušová K. 54, 127P, 323P Milla A. 314P Miller R. 196P Minato Y. 319P Minch K.J. 145P Minnikin D.E. 82P Miotto P. 108P, 317P Mirsaeidi M. 324P Mitra A. 135P Mizrahi V. 19, 81P, 149P, 156P, 340P Möckl L. 225P Modlin S. 76P, 83P Mohan M. 350P Molle V. 150P Mollenkopf H.J. 128P, 157P Möllmann U. 341P Mondesert G. 19, 147P Monk I. 3 Montamat-Sicotte D. 41, 251P Moody B. 79P, 132P Moores A. 96P Moosa A. 345P Moraña E.J. 306P Moraski G. 333P Moreira W. 140P, 312P, 334P Moreno P. 39 Mori G. 54 Morozova T. 268P Morris-Jones S. 196P Movert E. 297P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Mukamolova G. Mukherjee D. Muller T.L. Müller R. Munier-Lehmann H. Munshi T. Murugesan D. Musters R. Muvva J. Myllymäki H. 116P, 136P, 254P, 286P 335P 271P 129P 222P 137P 340P 163P 300P 189P N Author/Co-Author 150P 322P 36, 138P 299P 247P, 264P 139P, 144P 210P 151P, 250P 281P 242P 219P 37, 146P, 147P, Nathan C. 154P Navarro F. 330P Navas V.H. 106P Naya H. 4 Nebreda-Mayoral T. 272P Nedialkova L. 68P Negatu D. 336P Němeček J. 323P, 342P Neo H. 269P Neres J. 213P Netea M. 308P Neyrolles O. 43, 46, 113P, 221P, 298P, 306P Ngan G.J.Y. 140P Nicol M. 81P Niederweis M. 16, 135P, 166P, 332P Niemann S. 68P, 84P, 346P Nigou J. 10, 122P, 127P, 221P Niskanen M. 189P Noack S. 122P Noh K. 102P North E.J. 321P Notebaart R. 308P Notelaers K. 172P, 173P Noursadeghi M. 196P Novais Bastos H. 276P Ntoumi F. 84P Nuermberger E.L. 130P Nukdee K. 113P Nur M. 286P Nurani A. 278P Nyantakyi S. 334P Naandicoori V.K. Naftalin C. Nagaraja V. Naicker M. Nalpas N. Nambi S. Nanda R. Nandi M. Nandicoori V. Nanduri R. Nartey W. 401 O Obrien S. Odermatt N. Ofori-Anyinam B. O'garra A. Oh D. O'hare H. O'leary S. Omari S. Omondi F. Ong R.T.H. Oni T. Oom L. Oravcova K. Ordway D. Orena B.S. Ortega F. Osei-Wusu S. Ostrovsky D. O'sullivan M. Otchere I.D. Ottenhoff T. Otter A. Oualim A. Ouerfelli O. Ouni R.Y.M. P Pacitto A. Pain A. Palencia A. Palló A. Palmer T. Palucci I. Pan M.M. Pancani E. Pandey S. Paquet M. Parasa V.R. Pardini M. Parikka M. Parish T. Park S. Park Y. Parkhill J. Parret A. Pasca M.R. Pato J. Paton N. Pavan F. Pávek P. Pawlik A. Péan C. Pedre B. Pellet A. Penlap Beng V. Perdih A. 402 Author/Co-Author 73P, 74P 85P 141P 46 77P 35 112P 142P, 349P 244P 160P 81P, 195P, 271P 299P 158P 39 54 354P 66P, 86P, 142P, 349P 228P 112P 66P, 86P, 142P, 349P 22 87P 314P 154P 283P Author/Co-Author 19 13 55 206P 217P 253P 161P 318P 111P, 164P, 198P, 255P 41 300P 291P 189P 194P 146P 60, 337P 39, 66P, 81P, 86P 15, 113P, 165P, 207P 54 19 322P 132P 323P 106P, 175P, 233P, 304P 288P 206P 314P 84P 211P Pérez Sánchez I. Perez-Herran E. Peters P. Peterson E. Petrella S. Petrone L. Petrova E. Petruccioli E. Pettigrove M. Pham A. Pham H. Phan T. Phelan J. Picavet D. Pidot S. Pilátová M. Pillay K. Pingris K. Pióro M. Pissis C. Pitard B. Piton J. Plattner J.J. Plets E. Poce G. Pogenberg V. Poincloux R. Poquet Y. Portela M. Porter J. Posey J. Poulsen A. Pourakbari B. Prados-Rosales R. Prandi J. Prasad A. Prideaux B. Priestman M. Prieto A. Pritchard J. Prosser G. Pyle C.J. Pym A. 30, 190P 55, 133P, 350P 172P, 173P, 352P 99P 222P 253P 263P 253P 91P 72P 10 168P 238P 25, 163P 3 288P 299P 306P 121P 222P 234P 213P 350P 105P, 180P 32 113P 298P 221P 215P 3 317P 310P, 343P 188P 191P, 192P 221P 279P 60 143P 69P 7, 223P 338P 49 61, 299P Q Author/Co-Author 321P Quémard A. R Rae Sajorda D. Raffetseder J. Ragan T. Ragunathan P. Rahman M. Rajaram M. Rajwani R. Rakotosamimanana N. Ramakrishnan L. Author/Co-Author 101P 171P 132P 208P 131P 49 88P 193P 63, 284P, 302P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Rämet M. 189P Ramirez-Busby S. 91P Ramon Garcia S. 339P Ramos A. 276P Rampersad S. 299P Ramsheh M. 280P Ranganathan A. 275P Rao S. 224P Raposo-García S. 272P Rasolofo V. 193P Ravelli R. 172P, 173P Ravi J. 20 Rawkins A. 58 Ray P. 337P, 338P, 340P Rayasam G. 211P Raynaud-Messina B. 306P Raze D. 283P Read K. 340P Redinger N. 194P Redmond S. 302P Reed M. 79P Reed S.G. 200P Regad L. 222P Rego E.H. 161P Rego H. 11 Rehwinkel J. 46 Reiche M. 156P Reiling N. 225P, 243P, 245P, 301P Reimer R. 287P Remuinan M. 354P Repnik U. 287P, 296P Resende M. 270P Reseqtb Consortium C. 317P Ressami E. 95P Reynolds E. 204P Rhee K. 337P Riaz T. 103P Riccardi G. 54 Richard V. 193P Richard-Greenblatt M. 98P Richter A. 341P Riesco A. 96P Rigouts L. 346P Ring S. 273P Riou C. 195P, 244P, 271P Rittershaus E. 144P Rivera B. 215P Rivero-Lezcano O. 272P Rizal Ganiem A. 89P Rizzi M. 19, 337P Robbins K.K. 60 Robello C. 4 Roberts J. 146P, 147P Robertson B. 307P Roca Soler F. 302P Rock F.L. 350P Rock J. 7, 33 Rockwood N. 51 Tuberculosis 2016 – September 19-23, 2016 – Paris, France Rodrigues F. 276P Rodriguez-Beltran J. 69P Rodriguez-Rincon D. 39 Rodriguez-Rivera F. 226P Rodwell T. 91P, 317P Roe J. 196P Roh J. 323P, 342P Rohde M. 296P Rojas A. 69P Romagnoli A. 253P, 291P Ronacher K. 23 Ronning D.R. 321P Rossello J. 215P Rotkevich M. 73P, 74P Rottman M. 235P Rouanet C. 283P Roubert C. 147P Rousu J. 23 Roux A. 235P Rubin E. 7, 11, 21, 107P, 169P, 202P Rue-Albrecht K. 264P Ruesen C. 89P, 308P Rullas J. 355P Rullas-Trincado J. 133P Rüsch-Gerdes S. 84P Russell D. 52 Russell M. 296P Russell S. 131P Rustad T. 145P Ryan L. 292P Rybniker J. 213P S Sacchettini J. Sachs N. Saelens J. Saeys Y. Saini A. Saito K. Sajid A. Sakwa I. Sala A. Sala C. Salina T. Salzano G. Sampson S. Sander P. Sans S. Santos Villarejo A. Sapriel G. Saqib M. Saraiva M. Sarathy J. Šarkan M. Sarkar D. Sasiain M.C. Sassetti C. Author/Co-Author 337P 352P 303P 105P 242P 37, 146P 150P 46 113P 19, 138P, 174P, 213P 268P 318P 97P 28 129P, 130P, 147P 339P 252P 198P 276P 60, 216P, 227P, 227P 127P 148P 298P, 306P 7, 107P, 139P, 144P 403 Sayes F. 304P, 305P Schaaf K. 277P Schaible U. 245P, 256P, 287P Schena E. 68P Schiebler M. 288P Schierloh P. 46 Schito M. 317P Schlesinger L.S. 49 Schlussel S. 119P Schnappinger D. 7 Schneider B. 273P Schneider T. 113P Scholl E. 25 Schoolnik G. 141P Schubert O. 67P Schurr E. 258P Schütze S. 301P Schwudke D. 243P Scott M. 158P Scullion P. 340P Scutigliani E. 25 Seeger Markus. 