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Leibniz Institute for Natural Product Research and Infection Biology Hans Knöll Institute Research Report 2012 | 2013 HKI Research Report 2012/2013 1 As in the previous years, the Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI) continuously developed, networked and expanded further in the years 2012 and 2013. In the previous years, the number of staff and research groups increased to such an extent that we had to look for more office and laboratory space. Our new research building that we opened up in 2012, offers not only sufficient space but also features state-of-the art technology in stunning architecture. Since 2012, the team of the “Centre for Innovation Competence (ZIK) Septomics” has been performing their research in a new research building adding distinction to the profile of the Beutenberg Campus. In the years 2012 and 2013 we were pleased to welcome two new associated research groups originating from the Jena University Hospital. These include the working group “Infections in haematology / oncology” managed by Prof. Marie von Lilienfeld-Toal that studies infectious diseases on patients suffering from malign neoplasia. The working group “Network modelling”, headed by Prof. Rainer König, investigates different clinical progressions of infections on a molecular level by means of network analyses. In addition to that, we were proudly able to win two young scientists from the Harvard Medical School in Boston as the heads of junior research groups. Dr. Christine Beemelmanns and her group “Chemical biology of microbe – host interactions” researches into the interconnectivities between bacteria, fungi and insects together with the involved natural products. Finding out about the communication between bacteria and other organisms through chemical signal molecules I N T R O D U C T I O N | VO RWO R T Auch in den Jahren 2012 und 2013 hat sich das Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie – Hans-Knöll-Institut (HKI) kontinuierlich weiter entwickelt, vernetzt und vergrößert. In den vergangenen Jahren ist die Zahl der Mitarbeiter und Forschungsgruppen gar so weit angestiegen, dass wir uns eine neue Bleibe suchen mussten. In unserem 2012 eingeweihten, neuen Forschungsgebäude haben wir nicht nur ausreichend Raum, sondern auch optimal ausgestattete Arbeitsplätze verpackt in reizvoller Architektur gefunden. Das Team des „Zentrums für Innovationskompetenz Septomics“ forscht seit 2012 ebenso in einem neuen Forschungsbau, der das Gesicht des Beutenberg-Campus mitprägt. In den Jahren 2012 und 2013 durften wir zwei neue assoziierte Forschungsgruppen vom Universitätsklinikum Jena bei uns begrüßen: Die 2 HKI Research Report 2012 / 2013 Arbeitsgruppe „Infektionen in der Hämatologie / Onkologie“ unter der Leitung von Prof. Marie von Lilienfeld-Toal beschäftigt sich mit der Erforschung von Infektionskrankheiten bei Patienten mit malignen Neoplasien. Prof. Rainer Königs Gruppe „Netzwerkmodellierung“ klärt unter Zuhilfenahme von Netzwerken verschiedene klinische Verläufe von Infektionen auf molekularer Ebene auf. Des Weiteren konnten wir zwei junge Wissenschaftler von der Harvard Medical School in Boston als Nachwuchsgruppenleiter gewinnen: Dr. Christine Beemelmanns forscht mit ihrer Gruppe „Chemische Biologie der Mikroben-Wirt Interaktionen“ an den Wechselwirkungen zwischen Bakterien, Pilzen und Insekten und den daran beteiligten Naturstoffen. Wie Bakterien und andere Organismen durch chemische Signalmoleküle miteinander kommunizieren, möchte Dr. Pierre is the focus of Dr. Pierre Stallforth’s group “Chemistry of microbial communication”. PD Ilse Jacobsen, PhD has now started to head an independent research group – “Microbial Immunology”. What is becoming noticeably more important for us is the information exchange with colleagues across Germany and also with representatives from other disciplines. Within the scope of the Collaborative Research Center / Transregio 124 “Pathogenic fungi and their human hosts: networks of interaction – FungiNet”, which is funded by the Deutsche Forschungsgemeinschaft, scientists from Jena and Würzburg work together to understand the complex mechanisms of fungal infections in order to develop a virtual infection model. We expand our horizons by means of the “InfectoGnostics research campus Jena”, which is funded by the Federal Minis- Stallforth mit seiner Gruppe „Chemie Mikrobieller Kommunikation“ herausfinden. Eine eigene Forschungsgruppe leitet nun PD Ilse Jacobsen, PhD, – „Mikrobielle Immunologie“. Zusehends wichtiger wird für uns der Austausch mit Kollegen in ganz Deutschland und Vertretern anderer Disziplinen. Im Sonderforschungsbereich / Transregio 124 „Pathogene Pilze und ihr menschlicher Wirt: Netzwerke der Interaktion – FungiNet“, gefördert von der Deutschen Forschungsgemeinschaft, arbeiten Wissenschaftler aus Jena und Würzburg daran, die komplexen Mechanismen von Pilzinfektionen zu verstehen und ein virtuelles Infektionsmodell zu entwickeln. Über den Tellerrand der Disziplingrenzen hinaus blicken wir im „InfectoGnostics Forschungscampus Jena“, der vom Bundesministerium für Bildung und Forschung unterstützt wird. Gemeinsam mit Partnern aus try for Education and Research. Together with partners from science, medicine and industry we develop point-of-care tests for acute infections. We even proceed one step further in the project “InfectControl 2020 – New Anti-Infective Strategies – Science • Society • Economy”, which is also funded by the Federal Ministry for Education and Research. Especially East German research institutions, companies and interest groups from several disciplines work together in this medically and socially relevant enterprise aiming at preventing and fighting infectious diseases effectively in the future. Thanks to the extensive global networking of our scientists, we were able to host internationally renowned conferences and meetings in 2012 and 2013 that helped us to foster relations with scientists from all over the world. Prof. Bernhard Hube hosted the ISHAM-con- Wissenschaft, Medizin und Wirtschaft entwickeln wir Vor-Ort-Analysen akuter Infektionen. Noch einen Schritt weiter gehen wir mit „InfectControl 2020 – Neue Antiinfektionsstrategien – Wissenschaft – Gesellschaft – Wirtschaft“, das ebenfalls vom Bundesministerium für Bildung und Forschung gefördert wird. Größtenteils ostdeutsche Forschungsinstitute, Unternehmen und Interessengruppen verschiedenster Disziplinen arbeiten hier an dem medizinisch und gesellschaftlich relevanten Vorhaben, Infektionskrankheiten in Zukunft umfassend zu vermeiden und effektiv zu bekämpfen. Dank der guten weltweiten Vernetzung unserer Wissenschaftler, konnten wir auch 2012 und 2013 international renommierte Kongresse und Tagungen ausrichten sowie Wissenschaftler aus aller Welt bei uns begrüßen. Prof. Bernhard Hube richtete den HKI Research Report 2012 / 2013 3 ference and provided an insight into the work of our institute to young academics within the scope of the FEBS Advanced Course. Prof. Peter F. Zipfel co-operated in organising the specialist conference “14th European Meeting on Complement in Human Disease”. Since exceptional research results can only be achieved in a team that enjoys their working environment, it is in our utmost interest to promote gender equality between men and women as well as balancing work and family life. We proudly signed the „Charta der Vielfalt“ (“Charta of Diversity”) and we are a partner of the „Jenaer Bündnis für Familie“ (“Jena Association for Families”). This commitment was honoured by the „Total E-Quality“. ISHAM-Kongress aus und bot innerhalb des FEBS Advanced Courses Nachwuchswissenschaftlern Einblicke in die Arbeit unseres Instituts. Prof. Peter F. Zipfel organisierte die Fachtagung „14th European Meeting on Complement in Human Disease“ mit. Da herausragende Forschungsergebnisse nur mit einem Team erreicht werden können, das seinen Arbeitsplatz als angenehm empfindet, setzen wir uns verstärkt für die Gleichstellung von Frauen und Männern sowie die Vereinbarkeit von Familie und Beruf ein. So unterzeichneten wir die „Charta der Vielfalt“ und sind Teil des „Jenaer Bündnisses für Familie". Quittiert wurde dieses Engagement mit dem Prädikat „Total E-Quality“ vom Bundesministerium für Familie, Senioren, Frauen und Jugend. 4 HKI Research Report 2012 / 2013 I would like to thank all colleagues at the HKI for their excellent cooperation. It is this outstanding collaboration that makes it possible to maintain and further develop the high quality of the scientific work at the HKI. I would like to express my special thanks to Dr. Michael Ramm and Dr. Christine Vogler for their contribution to this Report 2012/2013. Jena, June 2014 Axel Brakhage Director of the HKI Für die ausgezeichnete Kooperation möchte ich mich bei allen Kolleginnen und Kollegen des HKI sehr herzlich bedanken. Nur durch die hervorragende Zusammenarbeit ist es möglich, die hohe Qualität der wissenschaftlichen Leistung des HKI zu halten und weiter zu steigern. Für die Mitarbeit an diesem Report 2012/13 danke ich allen Mitarbeitern des HKI, insbesondere Dr. Michael Ramm und Dr. Christine Vogler. Jena, June 2014 Axel Brakhage Director of the HKI HKI Research Report 2012 / 2013 5 Contents | Inhalt 2 Introduction | Vorwort 6 Contents | Inhalt 8 Organization of the HKI | Organisation des HKI Departments | Abteilungen 11 Biomolecular Chemistry | Biomolekulare Chemie 27 Infection Biology | Infektionsbiologie 43 Microbial Pathogenicity Mechanisms | Mikrobielle Pathogenitätsmechanismen 61 Molecular and Applied Microbiology | Molekulare und Angewandte Mikrobiologie 79 Cell and Molecular Biology | Zell-und Molekularbiologie Research Groups | Forschungsgruppen 95 Applied Systems Biology | Angewandte Systembiologie 105 Fungal Septomics | Fungal Septomics 115 Microbial Immunology | Mikrobielle Immunologie 125 Systems Biology and Bioinformatics | Systembiologie und Bioinformatik Independent Junior Research Group | Unabhängige Nachwuchsgruppe 133 Secondary Metabolism of Predatory Bacteria | Sekundärmetabolismus räuberischer Bakterien Cross-sectional Units | Querschnittseinrichtungen 143 Bio Pilot Plant | Biotechnikum 157 ILRS – International Leibniz Research School 165 JSMC – Jena School for Microbial Communication 169 JMRC – Jena Microbial Resource Collection 177 CRC/Transregio FungiNet | SFB/TR FungiNet Associated Groups | Assoziierte Gruppen 181 Infections in Hematology / Oncology | Infektionen in der Hämatologie / Onkologie 189 Microbial Biochemistry and Physiology | Mikrobielle Biochemie und Physiologie 199 Pharmaceutical Microbiology | Pharmazeutische Mikrobiologie 6 HKI Research Report 2012 / 2013 Appendix | Anhang 208 Peer Reviewed Articles 2012 | Originalarbeiten 2012 213 Peer Reviewed Articles 2013 | Originalarbeiten 2013 218 Reviews, Monographs, Book chapters 2012/2013 | Übersichtsarbeiten, Monographien, Sammelwerke 220 Memberships in Editorial Boards 2012/2013 | Mitgliedschaften in Editorial Boards 220 Lectures at the HKI 2012/2013 | Kolloquien am HKI 221 Scientific Awards 2012/2013 | Preise und Auszeichnungen 222 Meetings, Workshops, Symposia 2012/2013 | Wissenschaftliche Veranstaltungen 222 Participation in Research Networks 2012/2013 | Beteiligung an Netzwerken und Verbundprojekten 224 Calls for Appointments 2012/2013 | Rufe 224 Postdoctoral Lecture Qualifications 2012/2013 | Habilitationen 224 Graduations 2012/2013 | Promotionen 225 Bachelor / Master / Diploma Theses 2012/2013 | Bachelor- / Master- / Diplomarbeiten 228 Inventions and Patents 2012/2013 | Erfindungen und Schutzrechte 229 External Funding 2012/2013 | Drittmittel 230 Maps | Lagepläne HKI Research Report 2012 / 2013 7 Organization of the HKI Board of Trustees | Kuratorium Dr. W. Eberbach Thuringian Ministry of Education, Science and Culture (Head) N. Graf zu Stolberg Medac GmbH, Wedel (Deputy Head) Prof. Dr. G. Diekert Friedrich Schiller University Jena Prof. Dr. F. Laplace Federal Ministry of Education and Research, Berlin Prof. Dr. T. Heinzel Vice-Rector of Friedrich Schiller University Jena Prof. Dr. W. Rosenthal Max Delbrück Center for Molecular Medicine Berlin-Buch Dr. T. Maier Wacker Biotech Jena GmbH Scientific Council | Wissenschaftlicher Beirat Prof. Dr. W. Rosenthal Max Delbrück Center for Molecular Medicine Berlin-Buch (Head) Prof. Dr. G. Diekert Friedrich Schiller University Jena (Deputy Head) Prof. Dr. E. Dittmann University Potsdam Prof. Dr. B. Fleischer Bernhard Nocht Institute for Tropical Medicine, Hamburg Prof. Dr. H. Haas Innsbruck Medical University Prof. Dr. J. Hacker German National Academy of Sciences Leopoldina, Halle (Saale) Prof. Dr. P. Neubauer Technical University Berlin Prof. Dr. G. Pohnert Friedrich Schiller University Jena Prof. Dr. J. Selbig University Potsdam Executive Board | Vorstand Prof. Dr. Axel A. Brakhage Scientific director Elke Jäcksch Administrative director Scientific Coordination | Wissenschaftliche Koordination Dr. Michael Ramm Scientific coordination Dr. Christine Vogler Scientific coordination Angelika Rauchmaul Assistant of the scientific director Administration | Verwaltung Elke Jäcksch Administrative director Renate Becher Sandra Ertel Julia Große (since 07/2013) Fabian Grunert (since 08/2013) Nils Haußner (since 08/2013) Eckhard Hemme Carmen Henze Michael Hetz Michael Kind Britta Kammer-Rattey Jan Kösling-Wettstaedt Ilona Lux 8 HKI Research Report 2012 / 2013 Andrea Matthies Frank Schebitz Reinald Schorcht Michael Schunk Christine Serfling Kerstin Siegmund Ekkehard Tittelbach Jens Trotzer (since 05/2013) Scientific Advisory Board | Board of Trustees | General Meeting Administration Elke Jäcksch Scientific Coordination Public Relations Dr. Michael Ramm Departments Biomolecular Chemistry Prof. Christian Hertweck Executive Board Prof. Axel Brakhage Elke Jäcksch Research Groups Independent Junior Research Groups Applied Systems Biology Prof. Marc Thilo Figge Chemistry of Microbial Communication Dr. Pierre Stallforth Ombudsman Prof. Reinhard Guthke Equal Opportunities Graduate Schools Dr. Christine Vogler Cross Sectional Units Associated Groups Bio Pilot Plant Dr. Uwe Horn Infections in Hematology / Oncology Prof. Marie von Lilienfeld-Toal (UKJ) (since 12/2013) Infection Biology Prof. Peter F. Zipfel Fungal Septomics Prof. Oliver Kurzai (since 04/2013) Chemical Biology of Microbe-Host Interactions Dr. Christine Beemelmanns International Leibniz Research School Prof. Peter F. Zipfel Microbial Biochemistry and Physiology Dr. habil. Matthias Brock (FSU) Jena Microbial Resource Collection PD Dr. Kerstin Voigt Network Modeling Prof. Rainer König (UKJ) (since 12/2013) Microbial Pathogenicity Mechanisms Prof. Bernhard Hube Microbial Immunology PD Ilse Jacobsen, PhD Molecular and Applied Microbiology Prof. Axel Brakhage Systems Biology and Bioinformatics Prof. Reinhard Guthke (since 08/2013) Secondary Metabolism of Predatory Bacteria Dr. habil. Markus Nett National Reference Center for Invasive Fungal Infections Prof. Oliver Kurzai (since 01/2014) Cell and Molecular Biology Prof. Hans Peter Saluz HKI Research Report 2012 / 2013 9 Pharmaceutical Microbiology Prof. Dirk Hoffmeister (FSU) 10 HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 11 Department of Biomolecular Chemistry The Department of Biomolecular Chemistry, headed by Christian Hertweck, focuses on various aspects of microbial secondary metabolism. The group covers the chemical and biochemical expertise in modern natural product research (structure elucidation, biosynthesis, enzymology, synthesis, interactions through biomolecules). In addition to the basic research projects, the department operates the institute’s state-of-the-art analytical facilities (NMR, MS-techniques, MALDI-TOF) and supports other HKI departments and FSU groups with metabolite and proteome analyses. The department is specialised in the genomebased isolation and structural elucidation of natural products. This work is funded by the Pakt für Forschung und Innovation, the DFG and BMBF, and supported by various collaborations with industrial partners. For the discovery of new potential therapeutics we investigate primarily neglected microorganisms (endophytes, symbionts, little explored microorganisms) and apply genome-mining strategies (activation of silent gene clusters). A major research area of the department is the investigation of biosynthetic pathways by a combination of chemical and biological methods. In nature, structural and functional diversity is governed by multienzyme processing lines that lead to highly complex compounds. To gain insights into the I N T R O D U C T I O N | E I N LE I T U N G Die Abteilung Biomolekulare Chemie befasst sich unter der Leitung von Prof. Christian Hertweck mit der Chemie und Biologie des facettenreichen Sekundärmetabolismus von Mikroorganismen – Bakterien und Pilzen. Die Abteilung integriert die chemischen und biochemischen Arbeitsgebiete der modernen Naturstoff-Forschung integriert (Strukturaufklärung, Biosynthese, Enzymmechanismen, Synthese und Naturstoff-basierte Interaktionen). Die für das Institut essenzielle Hochleistungsanalytik (NMR, MS, MALDI-TOF) wird in der Abteilung Biomolekulare Chemie betrieben. Dort ist ein breites Methodenspektrum zur Strukturaufklärung von niedermolekularen Verbindungen und zur Proteomanalyse verfügbar, durch das insbesondere die HKI-Abteilungen und FSU-Arbeitsgruppen unterstützt werden. Head Prof. Dr. Christian Hertweck Die Abteilung Biomolekulare Chemie ist auf die Genom-basierte Isolierung und Struk- 12 turaufklärung von Naturstoffen spezialisiert. Diese Arbeiten werden vom Pakt für Forschung und Innovation, der DFG und dem BMBF gefördert und in zahlreichen Industriekooperationen durchgeführt. Zur Suche nach neuen Naturstoffen werden vorwiegend neue Organismentypen (Endophythen, Symbionten, wenig untersuchte Mikroorganismen) verwendet und genome mining-Strategien eingesetzt. Ein Schwerpunkt der Abteilung Biomolekulare Chemie liegt in der Aufklärung von mikrobiellen Biosynthesewegen über biologische und chemische Methoden. Die strukturelle und funktionelle Diversität entsteht in der Natur über Multi-Enzym-Prozesslinien, die zu hochkomplexen Verbindungen führen. Um die Mechanismen und die Evolution der Synthesemaschinerien biologischer Systeme zu verstehen, studiert die Arbeitsgruppe die Biosynthese-Gencluster und ausgewählte HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry mechanisms and the evolution of the synthetic machineries the group investigates the functions of biosynthesis gene clusters and selected intriguing enzymes. For this purpose, we also do structural studies in collaboration. The majority of our projects is conducted in national and international scientific networks. In addition to understanding natural evolution of metabolic diversity we aim at harnessing the biosynthetic potential to produce novel natural product derivatives. In particular, biosynthetic pathways of pharmacologically relevant polyketides are investigated. Modern organic synthetic methods complement the microbes’ biosynthetic capabilities. Natural products represent mediators of biological communication, and their specific functions have developed during evolution. They play a key role in the interaction of microorganisms, in symbiosis and also in pathogenesis, e.g. in fungal infections. The group also specialises on fungal-bacterial interactions and the molecular basis of selected symbiotic and pathogenic relationships. The biomolecular interactions in fungalbacterial associations are jointly investigated with research groups at the HKI, the FSU, and the Max Planck Institute for Chemical Ecology in the context of the graduate schools ILRS and JSMC. Enzyme. In Kollaboration werden hierzu auch strukturbiologische Projekte durchgeführt. Die meisten dieser interdisziplinären Arbeiten werden in überregionalen Netzwerken bearbeitet. Die Projekte haben nicht nur zum Ziel, ein besseres Verständnis von der Evolution metabolischer Diversität zu bekommen, sondern auch, das natürliche Biosynthesepotential zu nutzen, um neue Naturstoff-Derivate darzustellen. Bei den hierzu angewandten biokombinatorischen Techniken stehen Biosynthesewege pharmakologisch relevanter Polyketide im Mittelpunkt. Moderne Synthesemethoden und Biotransformationen komplementieren die Biosyntheseleistung der Mikroorganismen. als Virulenzdeterminanten, zum Beispiel bei Pilzinfektionen eine wichtige Rolle. Die Abteilung Biomolekulare Chemie hat sich auf Pilz-Bakterien-Interaktionen spezialisiert und studiert die molekulare Basis von symbiotischen und pathogenen Beziehungen. Die biomolekularen Interaktionen in Pilz-Bakterien-Symbiosen sind Themen stark vernetzter Gemeinschaftprojekte der Arbeitsgruppen am HKI, der FSU und des Max-Planck-Instituts für chemische Ökologie im Rahmen der Graduiertenschulen ILRS und JSMC. Naturstoffe sind Mediatoren der biologischen Kommunikation, die im Laufe der Evolution auf biologische Aktivität selektioniert worden sind. Sie spielen in der Interaktion von Mikroorganismen, bei Symbiosen und auch Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 13 Scientific Projects Figure 1 Activation of a silent biosynthesis gene cluster in Burkholderia thailandensis. Figure 2 Structure and siderophore action of malleobactin. 