167P Seemann T. 3, 106P Sefrioui H. 313P Seldon R. 81P Seng K.Y. 322P Serebryakova M. 201P Serrano I. 330P Seth A. 114P Severa M. 291P Sewgoolam B. 149P Shah S. 348P Shahrezaei V. 143P Shanmugan R. 91P, 93P Sharma A. 111P, 150P, 164P, 197P, 198P, 289P Sharma C. 242P Sharples K. 278P Sheikh J.A. 198P Shen L. 220P Sherman D.R. 145P Shetty A. 343P Shi L. 10 Shin E. 184P Shin S.J. 179P, 185P, 199P, 200P, 236P, 246P, 261P, 274P Shleeva M. 228P Shokrgozar M. 183P Shulgina M. 73P, 74P Siadat S. 183P Sidibe A. 84P Sigal A. 257P, 299P Sikri K. 151P, 250P Silve S. 130P Simeone R. 235P, 305P Simpson N. 196P Sina A.H. 324P Singh A. 99P, 249P, 281P Singh D.K. 275P 404 Singh H.N. Singh L.K. Singh N. Singh R. Singh S. Singh V. Singh Y. Singhal A. Sinigaglia A. Siroy A. Sisk D. Sitaraman R. Siu G. Sizemore C.F. Škovierová H. Skroch S. Slizen V. Smith C. Smith S.R. Smyth A. Sogi K. Sohst V. Soldati T. Soler P. Solovieva N. Somersan-Karakaya S. Sommer R. Somody L. Song O. Song S. Sørensen K. S. Osório N. Soualhine H. Sougakoff W. Souriant S. Sousa J. Sparrius M. Speer A. Sreejit G. Staack S. Stacey H. Stafford S. Stanley S. Steigedal M. Steinhäuser C. Stendahl O. Stewart G. Steyn A. Steyn A.J.C. Stinear T. Stolaříková J. Stout J. Stramer B. Strand T. Street L. Stucki D. Sturegård E. Subbarao S. 328P 150P 148P 152P, 249P 293P 19, 187P 150P, 289P 269P 291P 172P, 173P 303P 118P 88P 353P 10 299P 153P 14 277P 264P, 356P 44 225P 50, 282P, 295P 174P 73P, 74P 37, 146P 19, 357P 314P 305P 9 229P 276P 187P 72P 306P 276P 315P 135P, 315P 299P 207P 158P 196P 44 170P, 260P 301P 266P 279P, 295P 98P, 131P 248P 3, 106P 323P, 342P, 347P 303P 288P 170P 345P 81P 297P 307P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Sun J. Supply P. Surkova L. Suter M. Sweet G. Szabó N. Székely R. T 166P, 277P 106P 153P 208P 339P 326P 323P Tagliani E. Tailleux L. Takiff H. Talaat A. Tan S. Taylor S. Tchikov V. Tekwu E.M. Teo Y.Y. Tessema B. Thakur P. Tholey A. Thomas N. Thomas S. Thompson C. Thomson N.R. Thygesen M. Tiago Guimarães J. Tientcheu L. Tiraby G. Tischler A. Tiwari D. Tiwari P. Tobin D. Togun T. Tomar A.K. Tønjum T. Torta F. Totolian A. Toulza F. Trauner A. Tria G. Triglia D. Tripathi D. Troegeler A. Trovato A. Trutneva K. Tsaliki E. Tsang P. Tsodikov O. Tufariello J. Turapov O. Turner S. Tyagi A.K. Tyagi J. Author/Co-Author 317P 46 175P 90P 288P 240P 301P 84P 160P 317P 162P 301P 196P 240P 339P 125P 229P 276P 177P, 267P 221P 290P 242P 152P 303P 177P 328P 69P, 103P 269P 201P 239P 67P 172P, 173P 112P 111P, 114P, 255P 43, 298P 68P 228P 196P 340P 344P 230P 116P, 136P 351P 293P 151P, 250P U Ummels R. Author/Co-Author 13, 168P Tuberculosis 2016 – September 19-23, 2016 – Paris, France Upton A. Ussher J. Utpatel C. 129P, 130P 278P 68P V Author/Co-Author Valášková L. 323P Valafar F. 76P, 77P, 83P, 91P, 92P, 93P Valaskova L. 342P Valfridsson C. 297P Van T. 141P Van Bergen L. 206P Van Crevel R. 89P, 278P Van De Weerd R. 163P, 315P Van Der Sar A. 286P Van Der Wel N. 25, 163P Van Der Westhuyzen R. 345P Van Der Woude A. 163P Van Helden P. 23 Van Laarhoven A. 89P, 308P Van Molle I. 206P Van Stempvoort G. 163P Van Veen H. 25 Van Wyk N. 94P, 169P, 209P, 229P Vanderschaeghe D. 180P Vandewalle K. 105P, 180P Vasileva E. 201P Vaubourgeix J. 9, 37, 154P Vaziri F. 183P Veerapen N. 122P Veerasami M. 279P Verástegui M. 266P Verbov V. 201P Vercellone A. 221P Vergne I. 235P Vergnolle O. 205P, 230P Verma D. 39 Verma R. 322P Vermet H. 19 Vérollet C. 306P Verrall A. 278P Verreck F. 298P Veyrier F. 356P Veziris N. 72P Via L.E. 60, 337P Vieira C. 276P Vieira J. 276P Vilcheze C. 155P Viljoen A. 220P, 233P, 235P, 237P, 252P Villarreal-Ramos B. 247P, 279P Villaume S. 220P Villellas C. 346P Vinšová J. 326P Vincent S. 220P Vinsova J. 347P Visweswariah S. 104P Vitoria A. 309P Vocat A. 19, 331P Voigt K. 341P 405 Vollmer W. Vordermeier H. Vordermeier M. Voskuil M. Vouvoungui J.C. Vuylsteke M. 136P 247P 279P 141P 84P 105P W Author/Co-Author Waddell S. 23, 69P, 96P, 136P Wagner J. 202P Wahni K. 206P Wainwright H. 296P Walker B. 202P Walter N. 141P Walters-Morgan H. 204P Walzl G. 23 Wampande E. 71P Wang F. 101P Wang J. 78P, 79P Wang R. 17 Wang Y. 269P Warner D. 19, 81P, 149P, 156P, 340P, 345P Warren R. 13, 97P Warrier T. 37, 146P Webb P.R. 181P Wenk M. 269P West N. 3 Wetzlar T. 341P Wheat W. 10 Wilkinson K.A. 195P Wilkinson R. 24, 51, 81P, 195P, 244P, 271P, 296P Willand N. 318P Williams C. 280P Williams K. 279P Williams M. 97P Wilmanns M. 15, 113P, 165P, 207P Wiredu A. 231P Witt K. 196P Wolschendorf F. 348P Wooff E.E. 181P Wu H. 279P Wu M.L. 157P Wu Y. 124P Wyatt P. 340P X Xi L. Xiao H. Xu H. Xu W. Xu Z. Y Yadav S. Yam J. 406 Yam W.C. Yamada Y. Yang G. Yang T. Yang T.J. Yari S.H. Yeboah-Manu D. Yee M.M.K. Yimer S. Yoo J. Young D. Yun Y. Yurchenko A. Z Zalutskaya A. Zech G. Zelmer A. Zhang D. Zhang J. Zhang M. Zhang Y.W. Zheng X.H. Zhou Y. Zhu J.H. Zhuravlev V. Zimmerman M. Zoltner M. Zou H. Zou W. Zuccotto F. Zumla A. 88P 159P, 310P, 343P 154P 334P 130P 183P 66P, 86P, 142P, 349P 160P, 216P 103P 184P 51, 79P, 96P 256P 73P, 74P Author/Co-Author 153P 129P 194P 147P 130P 174P 124P 124P 55, 350P 124P, 161P 73P, 74P 60 217P 335P 350P 340P 193P Author/Co-Author 351P 158P 230P 9 32, 176P Author/Co-Author 328P 232P Tuberculosis 2016 – September 19-23, 2016 – Paris, France PARTICIPANT LIST September 2nd, 2016 ABEL Laurent Hôpital Necker Enfants Malades Paris, France [email protected] ANDERSEN Peter Lawætz Statens Serum Institut Coppenhagen S, Denmark [email protected] ADOLF Günther Austrianni GmbH Vienna, Austria [email protected] ANDREAS Nicholas Imperial College London London, United Kingdom [email protected] AGARWAL Nisheeth Translational Health Science and Technology Institute Faridabad, India [email protected] ANDREU Nuria London School of Hygiene and Tropical Medicine London, United Kingdom [email protected] AGUILAR PÉREZ Clara University of Zaragoza Zaragoza, Spain [email protected] ANWAR Shaheda London School of Hygiene and Tropical Medicine London, United Kingdom [email protected] AINHOA Lucia University of Zaragoza Zaragoza, Spain [email protected] AKABAYOV Barak Ben-Gurion University of the Negev Be'er Sheva, Israel [email protected] ALBEL Singh University of Birmingham Birmingham, United Kingdom [email protected] ALEMPARTE Carlos GlaxoSmithKline Tres Cantos, Spain [email protected] ALLEY Dickon Anacor Pharmaceuticals Palo Alto, United States [email protected] ALVAREZ ARGUEDAS Samuel University of Zaragoza Zaragoza, Spain [email protected] ALZARI Pedro Institut Pasteur Paris, France [email protected] AMORIM FRANCO Tathyana Mar Albert Einstein College of Medicine Bronx, United States [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France ARAGAW Wassihun NUS Singapore [email protected] ARNVIG Kristine University College London London, United Kingdom [email protected] ARORA Divya National Institute of Immunology New Delhi, India [email protected] ARRIES Jesmine Stellenbosch University Cape Town, South Africa [email protected] ARTHUR Michel UMRS 1138-UPMC INSERM UPD Paris, France [email protected] ASTOLFI ROSADO Leonardo Vrije Universiteit Brussel Brussels, Belgium [email protected] ATES Louis Institut Pasteur Paris, France [email protected] AVELINO Asia Stanford University Stanford, United States [email protected] 409 AV-GAY Yossef University of British Columbia Vancouver, Canada [email protected] BASU Piyali University of Surrey