1 Genomics-driven natural product discovery Group Leaders: Keishi Ishida, Sacha Pidot, Christian Hertweck The current crop of antibiotics in clinical use are either natural products or their derivatives. However, the rise of a multitude of different antibiotic resistant human pathogens means that new antibiotics are urgently needed. Unfortunately, the search for new antibiotics from traditional bacterial sources often results in a high rediscovery rate of known compounds and a low chance of identifying truly novel chemical entities. To overcome this, previously unexplored (or underinvestigated) bacterial sources are being tapped for their potential to produce novel compounds with new activities. Furthermore, in the postgenomic era it has become increasingly obvious that the biosynthetic potential of microorganisms has 14 been greatly underexplored. In many organisms the majority of genes potentially coding for the biosynthesis of biologically active compounds remain silent, and new methods are required to bring such ‘cryptic natural products’ to light. Our aim is to develop new ways to specifically activate such cryptic gene clusters. Manipulation of the promotor for the quorum sensing regulator gene in the model organism Burkholderia thailandensis caused a significant change in the metabolic profile. This genetic manipulation led to the activation of the thailandamide biosynthesis gene cluster. We succeeded in a stereochemical analysis to unravel the structure of the short-lived polyene, which likely is a signalling molecule. Using a similar genetic approach we unearthed the product of a cryptic NRPSPKS gene cluster that seems to be a hallmark of well-studied pathogenic bacteria belonging to the B. mallei group. Notably, Burkhold- HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry eria mallei and Burkholderia pseudomallei are infamous animal and human pathogens. Overall, surprisingly little is known about the biosynthetic potential and small-molecule virulence factors of these pathogens. Using the phylogenetically related albeit nonvirulent Burkholderia thailandensis strain as model organisms for pathogenesis, we succeeded in the isolation and structure elucidation of burkholderic acid, a highly oxygensensitive metabolite featuring an unusual polyketide scaffold. Using the same model organism, we have elucidated the structure of malleobactin A, the long searched for siderophore of animal and human pathogenic bacteria of the B. mallei complex. Through gene cluster analyses and targeted knock-outs we have unequivocally correlated siderophore activity to the mba gene cluster in B. thailandensis, the less-virulent model organism of the B. mallei family. In addition to disclosing an extraordinary siderophore variant featuring a nitro group, we report the unusual nitro amino acid ANPA for the first time as part of a natural product. With respect to natural products, one group of highly neglected bacteria are those that grow without oxygen: the anaerobes. Anaerobic bacteria are ubiquitous in the environment and play major roles in the natural cycling of both carbon and sulfur, and in the degradation of organic matter, are important in biofuel manufacture, and certain species are also well known pathogens. A major obstacle to the investigation of natural products in anaerobes comes from the long held view amongst secondary metabolite researchers that these bacteria are incapable of producing secondary metabolites. However, the isolation of the first antibiotic from an anaerobe, closthioamide from Clostridium cellulolyticum in the BMC, has turned this supposition on its head. Encouraged by this finding, a total of 211 complete and published genomes from anaerobic bacteria are analysed for the presence of secondary metabolite biosynthesis gene clusters, in particular those tentatively coding for polyketide synthases (PKS) and non-ribosomal peptide Figure 3 Cover art for Nat Prod Rep showing a summary of the genome analysis of anaerobic bacteria. Figure 4 Structure of the antibiotic closthioamide from Clostridium cellulolyticum and crystal structure of the Cu(I) complex; background: visialised Cu depletion in plate culture. synthetases (NRPS). We investigate the distribution of these gene clusters according to bacterial phylogeny and, if known, correlate these to the type of metabolic pathways they encode. Interestingly, we noted that PKS gene clusters were silent in representative strains. However, we successfully employed an antiterminator gene, nusG, for the induction of a cryptic biosynthetic pathway in Clostridium cellulolyticum. In this way we generated a mutant that can be harnessed for the sustainable production of the potent polythioamide antibiotic. Using this mutant, we also succeeded in the isolation of seven novel thioamide metabolites, which by far exceed the number of currently known natural products featuring this rare functional group. We also found that closthioamide is a selective metal chelator that forms a binuclear Cu(I) complex and disclosed an unparalleled architecture of the complex. Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 15 Figure 5 Structure of aureothin (1) and multifactorial control of chain assembly. 2 Natural product biosynthesis and pathway engineering Group Leader: Christian Hertweck Complex bacterial polyketides such as polyenes, macrolides and polyethers are a rich source of clinically used therapeutics. Despite their highly diverse structures, these valuable compounds are assembled from simple acyl and malonyl building blocks in an enzymatic assembly line. The polyketide synthases (PKS) in charge of selecting, fusing and processing the building blocks are organised in modules that consist of individual catalytic domains. Typically, there is a unidirectional progress of chain elongation, where each domain is only used once in the biosynthesis of one polyketide molecule. Thus, for most synthases there is a strict co-linearity between the number and architecture of the modules and the number of elongations and degree of β-keto pro- 16 cessing. However, there are more and more deviations from this textbook knowledge; in various modular PKS single domains or entire modules are skipped and individual modules may be used more than once. Despite the growing number of non-colinear PKS, to date the mechanisms that control the iteration processes in non-colinear modular PKS have remained obscure. We have obtained first insights into the factors governing the iteration processes in a noncolinear PKS involved in the biosynthesis of aureothin. Through gene deletion and complementation with a synthetic surrogate we found that the KS1 of the iterating module is exclusively in charge of priming the PKS. This is a prerequisite to warrant a vacant position for the retrotransfer of the diketide. The model of a kinetic control is fully in line with the observed complete loss of iteration in an engineered AurA variant harboring the designated loading module of the avermectin PKS. Finally, through dele- HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry Figure 6 Enzymatic chain branching reaction to install a pharmacophoric group of rhizoxin (left) and structure of the module (right, x-ray: G. Zocher, Tübingen). tion of the TE domain and the use of synthetic reference compounds we succeeded in detecting the polyketide intermediates of iterative and non-iterative aur PKS modules. Whereas this experiment confirmed a high fidelity of the programmed events, the iterative module alone produces polyketides with varying lengths that correspond to one to four rounds of elongations. Thus, we provide strong experimental support for a scenario where multiple factors are responsible for the rare iteration process. It is clearly an inherent quality of the KS1 to mediate priming, chain propagation and intermediate retrotransfer, but the downstream KS2 functions as a gatekeeper that excludes the propagation of intermediates that are either too short or too long. Beyond new insights into the hidden programme of a non-canonical multimodular assembly line our findings also provide valuable information for the rational design of complex polyketides by pathway engineering. A hallmark of all polyketide synthases studied to date is the head-to-tail fusion of acyl and malonyl building blocks, exclusively yielding linear chains. We recently discovered an unprecedented, non-canonical PKS module that mediates a chain bifurcation, thus enabling the formation of novel branched metabolites. This particular chain branching plays a key role in the biosynthesis of rhizoxin, one of the strongest antimitotic agents known. Notably, the biological activity of the molecule critically depends on the branch, and also in numerous other compounds similar pharmacophores are found. We experimentally support our findings by the successful full in vitro reconstitution of a non-canonical PKS module and infer the exact course of the reaction from protein structure data, point mutagenesis, isotope labelling, transformation of synthetic surrogates and NMR/MS analysis of ACP-bound and liberated products. We unveiled a novel role for a ketosynthase (KS) in polyketide Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 17 Figure 7 Unusual malonyl transfer reaction by the ketosynthase CerJ (X-ray: G. Zocher, Tübingen). biosynthesis. In light of the countless known KS domains that uniformly produce linear carbon chains, our finding of a KS that generates a branch is a rare case for a substantially different KS-mediated reaction channel. The crystal structure of the branching module provides new structural insight into the functioning of these important assembly lines. Moreover, we pioneered a new NMRbased method for analyzing ACP-bound enzyme products. This method will be of great value for many other studies as it allows the unequivocal assignment of hard-to-detect intermediates or products. In a different study we discovered another non-canonical ketosynthase homologue, CerJ, which unexpectedly forms malonyl esters during the biosynthesis of the anti-MRSA glycoside antibiotic cervimycin from the cave-derived bacterium Streptomyces tendae. Through a series of experiments we found that CerJ is uniquely capable of transferring 18 malonyl-, methylmalonyl-, and dimethylmalonyl units onto the glycoside. Elucidation of the protein crystal structure of CerJ revealed that this unusual enzyme is functionally and structurally positioned between KS catalyzing Claisen condensations and acyl-ACP shuttles. A non-canonical catalytic triad has been assigned to the aberrant malonyl transfer. Furthermore, we generated a mutant that produces a significantly more active cervimycin variant lacking the malonyl side chain. Chartreusin is a well-known antibacterial and cytotoxic agent equipped with a DNAintercalating aglycone. As to date, there has been a complete lack of information on the impact of the aglycone (chartarin) ring substitution on antitumoral and antibacterial activities. Notably, there are no reported efficient synthetic methods available for altering the ring substitution pattern, and to date the total synthesis of chartreusin has not been achieved. We have rationally designed HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry Figure 8 Mutasynthesis of chartreusin derivatives. improved chartreusin analogs by merging the strengths of chemical synthesis with biosynthesis. Specifically, we deleted the PKS genes and cross-complemented the mutant with anthracycline biosynthesis genes or with fully synthetic aglycones. In this way we succeeded in generating a library of chartreusin derivatives with altered aglycone structures. The blend of biological and synthetic methods not only granted access to a library of chartreusins that are otherwise not accessible, but also yielded promising candidates for further development as antitumoral or antibacterial agents. Specifically, we synthesised a novel vinyl-substituted chartreusin analogue, which forms covalent links to DNA upon mild photoactivation with visible light and has a markedly higher antitumoral potency than the parent compound. The novel apoptosis-inducing photoreactive DNA intercalator can be activated by visible light, a lead that holds promise for curing skin cancers and tumors that are ac- Figure 9 Cover art for Angew Chem illustrating photoactivation of a chartreusin derivative. cessible with light probes. Furthermore, we found that the lack of the methyl substituent causes a dramatic decrease in cytotoxicity, but strongly enhances antibacterial activities. Specifically, the desmethyl variant is ac- Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 19 Figure 10 Biosynthesis gene cluster and structures of a family of bacterial indoloterpenes. tive against mycobacteria, which are in the focus of antibiotic research due to the rise of tuberculosis and other life-threatening infectious diseases. Indole terpenoids encompass a highly diverse group of natural products, including infamous psychotropic agents such as lysergic acid derivatives, the aphrodisiac yohimbine, and the potassium channel blockers paxilline and lolitrem. What is remarkable about this multifarious class is that practically all indole terpene alkaloids have been isolated from plants and fungi. However, to date nothing is known about the biosynthesis of indole terpenes in bacteria. We have unveiled the molecular basis for the first indole terpenoid pathway in a bacterium. Through heterologous reconstitution of the xiamycin pathway we elucidated the set of xia genes that are sufficient for xiamycin biosynthesis and demonstrated the biogenetic relationship of the diverse alkaloids. Since 20 genetic engineering of the xia gene cluster resulted in increased metabolite production, we succeeded in the characterisation of yet overlooked, structurally unique N-N- and C-N-coupled xiamycin dimers. Through in silico studies, mutational analysis of all pathway genes and metabolite analyses we succeeded in dissecting an unparalleled bacterial alkaloid pathway. Our results reveal the involvement of two prenyl transferases, XiaK and XiaM for FPP formation and indole prenylation, respectively, and suggest concomitant epoxidation by a novel hybrid enzyme, XiaL. On biosynthetic grounds the most important discovery were two novel cyclases that play key roles in indole terpenoid cyclisation: XiaE, which promotes the cyclisation of the epoxidized farnesyl side chain, and the indole oxygenase XiaF, which paves the way for diverging cyclisation schemes. Because of the unusual IS architectures and the novel mechanisms involved, the xia biosynthesis enzymes are an important addition HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry to the emerging family of bacterial terpene synthases and modifying enzymes. Thus, this study not only sets the stage for investigating mechanistically intriguing reactions, but also is a prerequisite for engineering indoloterpenoids in bacteria. 3 Natural products in microbial interactions Group Leaders: Kirstin Scherlach, Christian Hertweck Natural products play a key role in symbiotic interactions between microorganisms and higher organisms, covering all kingdoms of life. In many cases the interactions involve multiple partners and thus the biogenetic basis of chemical mediators can be quite complex. This is well exemplified by the unparalleled tripartite relationship between the rice seedling blight fungus Rhizopus microsporus, its host plant Oryza sativa and endosymbiotic bacteria that reside in the fungal cytosol. The bacterial symbionts (Burkholderia spp.) produce a phytotoxin complex (rhizoxin and congeners) to assist the phytopathogenic fungus in colonising rice seedlings. Whereas the biosynthesis of the macrolide backbone has been decoded by mutational analyses, polyketide tailoring mechanisms and the biological role of the bis-epoxidation have remained elusive. Through a combination of genetic and chemical analyses we have now solved the riddle of the dual epoxidation in rhizoxin biosynthesis. Sequencing and comparison of rhizoxin biosynthesis gene clusters and engineering of a mutant producing didesepoxy variants of rhizoxin revealed that the macrolide is first epoxidized by the cytochrome P450 monooxygenase RhiH. By whole-cell biotransformation and cross-infection experiments we could unequivocally demonstrate that the 2,3-oxirane ring is introduced by the fungal host to specifically tailor the rhizoxin scaffold. According to the rice seedling swelling assays, the additional epoxide moiety substantially increases phytotoxic potency. From an ecological point of view, this finding is fully plausible since the second epoxidation is a specific trait of fungi belonging to the clade of rice seedling blight fungi. We therefore report for the first time on symbiotic synergism in the biosynthesis of a secondary metabolite that has a biological significance. A related bacteria-fungi interaction plays a key role in the context of food fermentations. The respiratory toxin bongkrekic acid (BA) caused countless health-threatening and even lethal intoxications after consumption of the Southeast-Asian specialty tempe bongkrek. Whereas it was long known that the toxin producer is a contaminant of the fungus used for fermentation, the genetic basis for BA biosynthesis has remained obscure. Through whole-genome sequencing of B. gladioli pv. cocovenenans we identified the gene locus for a giant trans-AT PKS and accessory enzymes catalysing assembly of the polyunsaturated tricarboxylic acid. Considering that BA is one of the most potent respiratory toxins known, these insights are of significance for pinpointing toxinogenic B. gladioli pathovars in the context of food processing, agriculture and medicine. On pharmacological grounds knowledge of the molecular basis for BA biosynthesis paves the way for engineering specific ligands for adenine nucleotide translocator, a promising target for antiapoptosis and chemotherapy. Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 Figure 11 Symbiotic cooperation in the biosynthesis of the phytotoxin rhizoxin. 21 Figure 12 Structure of bongkrekic acid (BA), micrograph of fungal-bacterial coculture and formation of BA at the bacterial-fungal interface. Soft rot diseases caused by a variety of bacteria account for severe losses in agriculture, devastating fruits, vegetables and cultivated mushrooms. After bacterial infection, often due to direct contact or transmission by insects, factors and lytic enzymes cause degradation of plant and mushroom tissues, turning crop into mush. In many cases, the chemical mediators of soft rot diseases are unknown, as in the long-known mushroom pathogen Janthinobacterium agaricidamnosum. We have discovered a new antifungal virulence factor, jagaricin, from the mushroom pathogen. The isolation proved to be challenging because the jag biosynthesis genes are downregulated or silenced under regular cultivation conditions. In the pathobiological context jagaricin production is triggered upon contact of the bacterium (J. agaricidamnosum) with the mushroom (A. bisporus). To elucidate the cryptic natural product we employed a combination of imaging mass spectrometry and genome mining. 22 Imaging mass spectrometry has proven to be a valuable tool for the discovery of microbial and plant natural products, in particular in symbioses. Coupling this technique to genome mining is a new approach, and to the best of our knowledge we describe the first case where this combination enabled the systematic discovery and full characterisation of a cryptic natural product. Furthermore, the revelation of a virulence factor by imaging mass spectrometry and genomics is unprecedented. For the rigorous elucidation of the antifungal principle the combination of physicochemical methods, chemical derivatisation and bioinformatic analysis of the encoded NRPS assembly line was most effective. Herein, predicting the lactone ring size by a TE phylogeny proved to be a particularly valuable, new approach that will certainly aid in solving related structural riddles. Furthermore, comparison of the jagaricin structure and the thiotemplate system revealed several unusual features of the jag NRPS. HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry Figure 13 Gene cluster and structure of jagaricin, a virulence factor of mushroom soft rot and illustration of imaging mass spectrometry. On ecological and agricultural grounds the discovery of the virulence factor and the corresponding genes is significant as it helps to understand the pathobiology of bacteriainduced soft rot. From a chemical point of view, our study highlights the impact of blending modern analytics with genetics to unveil cryptic natural products from neglected microorganisms. The general approach of combining imaging mass spectrometry with genome mining holds promise to be generally applicable to the discovery of cryptic natural products including chemical mediators such as virulence factors that are only produced in a particular (patho)biological context. pathogens. Its high pathogenicity combined with an almost worldwide spread constitutes serious ecological and economical problems. Thus, deciphering the molecular mechanisms Fire blight is one of the most devastating plant diseases that can eradicate entire orchards. It mainly affects apple and pear trees, among other members of the Rosaceae family. The causative agent of this rapidly spreading disease, the bacterium Erwinia amylovora, ranks among the top ten plant Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 Figure 14 Cover art for Angew Chem showing the structure of 6-thioguanine from Erwinia amylovora and effect on apple plants. 23 Figure 15 Biosynthesis of gliotoxin, a virulence factor of the human pathogen Aspergillus fumigatus. responsible for the success of this bacterium is of utmost importance. Interestingly, E. amylovora is capable of producing 6-thioguanine (6TG). Thioguanine, initially discovered and synthesised in the 1950s by Elion and Hitchings (Nobel Prize in Physiology or Medicine for their discovery of antimetabolite drugs) is a cytotoxic agent in clinical use. The natural role of 6TG and its biosynthesis in E. amylovora, however, have remained enigmatic until now. We have unveiled the molecular basis of 6TG biosynthesis in the plant pathogen, gained first insights into the formation of a peculiar enzymatic carbon-sulfur bond formation and demonstrate for the first time the crucial role of 6TG in fire blight pathogenesis. sulfide bridge. This signature residue for epidithiodiketopiperazines (ETPs) inactivates various proteins by thiol conjugation and generates reactive oxygen species by redox cycling. Gliotoxin, the archetypal ETP, has been in the focus of much research endeavor as it has been implicated as a virulence factor of Aspergillus fumigatus, a human fungal pathogen causing life-threatening systemic mycoses. In collaboration with the department MAM we have elucidated intriguing enzymatic steps for sulfur introduction in the gliotoxin biosynthetic pathway (for details see report MAM). A broad range of toxic fungal secondary metabolites that have been implicated in human, animal and plant diseases share a diketopiperazine scaffold that is overarched by a di- 24 HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry Group members Head Prof. Dr. Christian Hertweck Phone +49 3641 532-1100 Fax +49 3641 532-0804 [email protected] Trainees Laura Broschat (since 08/2013) Secretary Hiltrud Klose (until 5/2012) Lab Manager/Executive Assistant Caroline Rückert (since 10/2012) Visiting Scientists Scientists Dr. Emma Barnes (since 04/2013) Dr. Daniela Böttger-Schmidt (until 12/2013) Dr. Sebastien Coyne (until 12/2012) Dr. Ling Ding (maternity leave since 08/2013) Dr. Andreas Habel (until 12/2013) Dr. Keishi Ishida Dr. Mie Ishida-Ito Dr. Juangjun Jumpathong (since 12/2012) Dr. Sacha Pidot (until 07/2013) Dr. Severin Sasso (until 02/2012) Dr. Kirstin Scherlach Dr. Zhongli Xu (until 01/2013) Ph.D. Students Martin Baunach (since 11/2012) Swantje Behnken (until 02/2012) Nicole Brendel (until 11/2012) Sabrina Boldt (since 01/2012) Daniela Böttger-Schmidt Tom Bretschneider (until 11/2013) Pranatchareeya Chankhamjon Jakob Franke Katharina Graupner (maternity leave since 10/2013) Daniel Heine Florian Kloss Anne-Catrin Letzel Agnieszka Litomska Florian Meyer (since 01/2013) Nadine Möbius (until 03/2013) Claudia Roß Gülmire Shabuer (since 08/2012) Yuki Sugimoto Srividhya Sundaram (since 07/2012) Nico Ueberschaar Zerrin Üzüm (since 10/2012) Christoph Zähle (until 12/2012) Diploma / Bachelor / Master Students Philipp Balzer (since 02/2012) Tim Baumeister (04–07/2013) Martin Baunach (until 11/2012) Marco Böttger (03/2012–03/2013) Jan Dworschak (since 01/2013) Elena Geib (09/2012–12/2013) Stefanie Groening (since 2/2012) Florian Meyer (until 12/2012) Anja Hartmann (03–09/2013) Eric Helfrich (since 6/2012) Toni Neuwirth (since 1/2012) Viktoria Opel (04–07/2013) Elena Seibel (04–07/2013) Gülmire Shabuer (until 08/2012) Zerrin Üzüm (until 09/2012) Research Assistants Heike Heinecke Carmen Karkowski Dipl.-Troph. Barbara Urbansky Ing. Andrea Perner Ing. Ulrike Valentin (until 7/2012) Dr. Songsak Wattanachaisaereekul Bangkok University, Thailand 01/2012 Dr. Anton Liaimer University of Tromsoe, Norway 07/2012; 12/2012 Irshad ul Haq Universty of Groningen, The Netherlands 10/2013 Dr. Katherine Watts Standford University, USA 10/2013–11/2013 External funding Bundesministerium für Bildung und Forschung GenBioCom – Genombasierte Produktion bioaktiver Verbindungen aus Aktinomyceten: Komplexe Polyketide aus Aktinomyceten: Genom-Analyse, Gensynthese und Aktivitätsbestimmung Christian Hertweck Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Endosymbiosis – Molecular Basis of Bacterial-Fungal Symbioses Christian Hertweck Selected publications Baunach M, Ding L, Bruhn T, Bringmann G, Hertweck C (2013) Regiodivergent N,C- and N,N-Aryl couplings of indoloterpenes and cycloether formation mediated by a single bacterial flavoenzyme. Angew Chem Int Ed 52 9040-9043. Bretschneider T, Zocher G, Kusebauch B, Heine D, Winkler R, Stehle T, Hertweck C (2013) Vinylogous chain branching catalyzed by a dedicated polyketide synthase module. Nature 502, 124-128. Coyne S, Chizzali C, Khalil MNA , Litomska A, Richter K, Beerhues L, Hertweck C (2013) Biosynthesis of the antimetabolite 6-thioguanine in Erwinia amylovora plays a key role in fire blight pathogenesis. Angew Chem Int Ed 52 1056410568. Franke J, Ishida K, Ishida-Ito M, Hertweck C (2013) Nitro versus hydroxamate in siderophores of pathogenic bacteria: effect of missing hydroxylamine protection in malleobactin biosynthesis. Angew Chem Int Ed 52, 8271-8275. Scharf D, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis: a four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges. Angew Chem Int Ed 52, 11092-11095. Collaborations Centers for Disease Control and Prevention, Atlanta, USA Prof. Dr. Jay E. Gee Chalmers University of Technology, Göteborg, Sweden Prof. Dr. Jens Nielsen Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Symbiosis – Bacterial endosymbionts in plant-pathogenic fungi Christian Hertweck Friedrich Schiller University Jena, Germany Dr. Helmar Görls Prof. Dr. Maria Mittag Prof. Dr. Georg Pohnert Prof. Dr. Severin Sasso Deutsche Forschungsgemeinschaft Microviridine – Ribosomale Biosynthese trizyklischer Microviridine in Cyanobakterien: Biochemie, Evolution und Funktion Christian Hertweck INSA Rouen, Mont Saint Aignan, France Dr. Michael DePaolis Prof. Dr. Jacques Maddaluno Freistaat Thüringen ProExzellenz Initiative NanoConSens – Nanocontainer und nanostrukturierte Trägermaterialien für Sensorik und Wirkstofftransport Axel Brakhage, Christian Hertweck Pakt für Forschung und Innovation Genome Mining – Genome Mining zur Wirkstoffsuche in Mikroorganismen Christian Hertweck Max Planck Institute for Chemical Ecology Jena, Germany Prof. Dr. Jonathan Gershenzon Dr. Martin Kaltenpoth Dr. Bernd Schneider Dr. Aleš Svatoš John Hopkins University, Balitmore, USA Prof. Dr. Craig A. Townsend Medical University Innsbruck, Austria Prof. Dr. Hubertus Haas Department of Biomolecular Chemistry HKI Research Report 2012 / 2013 25 Oncotest GmbH, Freiburg, Germany Prof. Dr. Heinz-Herbert Fiebig Dr. Armin Maier TU Dortmund University, Germany Prof. Dr. Michael Spiteller UC San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences, USA Prof. Dr. Bradley S. Moore University of Bonn, Germany Prof. Dr. Jörn Piel University of Kentucky, USA Prof. Dr. Jurgen Rohr University of Lausanne, Switzerland Prof. Dr. Uta Paszkowski University of Napoli, Italy Prof. Dr. Anna Molinaro University of Potsdam, Germany Prof. Dr. Elke Dittmann University of Tromsoe, Norway Dr. Antong Liaimer University Tübingen, Germany Prof. Dr. Thilo Stehle Prof. Dr. Georg Zocher 26 HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry Department of Infection Biology HKI Research Report 2012 / 2013 27 Department of Infection Biology The fungal pathogen Candida albicans uses an arsenal of immune inhibitors to control the hostile immune attack generated by the immunocompetent human host. Upon infection the activated human immune system generates potent effector molecules that attack the invader C. albicans. However as a pathogen C. albicans controls, modulates and blocks the damaging and inflammatory actions of the host immune response. The aim of the Department of Infection Biology is to identify the microbial and human molecules that act on this fungal-host immune interface. To better understand the infection process we identify fungal immune evation proteins and also characterise the human immune proteins that participate in this immune battle. The identification and functional characterisation of fungal immune evasion proteins is a prerequisite to understand fungal evasion strategies in molecular detail, to define new targets and ultimately to develop new antifungal strategies. Four major topics are followed in the individual project groups in the Department of Infection Biology: I N T R O D U C T I O N | E I N LE I T U N G Die human-pathogene Hefe C. albicans nutzt ein breites Arsenal von Immuninhibitoren und Regulatoren um die schädigende Immunantwort des menschlichen Wirts zu umgehen. Im Laufe einer Infektion versucht C. albicans die Immunantwort des Menschen direkt und sofort zu kontrollieren, zu modulieren, und zu blockieren. Ziel der Arbeiten in der Abteilung Infektionsbiologie des HKI ist es, Immunevasionsproteine der pathogenen Hefe zu identifizieren, welche die humane Immunantwort steuern. Die funktionelle Charakterisierung dieser Immunevasionsproteine und die Beschreibung ihrer Inter- Head: Prof. Dr. Peter F. Zipfel 28 aktion mit den entsprechenden Wirtsproteinen erlaubt die detaillierte Analyse der Immuninteraktion auf molekularer Ebene. Die Identifizierung und Charakterisierung von Immunevasionsproteinen des Pathogens C. albicans bei der Immuninteraktion ist ein wichtiger Ansatz um neue Targets zu identifizieren und um eine C. albicans Infektion präventiv zu verhindern. Vier zentrale Themen werden in den Projektgruppen der Abteilung Infektionsbiologie bearbeitet: HKI Research Report 2012 / 2013 Department of Infection Biology 1) Identification of multifunctional C. albicans virulence factors, 2) Characterisation of general immune escape principles that are used by fungi, and other infectious pathogenic microbes 3) A n indepth understanding of the human innate immune response and its influence on the adaptive immunity during the infection process This approach allows to define general principles of microbial immune escape which is shared by pathogenic fungi, Gram-negative as well as Gram-positive bacteria, protozoa and viruses. Understanding the detailed action of human immune regulators also provides a platform to identify disease causing mechanisms of severe human disorders and to translate these results into novel diagnostic and therapeutic strategies. 4) Characterisation of protection mechanisms for self-structures against toxic immune reaction products 1) die Identifizierung von multifunktionalen Virulenzfaktoren von C. albicans 2) die Entschlüsselung eines allgemeinen Immunevasion-Prinzips, das auch von anderen Pilzen und weiteren infektiösen pathogenen Erregern genutzt wird 3) ein vertieftes Verständnis der angeborenen humanen Immunantwort sowie der Interaktion zwischen angeborener und erworbener Immunität Mit diesem Vorgehen werden zentrale Prinzipien der mikrobiellen Immunevasion definiert, die sowohl von C. albicans, als auch von weiteren pathogenen Pilzen, von Gram-negativen und Gram-positiven bakteriellen Erregern, Protozoen und Viren genutzt werden. Das detaillierte Verständnis der Immunreaktion hilft, Krankheitsmechanismen zu verstehen und neue Strategien für die Diagnose und Therapie schwerer menschlicher Erkrankungen zu entwickeln. 4) die Charakterisierung der Schutzmechanismen von körpereigenen Zellen vor toxischen Immunaktivierungprodukten Department of Infection Biology HKI Research Report 2012 / 2013 29 Scientific Projects 1 Immune and complement escape of the human pathogenic fungus C. albicans: Gpd2 (Glycerin-aldehyde 3 phosphatase) is a novel fungal moonlighting protein and immune evasion factor Group Leader: Peter F. Zipfel C. albicans is an opportunistic and medically important human fungal pathogen. Infections with C. albicans can range from superficial to systemic disorders. Resistant C. albicans strains are continuously developing and thus new prophylactic and therapeutic strategies in treatment of fungal infections are urgently needed. The identification of novel yeast virulence factors or determinants that substantially contribute to pathogenic growth inside an infected host represent ideal targets to ultimately fight C. albicans infections. Figure 1 C. albicans binds to human endothelial cells. C. albicans upon co cultivation with human endothelial cells (HUVEC) binds to the surface of HUVEC cells. C. albicans when opsonised with the complement activation product C3b binds strongly to the endothelial cells, and in consequence the phagocytic uptake of the pathogen by the human cells is increased. 30 C. albicans infections, similar to other microbial infections, activate the immune response of their host. The immediately responding innate immune system of the infected host uses pattern recognition molecules to recognise and damage the surface of the foreign invader. Candida infections are a particular challenge for immunocompromised individuals. A pathogenic microbe upon overcoming host immune reactions can propagate and bind to host cellular and extracellular matrices. The pathogens even degrade these tissue matrices to disseminate into deeper tissue layers. Complement is a central part of the human immune response and provides one of the first defense lines of host immunity. The complement cascade, which is activated by three major pathways, forms an immediate barrier against invading microbes. The alternative pathway is initiated spontaneously and is activated strongly on foreign surfaces. The lectin pathway is activated upon binding of mannose-binding lectin to mannan and carbohydrate structures on microbial surfaces. The classical pathway is activated via antigen-antibody complexes or by C-reactive protein. All three pathways merge on the level of the C3 convertase, which is a newly formed enzymatic complex that cleaves C3 into C3a and C3b. The cleavage product C3b binds to microbial surfaces where it acts as opsonin and mediates recognition and phagocytic uptake by human immune effector cells. C3a has antifungal, antimicrobial, chemotactic as well as inflammatory activities. C. albicans and also other infectious pathogens express a series of immune escape proteins, including complement inhibitors, proteases as well as adhesion proteins. Furthermore C. albicans recruits and exploits protective human plasma proteins with the purpose of controlling the innate immune attack, as well as mediating adhesion and tissue interaction and degradation (Fig. 1). C. albicans expresses four immune evasion proteins that bind the central human complement inhibitor Factor H (i.e. CRASP1/Gpm1, CRASP2/Pra1, CRASP3/Gpd2 and Hgt) and exposes a total of 12 surface proteins that bind the human coagulation zymogen plasminogen. Notably these fungal immune evasion proteins control many host immune reactions and represent multifunctional innate immune inhibitors. A single C. albicans immune escape protein can bind different human complement components or regula- HKI Research Report 2012 / 2013 Department of Infection Biology Figure 2 Pra1, the pH regulated antigen of C. albicans is a multifarious fungal immune escape protein. Pra1 binds and interacts with many human complement components, complement regulators Factor H, FHL1, C4BP, C3 and the C3 activation fragments C3b, iC3b, C3d as well as C3a. In addition Pra1 binds the extracellular matrix components lamin, fibrinogen and the proenyzme plasminogen. Plasminogen attached via Pra1 to the surface of C. albicans is accessible for the activator and is converted to the active protease which in consequence cleaves fibrinogen. Pra1 also binds to cellular integrin receptors, i.e. CD11b/CD18 (CR3) and to CD11c/CD18 (CR4) which are expressed by human macrophages and neutrophils. In addition Pra1 is a zinc chelator and assists in fungal zinc metabolism. Pra1 acts at many locations: the immune regulator is present and exposed at the fungal surface, is secreted by the pathogen, binds back to the fungal surface and also interacts with specific receptors that are expressed on human immune cells. tors, adhesion proteins or coagulation factors. CRASP2 of C. albicans, also termed Pra1 (pH regulated antigen 1), is a prime example for such a multifunctional immune evasion protein. Pra1 binds many soluble human immune proteins, including the complement components and regulators Factor H, FHL1, C4BP, C3, the C3 activation fragments, C3b, iC3b, C3d and C3a. Furthermore Pra1 binds the adhesion components fibrinogen, laminin as well as plasminogen. Plasminogen when bound to the fungal surface via Pra1 is accessible for activators and is converted to the active protease plasmin. In addition Pra1 is a zinc chelator and binds to the human integrin receptors i.e. CD11b/CD18 (CR3) as well as CD11c/CD18 (CR4) (Fig. 2). Upon binding to these human integrin receptors at the surface of human macrophages and neutrophilic granulocytes Pra1 modulates the response of these human innate immune effector cells. The Department of Infection Biology is interested in defining the whole immune escape repertoire that the fungal pathogen C. albicans uses to control human complement attack and to direct the host immune response. Therefore we aimed to identify new fungal complement escape proteins. Using a systematic screening approach C. albicans cell extract was adsorbed to a matrix to which the human plasma protein Factor H was attached. A 52 kDa C. albicans protein was eluted and identified by mass spectrometry as Glycerol3-phosphate dehydrogenase 2, Gpd2. Consequently the Gpd2 gene from C. albicans was amplified by PCR, cloned and the protein was recombinantly expressed in Pichia pastoris. Recombinant Gpd2 binds the three human immune regulators, Factor H, FHL-1 and also plasminogen. Each of the three human regulators when attached to Candida Gpd2 is functionally active and assists in complement inactivation as well as fibrinogen degradation. FHL1 and FHL1 inhibit the comple- Department of Infection Biology HKI Research Report 2012 / 2013 31 Gpd2 candida Figure 3 Gpd2 (Glycerinaldehyde 3 phosphatase) is expressed at the surface of C. albicans yeast cells and hyphae. Gpd2 is a novel fungal complement regulator binding protein of the human pathogenic fungus C. albicans. A new polyclonal antiserum was generated by immunising rabbits with recombinant Gpd2. This antiserum identified Gpd2 on the surface of C. albicans yeast cells (top panel) and hyphae (bottom panel). References Luo S, Hoffmann R, Skerka S, Zipfel PF (2013) Glycerol-3-phosphate Dehydrogenase 2 Is a Novel Factor H, FHL-1 and Plasminogen Binding Surface Protein of C. albicans albicans. J Infect Dis 207, 594-603. hyphae ment cascade at the level of the C3 convertase by acting as cofactors for the serine protease Factor I, which cleaves and degrades C3b into iC3b. In addition Factor H and FHL1 compete with Factor B for C3b binding and both proteins also accelerate the decay of a preformed alternative pathway C3 convertase. Native Gpd2 is identified at the surface of C. albicans yeast cells, as well as hyphae by a polyclonal anti-serum to Gpd2 (Fig. 3). Recombinant Gpd2 also binds to human non – phagocytic cells including endothelial HUVEC cells and human keratinocytes (HACAT) (Fig. 4). This specific binding to human endothelial cells and to keratinocytes indicates an additional role of the fungal protein for directing the host cellular response. In summary the C. albicans surface protein Gpd2 is a new fungal immune escape protein which binds several human immune regulators and also binds to the surface of human endothelial cells and keratinocytes. 32 Altogether evasion strategies to control complement action used by C. albicans are very similar to those used by certain other microorganisms and thus reflect rather general principles which are shared by pathogens derived from different species. 2 Identification and characterisation of human complement regulators as microbial evasion molecules (Group Immunoregulation) Group Leader: Christine Skerka Infectious microorganisms are immediately recognised and attacked by the innate immune system of the human host. This innate immune response results in the generation of antimicrobial peptides, the generation of complement activation products as well as the activation of immune cells. The complement system is a central part of the immune HKI Research Report 2012 / 2013 Department of Infection Biology Figure 4 The C. albicans Gpd2 protein binds to the surface of human endothelial cells and keratinocytes. Gpd2 binding to human endothelial cells and keratinocytes was analysed by flow cytometry (A) and by confocal laser scanning microscopy (B). Gpd2 binds to the surface of nonphagocytic endothelial cells and keratinocytes, but not to the surface of monocytic U937 cells. system and upon activation generates C3b, which then deposits onto nearby surfaces. On the surface of human cells, this newly generated C3b is immediately inactivated by regulators, but on microbial surfaces C3b deposition is amplified, leading to opsonisation. Such C3b covered foreign microbial surfaces are recognised and their uptake by phagocytes is enhanced. The complement activation split products C3a and C5a act as anaphylatoxins which recruit additional effector cells and initiate the adaptive immune response. In order to evade these potent and damaging host immune reactions C. albicans and other pathogenic microbes exploit human complement regulators to control the host response. In addition, these human regulators play a major role in the complement mediated noninflammatory phagocytosis and clearance of modified and dead cells from the circulation. Therefore many human complement regulators harbour a surface recognition region and a complement regulatory region which enables inhibition of different levels of activation on certain surfaces. As these proteins regulate important steps of the human immune response they represent attractive targets for infectious microbes like C. albicans (Fig. 1). The aim of the Group of Immunoregulation is to identify and characterise novel human complement regulators and to investigate the role of these regulators in the infection process with C. albicans. The group focuses on three human proteins: CFHR2 (complement factor H related protein 2), CFHR3, as well as plasminogen. The role of the individual proteins in complement activation and regulation is poorly defined and genetic variations in individual genes are associated with several immune diseases, including the retinal disorder AMD (Age Related Macular Degeneration). Department of Infection Biology HKI Research Report 2012 / 2013 33 Figure 5 CFHR2, the human plasma protein is a complement inhibitor. (A) Structure and domain composition of CFHR2, CFHR1 and Factor H and of the two deletions mutants CFHR2/1-2 and CFHR2/3-4. The numbers between the proteins indicate amino acid homology. (B-D) CFHR2 forms dimers in solution. Static light scattering in combination with size exclusion chromatography was used to determine the molecular mass of the complex in solution. The molar mass of each protein is indicated, as well as the status of dimerisation, in the panel above each experimental figure. The light scattering signals (LS) and the mass distribution (Mw) in each of the peaks in the elution profiles monitored by the absorbance at 280 nm (UV) and changes of the refractive index (dRI). (E) CFHR2 inhibits the alternative complement pathway in NHS which was activated on foreign surfaces. Factor H was used a positive control for alternative pathway inhibition. (F) CFHR2 did not affect classical pathway activation in NHS. Terminal complement complex (TCC) deposition in untreated NHS was set as 100 %. C4BP the classical pathway inhibitor was used as a positive control. CFHR2 and CFHR3 circulate in human plasma as differently glycosylated variants and both proteins bind the central activated complement component S3b and C3d. CFHR2 and CFHR3 also bind via specific binding domains to glycosaminoglycans on human cell surfaces, indicating a complement regulatory role of CFHR2 and CFHR3 on human cell surfaces (Fig. 5). In addition CFHR2 is bound by C. albicans and also by the Gram negative bacterium Borrelia burgdorferi, thus suggesting that these pathogenic microbes use the human protein for immune escape. The hypothesis that CFHR2 and CFHR3 are important immune modulators is further supported by the fact that mutations or deletions of the encoding genes enhance the risk to develop autoantibodies and kidney damages. 