Slough, United Kingdom [email protected] AZIZ Dinah National University of Singapore Singapore [email protected] BASU ROY Robin Imperial College London London, United Kingdom [email protected] BACON Joanna Public Health England, National Infection Service Salisbury, United Kingdom [email protected] BAUGHN Anthony University of Minnesota Minneapolis, United States [email protected] BAILO VERGARA Rebeca University of Birmingham Birmingham, United Kingdom [email protected] BALLELL Lluis GSK Miraflores De La Sierra, Spain [email protected] BANAEI ESFAHANI Amir Institute of Molecular Systems Biology, ETH Zurich Zurich, Switzerland [email protected] BARBIER Thibault T.H. Chan Harvard School of Public Health Boston, United States [email protected] BARCZAK Amy Massachusetts General Hospital Cambridge, United States [email protected] BARISCH Caroline University of Geneva Geneva 4, Switzerland [email protected] BARKAN Daniel Hebrew University Rehovot, Israel [email protected] BARRY Clifton NIH Bethesda, United States [email protected] BASU Nirmalya Max anck Institute for Molecular Cell Biology Dresden, Germany [email protected] 410 BAULARD Alain Institut Pasteur de Lille Lille, France [email protected] BAUMANN Christoph Austrianni GmbH Vienna, Austria [email protected] BECKHAM Kate EMBL Hamburg Hamburg, Germany [email protected] BEHAR Samuel University of Massachusetts Medical School Worcester, United States [email protected] BEITES Tiago Weill Cornell Medicine New York, United States [email protected] BELLINZONI Marco Institut Pasteur Paris, France [email protected] BENABDESSALEM Chaouki Institut Pasteur de Tunis Tunis, Tunisia [email protected] BENTALEB El Mehdi Moroccan Foundation for Advanced Science, Innovation and Research (MAScIR) Rabat, Morocco [email protected] BERNARD Christine Université Pierre et Marie Curie Paris, France [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France BERNARD Lucie CNRS Toulouse, France [email protected] BLAZQUEZ Jesus Centro Nacional de Biotecnologia Madrid, Spain [email protected] BERNEY Michael Albert Einstein College of Medice Bronx, United States [email protected] BOECK Lucas University of Cambridge Fulbourn, United Kingdom [email protected] BERNUT Audrey CNRS Montpellier, France [email protected] BOISSON-DUPUIS Stéphanie INSERM Hôpital Necker/Rockefeller University United States [email protected] BESTE Dany University of Surrey Guildford, United Kingdom [email protected] BONGAERTS Nadine Institut Cochin Paris, France [email protected] BHATT Apoorva University of Birmingham Birmingham, United Kingdom [email protected] BOOT Maikel VU medical center Amsterdam, The Netherlands [email protected] BIRHANU Alemayehu Godana University of Oslo Oslo, Norway [email protected] BORGERS Katlyn VIB - Ghent University Zwijnaarde, Belgium [email protected] BISWAS Priyanka Indian Institute of Science Bangalore, India [email protected] BORITSCH Eva Institut Pasteur Paris, France [email protected] BITTER Wilbert Vu Medical Centre Amsterdam Amsterdam, The Netherlands [email protected] BORRELL Sonia Swiss Tropical and Public Health Basel, Switzerland [email protected] BIUKOVIC Goran National University of Singapore, Yong Loo Lin School of Medicine Singapore [email protected] BOSHOFF Helena National Institutes of Health Bethesda, United States [email protected] BLAISE Mickael CNRS Montpellier, France [email protected] BLANC Isabelle SANOFI Marcy L'etoile, France [email protected] BLANC-POTARD Anne CNRS Montpellier, France [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France BOTELLA Helene Weil Cornell Medicine New York, United States [email protected] BOTTAI Daria University of Pisa Italy [email protected] BOULON Richard Institut de pharmacologie et biologie structurale de Toulouse Toulouse, France [email protected] 411 BOUTTE Cara Harvard TH Chan School of Public Health Somerville, United States [email protected] BRANDENBURG Julius Research Center Borstel Borstel, Germany [email protected] BRENNAN Patrick Colorado State University Fort Collins, United States [email protected] BRESCIANI Nadia San Raffaele Scientific Institute Milan, Italy [email protected] BRITES Daniela Swiss Tropical and Public Health Institute Basel, Switzerland [email protected] BRODIN Priscille Institut Pasteur de Lille / INSERM U10109 Lille, France [email protected] BROSCH Roland Institut Pasteur Paris, France [email protected] BROSET Esther Universidad De Zaragoza Zaragaoza, Spain [email protected] BROSSIER Florence Pitié-Salpêtrière Hospital Paris, France [email protected] BRUCH Eduardo Institut Pasteur Paris, France [email protected] BRYANT Josephine University of Cambridge Cambridge, United Kingdom [email protected] BUCHIERI Maria Virginia Institut Pasteur Sartrouville, France [email protected] 412 BUCHRIESER Carmen Institut Pasteur Paris, France BULL Tim St George's University of London London, United Kingdom [email protected] BUNDUC Catalin Vrije Universiteit, Amsterdam Amsterdam, The Netherlands [email protected] BURBAUD Sophie Cambridge University Cambridge, United Kingdom [email protected] BURGERS Wendy University of Cape Town Cape Town, South Africa [email protected] BURMEISTER Maike Research Center Borstel Borstel, Germany [email protected] CAMBIER Cj Stanford Stanford, United States [email protected] CANDEL Sergio Cambridge University Cambridge, United Kingdom [email protected] CAPTON Estelle UPMC -Cimi Paris Paris, France [email protected] CARRAT Christophe IPBS Toulouse, France [email protected] CASCIOFERRO Alessandro Institut Pasteur Paris, France [email protected] CASTAÑEDA GARCIA Alfredo Instituto de Biomedicina Sevilla Sevilla, Spain [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France CAVALIER Jean-François CNRS Marseille, France [email protected] COLLINS Ben ETH Zurich Zurich, Switzerland [email protected] CHAE Hansong Yonsei University Seoul, South Korea [email protected] COMAS Iñaki Institute of Biomedicine of Valencia Valencia, Spain [email protected] CHARLOTTE Passemar University of Cambridge Cambridge, United Kingdom [email protected] COMMANDEUR Susanna VUmc Weesp, The Netherlands [email protected] CHERNYAEVA Ekaterina St. Petersburg State University St. Petersburg, Russia [email protected] COOPER Andrea Trudeau Institute/University Leceister Leicester, United Kingdom [email protected] CHIARADIA Laura Institut de Pharmacologie et de Biologie Structurale Toulouse, France [email protected] COPPENS Rene TBVI Lelystad, The Netherlands [email protected] CHINER-OMS Álvaro University of Valencia Valencia, Spain [email protected] CHIODARELLI Giacomo GlaxoSmithKline Tres Cantos, Spain [email protected] CHNG Shu Sin National University of Singapore Singapore [email