34 Based on the recruitment of CFHR2 to the surface of C. albicans and an association of genetic mutations in the CFHR2 gene with kidney diseases, we were interested to define the function of CFHR2. To this end the CFHR2 gene was amplified, cloned and cloned and the protein recombinantly expressed in Pichia pastoris. The secreted protein was purified by chromatography and analysed by multi-angle laser scattering. Interestingly recombinant CFHR2 exists exclusively in a dimeric form. To determine the dimerisation domain, two fragments of CFHR2 were recombinantly expressed and analysed accordingly. Dimers were formed via the N-termini and the C-termini bound to C3b and C3d. Thus one CFHR2 dimer can bind via the two C-termini two C3b or C3d molecules at the same time. HKI Research Report 2012 / 2013 Department of Infection Biology The complement regulatory activity of CFHR2 was evaluated in hemolysis assays, where the effect of CFHR2 on the complement mediated lysis of erythrocytes was assayed. CFHR2 inhibited hemolysis when complement was activated via the alternative, but not via the classical pathway. Further studies revealed that CFHR2 inhibits the C3 convertase, the central enzyme of complement. The CFHR2 homodimer binds to the C3 convertase and blocks convertase function(s). Thereby the CFHR2 homodimer likely complexes the C3 convertases so that the substrate C3 is no longer cleaved and no activation products, i.e. C3b and C3a, are formed. In addition CFHR2 inhibits a further activation step of the complement cascade: the proteine blocks the terminal complement pathway. Each complement pathway generates the terminal complement complex, TCC, which forms a lytic pore, composed of polymerised C9 molecules and which integrates into the target cell membrane. CFHR2 binds to C9 and inhibits TCC assembly and pore formation. Thus CFHR2 is a complement regulator and protects self surfaces from complement damages. Pathogenic microbes like C. albicans and B. burgdorferi bind CFHR2 to their surfaces and thereby recruit this human complement regulator which can inhibit C3 convertase activity and TCC formation. Complement inhibition helps C. albicans and B. burgdorferi to survive the strong innate immune response of the human immune system. Plasminogen is another human plasma protein which is very frequently recruited by pathogens. Human plasminogen is a proenzyme that is proteolytically processed and cleaved to the active protease plasmin. Plasmin controls several relevant biological reactions, including fibrinolysis, wound healing and tissue remodelling. C. albicans, similar to many other pathogenic microbes, binds plasminogen to its surface and the inactive zymogen when attached to the fungal surface can be converted to the active protease plasmin. Plasmin in consequence degrades components of the extracellular matrix (ECM) and fibrinogen which allows pathogens to disseminate into deeper tissue layers. Besides the fibrinolytic activity, plasmin also influences the complement cascade, as newly generated plasmin cleaves native C3, via the intermediates C3b and C3a. However, plasmin further processes and degrades both complement activation fragments, thereby blocking their effector functions. This degrading activity is of interest for pathogens and explains why many pathogens recruit plasminogen to their surface. The pathogenic microbe Staphylococcus aureus as a master of immune evasion binds C3 to its surface via the microbial proteins Sbi and Efb. Whether S. aureus binds plasminogen to target the host C3 molecule for complement evasion was still unclear. To identify new plasminogen binding proteins of S. aureus we analysed whether the staphylococcal surface proteins Sbi and Efb also bind plasminogen. Plasminogen bound to both Sbi and Efb. Bound plasminogen is subsequently converted to plasmin by S. aureus secreted staphylokinase and newly generated plasmin is proteolytically active and cleaves the synthetic substrate and also the complement components C3 and C3b. This proteolytic activity is modulated and enhanced by the conformational changes induced of C3 and C3b upon interaction with Sbi or Efb. Plasmin generated on the surface of S. aureus cleaves and degrades additional substrates, like C3a and thereby reduces the chemoattractant and antimicrobial activities of this complement activation product. Thus, recruitment and activation of plasminogen by S. aureus is highly coordinated to maximise complement inhibition at the level of C3. 3 References Koch TK, Reuter M, Barthel D, Böhm S, van den Elsen J, Kraiczy P, Zipfel PF, Skerka C (2012) Staphylococcus aureus proteins Sbi and Efb recruit human plasmin to degrade complement C3 and C3b. PLoS One 7, e47638. Eberhardt HU, Buhlmann D, Hortschansky P, Chen Q, Böhm S, Kemper MJ, Wallich R, Hartmann A, Hallström T, Zipfel PF, Skerka C (2013) Human factor H-related protein 2 (CFHR2) regulates complement activation. PLoS One, 8:e78617. The role of complement regulators in human kidney diseases Group Leader: Peter F. Zipfel Human complement regulators are essential for the protection of self-cells and tissues against toxic complement activation prod- Department of Infection Biology HKI Research Report 2012 / 2013 35 Figure 6 C. albicans expresses proteins to bind and exploit human regulators which play a central role in self-cell protection and prevention of diseases. Aim of the human immune studies is to define new complement regulators that C. albicans recruits for the fungal surface and uses immune evasion and for dissemination. The acquired human proteins are linked to human diseases. ucts and for the non-inflammatory phagocytosis of modified or damaged self-cells. Such non-inflammatory action of the human regulators is apparently highly attractive for pathogenic microbes and another reason why such host immune regulators are acquired by pathogens. Pathogenic microbes express specific surface proteins or secrete moonlighting proteins to recruit human regulators. However, the exact role of recruited individual human proteins in the immune response is often not precisely defined. Even for known human regulators like Factor H new functions are postulated and identified. Genetic variations in form of mutations, polymorphic variations, as well as gene copy number variations often affect or disrupt the function of the individual proteins and are frequently associated with human diseases. In consequence such genetic variations interfere with the fine-tuned action and regulation of innate immunity and of the complement cascade and cause pathology in single organs. 36 The pathological effects of the genetic variations confirm the important role of single complement proteins or regulators for host immune homeostasis and for restricting inflammation. In this context defining how single gene mutations, genetic copy number variations or even genetic polymorphisms infuence the action of a single regulator and in consequence how variant proteins affect the tightly controlled innate immune and complement network helps to understand disease pathology. Ultimately this also allows us to understand why pathogenic microbes exploit the regulators of their human host and how the acquired regulators are used for microbial immune escape (Fig. 6). The human kidney disorder C3 Glomerulopathy with Dense Deposit Disease pattern (C3G-DDD) is a renal disease that is associated with mutations in complement regulatory genes. C3G-DDD primarily affects children and young adults, is difficult to treat and at present no established and safe therapy exists. HKI Research Report 2012 / 2013 Department of Infection Biology Figure 7 Kidney biopsy of a patient with C3 Glomerulopathy with Dense Deposit Disease pattern (C3G-DDD). Hematoxylin and eosin staining in a kidney biopsy of patient #635 reveals the presence of foam cells (asterix) and shows hypercellularity and mesangial cell proliferation (arrow). C3G-DDD patients present with nephrotic or nephritic syndrome or with asymptomatic renal disease. The disease often progresses to end-stage renal disease (ESRD) and tends to recur in the transplanted kidney. Diagnosis of C3G-DDD is primarily based on a histological evaluation of a kidney biopsy with prominent C3 staining, the absence of immunoglobulins and the presence of dense deposits along the glomerular basement membrane (GBM) combined with mesangial hypercellularity by electron microscopy. Numerous diverse genetic, as well as autoimmune causes are reported for C3G-DDD patients, including mutations of single complement genes, genetic deficiencies, copy number variations. Also autoimmune forms exist which include various autoantibodies to the central complement enzyme C3 convertase. In collaboration with M. Wiesener from the University Hospital Erlangen we analysed two related C3G-DDD patients who presented with low C3 plasma levels, end stage renal disease and who have received dialysis for over 10 years (Fig. 7). Genetic analyses were performed by MLPA (multiplex ligation dependent probe amplification) and revealed a new heterozygous chromosomal deletion in the cluster of complement regulators on chromosome 1q32. Subsequently, genomic DNA isolated from both patients was amplified by nested PCR in order to identify and localise the chromosomal breakpoint. Both patients had the same 24804 nucleotide fragment in the CFHR gene cluster deleted and this deleted chromosomal fragment spans from exon II of the CFHR2 gene to the promoter region of CFHR5 gene (Fig. 8). This chromosomal deletion resulted in expression of a hybrid transcript that had the signal peptide and SCRs1-2 of the CFHR2 gene fused to the CFHR5 coding exons. This novel transcript encoded a CFHR2CFHR5 hybrid protein which was secreted Department of Infection Biology HKI Research Report 2012 / 2013 37 Figure 8 A new genetic deletion in the CFHR gene cluster on human chromosomes 1q32 results in expression of a CFHR2-CFHR5 hybrid protein. Two related C3G - DDD patients #635 and #638 showed the same deletion. The breakpoint was identified by sequencing and a 24805 bp region is deleted in the patients. The deletion brings the cFHR2 and cFHR5 gene in close proximity, results in a hybrid transcript that links exon I to exon III of the CFHR2 gene to the whole CFHR5 transcript. The protein product is a hybrid CFHR2-CFHR5 protein that is expressed, secreted and identified in plasma of both patients. into plasma and was identified as two bands with mobilities of ca. 65 and 70 kDa. The CFHR2-CFHR5 hybrid was composed of 11 SCR domains comprising the two N terminal domains of CFHR2, i.e. SCRs1-2 linked to SCRs1-9 of the CFHR5 gene. To allow a detailed characterisation a recombinant CFHR2-CFHR5 hybrid protein was generated, expressed in HEK 293 cells and purified. This recombinant hybrid protein bound to C3b and C3 activation fragments iC3b and C3d. Given this C3 binding profile and upon intensive discussions with the clinicians to obtain appropriate treatment options, one patient first received a plasma infusion. The rational was to dilute the hybrid protein in plasma, to reconstitute complement components like C3 and also to increase the levels of the CFHR2, CFHR5 regulators. Unexpectedly this treatment resulted in enhanced complement activation, as upon plasma addition the levels of C3 38 activation fragments and terminal complement complexes increased. In consequence this problem was moved back to the laboratory, with the goal to identify why and how the hybrid protein deregulated complement. The subsequent functional characterisation revealed that the purified recombinant hybrid protein deregulated the C3 convertase of complement in fluid phase. CFHR2-CFHR5 stabilised the C3 convertase, reduced the dissociation of the enzymatic complex and blocked the access or binding of the natural inhibitor Factor H. Now we can explain how the novel hybrid protein overactivates complement in plasma of both patients. This functional characterisation combined with the understanding how the hybrid protein deregulated complement on the level of the C3 convertase resulted in a treatment switch to plasma exchange. Plasma exchange reduced the levels of the deregulating, hybrid protein and this resulted in lower comple- HKI Research Report 2012 / 2013 Department of Infection Biology Figure 9 Evaluating complement inhibitors as treatment option for patients who express a novel CFHR2-CFHR5 hybrid protein. The inhibitory effect of the complement inhibitors Eculizumab, the tagged version of the C3 inhibitor Compstatin and soluble CR1 on the enhanced complement activation in substituted serum of patient #635 was evaluated. C3b surface deposition was monitored. sCR1 and tagged Compstatin inhibited C3 convertase action and C3b deposition, Eculizumab which acts downstream of the C3 convertase had no effect. ment activation, as revealed by reduced levels of the activation marker Ba. However this effect was short lived. Already 12 hours after treatment the activation marker Ba increased and reached previous levels after 24 hours. Thus complement activation correlated directly with the levels of the damaging hybrid protein in the patient’s plasma. In order to evaluate additional treatment options complement inhibitors were tested in vitro in the laboratory. Various complement inhibitors were screened for their effect on complement activation in plasma of patient #635 which was substituted with normal plasma. Eculizumab the established complement and C5 convertase inhibitor did not block the damaging action of the hybrid protein (Fig. 9). Therefore additional inhibitors were tested, including the complement inhibitor Compstatin, which binds directly C3 and blocks C3 activation. Compstatin when equipped with a surface anchoring domain blocked the defective complement action by the hybrid protein in patient’s plasma. Similarly sCR1, the soluble complement inhibitor also overruled the CFHR2-CFHR5 mediated defective complement regulation. In consequence this soluble complement inhibitor provides an option for therapy of the patient. The identification of a new genetic cause that resulted in C3 glomerulopathy with DDD pattern provides a basis to understand how the newly generated CFHR2-CFHR5 hybrid protein deregulates the complement convertase C3 in fluid phase and allows to evaluate and to develop therapeutic approaches for these patients. These findings also provide insights into the role of CFHR proteins that in general are frequently recruited by pathogenic microbes including C. albicans. Department of Infection Biology HKI Research Report 2012 / 2013 References Chen Q, Wiesener M, Eberhardt HU, Hartmann A, Uzonyi B, Kirschfink M, Amann K, Buettner M, Goodship T, Hugo C, Skerka C, Zipfel PF (2014) Complement factor H-related hybrid protein deregulates complement in dense deposit disease. J Clin Invest 124, 145-155. 39 Group members Prof. Christoph Licht Hospital for Sick Children, Toronto, Canada 03/2008, 03/2009, 10/2009 Head Prof. Dr. Peter F. Zipfel Phone +49 3641 532-1300 Fax +49 3641 532-0807 [email protected] Prof. Kristian Riesbeck University Malmö, Sweden 07/2008 Secretary Heike Gäbler Prof. Richard Smith The University of Iowa, USA 10/2009 Scientists Dr. Hans-Martin Dahse Dr. Prasad Dasari (since 10/2013) Dr. Teresia Hallström Dr. Nadine Lauer (until 02/2012) Dr. Shanshan Luo (until 07/2012) Dr. Michael Reuter (until 08/2012) Dr. Malik J. Reza (since 02/2013) PD Dr. Christine Skerka External funding Bundesministerium für Bildung und Forschung Verbundvorhaben Medizinische Infektionsgenomik: Wirt-Pathogen-Interaktion: Wirkung sekretierter Proteine von Staphylococcus aureus auf Zellen und Komponenten des Immunsystems Peter F. Zipfel Deutsche Forschungsgemeinschaft AMD des Auges – Rolle der Komplementregulatoren Faktor H, CFHL1, CFHR1 und CFHHR3 bei der Altersabhängigen Makuladegeneration (AMD) des Auges Christine Skerka Deutsche Forschungsgemeinschaft AMD des Auges II – Rolle der Komplementregulatoren Faktor H, FHL1, CFHR1 und CFHR3 bei der Altersabhängigen Makuladegeneration (AMD) des Auges Christine Skerka Diploma / Bachelor / Master Students Research Assistants Nadine Flach Steffi Hälbich Andrea Hartmann Monika von der Heide Ina Löschmann Susanne Lorenz Eva-Maria Neumann Silvya Spielmann Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project C04: Mechanismen der Adhäsion und Modulation humaner Immunzellen durch Candida albicans Christine Skerka Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project C06: Einfluss sekretierter Proteine von Candida albicans bei der Immunevasion und der Pathogenität Peter F. Zipfel Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Immunevasion of the Gram positive bacterium Streptococcus pneumoniae Peter F. Zipfel Deutsche Forschungsgemeinschaft Molekulare Genetik IV – Molekulare Genetik und Pathogenese des Hämolytisch-Urämischen Syndroms im Erwachsenenalter Peter F. Zipfel Trainee Alexandra Rülke (since 08/2013) Visiting Scientists Punet Juneja Himachal Pradesh University, India 07/2008–09/2008 40 Pro Retina Stipendium Die Rolle des ARMS2 Proteins in der Immunregulation bei der AMD Christine Skerka Selected publications Anika Westphal Ernst Moritz Arndt University Greifswald, Germany 01/2012–02/2012 Ph.D. Students Susanne Ackermann (since 10/2012) Sascha Böhm (until 06/2013) Denise Buhlmann (since 08/2012) Qian Chen Nadia Döring (until 08/2012) Hannes Eberhardt (until 06/2013) Selina Icke (until 06/2012) Emeraldo Jo (since 10/2013) Isabell Kopka (until 06/2013) Justus Linden (since 08/2013) Susanne Lindner Crisanto Lopez (until 06/2013) Christian Meinel (since 10/2013) Sven Micklisch Christiane Pilz Sarbani Sarkar Anika Westphal (since 10/2013) Fei Zhao (since 09/2012) Florian Bredy (until 08/2013) Denise Buhlmann (until 07/2012) Karolin Hess (until 08/2013) Susanne Jacksch (until 09/2012) Tom Kretzschmar (until 08/2013) Melanie Manzke (until 08/2013) Iris Koske (10/2012–12/2013) Christian Meinel (10/2012–09/2013) Marina Raguse (until 09/2012) Kristin Schult (09/2012–09/2013) Rajiuddin Sheikh MD (until 06/2012) Corinna Weist (until 09/2012) Jana Ziereisen (until 09/2012) National Institutes of Health / University of Iowa Collaborative Study of Membranoproliferative Glomerulonephritis Type II Peter F. Zipfel European Union Framework Programme 7: Large-scale Integrating Project EURenOmics – European Consortium for HighThroughput Research in Rare Kidney Diseases; WP 05: Complement Disorders Peter F. Zipfel Chen Q, Wiesener M, Eberhardt HU, Hartmann A, Kirschfink M, Uzonyi B, Amann K, Buettner M, Goodship T, Hugo C, Skerka C, Zipfel PF (2014) Complement factor H-related hybrid protein deregulates complement in dense deposit disease. J Clin Invest 124, 145-155. Hallström T, Siegel C, Mörgelin M, Kraiczy P, Skerka C, Zipfel PF (2013) CspA from Borrelia burgdorferi inhibits the terminal complement pathway. MBio 4, e00481-13. Luo S, Hoffmann R, Skerka C, Zipfel PF (2013) Glycerol-3-phosphate dehydrogenase 2 is a novel factor H-, factor H-like protein 1-, and plasminogen-binding surface protein of Candida albicans. J Infect Dis 207, 594-603. Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G (2013) Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13, 29-41. Ueberschaar N, Dahse HM, Bretschneider T, Hertweck C (2013) Rational design of an apoptosisinducing photoreactive DNA intercalator. Angew Chem Int Ed 52, 6185-6189. Collaborations Albert Ludwigs University Freiburg, Germany PD Dr. Eva Decker Prof. Dr. Hans-Hartmut Peter Prof. Dr. Ralf Reski Centro de Investigaciones Biologicas (CSC), Madrid, Spain Prof. Dr. Santiago R. de Córdoba Charité Universitätsmedizin Berlin, Germany PD Dr. Dominik Müller Prof. Dr. Antonia Joussen Prof. Dr. Olaf Strauß Ernst Moritz Arndt University Greifswald, Germany Prof. Dr. Sven Hammerschmidt Prof. Dr. Susanne Engelmann Technische Universität Braunschweig, Institut für Mikrobiologie, Germany University Hospital Jena, Germany Prof. Dr. Dagmar Barz PD Dr. Uta-Christina Hipler PD Dr. Ulrike John Prof. Dr. Iver Petersen Prof. Dr. Gunter Wolf Heinrich Heine University Düsseldorf, Germany Prof. Dr. Joachim F. Ernst HKI Research Report 2012 / 2013 Department of Infection Biology Hôpital Européen Georges-Pompidou, Paris, France Prof. Dr. Veronique Fremeaux-Bacchi University of Lübeck, Germany Prof. Dr. med. Jörg Köhl Prof. Dr. Marc Ehlers Prof. Dr. Rolf Hilgenfeld Instituto di Recherche Farmacologoche “Mario Negri”, Bergamo, Italy Prof. Dr. Marina Noris Prof. Dr. Giuseppe Remuzzi University of Massachussetts, USA Prof. Dr. Sanjay Ram University of Pennsylvania, USA Prof. Dr. John Lambris Prof. Dr. Wenchao Song Johann Wolfgang Goethe University Frankfurt, Germany Prof. Dr. Peter Kraiczy University of Regensburg, Germany Prof. Dr. Bernhard Weber Dr. Diana Pauly Lund University Malmö, Sweden Prof. Dr. Anna Blom Prof. Dr. Kristian Riesbeck Prof. Dr. Matthias Mörgelin University of Rostock, Germany Prof. Dr. Marianne Wigger Medical University of Graz, Austria Prof. Dr. Christoph Mache Western General Hospital Edinburgh, Edinburgh, UK Prof. Dr. Alan Wright Medical University of Vienna, Austria Prof. Dr. Christoph Binder West Viginia University Morgantown, USA Prof. Dr. Slawomir Lukomski Queen's University Belfast, Ireland Prof. Anne E. Hughes Dr. Declan T. Bradley University Uppsala, Sweden Bo Nilsson Rheinisch-Westfaelische Technische Hochschule Aachen, Germany PD Dr. Gabriele Pradel University Kalmar, Sweden Kristina Nilsson-Eckdahl Rupprechts Karl University Heidelberg, Germany Prof. Dr. Michael Kirschfink Prof. Dr. Reinhard Wallich Sir William Dunn School of Pathology, Oxford, UK Prof. Dr. Christoph M. Tang The Hospital for Sick Children, Toronto, Canada Prof. Dr. Christoph Licht The Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA Prof. Dr. Hendrik Scholl University of Aarhus, Denmark Prof. Dr. Gregers R. Andersen Universitäts-Augenklinik Bonn, Germany Prof. Dr. Frank G. Holz University Hospital Heidelberg, Germany Prof. Dr. Franz Schäfer University Medical Center/Institute for Cardiovascular Research, Amsterdam, Netherland Prof. Dr. Hans W. M. Niessen University Newcastle upon Tyne, U.K. Prof. Dr. Tim Goodship University of Colorado Denver, Denver, USA Dr. Nirmal K. Banda University of Erlangen Nürnberg, Germany Prof. Dr. Michael Wiesener Prof. Dr. Kerstin Amann University of Iowa, USA Prof. Dr. Richard Smith University of Kentucky, USA Prof. Dr. Brian Stevenson Department of Infection Biology HKI Research Report 2012 / 2013 41 42 HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012/ 2013 43 Department of Microbial Pathogenicity Mechanisms The Department of Microbial Pathogenicity Mechanisms (MPM) is investigating infections caused by human pathogenic fungi. The research is focussed on the most common pathogenic yeasts Candida albicans, and C. glabrata, which has been recognised as an emerging pathogenic yeast. In addition, projects deal with C. dubliniensis, C. tropicalis and C. parapsilosis. We investigate all aspects of pathogenicity mechanisms of these yeasts using genomics, in silico analyses, microbiology, cell biology, biochemistry, infection biology and immunology approaches. Projects deal with (1) infection associated genes of Candida, including unknown function genes, genes required for osmolarity and encoding damage factors, and genes unique for C. albicans, C. glabrata or C. dubliniensis; (2) macro- and micronutrients of Candida, including nitrogen, iron and zinc sources within the host; (3) evolution, adaptation and pathogenicity of Candida, including microevolution, flexibility of transcriptional networks and transcriptional profiling of Candida within blood; and (4) interaction of Candida with immune cells, including neutrophils and macrophages. The research group Microbial Immunology, headed by Ilse D. Jacobsen, which carries out I N T R O D U C T I O N | E I N LE I T U N G Head: Prof. Dr. Bernhard Hube Die Abteilung Mikrobielle Pathogenitätsmechanismen (MPM) erforscht die Ursache und Entwicklung von Infektionen durch humanpathogene Pilze. Die Forschung konzentriert sich auf die häufigsten humanpathogenen Hefen Candida albicans und C. glabrata. Bei weiteren Projekten werden auch Aspekte der Pathogenität von C. dubliniensis, C. parapsilosis und C. tropicalis bearbeitet. Wir untersuchen alle Aspekte der Pathogenitätsmechanismen dieser Hefen mit Hilfe von genomischen, molekularbiologischen, mikrobiologischen, zellbiologischen, biochemischen, infektionsbiologischen und immunologischen Methoden, sowie in silico-Analysen. 