protected] CORREIA Carolina University College Dublin Dublin, Ireland [email protected] CORTES Teresa London School of Hygiene & Tropical Medicine London, United Kingdom [email protected] COSCOLLA Mireia SwissTPH Basel, Switzerland [email protected] CHOI Hong-Hee Yonsei University College of Medicine Seoul, South Korea [email protected] COSTA-GOUVEIA Joana Univ. Lille, CNRS, Inserm, CHU Lille Institut Pasteur de Lille Lille, France [email protected] CIMINO Mena Institut Pasteur Paris, France [email protected] COUGOULE Céline IPBS CNRS UMR5089 Toulouse, France [email protected] COHEN-GONSAUD Martin Centre de Biochimie Structurale Montpellier, France [email protected] COX Jeffery Berkeley University San Francisco, United States [email protected] COLE Stewart EPFL Lausanne, Switzerland [email protected] COX Jonathan Aston University Birmingham, Uk, United Kingdom [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France 413 COYA RABOSO Juan Manuel Institut Pasteur Paris, France [email protected] CUMMING Bridgette KwaZulu-Natal Research Institute for Tuberculosis and HIV Durban, South Africa [email protected] DHIMAN Rohan National Institute of Technology, Rourkela, Odisha, India Rourkela, India [email protected] DHINGRA Sakshi All India Institute of Medical Sciences Delhi, India [email protected] DALLENGA Tobias Research Center Borstel Borstel, Germany [email protected] DICK Thomas National University of Singapore Singapore [email protected] DARTOIS Veronique Rutgers University Newark, United States [email protected] DILLON Nicholas University of Minnesota Lakeville, United States [email protected] DARWIN Heran New York University School of Medicine New York, United States [email protected] DODD Claire The Ohio State University Columbus, United States [email protected] DE ROSSI Edda University of Pavia Pavia, Italy [email protected] DOORNBOS Kathryn University of Alabama at Birmingham Birmingham, United States [email protected] DEBOOSERE Nathalie Institut Pasteur de Lille Lille, France [email protected] DOU Horng-Yunn National Health Research Institutes Miaoli County, Taiwan [email protected] DEBRÉ Patrice AP-HP Paris, France [email protected] DUBAR Faustine Université Lille 1 Villeneuve D'ascq, France [email protected] DEMANGEL Caroline Institut Pasteur Paris, France [email protected] DUBE Jean-Yves McGill University Montreal, Quebec, Canada [email protected] DEMARET Julie Queen Mary University of London Lyon, France [email protected] DUBEY Neha University of Delhi Delhi, India [email protected] DEMOITIE Marie-Ange GlaxoSmithKline Vaccines Wavre, Belgium [email protected] DUBOIS Violaine UVSQ La Celle-Saint-Cloud, France [email protected] DERBYSHIRE Keith Wadsworth Center, New York State Department of Health Albany,, United States [email protected] DUMA Bogdan GlaxoSmithKline Tres Cantos, Spain [email protected] 414 Tuberculosis 2016 – September 19-23, 2016 – Paris, France DUMAS Alexia CNRS Toulouse, France [email protected] EPEMOLU Ola University of Dundee Dundee, United Kingdom [email protected] DUPIN marilyne 1979 Vaugneray, France [email protected] ESKANDARIAN Haig Alexander École Polytechnique Fédérale de Lausanne Lausanne, Switzerland [email protected] DUPONT Christian Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé Montpellier, France [email protected] ETNA Marilena Paola Istituto Superiore di Sanità Roma, Italy [email protected] DURAN Rosario Institut Pasteur de Montevideo Montevideo, Uruguay [email protected] EDELSTEIN Paul University of Pennsylvania Philadelphia, United States [email protected] EHRT Sabine Weill Cornell Medical College New York, United States [email protected] EL ACHKAR Salam Institut Pasteur de Lille Lille, France [email protected] EVANGELOPOULOS Dimitrios The Francis Crick Institute London, United Kingdom [email protected] EVERALL Izzy Wellcome Trust Sanger Institute Cambridge, United Kingdom [email protected] EYANGOH Sara Centre Pasteur du Cameroun Yaoundé, Cameroon [email protected] FAVROT Lorenza Albert Einstein College of Medicine Bronx, Ny, United States [email protected] EL DAKER Sary K-RITH Durban, South Africa [email protected] FEILCKE Ruth Martin-Luther-University Halle-Wittenberg, Germany Halle (saale), Germany [email protected] ELAYATI Karima TCD Dublin, Ireland [email protected] FESTJENS Nele VIB - Ghent University Zwijnaarde, Belgium [email protected] ELGHRAOUI Afif San Diego State University San Diego, United States [email protected] FLO Trude Helen Norwegian University of Science and Technology Trondheim, Norway [email protected] ELLA Bhagyaraj CSIR Chandigarh, India [email protected] ELLIOTT Sarah University of Minnesota - Twin Cities Burnsville, United States [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France FLOTO Andres University of Cambridge Cambridge, United Kingdom [email protected] FOO Shi-Yan Caroline EPFL Lausanne, Switzerland [email protected] 415 FOTOUHI Nader TB Alliance New York, United States [email protected] GHYS Anne Janssen Pharmaceuticals Beerse, Belgium [email protected] FREIRE Diana EMBL-Hamburg Hamburg, Germany [email protected] GICQUEL Brigitte Institut Pasteur Paris, France [email protected] GAEVAYA Ludmila Bogomolets National Medical University Kiev, Ukraine [email protected] GIJSBERS ALEJANDRE Abril The Maastricht Multimodal Molecular Imaging institute (M4I) Maastricht, The Netherlands [email protected] GAGNEUX Sébastien Swiss Tropical & Public Health Institute Basel Basel, Switzerland [email protected] GAO Qian Fudan University Shangai, China [email protected] GARCIA-PELAYO M. Carmen Animal & ant Health Agency (APHA) Addlestone, United Kingdom [email protected] GARG Rajni Amity University Gurgaon Haryana New Delhi, India [email protected] GEIBEL Sebastian University of Wuerzburg Wuerzburg, Germany [email protected] GENGENBACHER Martin National University of Singapore Singapore [email protected] GENNARO Maria Laura Rutgers University Newark, United States [email protected] GERRICK Elias Harvard School of Public Health Boston, United States [email protected] GHANEM Marwan McGill University Montreal, Canada [email protected] 416 GIL Magdalena Institut Pasteur de Montevideo Montevideo, Uruguay [email protected] GIRAUD GATINEAU Alexandre Institut Pasteur Dreux, France [email protected] GLENN Sarah University of Leicester Leicester, France [email protected] GOIG SERRANO Galo Adrián Instituto de Biomedicina de Valencia - CSIC Valencia, Spain [email protected] GONZALO-ASENSIO Jesús University of Zaragoza Zaragoza, Spain [email protected] GOPAL Pooja National University of Singapore Singapore [email protected] GORDON Stephen University Colloge of Dublin Ireland [email protected] GOUZY Alexandre Weill Cornell Medecine New