44 Die Projekte der Abteilung bearbeiten folgende Themen: (1) infektionsassoziierte Gene von Candida (Gene mit unbekannter Funktion, für Osmolarität, gewebeschädigende Faktoren sowie Gene, die spezifisch für C. albicans, C. glabrata oder C. dubliniensis sind); (2) Makro- und Mikronährstoffe von Candida (einschließlich Stickstoff-, Eisen- und Zinkquellen innerhalb des Wirts); (3) Evolution, Adaptation und Pathogenität von Candida (einschließlich Mikroevolution, Flexibilität von transkriptionellen Netzwerken und Transkriptionsprofile im Blut); und (4) Interaktionen von Candida mit Immunzellen (zum Beispiel Neutrophile und Makrophagen). Die Arbeitsgruppe von Ilse D. Jacobsen, die experimentelle Pilzinfektionen durchge- HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms experimental fungal infections and investigates the pathogenesis of systemic candidiasis and other fungi, was integrated into the department of MPM. Since August 2013, this group is an independent research group of the HKI (see report Microbial Immunology). From 2012 to 2013, the research of MPM was supported by the Jena School for Microbial Communication (JSMC), the International Leibniz Research School (ILRS), the Deutsche Forschungsgemeinschaft (DFG), the Studienstiftung des Deutschen Volkes, the Federal Ministry of Education and Research (BMBF), the European Union and Bayer Vital GmbH. Projects are integrated into the Center for Sepsis Control and Care (CSCC), the DFG Pri- führt hat und welche die Pathogenese von systemischen Candida-Infektionen and Infektionen mit anderen Pilzen untersuchte, war in die Abteilung MPM integriert. Seit August 2013 arbeitet diese Arbeitsgruppe am HKI selbständig (siehe Bericht „Mikrobielle Immunologie“). In den Jahren 2012 bis 2013 wurde die Forschung der Abteilung MPM durch folgende Institutionen unterstützt: Jena School for Microbial Communication (JSMC), International Leibniz Research School (ILRS), Deutsche Forschungsgemeinschaft (DFG), Studienstiftung des Deutschen Volkes, Bundesministeriums für Bildung und Forschung (BMBF), Europäische Union und Bayer Vital GmbH. Mehrere Projekte waren in die folgenden ority Programmme SPP1580 “Intracellular compartments as places of pathogen-host-interactions”, the DFG Collaborative Research Center / Transregio 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet”, the project “Microevolution of pathogenic yeasts during interactions with the host” within the DFG DACH programme, the European CandiCol consortium within the ERA-Pathogenomics programme and the European Marie Curie Research Training Network FINSysB. Netzwerke integriert: Center for Sepsis Control and Care (CSCC), DFG-Schwerpunktprogramm SPP1580 „Intracellular compartments as places of pathogen-host-interactions“, DFG-Sonderforschungsbereich / Transregio 124 „Pathogenic fungi and their human host: Networks of interaction – FungiNet“, das Verbundprojekt „Microevolution of pathogenic yeasts during interactions with the host” innerhalb des DFG DACH-Programms, das Europäische CandiCol-Consortium innerhalb des ERA-Pathogenomics Programmes und das European Marie Curie Research Training Netzwerks FINSysB. Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 45 Scientific Projects 1 Infection-associated genes of Candida albicans Group Leader: Duncan Wilson Transcriptional profiling of fungal pathogens during interactions with their hosts can provide important insights into the biology of infection. We have performed global gene expression analysis of Candida albicans as it infects a range of anatomical niches: oral tissue (representing superficial mucosal infection); blood and blood components (reflecting dissemination of the fungus throughout the host) and invasion of internal organs, such as the liver (representing deep seated infections). The expression patterns of known genes have provided detailed information on the environmental conditions faced by the pathogen in these diverse niches. However, a large proportion of genes, upregulated during infection encode proteins with no known function. To address this, we have systematically deleted over 40 such “infection associated genes” and, from these, identified a number of novel C. albicans pathogenicity mechanisms. With these approaches, we are elucidating, at the molecular level, the mechanisms employed by C. albicans to survive, grow within and damage its host. Evolutionary adaptation: C. albicans unique genes The major fungal pathogens of humans are distantly related to each other and often more closely related to non-pathogenic species. Therefore, fungal pathogenicity is a trait which has been selected independently, multiple times during evolution. To test the hypothesis that species-specific genes represent candidate virulence factors, we have performed genome subtraction to identify C. albicans unique genes. Subsequent functional analysis of seven C. albicans unique genes 46 demonstrated that they are dispensable for in vitro growth, but play specific roles during host pathogen interactions. Detailed analysis of one gene demonstrated a role in both superficial and invasive candidiasis. Survival: counteracting the stressful environment during infection Immune cells are known to impose significant stress on engulfed microbes (see “Interaction with immune cells”). We have deleted a set of genes which are transcriptionally upregulated upon interactions with phagocytes. These genes are required for resisting attack by environmental stresses. For example, the small heat shock protein Hsp21 governs tolerance to fluctuations in the environment by modulating MAP kinase signalling and controlling homeostasis of the stress protectant, trehalose (Fig. 1). We found that Hsp21 was not only required for C. albicans growth under elevated temperatures and in the presence of oxidants, but also for surviving attack by human neutrophils and for virulence. We have also demonstrated that C. albicans synthesises lipid droplets. These intracellular organelles act as storage depots for neutral lipids and contain high levels of triacylglycerols. These esters consist of three fatty acids joined to a glycerol backbone. As glycerol is the major cellular protectant against osmotic stress, we reasoned that lipid dropletassociated triacylglycerides may serve as a novel glycerol source and osmoprotectant pathway. Indeed, salt stress resulted in the rapid degradation of lipid droplets in wild type C. albicans cells. We identified and deleted a putative acylglyceride hydrolase. Interestingly, the hydrolase deficient mutant was unable to degrade lipid droplets in response to cationic stress, defective in osmotic stress-induced accumulation of intracellular glycerol and unable to grow in the presence of cationic stressors. HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms Figure 1 The novel small heat shock protein, Hsp21, is required for regulating MAP kinase signalling and growth under environmental stress. Figure 2 A fungal zincophore system. Invasive C. albicans hyphae secrete a zincbinding protein, Pra1, which sequesters this essential metal from host cells before reassociating with the fungus via a cognate receptor, Zrt1. Growth: feeding on the host with an extracellular micronutrient scavenger, the zincophore The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. We have demonstrated endothelial zinc sequestration by Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited specific zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and this was confirmed experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell (Fig. 2). Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution. Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 47 Figure 3 Model of Ece1 peptide pore formation by a novel C. albicans toxin in host cell membranes. 48 Damage: a dual substrate transporter and the first fungal pore-forming peptide toxin Although hypha formation is known to play a crucial role in pathogenicity at epithelial surfaces, the exact molecular mechanisms by which C. albicans destroys these host cells has remained enigmatic. We have made recent progress in characterising epithelial damage at both the molecular and cellular level. amines from the environment and functions in the extracellular alkalinisation pathway. Importantly, this gene was essential for epithelial damage, resisting attack by immune effector cells and virulence. Intriguingly, Dur31 also imports the deadly antimicrobial peptide, histatin-5. Therefore, a single gene product can serve to both promote virulence and kill the pathogen, depending on the host niche in question. This represents a novel example of pathogen-host co-evolution. The import and subsequent metabolism of amine-containing compounds by C. albicans allows the release of ammonia from the cell and active alkalinisation of the surrounding environment. This sequence of events is known as the hyphal auto-induction pathway because an alkaline environmental pH serves as a strong inducer of C. albicans hyphal filamentation. We have shown that a previously unknown function gene, DUR31, encodes a transporter, which imports poly- Although hypha formation is known to play a crucial role in pathogenicity at epithelial surfaces, the exact molecular mechanisms by which C. albicans destroys these host cells has remained enigmatic. We have identified a peptide toxin, secreted by C. albicans, which has remarkable similarities with mellitin – the major component of bee venom. By deleting the encoding gene and in vitro synthesis of the fragment, we have shown that this peptide is, in itself, essential and sufficient HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms for lysis of host cells. In collaboration with Julian Naglik, Kings College London/UK, and Thomas Gutsmann, Forschungszentrum Borstel/Germany, we are now examining the mechanism by which this potent C. albicans cytolysin disrupts host cell integrity (Fig. 3). These projects were supported by the Federal Ministry of Education and Research (BMBF) within the ERA-Pathogenomics programme (CandiCol 0315901B), the European Marie Curie Research Training Network FINSysB (MCRTN FP7-214004), and the International Leibniz Research School (ILRS). 2 Nutrient acquisition in infections Group Leader: Sascha Brunke The ability to acquire nutrients during infection is essential for the survival and proliferation of pathogenic microbes. In return, the host fights back by limiting access to these nutrients, in a process termed “nutritional immunity”. We are interested in how Candida species still manage to obtain their essential nutrients in the host, and how this process is regulated via the transcriptional networks of the fungi. One focus of our work is the acquisition of trace metals, especially iron and zinc. As transition metals, both play indispensable roles in a plethora of biological processes, especially as co-factors in many essential enzymes and transcription factors. We mainly use in vitro models of single metal limitation to obtain transcriptome data from zinc or iron starved Candida cells. In close cooperation with the research group Systems Biology / Bioinformatics, we were able to infer gene regulatory networks from these conditions and define possible gene-gene interactions. Regulation of C. glabrata and C. albicans metal acquisition systems Interestingly, C. glabrata has the ability to survive and even replicate inside the phago- some of macrophages (Seider, Brunke et al. 2011), which is generally considered to be strongly depleted for iron and other metals. We are therefore especially interested in the mechanisms C. glabrata employs to obtain metals in this organelle. Our data indicated that C. glabrata is able to actively manipulate the pH in the phagosome, which has a direct influence on the solubility and hence accessibility of iron. Experimental manipulation of the phagosomal pH indeed inhibits growth effectively, and C. glabrata mutants with disrupted iron homeostasis show a reduced survival in macrophages (Seider, Gerwien et al. report 2012/2013). Furthermore, transcription data suggest that C. glabrata has iron available inside the phagosome. Detailed investigations into the type of iron source and the C. glabrata factors needed for both the phagosomal manipulation and the iron uptake are underway. For C. albicans, we focus on the interconnection of metal acquisition systems, exemplified by zinc and iron. Data obtained with deletion mutants and transcriptome analyses indicate strong links in detecting the levels of these two metals. In fact, many transcriptional pathways are shared between the two metal homeostasis systems, but the precise response is modulated depending on the specific absent metal. The molecular basis for this regulation is under investigation using deletion mutant libraries and transcriptome analyses (Citiulo, Jacobsen et al. 2012). This effort to investigate the regulation of C. albicans metal acquisition in the host is a joint project with the team “Infection Associated Genes” (see below for more details). Regulation of nitrogen and carbon acquisition Furthermore, in a joint project with the research groups Microbial Biochemistry and Physiology and Systems Biology / Bioinformatics, we investigate the signal transduction pathways which allow C. albicans to adapt to available nitrogen sources in the host. Using the network inference approach, we investigated the transcriptional response Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 49 Figure 4 Network of iron homeostasis in C. albicans. Extensive transcriptional data was used to infer a regulatory network model for C. albicans under iron starvation. The hub-like function of the HAP complex is clearly visible, and several novel gene-gene connections were detected. of C. albicans to typical host-associated nitrogen availability scenarios: nitrogen starvation, presence of free amino acids, and presence of proteins. We obtained transcription network models of selected genes for all these conditions (Ramachandra, Linde et al. 2014), which indicate novel network connections involved in nitrogen sensing and uptake. Furthermore, we found that the C. albicans transcription networks themselves are flexible. Depending on the available nitrogen source, gene-gene transcriptional interactions were present or absent, shaping the overall response of the fungus to nutrient availability. In a related project, we have analysed the function of a transcription factor with presumed roles both in iron and carbon uptake. Here, we were able to describe new functions for this factor, which connect the use of alternative (non-glucose) carbon sources with 50 available oxygen. Under conditions which resemble growth in the human gut, this transcription factor evidently plays a central role in carbon source uptake and utilisation. Altogether, our data indicate a complex and interconnected transcriptional regulation in nutrient acquisition. Deciphering these networks will help to understand how the fungus survives and multiplies inside the host during infection. This knowledge will be helpful in developing novel antifungal strategies aimed at the metabolism of the pathogens. These projects were supported by the Jena School for Microbial Communication (JSMC) and by the Deutsche Forschungsgemeinschaft (Hu528/15-1 and 16-1) within the DFG Priority Progamme SPP1580: Intracellular compartments as places of pathogen-host interaction (Fig. 4). HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms 3 Evolution, adaptation and pathogenicity Group Leader: Sascha Brunke The ability to adapt to the different niches in the host is realised in the short term mainly by changes in the transcriptional programme of the infecting microbe. In the long term, however, an optimal adaptation to the host requires evolutionary processes to take place. In fact, the host is a highly stressful environment, with few freely available resources and many immune-related antimicrobial activities. In addition, antifungal treatments in the clinical setting provide even more selection pressures on the fungus. We are interested in the ability of Candida species to cope with these stresses by microevolutionary changes, and how these adaptive mechanisms can shed light on pathogenicity factors of the fungus. Several researchers have previously shown the ability of fungi to adapt to antifungal stresses both in vitro and in vivo. Often, a few or a single nucleotide mutations are sufficient to render a fungus comparatively resistant to a specific class of antifungals – commonly via increased export of the drug or subtle changes in the target macromolecules. These strong, but simple abiotic selection pressures are evidently counteracted by fast microevolutionary adaptations. Therefore, to simulate biologically relevant, directly hostrelated stresses, we performed long-term co-incubation experiments of Candida species with macrophages. These phagocytes provide a complex selection pressure on the fungi, which were essentially replicating inside the phagosome for several weeks during the experiment. Analogous to the antifungal experimental regimes, we expected adaptations to cope with the prevailing stresses. C. glabrata and C. albicans adaptation to macrophage exposure In a first round, we continuously exposed C. glabrata to macrophages (Fig. 7). After several months of constant coincubation, the fungus changed from its typical yeast growth to a highly unusual filament-like form. In this form, it was able to damage the hosting macrophages quickly and escape. We tested this evolved variant in our infection models and found an increased virulence in mice, with a surprisingly high fungal burden in the brain and a concomitant increase in inflammatory markers in this organ. By whole genome sequencing we were able to detect all mutations in the evolved strain. Via genetic manipulation of the parental wild type, we finally determined a single nucleotide exchange as the basis of the novel phenotype. Hence, the complex selection pressure of macrophages can be circumvented by a single mutation event, with striking consequences for the growth morphology. In a similar experiment, we used a non-filamentous mutant of C. albicans which is missing two central transcription regulators of hyphal formation. Normally, C. albicans can pierce macrophages from the inside by hyphae production (Fig. 5), and hence this strain was unable to escape. After several weeks of coincubation, this strain regained its ability to filament, and formed hyphae under most of the tested classically hyphae inducing conditions. Evidently, the need for the deleted transcription factors was abolished via a transcriptional rewiring event. Consequently, the evolved strain regained most of its virulence, which had been nearly lost in the parental deletion mutant strain. Like for C. glabrata, whole genome sequencing in combination with RNAseq approaches to determine the transcribed alleles allowed us to determine the mutation events during the experiment. Again, a single nucleotide could be found which is associated with the striking phenotypic changes. We conclude that C. albicans has robust ways to rewire the signal transduction pathways of its hyphal programme, and that filament formation presents a huge selection advantage under our experimental conditions. Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 51 Figure 5 Giemsa staining of a blood smear after four hours of co-incubation. Several C. albicans form hyphae to escape from macrophages after phagocytosis. The transcriptomes of both host and fungus are analysed during these events in our whole blood model. Evolution of pathogenicity and patho-genicity-associated genes To measure whether C. albicans can adapt even further to the host during long-term infections, we performed a similar serial passage experiment with mice. After the initial infection, we re-isolated C. albicans from the target organ kidney and used these fungi for the next round of experimental infections. After eight passages, we detected no evident changes in virulence or in the in vitro phenotypes, indicating that the fungus is already well adapted to establish and maintain kidney infections (Lüttich, Brunke et al. 2013). In another approach, we want to determine the evolutionary events which allowed certain fungi to become pathogenic. As models, we use the closely related pairs C. glabrata and S. cerevisiae (a pathogenic and mostly non-pathogenic yeast) and C. albicans and C. dubliniensis (a frequent and an infrequent human pathogen). In these systems, we 52 search for genes which are either unique to the pathogen, or have different functions in the two species. For C. glabrata, genes were selected which are a) unique to C. glabrata and not found in other species like S. cerevisiae, C. albicans, C. parapsilosis or S. pombe and b) expressed under infection-simulating conditions, as determined by existing and specifically generated microarry and RNAseq transcriptome data (Fig. 6). These analyses were performed in cooperation with the research group Systems Biology / Bioinformatics. Unique, infection-associated genes are now being deleted, and the resultant mutants are tested both in vitro and in infection models to determine their function in pathogenicity. Similarly, for C. dubliniensis we used two gene banks: one with C. albicans genome fragments transferred into C. dubliniensis, and the other with a reciprocal transfer of HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms Figure 6 Meta-analyses of published transcriptome data on C. glabrata unique genes. Genes which are specific for C. glabrata were selected, and scored according to their expression under infection-simulating conditions. Heat map of the expression profiles under different conditions created in cooperation with the Research Group Systems Biology / Bioinformatics. C. dubliniensis to C. albicans. By selecting the recipients for phenotypes characteristic for either donor, we can determine the genetic basis for the strain differences. So far, genes were found which are for example involved in chlamydospore formation, a typical property of C. dubliniensis. Further analyses are under way to solve the riddle why these two very closely related species differ in their pathogenicity potential. These projects were supported by the Jena School for Microbial Communication (JSMC), the International Leibniz Research School (ILRS), and by the Deutsche Forschungsgemeinschaft via the DACH programme grant “Microevolution of pathogenic yeasts during interactions with the host immune system” (Hu 528 17-1). Figure 7 In vitro microevolution regime. Yeast cells (either C. glabrata or a C. albicans nonfilamenting mutant) were continuously exposed to macrophages, which were replenished daily. Constant selection inside the phagosome induced filamentous growth in both strains, either as pseudohyphae-like chains of yeasts (C. glabrata) or via recovery of hyphae formation (C. albicans). 4 Joint CSCC project: Pathogen-host interaction during infection with Candida albicans and its progression to sepsis (Project D1.5) Group Leader: Sascha Brunke The relevance of fungal pathogens in the nosocomial setting is steadily increasing. As causative agent, the opportunistic pathogenic yeast Candida albicans is the predominant species, with a high mortality due to sepsis and organ failure despite antifungal therapy. We aimed to analyse the initial steps during fungal sepsis, after the pathogen has entered the blood-stream and dispersed to its target organs. To this end, we employed an intravital microscopy model of murine systemic C. albicans infections to gain insight into the early interaction between the fungus and the murine liver (Fig. 9). Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 53 References Citiulo F, Jacobsen ID, Miramón P, Schild L, Brunke S, Zipfel P, Brock M, Hube B, Wilson D (2012) Candida albicans scavenges host zinc via Pra1 during endothelial invasion. PLoS Pathogens 8, e1002777. Lüttich A, Brunke S, Hube B, Jacobsen ID (2013) Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney. PLoS One 8, e64482. Ramachandra S, Linde J, Brock M, Guthke R, Hube B, Brunke S (2014) Regulatory networks controlling nitrogen sensing and uptake in Candida albicans. PLoS One 9, e92734. Seider K, Brunke S, Schild L, Jablonowski N, Wilson D, Majer O, Barz D, Haas A, Kuchler K, Schaller M, Hube B (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation. J Immunol 187, 3072-3086. Seider K, Gerwien F, Kasper L, Allert S, Brunke S, Jablonowski N, Schwarzmüller T, Barz D, Rupp S, Kuchler K, Hube B (2014) Immune evasion, stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata within macrophages. Eukaryot Cell 13, 170-183. We developed a fluorescent C. albicans reporter strain suitable for use in vivo despite the strong tissue autofluorescence, and modified an established murine intravital microscopy system for use with systemic fungal infections. With this system, we gained first insight into the temporal and spatial kinetics of early C. albicans-liver interactions. For this murine intravital microscopy model, our main findings are: (1) The adhesion to the murine liver endothelium occurs much faster than expected. Adhesion of fluorescent yeasts was observed tens of seconds after initial infection via a central venous catheter. This adhesion explains the quick clearance of the fungus from the blood-stream, which is a well-known, but thus far not fully understood, event in C. albicans infection biology. For the first time, we could show the fate of these fungi in vivo (Fig. 9). (2) The binding of C. albicans to the sinusoids is spatially distinct, as the yeasts bind preferentially in the distal part of the hepatic lobule, and rarely close to the central vein. (3) The morphological transition from yeast to hyphae, an important virulence factor of C. albicans, is still under investigation for its role in escape from the blood-stream. To this end, fluorescent mutant strains were created which lack the key regulators of hyphae formation. This project was supported by the German Federal Ministry of Education and Research (BMBF) within the Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC) (FKZ 01EO1002). 5 Interaction with immune cells 5.1 Survival of C. glabrata in macrophages Group Leader: Katja Seider Candida glabrata is a well-adapted fungal species that establishes commensal relationships with its human host and beyond that causes infections under certain predisposing conditions. Previous studies suggest that immune evasion strategies may be key viru- 54 lence attributes of C. glabrata and that interactions with macrophages, important phagocytic cells of the host’s innate immunity, may play a critical role in the pathogenesis of C. glabrata infections (Jacobsen et al. 2010). Macrophages ingest and destroy microbes intracellularly, by exposing them to a mixture of acidic pH and highly toxic substances such as oxidants, proteases and antimicrobial peptides in a specialised compartment – the phago(lyso)some. However, C. glabrata can resist killing by macrophages in vitro, most probably by inhibiting antimicrobial properties like phagosome maturation, oxidative burst and cytokine production (Seider et al. 2011). Its intracellular replication results eventually in macrophage lysis, and is thus a possible escape mechanism of C. glabrata from macrophages. Screening a targeted gene deletion library of C. glabrata for macrophage survival To gain more insights into the interactions of C. glabrata with macrophages, we measured survival rates of 433 C. glabrata deletion mutants, phagocytosed by human primary macrophages (Seider et al. 2013). 23 survivaldefective mutants, with putative functions in response to stress, cell wall and membrane architecture, glycosylation and nutritional sensing and acquisition were identified. Extensive phenotypic analysis of survivaldefective mutants revealed a relationship between survival and growth under iron-, but not carbon- or nitrogen-limitation. Furthermore, stress resistance is crucial for an intra-phagosomal residence of C. glabrata. Genes involved in cell surface integrity appeared to be of particular importance for macrophage survival, too. In this context, alterations of cell wall composition induced both a higher production of reactive oxygen species and the pro-inflammatory cytokine TNFα by macrophages. These data highlight the importance of the cell wall as a barrier shielding yeasts from immune recognition and substantiate immune evasion as a possible virulence strategy of C. glabrata. HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms Figure 8 Phases of C. albicans infection. After adhesion, C. albicans penetrates the epithelial barrier by hyphae formation or via injuries to the tissue. After dissemination via the bloodstream, fungal cells enter the target organs. In this project, we investigate these early steps after dissemination. Furthermore, detailed in vitro characterisation of three selected mutants (cch1Δ, slm1Δ, vhr1Δ) point to a role of Ca2+/calcineurin- and sphingolipid/TORC2-dependent signalling as well as biotin-regulatory processes in coping with macrophage-generated stresses. Modulation of environmental and phagosomal pH by C. glabrata Because C. glabrata-containing phagosomes lack acidification, the possibility of active pH modulation by C. glabrata was analysed in vitro. In fact, C. glabrata is able to alkalinise its extracellular environment, when growing on amino acids as the sole carbon source. By screening the C. glabrata mutant library we identified genes important for extracellular alkalinisation, which were further tested for phagosomal pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinisation in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosyl- Figure 9 C. albicans in the murine liver. Yeast cells attach to the sinusoids of the liver lobule just seconds after injection into the blood-stream. Using a fluorescent reporter strain (mCherry), the fungus can be visualised in the living animal via intra vital microscopy. Blue, tissue autofluorescence; red, C. albicans mCherry. Hepatic lobule at 63×. ation may play a key role in alterations of phagosomal properties caused by C. glabrata (Fig. 10). 5.2 Interaction of Candida albicans with neutrophils Group Leader: Lydia Kasper Neutrophils are key phagocytic cells, which play an extremely important role in the innate immune response and are the phagocytes with the most potent candidacidal activity. In contrast to C. albicans cells phagocytosed by macrophages, C. albicans cells are unable to form filaments inside the neutrophil. This means that the fungus is unable to escape from this phagocyte, hence it is effectively killed. Antimicrobial mechanisms of neutrophils range from intracellular killing mechanisms, by exposing fungal cells to a mixture of highly toxic substances such as oxidants, Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 55 Figure 10 C. glabrata (green) internalised by macrophages (yellow). Red: LysoTracker staining showing acidic compartements within macrophages. Blue: macrophage nuclei. proteases and antimicrobial peptides, to extracellular inhibition of growth by releasing the content of their granules to the extracellular milieu. Single cell profiling The contributions of the intra- and extracellular antifungal activities of human neutrophils on the transcriptional response of C. albicans were studied. For this purpose, C. albicans strains expressing reporter gene fusions to the Green Fluorescent Protein (GFP) were used in a differential staining experiment to determine the fungal transcriptional response at the single cell level of internalised compared to non-phagocytosed cells. It was found that a carbohydrate starvation response, indicated by up-regulation of genes encoding proteins of the glyoxylate cycle, was induced in C. albicans only in the intracellular environment of the neutrophil. Likewise, only internalised fungal cells were 56 exposed and responded to nitrosative stress. On the other hand, the oxidative stress response was observed in both phagocytosed and non-phagocytosed fungal cells, demonstrating that this type of stress is encountered inside and outside of the neutrophil. Mutant analysis and oxidative stress response of C. albicans To assess the actual contributions of carbohydrate starvation, oxidative and nitrosative stress in the killing of C. albicans, mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes were tested in their susceptibility to neutrophil killing. The glyoxylate cycle was found to be crucial for normal survival of C. albicans cells exposed to neutrophils. In the context of oxidative stress, C. albicans expresses genes coding for detoxifying enzymes, in order to cope with the oxidative and nitrosative insult exerted by neutrophils HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms References Fradin C, De Groot P, MacCallum D, Schaller M, Klis F, Odds FC, Hube B (2005) Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol 56, 397-415. Frohner IE, Bourgeois C, Yatsyk K, Majer O, Kuchler K (2009) Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance. Mol Microbiol 71, 240-252. Jacobsen ID, Brunke S, Seider K, Schwarzmüller T, Firon A, d'Enfért C, Kuchler K, Hube B (2010) Candida glabrata persistence in mice does not depend on host immunosuppression and is unaffected by fungal amino acid auxotrophy. Infect Immun 78, 1066-1077. Figure 11 Microphotograph of a C. albicans reporter strain expressing a GFP-tagged actin1 protein (green) when incubated in RPMI medium supplemented with 5 % FBS. (Fradin et al. 2005). The oxidative stress response effectors contribute differentially to the normal outcome of the interaction. Mutants lacking superoxide dismutase 5 (Sod5), glutathione reductase 2 (Grx2) or a novel family of glutathione peroxidases (Gpx31-33), rendered C. albicans hypersensitive to neutrophil killing (Miramon et al. 2013, Frohner et al. 2009). In contrast, catalase (Cta1) and thioredoxin (Trx1) seem to be dispensable for coping with the neutrophilinduced oxidative stress. Using a C. albicans mutant lacking the nitric oxide dioxygenase Yhb1 it was demonstrated that nitrosative stress generated by neutrophils is a relevant stress that C. albicans faces as a killing mechanism. phil survival since mutants lacking this protein were clearly more susceptible to killing. In addition, proteins involved in the synthesis of trehalose and glycerol, which expression is coordinated by Hog1, were needed for full resistance of C. albicans to neutrophils. These projects were financed by the Deutsche Forschungsgemeinschaft (Hu528/15-1 and 16-1) within the DFG Priority Progamme SPP1580: Intracellular compartments as places of pathogen-host interaction; and the European Commission through the FINSysB Marie Curie Initial Training Network (PITNGA-2008-214004) (Fig. 11). Moreover, the contribution of fungal stress regulators to normal survival of C. albicans upon neutrophil attack was assessed. The stress regulator Hog1 and the oxidative stress regulator Cap1 play a pivotal role in neutro- Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 Miramón P, Dunker C, Windecker H, Bohovych IM, Brown AJ, Kurzai O, Hube B (2012) Cellular responses of Candida albicans to phagocytosis and the extracellular activities of neutrophils are critical to counteract carbohydrate starvation, oxidative and nitrosative stress. PLoS One 7, e52850. Miramón P, Dunker C, Kasper L, Jacobsen ID, Barz D, Kurzai O, Hube B (2014) A family of glutathione peroxidases contributes to oxidative stress resistance in Candida albicans. Med Mycol 52, 223-239. Seider K, Brunke S, Schild L, Jablonowski N, Wilson D, Majer O, Barz D, Haas A, Kuchler K, Schaller M, Hube B (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation. J Immunol 187, 3072-3086. Seider K, Gerwien F, Kasper L, Allert S, Brunke S, Jablonowski N, Schwarzmüller T, Barz D, Rupp S, Kuchler K, Hube B (2013) Immune evasion, stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata within macrophages. Eukaryot Cell 13, 170-183. 57 Group members Visiting Scientists Head Prof. Dr. Bernhard Hube Phone +49 3641 532-1401 Fax +49 3641 532-0810 [email protected] Inês Correia Universidad Complutense de Madrid, Spain 04/2013–07/2013 Josephine Fischer Westfälische Wilhelms-Universität Münster 11/2013 Secretary Petra Flemming Scientists Dr. Sascha Brunke Dr. Francesco Citiulo (until 03/2012) Dr. Betty Hebecker (joint project with MI, since 08/2013) PD Ilse D. Jacobsen, PhD (Head MI, since 08/2013) Dr. Lydia Kasper Dr. François Mayer (since 10/2013) Dr. Katja Seider Dr. Duncan Wilson Ph.D. Students Stefanie Allert (since 11/2013) Bettina Böttcher (since 01/2013) Christine Dunker (MI, since 06/2013) Daniel Fischer (since 04/2013) Toni Förster (since 10/2013) Franziska Gerwien Katharina Große (until 06/2012) Sarah Höfs Philipp Kämmer (since 11/2012) Anja Lüttich François Mayer (until 12/2012) Pedro Miramon (until 12/2013) Tony Pawlik (joint project with MI, since 04/2013) Melanie Polke (joint project with MI, since 08/2013) Shruthi Ramachandra (until 09/2013) Florian Roas (until 12/2012) Volker Schwartze Katja Seider Volha Skrahina (since 09/2012) Diploma / Bachelor / Master Students María Cristina Albán Proaño (10/2012–11/2013) Stefanie Allert (10/2012–10/2013) Erik Borchert (10/2012–11/2013) Christine Dunker (until 05/2013) Daniel Fischer (01/2012–03/2013) Alexa Lauer (since 10/2013) Camilo Juan Molina Rodriguez (until 01/2012) Christine Pöllath (since 11/2013) Maria Schreiner (until 04/2012) Marcel Sprenger (since 10/2013) Elisabeth Weiß (10/2012–11/2013) Cristina Zubiria Barrera (since 10/2013) Research Assistants Nadja Jablonowski Daniela Schulz (since 04/2013) Birgit Weber (MI, since 08/2013)) Stephanie Wisgott Trainees Dorothee Eckhardt (since 08/2013) Madlen Peisker (09/2013–10/2013) Jessica Pötschner (08/2013–08/2013) 58 Dr. Nayla Jbeily University Hospital Jena 05/2013–12/2013 Jessica Quintin Radboud University Nijmegen Medical Centre, The Netherlands 03/2013–04/2013 Barbara Schaarschmidt University Hospital Jena 03/2013–05/2013 External funding Bundesministerium für Bildung und Forschung ERA-Net – CandiCol Grundlagen zur Kolonisation und der Dissemination pathogener CandidaSpezies: Entwicklung früher Diagnose- und Therapieansätze Bernhard Hube Bundesministerium für Bildung und Forschung Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) Project: Pathogen-Wirt-Interaktion während der Infektion mit Candida albicans und die Entwicklung zur Sepsis Bernhard Hube, Sascha Brunke Bundesministerium für Bildung und Forschung Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) In-house Professorship Molekulare Mechanismen der Candida-Sepsis Bernhard Hube, Ilse Jacobsen Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: MicroCom – Host pathogen interactions of human pathogenic yeast Bernhard Hube Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project. Infection-associated genes of Candida glabrata Bernhard Hube Deutsche Forschungsgemeinschaft SPP 1580: Intracellular compartments as places of pathogen-host interaction Phagosom – survival and proliferation of human pathogenic Candida species within phagocytes Bernhard Hube Deutsche Forschungsgemeinschaft DACH Evolution – Microevolution of pathogenic yeasts during interactions with the host immune system Bernhard Hube Deutsche Forschungsgemeinschaft Chlamydosporen – Identifizierung und Analyse von Chlamydosporen-und Pathogenitätsassoziierten Genen in Candida albicans Bernhard Hube Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project C01 – Charakterisierung molekularer Mechanismen der Translokation von Candida albicans durch Epithelgewebe Bernhard Hube European Union Framework Programme 7 Marie Curie Action Initial Training Network FINSysB – Pathogenomics and Systems Biology of Fungal Infections – An Integrative Approach Bernhard Hube Selected publications Lüttich A, Brunke S, Hube B, Jacobsen ID (2013) Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney. PLoS One 8, e64482. Mayer FL, Wilson D, Hube B (2013) Hsp21 potentiates antifungal drug tolerance in Candida albicans. PloS One 8, e60417. Voigt J, Hünniger K, Bouzani M, Jacobsen ID, Barz D, Hube B, Löffler J, Kurzai O (2013) Human natural killer cells acting as phagocytes against Candida albicans and mounting an inflammatory response that modulates neutrophil antifungal activity. J Infect Dis 209, 616-626. Wilson D, Hebecker B, Moyes DL, Miramón P, Jablonowski N, Wisgott S, Allert S, Naglik JR, Hube B (2013) Clotrimazole dampens vaginal inflammation and neutrophil infiltration in response to Candida albicans infection. Antimicrob Agents Chemother 57, 5178-5180. Brunke S, Hube B (2013) Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol 15, 701-708. Collaborations Bayer Vital GmbH, Leverkusen, Germany Dr. Norbert-Heinz Becker Friedrich Schiller University Jena, Germany Prof. Dr. Alexander Berndt Georg August University Göttingen, Germany Prof. Dr. Uwe Groß HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms Heinrich Heine University Düsseldorf, Germany Prof. Dr. Joachim Ernst Humboldt University Berlin, Germany Prof. Dr. Edda Klipp INRA - Institut National de la Recherche Agronomique, Paris, France Dr. Mathias Richard Institut Pasteur, Paris, France Prof. Dr. Christophe d‘Enfert Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Departamento de Biotecnología, Spain Dr. Amparo Querol University of Vienna, Kuchler Laboratory, Austria Prof. Dr. Karl Kuchler VIB Department of Molecular Microbiology K.U. Leuven Laboratory of Molecular Cell Biology, Belgium Prof. Dr. Patrick van Dijck Westfälische Wilhelms University, Laboratory of Molecular Stem Cell Biology, Münster, Germany Prof. Dr. Frank Rosenbauer ZIK Septomics, Research group Host Septomics, Jena Prof. Dr. Hortense Slevogt King's College London Dental Institute, UK Dr. Julian Naglik Leibniz Institute for Age Research – Fritz-Lipmann-Institute, Jena, Germany Prof. Dr. Falk Weih Max von Pettenkofer-Institut, Ludwig Maximilians University, München, Germany Prof. Dr. Frank Ebel Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands Prof. Dr. Mihai Netea School of Dental Medicine, New York, USA Prof. Dr. Mira Edgerton University Hospital Jena, Germany Prof. Dr. Michael Bauer Prof. Dr. Konrad Reinhart Prof. Dr. Niels Riedemann University Hospital Tübingen, Germany Prof. Dr. Martin Schaller University of Aberdeen, UK Prof. Dr. Andrew Parker University of Aberdeen & Aberdeen Fungal Group, UK Dr. Alexandra Brand Prof. Dr. Alistair Brown Prof. Dr. Neil Gow University of Amsterdam, Netherlands Prof. Frans Klis University of Bonn, Institute for Cell Biology, Germany Prof. Dr. Albert Haas University Dijon, France Dr. Frédéric Dalle University of Perugia, Italy Prof. Dr. Anna Vecchiarelli University of Szeged, Dept. of Microbiology, Hungary Dr. Attila Gacser Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013 59 60 HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 61 Department Molecular and Applied Microbiology The Department of Molecular and Applied Microbiology (MAM) is devoted to research in the two main areas of the HKI, i.e. infection biology of fungi and natural product research (Fig. 1). The department studies the ascomycetes Aspergillus fumigatus and Aspergillus nidulans but also zygomycetes. Furthermore, the department MAM sequenced the genomes of dermatophytes, a group of fungi which cause infections of the skin, hair and nails by colonising keratin tissue. Recently the department started research on Streptomyces rapamycinicus, which induces distinct silent gene clusters in A. fumigatus and A. nidulans. Furthermore, the department is also producing compounds using innovative techniques for further testing in animal models. The departments is organised in seven subgroups reflecting the research areas (Fig. 2). Aspergillus fumigatus has become the most important airborne fungal pathogen in humans. It causes different diseases ranging from allergies to systemic, life-threatening infections. Individuals at risk for developing invasive aspergillosis are immunocompromised patients. This is particularly true for patients with haematological malignancies and patients undergoing bone marrow transplantation. On average, between 5 - 15 % of individuals of these patient groups acquire invasive aspergillosis during the course of their underlying diseases. Invasive aspergillosis I N T R O D U C T I O N | E I N LE I T U N G Die Abteilung Molekulare und Angewandte Mikrobiologie (MAM) widmet sich den beiden Forschungsschwerpunkten des HKI, d. h. der Infektionsbiologie von Pilzen und der Naturstoff-Forschung (Fig. 1). Die Abteilung untersucht die beiden Ascomyzeten Aspergillus fumigatus und Aspergillus nidulans, aber auch Zygomyzeten, ergänzt durch die Untersuchung des Bakteriums Streptomyces rapamycinicus, welches spezifisch stille Gencluster von A. nidulans und A. fumigatus induziert. Die Abteilung MAM führte außerdem die Genomsequenzierung und biologische Charakterisierung von Dermatophyten durch. Dermatophyten, sogenannte Hautpilze, können oberflächliche Infektionen der Haut, der Haare und Nägel auslösen und stellen den häufigsten Erreger pilzlicher Erkrankungen beim Menschen dar. Die Abteilung produziert mit Hilfe neuer Techniken Naturstoffe, die in verschiedenen Infektionsmodel- Head: Prof. Dr. Axel A. Brakhage 62 len getestet werden. Die Abteilung MAM ist in sieben Untergruppen gegliedert (Fig. 2). Aspergillus fumigatus ist medizinisch gesehen der wichtigste, über die Luft übertragbare human-pathogene Pilz. Er verursacht verschiedenste Krankheitsbilder beim Menschen. Das Spektrum umfasst allergische Symptome bis hin zu systemischen, lebensbedrohlichen Infektionen. Vor allem immunsupprimierte Patienten sind besonders anfällig für eine invasive Aspergillus-Infektion. Dies trifft insbesondere auf Patienten mit hämatologischen Tumoren und nach Knochenmarkstransplantation zu. Im Durchschnitt erkranken 5 – 15 % dieser Patientengruppe an einer invasiven Aspergillose, die in ca. 50 % der Fälle tödlich verläuft. Die hohe Sterblichkeit liegt vor allem darin begründet, dass die Diagnose invasiver Aspergillosen schwierig ist und nur eine geringe HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology is associated with high mortality because diagnostic and therapeutic options are inefficient. Furthermore, the pathophysiology of A. fumigatus infections is poorly understood. Scientists of the department MAM study diverse aspects of the biology and virulence of A. fumigatus including signal transduction, the biosynthesis of secondary metabolites, the improvement of genomic, transcriptomic and proteomic tools and host-pathogen interaction. Fungi produce numerous secondary metabolites including mycotoxins, antibiotics such as penicillin, immunosuppressants like cyclosporin and cholesterol lowering drugs, e.g. lovastatin (Brakhage 2013). By Zahl antimykotischer Therapeutika zur Verfügung steht. Zusätzlich sind die Pathogenitätsmechanismen von A. fumigatus bisher kaum verstanden. Wissenschaftler der MAM forschen an vielen Aspekten der Biologie und Virulenz des Schimmelpilzes A. fumigatus, so. z. B. an der Signaltransduktion, der Biosynthese von Sekundärmetaboliten und deren Bedeutung für die Virulenz, der Anwendung und Verbesserung genomischer, transkriptomischer und proteomischer Methoden sowie der Charakterisierung der Pathogen-Wirt Interaktion. Pilze produzieren eine Vielzahl an Sekundärmetaboliten, z. B. Mykotoxine, Antibiotika wie Penicillin, Immunsuppressiva und Cholesterin-senkende Wirkstoffe wie Lovastatin. Mit Hilfe des Modellorganismus Aspergillus nidulans versucht die Abteilung MAM aufzuklären, welche Regulationsmechanismen using Aspergillus nidulans as a model organism the department uncovers the regulatory pathways and physiological conditions which lead to the biosynthesis of secondary metabolites (Fig. 3). In collaboration with other departments of the HKI we focus on the search and characterisation of new secondary metabolite gene clusters and their molecular regulation. In this context, we have discovered a novel principle of activation based on the interaction of A. nidulans with a Gram-positive soil bacterium, Streptomyces rapamycinicus, which reprograms the histone modification machinery of the fungus (Schroeckh et al. 2009, Nützmann et al. 2011, Nützmann et al. 2013). und welche physiologischen Bedingungen dazu führen (Fig. 3), dass Sekundärmetabolite produziert werden (Brakhage 2013). In Zusammenarbeit mit anderen Abteilungen sucht die MAM nach neuen, unbekannten Sekundärmetaboliten, die auf genetischer und biochemischer Ebene charakterisiert werden. Mitarbeitern der Abteilung ist es gelungen, ein neues Prinzip der Aktivierung sogenannter stiller Gencluster zu entdecken: Bei der direkten Interaktion des Gram-positiven Bodenbakteriums Streptomyces rapamycinicus werden spezifische stille Gencluster sowohl in A. nidulans als auch A. fumigatus aktiviert, die zur Produktion neuer Substanzen führen. Zumindest für A. nidulans konnte gezeigt werden, dass das Bakterium in der Lage ist, die Histonmodifikationsmaschinerie im Pilz umzuprogrammieren (Schroeckh et al. 2009, Nützmann et al. 2011, Nützmann et al. 2013). Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 63 Scientific Projects Figure 1 Research areas of the Department of Molecular and Applied Microbiology. The research areas combine various aspects of infection biology and natural product research with emphasis on microbial communication and the importance of the produced compounds as infochemicals. 1 Infection biology of Aspergillus fumigatus Group Leaders: Thorsten Heinekamp, Axel A. Brakhage In this project, we attempt to identify pathogenicity determinants of A. fumigatus and their regulation. Also, the interaction between immune effector cells and the fungus has been analysed. Below, a selection of research topics is summarised. G protein-coupled receptors of A. fumigatus The capability to grow on a wide variety of nutrients and its arsenal of virulence factors enable A. fumigatus to infect and colonise the human body. For the response to changing environmental conditions during infection, A. fumigatus contains a number of sensing and signalling mechanisms including G protein-coupled receptors (GPCRs). 64 GPCRs form the largest group of membrane receptors among eukaryotic organisms. Until now, little is known about the stimuli and signal transduction mechanisms associated with the 15 predicted GPCRs in the genome of A. fumigatus (Gehrke et al. 2009). Therefore, to understand their impact on fungal growth, development and pathogenicity, all GPCR-encoding genes were deleted. Various phenotypes of the deletion mutants were observed such as a significant decrease in spore formation and growth rate. A major question concerning receptors of all organisms is the identification of agonists and antagonists modulating their activity. Using the “Biolog” System, we were able to identify metabolites that led to significantly changed growth of GPCR knock-out strains compared to the wild type, suggesting that the recognition and/or corresponding signal transduction processes might be altered in these mutant strains. Interestingly, addition of excess metabolites complemented the growth defect HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology Infec.on Biology of Aspergillus fumigatus Microbial Communica.on and Natural Products Thorsten Heinekamp Stress-‐ and Immunoproteomics Olaf Kniemeyer Volker Schroeckh Molecular and Applied Microbiology Transcrip.on Factors and Signal Transduc.on Molecular Biology of Natural Products Daniel Scharf Peter Hortschansky Zygomycetes – Pathogenicity and Biodiversity Molecular Biotechnology of Natural Products Kers:n Voigt Vito Valiante Special Methods: Proteomics platform Surface Plasmon Resonance Analyses of the respective mutants, giving a strong hint that the corresponding GPCRs are important for sensing these molecules. The ongoing analysis allows defining the function of different GPCRs in this important opportunistic filamentous fungus. Identification of a novel compound regulated by protein kinase A in A. fumigatus After perception of environmental stimuli by fungal receptors, intracellular signal transduction in A. fumigatus is mediated via several pathways. One of these signalling cascades is the cyclic AMP dependent protein kinase A (PKA) pathway, which was shown to be involved in virulence of A. fumigatus. In a transcriptomics approach putative targets of this signalling cascade were identified. Among these were several transcriptional regulators. The transcription factor gene, which showed highest up-regulation by PKA is located in a natural product gene cluster. Figure 2 Research groups of the Department of Molecular and Applied Microbiology. Deletion of the corresponding gene and of a non-ribosomal peptide synthetase (NRPS) gene located in the cluster led to reduced fungal growth and sporulation. Overproduction of the transcription factor resulted in formation of a brown substance identified as the novel compound fumipyrrole. Interaction of A. fumigatus with macrophages – Interference of A. fumigatus with phagolysosomal vATPase assembly Human pathogenic microorganisms have evolved a multitude of highly different immune evasion strategies for establishing their pathogenic life style inside the host (Brakhage et al. 2010). The pathogen mimics or alters host structures thereby preventing or diminishing the immune response. Especially professional phagocytes are the target of manipulation by intruding microorganisms, e.g. by reprogramming the host endocytic pathway to prevent the formation of a hostile phagolysosome and thereby generat- Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 65 Figure 3 Global regulatory proteins involved in the regulation of secondary metabolism gene clusters in various fungi. Environmental signals can influence the regulation of various secondary metabolism gene clusters through regulatory proteins that respond to these environmental stimuli and, in turn, modulate the expression of the clusters. Shown is a model secondary metabolism gene cluster containing a gene encoding a central non-ribosomal peptide synthetase (NRPS), a polyketide synthase (PKS) or a hybrid PKS–NRPS enzyme. CBC, CCAATbinding complex; CpcR1, cephalosporin C regulator 1; LaeA, loss of aflR expression A; RsmA, restorer of secondary metabolism A; SAGA-ADA, Spt–Ada–Gcn5–acetyltransferaseADA (taken from Brakhage 2013, Nat Rev Microbiol). ing a compartment in which the pathogen not only survives but also replicates until it is released. However, only little is known about how pathogenic filamentous fungi avoid intracellular killing by phagocytes. In lung alveoli, resident alveolar macrophages represent the first line of defence against inhaled conidia of A. fumigatus. Recently, we showed that depending on dihydroxynaphthalene (DHN) melanin, the gray green conidial pigment, A. fumigatus prevents acidification of phagolysosomes and thereby counteracts degradation by phagozytes (Thywißen et al. 2011, Heinekamp et al. 2013). We elucidated the mechanism contributing to the regulation of the host vATPase activity during phagosomal maturation. By interference of the DHN-melanin polymer with the recruitment of lipid rafts, small membrane microdomains rich in cholesterol and sphingolipids, A. fumigatus prevents assembly of the vATPase at the 66 phagolysosomal membrane. Lipid rafts, but not caveolae contribute as an intracellular platform essential for maturation of phagolysosomes. We showed that pathogenic fungi manipulate this system to generate a niche allowing them to survive within the host. Interaction of A. fumigatus with neutrophil granulocytes – Detoxification of reactive nitrogen intermediates by A. fumigatus and the role for nitric oxide in regulation of neutrophil extracellular trap formation In the lung, spores (conidia) of A. fumigatus are confronted with immune effector cells. Macrophages and neutrophil granulocytes phagocytose conidia and attack them intracellularly by various fungicidal mechanisms including antimicrobial proteins. Neutrophils also attack hyphae and employ extracellular mechanisms like degranulation or neutrophil extracellular trap (NET) formation (Bruns et al. 2010). Additionally, macrophages and HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology neutrophils form nitric oxide (NO) and other reactive nitrogen intermediates (RNI), whose function in defence against A. fumigatus is a matter of debate. Based on a dual RNAseq approach including the analysis of the transcriptome of both the fungus and neutrophils, we showed that A. fumigatus produces several enzymes potentially detoxifying RNI, namely two flavohemoglobins, FhpA and FhpB, and the S-nitrosoglutathione reductase GnoA. To elucidate the role of these enzymes, single and double deletion mutants of FhpA, FhpB and GnoA encoding genes were generated. Our data indicate that FhpA and GnoA are the primarily responsible enzymes in A. fumigatus to counteract RNI. Interestingly, based on the finding that human neutrophil granulocytes showed a significant reduction in NETosis when either confronted with hyphae of A. fumigatus mutants deficient for RNI-detoxification or by addition of exogenous NO donors, we were able to attribute NO a role as negative feedback regulator of NET formation. Interaction of Aspergillus fumigatus with Pseudomonas aeruginosa Cystic fibrosis (CF) is characterised by chronic airway infections with opportunistic pathogens. Pseudomonas aeruginosa is the predominant bacterial pathogen in CF-lungs and until now it is unclear how P. aeruginosa interacts with the airborne fungal pathogen A. fumigatus. To get first insights into putative inter-microbial communication processes between A. fumigatus and P. aeruginosa, co-incubation experiments were performed. A direct physical contact between both microorganisms was revealed by electron microscopy. Fungal growth was inhibited by P. aeruginosa culture filtrate. Interestingly, culture filtrate of the quorum sensing defective mutant ΔlasR was significantly less efficient in affecting fungal growth. Therefore, P. aeruginosa molecules regulated by LasR seem to be involved in fungal growth inhibition. In cell culture experiments the supernatant of the A. fumigatus-P. aeruginosa co-culture revealed reduced cytotoxicity and reduced anti-proliferative activity in comparison to the A. fumigatus control. These findings led to the hypothesis that both microorganisms are able to detoxify each other to enable prolonged coexistence during infection. 2 Stress and Immunoproteomics Group Leader: Olaf Kniemeyer The release of genome sequences of the filamentous fungi Aspergillus fumigatus, Aspergillus nidulans and the dermatophyte Arthroderma benhamiae (Burmester et al. 2011) have paved the way for global studies on the entire proteome and its dynamic changes during specific developmental stages, stress conditions or during the interaction with immune effector cells (Kroll et al. 2014). In the following, we report some exemplary research projects of the group “Stress and Immunoproteomics”. Analysis of the Aspergillus fumigatus response to low atmospheric oxygen levels (hypoxia) The ability to adapt to low oxygen levels seems to be an important trait of A. fumigatus to be able to cause infections in humans with impaired immune system. Therefore, together with co-operation partners in the USA (R. Cramer), we aimed at obtaining a comprehensive overview about this process on the transcript and protein level. We investigated the dynamic changes of the A. fumigatus transcriptome and proteome in hypoxia over a period of 24 hours utilising an oxygen-controlled fermenter system. Interestingly, significant increases in transcripts associated with iron and sterol metabolism, the cell wall and transcriptional regulators were observed in response to hypoxia. On the protein level an increase of enzymes involved in glycolysis was detected. Thus, the hypoxic response of A. fumigatus is regu- Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 67 Figure 4 Light microscopic image (left) and fluorescence emission (middle) from mycelium of Aspergillus fumigatus wild type and ΔhorA mutant strain stained with MitoTracker Red, which stains mitochondria in living cells. Its accumulation is dependent on the mitochondrial membrane potential. The right pictures show an overlay of the light microscopic and fluorescence microscopic image. lated at both the transcriptional and posttranscriptional level. During the short term response to hypoxic conditions, the observed induction of ergosterol biosynthesis genes may represent a strategy to compensate for a temporary deficit of sterols, whose biosynthesis requires molecular oxygen. Because the enzymes of the ergosterol pathway also need iron as a co-factor, the increase of transcripts of iron-uptake genes is consistent with an increased need for iron under hypoxia. In addition, the level of some proteins of the respiratory mitochondrial machinery increased in abundance during hypoxia. Besides the regulatory circuits controlling the hypoxic response, our study provides candidate genes for further analysis of their role in hypoxia adaptation and fungal virulence. This includes a flavohemoprotein which is known to be involved in the detoxification of NO and which was clearly induced at the early phase of hypoxia. To characterise its 68 role during hypoxic growth a deletion mutant was created. The mutant showed increased sensitivity towards reactive nitrogen species; however, growth under hypoxic condition was not reduced. This suggests that the level of reactive nitrogen species increases during hypoxia. However, the presence of the NOdetoxifying flavohemoprotein is not essential at low oxygen levels. A hypoxia-induced short-chain dehydrogenase HorA was another candidate protein, which was investigated in more detail. Deletion of the corresponding gene revealed interesting phenotypes: at atmospheric oxygen levels growth of the mutant was significantly reduced. Furthermore, the ∆horA mutant was attenuated in virulence, but showed increased resistance towards antifungal agents of the triazole group. These and further results along with the evidence of mitochondrial localisation suggest that HorA represents a new short-chain dehydrogenase HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology which is associated with the mitochondrial respiratory complex. HorA is essential for mitochondrial functionality (Fig. 4). promoted the adhesion of fungal spores and extracellular matrix production and led to an eDNA-specific structure of the biofilm (Shopova et al. 2013). Another hypoxia-induced gene was identified as a member of the large protein family of sensor globins. The function of these proteins is largely unknown, but heterologous expression and purification gave evidence for a functional heme-binding site. To characterise the protein’s contribution to hypoxic survival the corresponding gene was deleted in A. fumigatus. Interestingly, the mutant showed impaired growth in low oxygen atmospheres and hence, it may play role during adaptation to hypoxic conditions. In collaboration with the Aspergillus unit at the Institut Pasteur, Paris, we compared the different transcriptional and translational signatures of biofilm-grown mycelium in comparison to submerged A. fumigatus cultures. A. fumigatus biofilms produced more antigens, the hydrophobic surface protein RodB and mycotoxins. The overexpression of several efflux pumps may explain the increased resistance of A. fumigatus biofilms to antifungals. Functional genomic profiling of Aspergillus fumigatus biofilms Besides disseminated forms of disease in immunocompromised patients Aspergillus fumigatus is able to cause local infections in immunocompetent individuals. Particularly, cystic fibrosis patients are at risk for developing locally restricted respiratory tract infections. Airway inflammation is the typical symptom for this disease, which is referred to as allergic bronchopulmonary aspergillosis (ABPA). To establish such an infection, spores of A. fumigatus have to adhere to the surface of epithelial cells and, as a second step, start to germinate. Later, a biofilm-like structure is formed comprising dense hyphal networks embedded in an adhesive extracellular matrix built predominantly of polysaccharides (Bruns et al. 2010). Fungal biofilms are likely to be of clinical relevance, as they have been observed in patients. Interestingly, in vitro experiments also showed their drastically reduced susceptibility to antifungal drugs. Little is known about the environmental factors which trigger the formation of biofilms in A. fumigatus. Since extracellular DNA (eDNA) released from white blood cells is an abundant molecule in the mucus-rich airway surfaces of CF patients, we investigated the effect of extracellular DNA on A. fumigatus biofilm establishment. Using an in vitro biofilm model eDNA was identified as an efficient biofilm inducer. It The immunoproteome of Aspergillus fumigatus 2D-gel electrophoresis can be easily combined with Western blotting. The blotted proteins are then accessible for antibody binding and detection. Thus this method allows screening for pathogen-specific anti-bodies, which are produced in infected patients, in comparison to healthy controls. Many allergenic A. fumigatus proteins have already been detected in serum of patients with allergy-type Aspergillus diseases, such as asthma and allergic broncho pulmonary aspergillosis (ABPA). Only little is known about the A. fumigatus proteins produced during the course of invasive aspergillosis. Furthermore, it has not been investigated so far which of these proteins trigger the production of Aspergillusspecific IgG anti-bodies. Therefore, we separated mycelial proteins by 2D-gel electrophoresis followed by Western blotting using sera from patients with probable and proven invasive aspergillosis, and risk patients without evidence of invasive aspergillosis. Altogether, 57 different protein spots representing 46 different proteins gave a positive IgG antibody signal. Most of these protein antigens play a role in primary metabolism and the general stress response. In order to examine whether some protein antigens may be used as diagnostic or prognostic marker or exhibit a protective activity we employed a classification system based on supervised Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 69 References Schroeckh V, Scherlach K, Nützmann HW, Shelest E, Schmidt-Heck W, Schuemann J, Martin K, Hertweck C, Brakhage AA (2009) Intimate bacterial-fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans. Proc Natl Acad Sci U S A 106, 14558-14563. Nützmann HW, Reyes-Dominguez Y, Scherlach K, Schroeckh V, Horn F, Gacek A, Schümann J, Hertweck C, Strauss J, Brakhage AA (2011) Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation. Proc Natl Acad Sci U S A 108, 14282-14287. Nützmann HW, Fischer J, Scherlach K, Hertweck C, Brakhage AA (2013) Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans. Appl Environ Microbiol 79, 6102-6109. Gehrke A, Heinekamp T, Jacobsen ID, Brakhage AA (2010) Heptahelical receptors GprC and GprD of Aspergillus fumigatus are essential regulators of colony growth, hyphal morphogenesis, and virulence. Appl Environ Microbiol 76, 3989-3998. Brakhage AA, Bruns S, Thywißen A, Zipfel PF, Behnsen J (2010) Interaction of phagocytes with filamentous fungi. Curr Opin Microbiol 13, 409-415. Thywißen A1, Heinekamp T, Dahse HM, Schmaler-Ripcke J, Nietzsche S, Zipfel PF, Brakhage AA (2011) Conidial Dihydroxynaphthalene Melanin of the Human Pathogenic Fungus Aspergillus fumigatus Interferes with the Host Endocytosis Pathway. Front Microbiol 2, 96. Heinekamp T, Thywißen A, Macheleidt J, Keller S, Valiante V, Brakhage AA (2013) Aspergillus fumigatus melanins: interference with the host endocytosis pathway and impact on virulence. Front Microbiol 3, 440. 70 machine learning with decision tree as a predictive model. These analyses indicated that this method is helpful to improve diagnosis of IA and that some antigens may be protective and may have the potential to be used for immunotherapy of invasive aspergillosis. Some of these anti-gens have been used to test their capacity to induce T cell expansion (in collaboration with Alexander Scheffold, Berlin) (Bacher et al. 2013a, Bacher et al. 2013b). 