York, France [email protected] GRABOWSKA Anna London School of Hygiene and Tropical Medicine London, United Kingdom peliroja@wp. Tuberculosis 2016 – September 19-23, 2016 – Paris, France GRODE Leander Vakzine Projekt Management GmbH Hannover, Germany [email protected] HAFNEH Nor Azian University of Leicester Leicester, United Kingdom [email protected] GROESCHEL Matthias Institut Pasteur Paris, France [email protected] HANANE Issa Université Paris Sud - Paris Saclay Courbevoie, France [email protected] GRZEGORZEWICZ Anna Colorado State University Fort Collins, United States [email protected] HARTKOORN Ruben Institut Pasteur de Lille Lille, France [email protected] GUBELLINI Francesca Institut Pasteur Paris, France [email protected] HAUPTMANN Matthias Research Center Borstel Borstel, Germany [email protected] GUERIN Marcelo CIC bioGUNE Derio, Spain [email protected] HAWRYLUK Natalie Celgene Global Health San Diego, United States [email protected] GUILHOT Christophe CNRS, IPBS Toulouse, France [email protected] HE Le Centre d'immunologie Marseille Luminy Marseille, France [email protected] GUPTA Manish Jawaharlal Nehru University, Delhi, India [email protected] HEALY Claire Weill Cornell Medicine New York, United States [email protected] GUR Eyal Ben-Gurion University of the Negev Beer Sheva, Israel [email protected] HERNANDEZ Johana University of Surrey Guildford, United Kingdom [email protected] GURUMURTHY Meera National University of Singapore Singapore [email protected] HERRMANN Jean-Louis Université de Versailles, Hôpital Poincaré Versailles, France [email protected] GUSHCHIN Vladimir Federal State Budgetary Institution Moscow, Russia [email protected] HINGLEY-WILSON Suzie University of Surrey Guildford, United Kingdom [email protected] GUTIERREZ Claude University Toulouse Toulouse, France [email protected] HOHL Michael University of Zurich Zürich, Switzerland [email protected] HADIZADEH TASBITI Alireza Institut Pasteur of Iran Tehran, Iran [email protected] HOUBEN Edith Vrije Universiteit Amsterdam Amsterdam, The Netherlands [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France 417 HRABALEK Alexandr Charles University Hradec Králové, Czech Republic [email protected] JANKUTE Monika University of Birmingham Birmingham, United Kingdom [email protected] HUANG Wei Institut Pasteur Paris, France [email protected] JAVID Asma University of Birmingham Birmingham, United Kingdom [email protected] HUDRISIER Denis CNRS Toulouse, France [email protected] JAVID Babak Tsinghua University School of Medicine Beijing, China [email protected] HUGONNET Jean-Emmanuel UMRS 1138-UPMC INSERM UPD Paris, France [email protected] JONG Robyn UC Berkeley Berkeley, United States [email protected] HUNT Debbie The Francis Crick Institute London, United Kingdom [email protected] JOSE Ainsa Universidad de Zaragoza Zaragoza, Spain [email protected] HWA Shi-Hsia K-RITH Durban, South Africa [email protected] JOSEPHSON Robert University of Toronto Toronto, Canada [email protected] IAKOBACHVILI Nino Maastricht University Maastricht, The Netherlands [email protected] JUNG Juyeon Korea Research Institute of Bioscience and Biotechnology Daejeon, South Korea [email protected] JABOT-HANIN Fabienne INSERM Paris, France [email protected] JACKSON Mary Colorado State University Fort Collins, United States [email protected] JACOBS William R Jr Albert Einstein College of Medicine Bronx, United States [email protected] JÄGER Franziska University of Dundee Dundee, United Kingdom [email protected] JAKIMOWICZ Dagmara University of Wroclaw Wroclaw, Poland [email protected]. 418 KALSUM Sadaf Linköping University Linköping, Sweden [email protected] KAMARIZA Mireille Stanford University San Francisco, United States [email protected] KAMINSKI Lina Albert Einstein College of Medicine Bronx, United States [email protected] KANEKO Takushi TB Alliance Guilford, United States [email protected] KANNAN Nisha NTNU Trondheim, Norway [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France KANVATIRTH Panchali University of Birmingham Birmingham, United Kingdom [email protected] KOCH Anastasia University of Cape Town Cape Town, South Africa [email protected] KAAN Gilla Bill and Melinda Gates Foundation Seattle, United States [email protected] KOEKEN Valerie Radboud University Medical Centre Nijmegen, The Netherlands [email protected] KARABANOVICH Galina Charles University, Faculty of Pharmacy in Hradec Kralove Hradec Králové, Czech Republic [email protected] KORDULÁKOVÁ Jana Faculty of Natural Sciences, Comenius University Bratislava, Slovakia [email protected] KEISER Tracy Albert Einstein College of Med Bronx, United States [email protected] KOUL Anil Janssen Pharmaceutica NV Beerse, Belgium [email protected] KESTER Jemila Harvard University Cambridge, United States [email protected] KRAIGSLEY Alison NIH/NIAID North Bethesda, United States [email protected] KHANDELIA Arvind Kumar Nanyang technological University Singapore [email protected] KRÁTKÝ Martin Charles University in Prague, Faculty of Pharmacy in Hradec Králové Hradec Králové, Czech Republic [email protected] KHOSRAVI BOROUJENI Azar Dokht Ahvaz Jundishapur University of Medical Sciences Ahvaz, Iran [email protected] KREITMANN Louis Lille University Paris, France [email protected] KIM Hongmin Yonsei University Seoul, South Korea [email protected] KREMER Laurent CNRS Montpellier, France [email protected] KIM Jae Myung Animal and ant Quaratine Service Gyeongsangbuk-Do, South Korea [email protected] KREUTZFELDT Kaj Weill Cornell Medicine New York, United States [email protected] KIM Woo Sik Yonsei University College of Medicine Seoul, South Korea [email protected] KRISHNAMOORTHY Gopinath Max anck Institute for Infection Biology Berlin, Germany [email protected] KLIMESOVA Vera Charles University Hradec Kralove, Czech Republic [email protected] KUNDU Subhashri National University of Singapore Singapore [email protected] KNAUL Julia Vakzine Projekt Managment GmbH Hannover, Germany [email protected] LAANG Hannu European Commission Brussel, Belgium [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France 419 LABOUDIE Patricia Sanofi R&D Marcy L'etoile, France [email protected] LASSERRE Moira Institut Pasteur de Montevideo Montevideo, Uruguay [email protected] LACKNER Gerald Friedrich Schiller University Jena Jena, Germany [email protected] LAU Alice University of British Columbia Vancouver, Canada [email protected] LAGRANGE Sophie Sanofi R&D Marcy L'etoile, France [email protected] LAUGHON Barbara NIH/NIAID/DHHS/US Government Bethesda, United States [email protected] LAI Rachel The Francis Crick Institute London, United Kingdom [email protected] LAURSEN Mette EMBL Hamburg Hamburg, Germany [email protected] LAIR Christine Sanofi R&D Marcy L'etoile, France [email protected] LE Nguyen-Hung CNRS IPBS Toulouse, France [email protected] LALVANI Ajit Imperial College London London, United Kingdom [email protected] LE CHEVALIER Fabien Institut Pasteur Paris, France [email protected] LAMBERT Linda National Institute of Allergy, Immunology, and Transantation, NIH Bethesda, United States [email protected] LE MOIGNE Vincent Université Versailles St-Quentin-en-YvelinesINSERM U1173 Montigny-Le-Bretonneux, France [email protected] LAMERS Meindert MRC Laboratory of Molecular Biology Cambridge, United Kingdom [email protected] LEFEBVRE Cyril Institute of Pharmacology and Structural Biology Toulouse, France [email protected] LAMPRECHT Dirk K-RITH Durban, South Africa [email protected] LAPA E SILVA Jose Roberto Federal University of Rio De Janeiro Rio De Janeiro, Brazil [email protected] LARROUY-MAUMUS Gerald Imperial College London London, United Kingdom [email protected] LARSEN Michelle Albert Einstein College of Medicine Bronx, United States [email protected] 420 LEFRANÇOIS Louise University of Geneva Geneva, Switzerland [email protected] LELIEVRE Joel GlaxoSmithKline Tres Cantos, Spain [email protected] LERM Maria Linköping University Linköping, Sweden [email protected] LERNER Thomas The Francis Crick Institute London, United Kingdom [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France LEUNG Vivian Harvard School of Public Health Boston, United States [email protected] MAHAMED Deeqa K-RITH Durban, South Africa [email protected] LIENARD Julia Lund University Lund, Sweden [email protected] MAJLESSI Laleh Institut Pasteur France [email protected] LIENHARTD Christian OMS Geneva, Switzerland [email protected] MAKAROV Vadim A.N. Bach Institute of Biochemistry Moscow, Russia [email protected] LINGE Irina Central Institute for Tuberculosis Moscow, Russia [email protected] MALM Sven Research Center Borstel - Leibniz Center for Medicine and Biosciences Borstel, Germany [email protected] LOUNIS Nacer Janssen Pharmaceutica Beerse, Belgium [email protected] LOW Jian Liang National University of Singapore Singapore [email protected] LU Ping VU University Amsterdam Amsterdam, The Netherlands [email protected] LUGO Geanncarlo CNRS, IPBS Toulouse, France [email protected] LUKE Alderwick University of Birmingham Birmingham, United Kingdom [email protected] LUPIEN Andréanne EPFL Lausanne, Switzerland [email protected] MACHUGH David University College Dublin Dublin, Ireland [email protected] MAERK Mali Norwegian University of Science and Technology Trondheim, Norway [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France MALONE Kerri University College Dublin Dublin, Ireland [email protected] MANGANELLI Riccardo University of Padua Padova, Italy [email protected] MANINA Giulia Institut Pasteur Paris, France [email protected] MAREKOVIĆ Ivana University Hospital Centre Zagreb Zagreb, Croatia [email protected] MARTIN Anandi Ghent University Gent, Belgium [email protected] MARTIN Carlos University of Zaragoza Zaragoza, Spain [email protected] MARTINEZ Mariano Institut Pasteur Paris, France [email protected] MASERUMULE Charlotte Cambridge University Cambridge, United Kingdom [email protected] 421 MAZUREK Jolanta IPBS, CNRS, France and KI, Sweden Toulouse, France [email protected] MIZRAHI Valerie University of Cape Town Cape Town, South Africa [email protected] MDLULI Khisimuzi TB Alliance Brooklyn, United States [email protected] MODLIN Samuel San Diego State University San Diego, United States [email protected] MEIKLE Virginia University of Alabama at Birmingham Birmingham, United States [email protected] MONICA Monica GlaxoSmithKline Tres Cantos, Spain [email protected] MEILER Eugenia GlaxoSmithKline Tres Cantos, Madrid, Spain [email protected] MONTAMAT-SICOTTE Damien Research Institute of the McGill University Health Centre Montreal, Canada [email protected] MÉNDEZ Melissa Universidad Peruana Cayetano Heredia Lima, Peru [email protected] MENDOZA-LOSANA Alfonso GSK Tres Cantos, Spain [email protected] MENDUM Tom University of surrey Guildford, United Kingdom [email protected] MERY Alexandre University Lille1 Villeneuve D'ascq, France [email protected] MIKUSOVA Katarina University of Bratislava Slovakia [email protected] MINNIKIN David University of Birmingham Birmingham, United Kingdom [email protected] MITERMITE Morgane University College Dublin Dublin, Ireland [email protected] MITRA Avishek University of Alabama at Birmingham Birmingham, United States [email protected] 422 MOOLLA Nabiela University of Birmingham Birmingham, United Kingdom [email protected] MORASKI Garrett Montana State University Bozeman, United States [email protected] MOREIRA Wilfried National University of Singapore Singapore [email protected] MOYNIHAN Patrick University of Birmingham Birmingham, United Kingdom [email protected] MUKAMOLOVA Galina University of Leicester Leicester, United Kingdom [email protected] MUKHERJEE Devika NUS Singapore [email protected] MUNSHI Tulika St. George's, University of London London, United Kingdom [email protected] MURPHY Leanne University College Dublin Leixlip, Ireland [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France NAGARAJA V Indian Institute of Science Bangalore, India [email protected] NISKANEN Mirja University of Tampere Tampere, Finland [email protected] NAMBI Subhalaxmi Umass medical school Worcester, United States [email protected] NOURSADEGHI Mahdad University College London London, United Kingdom [email protected] NATHAN Carl Weill Cornell Medical College New York, United States [email protected] ODERMATT Nina EPFL Lausanne, Switzerland [email protected] NEGATU Dereje National University of Singapore Singapore [email protected] OFORI-ANYINAM Nana Akua Boatema Institute of Tropical Medicine Antwerpen, Belgium [email protected] NEMECEK Jan Charles University, Faculty of Pharmacy Hradec Králové, Czech Republic [email protected] O'HARE Helen University of Leicester Leicester, United Kingdom [email protected] NEYROLLES Olivier CNRS / IPBS Toulouse, France [email protected] OREN Rosenberg UCSF San Francisco, United States [email protected] NGAN Grace National University Singapore Singapore [email protected] ORGEUR Mickael Institut Pasteur Paris, France [email protected] NGUYEN Quang Huy L'Institut de recherche pour le développement (IRD) Montpellier, France [email protected] OSEI-WUSU Stephen Noguchi Memorial Institute for Medical Research, University of Ghana Accra, Ghana [email protected] NICOD Charlotte ETH Zürich Zürich, Switzerland [email protected] OSMAN Morwan University of Cambridge Cambridge, United Kingdom [email protected] NIEDERWEIS Michael University of Alabama at Birmingham Homewood, United States [email protected] O'SULLIVAN Mary Trinity College Dublin Dublin, Ireland [email protected] NIEL Van Wyk CNRS Montpellier, France [email protected] OTCHERE Isaac Darko Noguchi Memorial Institute for Medical Research, University of Ghana Accra, Ghana [email protected] NIEMANN Stefan Research Center Borstel Borstel, Germany [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France OTTENHOFF Tom Leiden University Medical Center Leiden, The Netherlands [email protected] 423 OTTER Ashley Royal Veterinary College London, United Kingdom [email protected] PETHE Kevin Nanyang Technological University Singapore [email protected] OUAKED Nadia GSK Rixensart, Belgium [email protected] PETRUCCIOLI Elisa National Institute for Infectiouse Diseases Lazzaro Spallanzani Roma, Italy [email protected] PALOMINO Juan Carlos Ghent University Gent, Belgium [email protected] PALUCCI Ivana università cattolica del sacro cuore Roma, Italy [email protected] PARASA Venkata Ramanarao Linkoping University Linkoping, Sweden [email protected] PARK Yu Mi NIH Bethesda, United States [email protected] PARRET Annabel EMBL Hamburg Hamburg, Germany [email protected] PASCA Maria Rosalia University of Pavia, Italy Pavia, Italy [email protected] PAWLIK Alexandre Institut Pasteur Paris, France [email protected] PÉREZ SÁNCHEZ Irene University of Zaragoza Zaragoza, Spain [email protected] PERRIN Pascale Université Montpellier Montpellier, France [email protected] PETERS Peter Maastricht University Amsterdam, The Netherlands [email protected] 424 PIETRETTI Danilo Institute Pharmacologie et Biologie Structurale IPBS - Centre National de la Recherche Scientifique Ramonville St Agne, France [email protected] PITON Jérémie EPFL Lausanne, France [email protected] POJER Florence EPFL Lausanne, Switzerland [email protected] PRADOS-ROSALES Rafael CICbioGUNE Derio, Spain [email protected] PRIESTMAN Miles Imperial College London London, United Kingdom [email protected] PRITCHARD Jennifer Institute of Structural Molecular Biology Chelmsford, Essex, United Kingdom [email protected] PROSSER Gareth NIH Bethesda, United States [email protected] PYM Alexander K-RITH Durban, South Africa [email protected] QUEVAL Christophe Institut Pasteur Paris, France [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France QUICK Isadora INSERM Paris, France [email protected] REGO Hesper Yale Boston, United States [email protected] RAFFETSEDER Johanna Linköping University Linköping, Sweden [email protected] REILING Norbert Research Center Borstel Borstel, Germany [email protected] RAJWANI Rahim The Hong Kong Polytechnic University Hong Kong, China [email protected] REMUINAN Modesto GSK Tres Cantos, Madrid, Spain [email protected] RAKOTOSAMIMANANA Niaina Institut Pasteur de Madagascar Antananarivo, Madagascar [email protected] RESENDE Mariana I3S/IBMC Porto, Portugal [email protected] RAMAKRISHNAN Lalita University of Cambridge Cambridge, United Kingdom [email protected] RICCARDI Giovanna University of Pavia Pavia, Italy [email protected] RAMON GARCIA Santiago The University of British Columbia Vancouver, Canada [email protected] RICHTER Adrian Martin Luther Universität Halle Wittenberg Rossleben, Germany [email protected] RAO Saroja Dayananda Sagar College of Engineering Bengaluru, India [email protected] RIOU Catherine University of Cape Town Cape Town, South Africa [email protected] RASTOGI Nalin Institut Pasteur de la Guadeloupe Abymes, France [email protected] RIVERO-LEZCANO Octavio Comejo Asistencial Universitario de León Leon, Spain [email protected] RAVELLI Raimond Maastricht University Maastricht, The Netherlands [email protected] ROBERTSON Brian Imperial College London London, United Kingdom [email protected] RAWKINS Ann Public Health England Salisbury, United Kingdom [email protected] ROCA SOLER Francisco Jose University of Cambridge Cambridge, United Kingdom [email protected] RAY Peter University of Dundee Stirling, United Kingdom [email protected] ROCK Jeremy Harvard School of Public Health Boston, United States [email protected] REDINGER Natalja Research Center Borstel Borstel, Germany [email protected] RODRIGO-UNZUETA Ane University of Cape Town Cape Town, South Africa [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France 425 RODRIGUEZ Daniela University of Cambridge Cambridge, United Kingdom [email protected] SAITO Kohta Weill Cornell Medicine New York, United States [email protected] RODRIGUEZ-RIVERA Frances Stanford University Stanford, United States [email protected] SANCHEZ MONTALVA Adrian Vall d'Hebron Hospital. Universitat autonoma de Barcelona Barcelona, Spain [email protected] ROE Jennifer University College London London, United Kingdom [email protected] ROH Jaroslav Charles University in Prague, Faculty of Pharmacy Hradec Králové, Czech Republic [email protected] SANDER Peter University of Zurich Zurich, Switzerland [email protected] SANS Stéphanie Sanofi R&D Marcy L'etoile, France [email protected] ROMBOUTS Yoann IPBS Toulouse, France [email protected] SANTOS Mariana The Francis Crick Institute London, United Kingdom [email protected] ROUBERT Christine Sanofi R&D Marcy L'etoile, France [email protected] SANTUCCI Pierre CNRS Marseille, France [email protected] RUBIN Eric Harvard TH Chan Scholl of Public Health Boston, Ma, United States [email protected] SARATHY Jickky Palmae National University of Singapore Singapore [email protected] RUESEN Carolien Radboud university medical center Nijmegen Nijmegen, The Netherlands [email protected] SAVINO Maria LABCYTE INC Sunnyvale, United States [email protected] RULLAS Joaquin GlaxoSmithKline Tres Cantos, Spain [email protected] SAWYER Elizabeth London School of Hygiene and Tropical Medicine London, United Kingdom [email protected] RUSSELL David Cornell University Ithaca, Ny, United States [email protected] RUSTAD Tige Center for Infectious Disease Research Seattle, United States [email protected] SAELENS Joseph Duke University Durham, United States [email protected] 426 SAYES Fadel Institut Pasteur Paris, France [email protected] SCHNAPPINGER Dirk Weill Cornell Medicine New York, United States [email protected] SCHNEIDER Bianca Research Center Borstel Borstel, Germany [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France SEEGER Markus University of Zurich Zurich, Switzerland [email protected] SIMEONE Roxane Institut Pasteur France SERAFINI Agnese The Francis Crick Institute London, United Kingdom [email protected] SINGH Ramandeep Translational Health Science and Technology Institute Haryana, India [email protected] SEWGOOLAM Bevika University of Cape Town Cape Town, South Africa [email protected] SIROY Axel Maastricht University Maastricht, France [email protected] SHANAHAN Jonathan University of Cambridge Cambridge, United Kingdom [email protected] SMYTH Alicia University College Dublin Dublin, Ireland [email protected] SHARMA Aditya Kumar Institute of genomic and integrative biology Delhi, India [email protected] SOLDATI Thierry University of Geneva Geneva, Switzerland [email protected] SHARMA Ashish Indian Institute of Technology New Delhi New Delhi, India [email protected] SOLER Paloma EPFL Lausanne, Switzerland [email protected] SHARMA Meenu Public Health Agency of Canada Winnipeg, Canada [email protected] SOMMER Raphael EPFL Lausanne, Switzerland [email protected] SHARON Kendall The Royal Veterinary College London, United Kingdom [email protected] SOMODY Laurence SANOFI AVENTIS R et D Marcy L'etoile, France [email protected] SHETTY Annanya National University of Singapore Singapore [email protected] SONG Ok-Ryul INSERM Lille, France [email protected] SHIN Sung Jae Yonsei University College of Medicine Seoul, South Korea [email protected] SOOD Sakshi The Francis Crick Institute London, United Kingdom [email protected] SHLEEVA Margarita A.N. Bach Institute of Biochemistry Moscow, Russia [email protected] SOTO Ramon University of Birmingham Birmingham, United Kingdom [email protected] SIKRI Kriti All India Institute of Medical Sciences New Delhi, India [email protected] SOURIANT Shanti IPBS, CNRS UMR 5089 Toulouse, France [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France 427 SOUSA Jeremy i3S - Instituto de Investigação e Inovação em Saúde Porto, Portugal [email protected] SPEER Alexander VU University Medical Center Amsterdam, The Netherlands [email protected] STAACK Sonja EMBL Hamburg Hamburg, Germany [email protected] STANLEY Sarah University of California, Berkeley Berkeley, United States [email protected] STEIGEDAL Magnus NTNU Trondheim, Norway [email protected] STENDAHL Olle Linköping University Linköping, Sweden [email protected] STINEAR Timothy University of Melbourne Merlbourne, Australia [email protected] STRAND Trine Norwegian University of Science and Technology Trondheim, Norway [email protected] SUBBARAO Vani Imperial College London, United Kingdom [email protected] SUN Jim University of Alabama at Birmingham Birmingham, United States [email protected] SUPY Philip CNRS Lille, France [email protected] TAILLEUX Ludovic Institut Pasteur Paris, France [email protected] 428 TAKIFF Howard IVIC/Pasteur Paris, France [email protected] TANVIR Zaid TB ALLIANCE New York, United States [email protected] TASSINARI Matteo Institut Pasteur Paris, France [email protected] TELEVANTOS Constantinos University of Cambridge Cambridge, United Kingdom [email protected] TOMLINSON Gillian University College London London, United Kingdom [email protected] TROFIMOV Valentin Insitut Pasteur de Lille Lille, France [email protected] TSODIKOV Oleg University of Kentucky Lexington, United States [email protected] VALAFAR Faramarz San Diego State University San Diego, United States [email protected] VALASKOVA Lenka Faculty of Pharmacy in Hradec Kralove, Charles University Hradec Kralove, Czech Republic [email protected] VAN DER MEEREN Olivier GlaxoSmithKline Vaccines Rixensart, Belgium [email protected] VAN DER WEL Nicole AMC Amsterdam, The Netherlands [email protected] VAN DER WESTHUYZEN Renier University of Cape Town Cape Town, South Africa [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France VARELA RAMIREZ Cristian University of Birmingham Birmingham, United Kingdom [email protected] VINSOVA Jarmila Faculty of Pharmacy, Charles University Hradec Kralove, Czech Republic [email protected] VASILEVA Elena Saint-Petersburg Pasteur Institute Saint-Petersburg, Russia [email protected] WADDELL Simon University of Sussex Brighton, United Kingdom [email protected] VAUBOURGEIX Julien Weill Cornell Medicine New York, United States [email protected] WAGNER Jeffrey Harvard T.H. Chan School of Public Health Boston, United States [email protected] VAZIRI Farzam Institut Pasteur of Iran Tehran, Iran [email protected] WANG Chuan Fudan University Shanghai, China [email protected] VEGA DOMINGUEZ Perla Jazmin University of Birmingham Birmingham, United Kingdom [email protected] WARNER Digby University of Cape Town Cape Town, South Africa [email protected] VERGNOLLE Olivia Albert Einstein College of Medicine Bronx, United States [email protected] WEHENKEL Anne Marie Institut Pasteur Paris, France [email protected] VERMA Amit Kumar University of Cambridge Cambridge, United Kingdom [email protected] WHITWORTH Laura University of Cambridge Cambridge, United Kingdom [email protected] VERRALL Ayesha University of Otago Wellington South, New Zealand [email protected] WILKINSON Robert Imperial College London United Kingdom [email protected] VIA Laura Ellen NIAID Bethesda, United States [email protected] WILLIAM Conrad Cambridge University Cambridge, United Kingdom [email protected] VILCHEZE Catherine Howard Hughes Medical Institute at AECOM Flushing, New York, United States [email protected] WILLIAMS Kerstin University of Surrey Guildford, United Kingdom [email protected] VILJOEN Albertus CNRS Montpellier, France [email protected] WILMANNS Matthias European Molecular Biology Laboratory Hamburg, Germany [email protected] VILLELLAS Cristina Janssen Pharmaceutica Beerse, Belgium [email protected] WILSON Colin Drug Discovery and Development Center, University of Cape Town Cape Town, South Africa [email protected] Tuberculosis 2016 – September 19-23, 2016 – Paris, France 429 WINTER Nathalie INRA Nouzilly, France [email protected] YAMADA Yoshiyuki National University of Singapore Singapore [email protected] WIREDU Adwoa Noguchi Memorial Institute for medical research, University of Ghana Greater Accra, Ghana [email protected] YEBOAH-MANU Dorothy Noguchi Memorial Institute for Medical Research, University of Ghana Legon, Accra, Ghana [email protected] WITT Karolina QMUL Feltham, United Kingdom [email protected] YEE Michelle National University of Singapore Singapore [email protected] WOLSCHENDORF Frank University of Alabama at Birmingham Birmingham, United States [email protected] ZHU Junhao Tsinghua University Beijing, China [email protected] WU Katherine Harvard University Cambridge, Ma, United States [email protected] ZHURAVLEV Viacheslav St.Petersburg Research Institute of Phthisiopulmonology Saint-Petersburg, Russia [email protected] WU Mulu National University of Singapore Singapore [email protected] XIAO Han University of St.Andrews St.andrews, United Kingdom [email protected] ZOUAOUI-FRIGUI Wafa Institut Pasteur Paris, France [email protected] XU Zhujun National University of Singapore Singapore [email protected] 430 Tuberculosis 2016 – September 19-23, 2016 – Paris, France