3 Microbial communication and natural products Group Leaders: Volker Schroeckh, Axel A. Brakhage Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans For the important model fungus A. nidulans we have shown that co-cultivation of the fungus with the soil-dwelling actinomycete Streptomyces rapamycinicus led to the specific activation of a cryptic polyketide gene cluster that codes for the biosynthesis of the archetypal polyketide orsellinic acid, as well as for a number of polyphenolic compounds with important pharmacological activities (Schroeckh et al. 2009). Further studies revealed that the bacterium is able to reprogram the histone modifying SAGA/ADA machinery (Nützmann et al. 2011) (Fig. 5). Our data showed that the histone H3 amino acid lysine residues 9 and 14 at distinct secondary metabolism genes were specifically acetylated during the bacterial fungal interaction, which furthermore was associated with the activation of the otherwise silent ors gene cluster. Lysine residues at this cluster were replaced by arginine or glutamine residues. As a result, the lysine to arginine exchange mimics a non-acetylatable lysine, whereas the incorporation of glutamine displays a constitutive acetylation of histone H3. Of all tested strains the exchange of lysine 14 and lysine 9/14 showed the strongest phenotype compared to the wild type. The mimic- ry mutants of the non-acetylatable lysine 14 as well as those of the lysine 9/14 double exchange showed strongly reduced transcription of the penicillin, sterigmatocystin and orsellinic acid gene clusters. These results revealed a causal linkage between histone acetylation, secondary metabolite gene cluster activation, and SAGA/ADA (Nützmann et al. 2013). Further open questions concern the identification and characterisation of upstream elements activating SAGA/ADA upon a bacterial signal and downstream elements regulated by SAGA/ADA including transcription factors and counteracting histone deacetylases. Bacteria-induced natural product formation in the fungus Aspergillus fumigatus leads to fumicycline formation Genome analyses indicated that bacteria and fungi are able to produce a much broader range of chemical entities than estimated from previous chemical analyses of microorganisms grown under standardised laboratory conditions. To access this cryptic metabolome it is necessary to elucidate the physiological conditions under which the compounds are produced. A strategy that has proven highly successful in inducing the expression of silent biosynthesis gene clusters is the simulation of a possible scenario occurring in the habitat. Interestingly, S. rapamycinicus has a similar impact on the human-pathogenic fungus A. fumigatus. An intimate, physical interaction between both organisms resulted in the activation of an otherwise silent polyketide synthase (PKS) gene-containing cluster in the fungus. We named the encoded compounds fumicyclines, which are unusual prenylated polyphenols belonging to the meroterpenoids (König et al. 2013). The biosynthesis of these compounds is regulated by a pathway-specific activator as well as by epigenetic factors. However, transduction of the yet unknown bacterial signal seems to be different in both Aspergillus species. HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology Figure 5 Intimate physical contact between S. rapamycinicus and A. nidulans leads to the activation of the previously silent ors gene cluster in A. nidulans and the formation of orsellinic acid and derivatives thereof. Thereby, S. rapamycinicus reprograms the histone-modifying SAGA/ADA complex of the model fungus A. nidulans by a yet unknown trigger. Transposon mutagenesis of Streptomyces rapamycinicus – a tool to elucidate how S. rapamycinicus targets Aspergillus nidulans and Aspergillus fumigatus To elucidate the activating principle of the streptomycete, we generated a mutant library of S. rapamycinicus using random Tnp(a) transposon insertion mutagenesis. For this purpose, the standard protocols for mutant library generation had to be adapted for the genetically difficult to access S. rapamycinicus. We modified the transposon vector pTNM. This plasmid contains a synthetic, codon usage-optimised transposase gene tnp(a). Previously, it was shown that the plasmid was randomly inserted into the genome of S. coelicolor. Successful conjugation of the modified vector into S. rapamycinicus was achieved. By analysing several mutants of S. rapamycinicus, we verified both the selective induction of the transposase gene expression as well as the random transposon insertion into the streptomycete genome. Moreover, we optimised the mutant library screening protocol, which is based on the non-appearance of a brownish colour when genes involved in the specific fungus-streptomycete interaction are ‘transposon’-mutated. 4 Transcription factors and signal transduction Group Leaders: Peter Hortschansky, Axel A. Brakhage CCAAT-binding complex structure reveals novel mode of DNA interaction by minor groove sensing and widening The CCAAT box is a frequent motif in eukaryotic promoters and its specific recognition by the heterotrimeric CCAAT-binding complex (CBC), which is strictly conserved among species, constitutes a key step in promoter organisation and transcriptional regulation of downstream-encoded genes. Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 71 Figure 6 Structure of the CBC:DNA complex. (A) Ribbon plot of the CBC bound to the CCAAT box in the cycA promoter. (B) Structural superposition of the CBC and the first turn of the nucleosome. The high similarity of the CBC to the histones H2A and H2B might allow their mutual substitution in nucleosomes and enable interactions with the histones H3 and H4. In Aspergillus nidulans the CBC consists of the subunits HapB, HapC and HapE. A multitude of A. nidulans genes has been found to be positively or negatively regulated by the CBC, including developmental genes as well as genes involved in oxidative stress adaptation, production of secondary metabolites and iron homeostasis (Hortschansky et al. 2007, Thön et al. 2010). It has long been an open question how the CBC employs the HapC / HapE histone fold motifs to gain sequence specificity for the CCAAT box. In collaboration with the group of Michael Groll (TU Munich) we determined the crystal structure of the CBC:DNA complex. HapC and HapE induce nucleosome-like DNA bending by interacting with the sugarphosphate backbone, whereas HapB tightly anchors the CBC to the CCAAT box by minor groove sensing and widening (Fig. 6). Interestingly, the tertiary structure of subunit 72 HapB displays no similarity to any known protein domains characterised today. It is composed of an N-terminal helix followed by a loop, a sensor helix S and an anchor A. Whereas most transcription factors insert into the major groove of the DNA, since it offers more contact sites with DNA bases, HapB binds into the minor groove of the DNA and widens it from 12 Å up to 19 Å. This structural deformation is not accomplished by the sensor helix S, but by the anchor A, which functions as a tentacle and inserts into the CCAAT-box. Surface Plasmon Resonance (SPR) measurements of structure-based truncated HapB mutants proved that the anchor A is essential for high-affinity binding of the CBC to the CCAAT-box. Furthermore, the CBC:DNA structure visualised for the first time the position of the HapE N-terminal helix (N) within the CBC and relative to the DNA backbone. This domain was previously shown to be important HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology Figure 7 (A) An evolutionary conserved, bipartite motif present in cccA promoters from 28 fungal species including A. fumigatus. (B) Surface Plasmon Resonance analysis of in vitro formation of the CBC/DNA-HapX ternary complex on the conserved cccA promoter motif from A. fumigatus. (C) Scheme of the regulatory mechanism mediating both adaptation to iron starvation and iron resistance by the same transcription factor complex, comprising the CBC and HapX. for the interaction with HapX, a fourth CBC subunit that regulates various iron-dependent pathways (e. g. heme and siderophore biosynthesis) (Huber et al. 2012). Aspergillus fumigatus HapX is a Janus transcription factor required for adaptation to both iron starvation and iron excess Iron is an essential but, in excess, toxic nutrient. As a consequence, balance of physiological levels of iron is crucial for every organism. In A. fumigatus and other fungal pathogens, the bZIP transcription factor HapX mediates adaptation to iron limitation and consequently virulence by repressing iron-consumption and activating iron uptake (Schrettl et al. 2010, Linde et al. 2012). Importantly, HapX activity depends on physical interaction with the above described heterotrimeric CCAAT binding factor (CBC), which is conserved in all eukaryotes. In collaboration with the group of Hubertus Haas (Inns- bruck Medical University), we demonstrated for the first time that HapX is not only crucial for adaptation to iron starvation but also for iron resistance via activating vacuolar iron storage, i.e. HapX is a Janus-type transcription factor acting as both an activator and repressor depending on iron availability. Consistently, we identified evolutionarily conserved protein domains within HapX that are exclusively essential for adaptation to either limitation or excess of iron, and are presumably involved in iron sensing. We further found that for promoter recognition, a HapX homodimer and the CBC cooperatively bind an evolutionarily conserved DNA motif in the promoter of the cccA gene, encoding for the vacuolar iron importer CccA (Fig. 7). As the CBC can also act independently of HapX, the latter reveals the mode of discrimination between CBC and HapX/CBC target genes. Collectively, our data uncovered a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 73 References Burmester A, Shelest E, Glöckner G, Heddergott C, Schindler S, Staib P, Heidel A, Felder M, Petzold A, Szafranski K, Feuermann M, Pedruzzi I, Priebe S, Groth M, Winkler R, Li W, Kniemeyer O, Schroeckh V, Hertweck C, Hube B, White TC, Platzer M, Guthke R, Heitman J, Wöstemeyer J, Zipfel PF, Monod M, Brakhage AA (2011) Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi. Genome Biol 12, R7. Kroll K, Pähtz V, Kniemeyer O (2014) Elucidating the fungal stress response by proteomics. J Proteomics 97, 151-163. Bruns S, Seidler M, Albrecht D, Salvenmoser S, Remme N, Hertweck C, Brakhage AA, Kniemeyer O, Müller FM (2010) Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin. Proteomics 10, 3097-107. Scherlach K, Nützmann HW, Schroeckh V, Dahse HM, Brakhage AA, Hertweck C (2011) Cytotoxic pheofungins from an engineered fungus impaired in posttranslational protein modification. Angew Chem Int Ed Engl 50, 9843-9847. Bacher P, Kniemeyer O, Schönbrunn A, Sawitzki B, Assenmacher M, Rietschel E, Steinbach A, Cornely OA, Brakhage AA, Thiel A, Scheffold A (2013) Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. Mucosal Immunol 7, 916-928. Bacher P, Schink C, Teutschbein J, Kniemeyer O, Assenmacher M, Brakhage AA, Scheffold A (2013) Antigen-reactive T cell enrichment for direct, high-resolution analysis of the human naive and memory Th cell repertoire. J Immunol 190, 3967-3976. König CC1, Scherlach K, Schroeckh V, Horn F, Nietzsche S, Brakhage AA, Hertweck C (2013) Bacterium induces cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus. ChemBioChem 14, 938-942. 74 the same transcription factor complex with activating and repressing functions depending on ambient iron availability. 5 6 Molecular biotechnology Group Leaders: Vito Valiante, Axel A. Brakhage Molecular biology of natural products Group Leaders: Daniel Scharf, Axel A. Brakhage Gliotoxin (GT) is the most important epidithiodioxopiperazine (ETP)-type fungal toxin. GT was the first ETP discovered and has been investigated for years. This low molecular mass secondary metabolite plays a critical role in the pathobiology of Aspergillus fimigatus (Scharf et al. 2012). It modulates the immune response and induces apoptosis in cell culture models. The toxicity is due to a disulfide bridge, which is the functional motif within this metabolite. Because of the extraordinary structure and activity of GT, we have studied the biosynthesis of this molecule genetically, biochemically and also by elucidation of the crystal structure of some of the biosynthesis enzymes (in collaboration with Michael Groll, TU Munich). The sulfur moieties are introduced into the diketopiperazine core by addition of glutathione (GSH) and its sequential degradation to the free thiol precursor. A flavin-adenine-dinucleotide (FAD)-dependent oxidoreductase, termed GliT, finally oxidises an intermediate to the disulfide containing GT, thereby conferring resistance of the fungus to its own toxin. It is very conceivable that GliT-type enzymes catalyse the final biosynthesis step of all fungal ETPs (Scharf et al. 2012, Scharf et al. 2011, Scharf et al. 2010). Recently, we were able to carry out most of the reactions in vitro using purified enzymes. This will open up the possibility to use these unusual enzymes to engineer novel ETP analogs in vitro (Scharf et al. 2013). Also, the crystal structure of the oxidoreductase GliT was solved together with the structures of two other oxidoreductases which allowed proposing a unifying reaction mechanism (in collaboration with Michael Groll, TU Munich). Signalling components in fungi For growth in all habitats, fungi need to be able to sense environmental stimuli and transduce signals via phosphorylation cascades. Previously, we showed that cyclic AMP signal transduction and the calcineurin pathway are involved in virulence of A. fumigatus. Comparative genomic analyses led to the identification of the member proteins of the different signalling pathways in pathogenic fungi. Many of them were found to be important for virulence in both plant and human pathogens (Horn et al. 2012). Among evolutionarily conserved pathways, mitogen-activated protein kinases (MAPK) cascades in all eukaryotes generally consist of three interlinked protein kinases. A. fumigatus contains four putative MAPKs named MpkA, MpkB, MpkC and SakA. MpkA regulated cell wall integrity signalling is involved in regulation of a plethora of genes ranging from those involved in cell wall repair and synthesis, defence against oxidative stress, pigment and toxin biosynthesis as well as siderophore production (Jain et al. 2011). MpkA appears to fine-tune the balance between the stress responses and the energy consumed in cellular processes required for growth, development and secondary metabolite biosynthesis. Transcriptomics data revealed that mutant strains of all MAPK showed different expression of gene clusters. For many of them, several MAPK pathways contribute to their activation / repression, revealing a cross-talk between the different signalling pathways. Another important signalling is the mTor pathway. Tor is an evolutionary conserved nutrient sensing protein kinase member of the phosphatidylinositol 3-kinase (PI-3K) superfamily that regulates cell growth and differentiation in response to nutrients. A HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology deletion mutant for Tor in A. fumigatus appears not to be viable, revealing the importance of this gene for the fungus. An inducible Tor mutant, generated using the xylose promoter, allowed us to characterise the role of Tor in the adaptation to different stress conditions. Proteome analysis of the Tor inducible mutant revealed that in A. fumigatus this gene is responsible for major metabolic activities, which include sugar metabolism, amino acid biosynthesis, and polyamines storage and consumption. Hitting the caspofungin salvage pathway of human-pathogenic fungi with the novel natural product humidimycin The dramatic increase of fatal fungal infections, coupled with the emergence of antifungal drug resistant strains, makes it an urgent task to search for new therapeutic strategies against invasive mycoses. An innovative concept relies on the combination of two drugs, which act on different targets or even target a salvage pathway of one of the drugs. The cell wall biosynthesis of the two most prominent human-pathogenic fungi, i.e. Candida albicans and Aspergillus fumigatus, is inhibited by the clinically used antifungal drug caspofungin (CAS). Here, by screening 20,000 microbial extracts, we discovered a novel compound, designated humidimycin, produced by a strain of Streptomyces humidus, which strongly enhanced the growth inhibitory effect of caspofungin and also another clinically used antifungal, i.e., itraconazole. The structural elucidation of the new molecule was accomplished using a combination of spectroscopic and chemical techniques, including 1D and 2D NMR, HRMS and MS/MS sequencing. Transcriptome analyses revealed that CAS mainly induced the expression of cell wall biogenesis genes of A. fumigatus. When both drugs were combined, the number of CAS-induced genes decreased, suggesting that humidimycin reduced the A. fumigatus stress response against CAS. Phenotypic characterisation of A. fumigatus mutants defective in different signal transduction pathways, measurements of the cell wall carbohydrate content and western blot analyses of protein kinases indicated that CAS affects the cell wall integrity pathway and the high osmolarity glycerol response (HOG) pathway, the latter acting as a salvage pathway of CAS-triggered disturbance of cell wall integrity. When used in combination, humidimycin silenced the HOG pathway and thus increased the antifungal activity of CAS. Additionally, humidimycin alone or in combination with CAS did not show any toxicity on human hepatocyte cell lines. Taken together, our data demonstrate that targeting compensatory signalling pathways provides an excellent basis to develop novel anti-infective strategies. The role of histone acetyltransferases in regulation of natural product biosynthesis In A. fumigatus the biosynthesis genes of natural products are found in specific biosynthesis gene clusters. Under standard laboratory conditions, many of these gene clusters are not activated, making it difficult to identify which secondary metabolites are biosynthesised from a particular gene cluster. One approach to identify silent gene clusters involves the use of epigenetics methods to manipulate the chromatin landscape. Previous studies in Aspergillus nidulans have demonstrated that histone acetyl transferases (HATs) play an important role in secondary metabolism, but little is known so far in A. fumigatus. There are approximately 46 putative HAT-encoding genes in A. fumigatus and current work aims at creating a knockout library of these HATs. Further work will help determine the function of HATs. Synthetic biology tools for bioprospecting of natural products in eukaryotes Most natural product biosynthesis pathways are silent, many in intractable organisms, and their products consequently unidentified. Viral 2A peptides have been shown to promote cotranslational cleavage of eukaryotic polycistronic mRNA. Expression of viral proteins often includes non-canonical decoding events during translation. 2A peptides are able to drive such events, termed Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 References Hortschansky P, Eisendle M, Al-Abdallah Q, Schmidt AD, Bergmann S, Thön M, Kniemeyer O, Abt B, Seeber B, Werner ER, Kato M, Brakhage AA, Haas H (2007) Interaction of HapX with the CCAAT-binding complex--a novel mechanism of gene regulation by iron. EMBO J 26, 3157-3168. Thön M, Al Abdallah Q, Hortschansky P, Scharf DH, Eisendle M, Haas H, Brakhage AA (2010) The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes. Nucleic Acids Res 38, 1098-1113. Schrettl M, Beckmann N, Varga J, Heinekamp T, Jacobsen ID, Jöchl C, Moussa TA, Wang S, Gsaller F, Blatzer M, Werner ER, Niermann WC, Brakhage AA, Haas H (2010) HapXmediated adaption to iron starvation is crucial for virulence of Aspergillus fumigatus. PLoS Pathog 6, e1001124. Linde J, Hortschansky P, Fazius E, Brakhage AA, Guthke R, Haas H (2012) Regulatory interactions for iron homeostasis in Aspergillus fumigatus inferred by a Systems Biology approach. BMC Syst Biol 6, 6. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Roth M, Brakhage AA, Hertweck C (2012) Epidithiol formation by an unprecedented twin carbon-sulfur lyase in the gliotoxin pathway. Angew Chem Int Ed 51, 10064-10068. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis: a four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges. Angew Chem Int Ed 52, 11092-11095. Scharf DH, Remme N, Habel A, Chankhamjon P, Scherlach K, Heinekamp T, Hortschansky P, Brakhage AA, Hertweck C (2011) A dedicated glutathione S-transferase mediates carbon-sulfur bond formation in gliotoxin biosynthesis. J Am Chem Soc 133, 12322-12325. 75 Figure 8 Summary of heterologous production of natural product biosynthesis pathways using virus 2A peptide. The polycistronic mRNA is translated by the ribosomes and the nascent peptide chain cotranslationally cleaved to the single biosynthesis proteins. References Scharf DH, Remme N, Heinekamp T, Hortschansky P, Brakhage AA, Hertweck C (2010) Transannular disulfide formation in gliotoxin biosynthesis and its role in self-resistance of the human pathogen Aspergillus fumigatus. J Am Chem Soc 132, 10136-10141. Horn F, Heinekamp T, Kniemeyer O, Pollmächer J, Valiante V, Brakhage AA (2012) Systems biology of fungal infection. Front Microbiol 3, 108. 76 ‘stop-carry on’ recoding. Nascent 2A peptides interact with the ribosomal exit tunnel to promote an unusual stop codon-independent termination of translation at the final Pro codon of 2A peptides. As synthetic tools viral 2A peptides therefore provide an opportunity to express pathways as polycistronic genes in eukaryotes. Obvious examples are large non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) proteins, which are often the key biosynthetic enzymes of secondary metabolite pathways, and are technically complicated to express heterologously. The increased in vivo cloning efficiency, and the use of viral 2A peptide sequences to yield a large polycistronic mRNA, has facilitated generation of large constructs used for expression from a single promoter of entire biosynthesis pathways. As a proof-of-concept, we described how the entire penicillin biosynthesis pathway from Penicillium chrysogenum can be produced in a heterologous host, allowing subsequent characterisation, using a combination of (i) improved yeast in vivo cloning technology, (ii) generation of polycistronic mRNA for the gene cluster under study and (iii) the use of an amenable and easily manipulated fungal host i.e. Aspergillus nidulans (Fig. 8) (Unkles et al. 2014). 7 Zygomycetes – Pathogenicity and Biodiversity Group Leader: Kerstin Voigt This project is described in more detail in the section Jena Microbial Resource Collection (JMRC) HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology Group members Visiting Scientists Head Prof. Dr. Axel Brakhage Phone +49 3641 532-1001 Fax +49 3641 532-0802 [email protected] Dafna Ben-Yaacov University of Tel Aviv, Israel 04/2013 Secretary Cornelia Schwab (until 12/2013) Daniela Wagner (since 12/2013) Scientists Dr. Falk Hillmann Dr. Nora Köster-Eiserfunke Dr. Thorsten Heinekamp Dr. Olaf Kniemeyer Dr. Thomas Krüger Dr. Kerstin Hoffmann Dr. Peter Hortschansky Dr. Daniel Scharf Dr. Volker Schroeckh Dr. Maria Strassburger (since 07/2012) Dr. Andreas Thywißen Dr. Vito Valiante PD Dr. Kerstin Voigt Ph.D. Students Clara Baldin Juliane Fischer Martin Föge Benjamin Hanf (since 10/2013) Christoph Heddergott (until 06/2012) Sophia Keller Claudia König Antje Kröber Kristin Kroll Katrin Lapp Ting Luo Juliane Macheleidt Derek Mattern Tina Netzker Vera Pähtz Hea-Reung Park (since 09/2012) Hella Schmidt (since 11/2012) Hanno Schoeler (since 10/2013) Volker Schwartze Elena Shekhova (since 05/2013) Iordana Shopova Katja Tuppatsch Jakob Weber Diploma / Bachelor / Master Students Jana Haueisen Patrick Pann Franziska Schmidt Iordana Shopova Lysett Wagner Lars Wallstabe Research Assistants Sylke Fricke Nancy Hannwacker (until 01/2012) Maria Pötsch Carmen Schult Caroline Semm (until 07/2012) Silke Steinbach Christina Täumer Dr. Grit Walther (since 06/2012) Christiane Weigel Dr. James Kinghorn University of St. Andrews, Scotland 04/2013 – 07/2013 Susana Pamela Mejia University of Antioquia, Medellín, Colombia 06/2012 – 07/2012 Dr. Shiela Unkles University of St. Andrews, Scotland 04/2013 – 07/2013 External funding Bundesministerium für Bildung und Forschung ERA-NET PathoGenoMics 2 Apergillosis – Die Zellwand als Target zur Verbesserung der antifungalen Therapie der Aspergillose Olaf Kniemeyer Bundesministerium für Bildung und Forschung ERA-NET – AspBIOmics Biomarker zur Therapie von invasiver Aspergillose Olaf Kniemeyer Bundesministerium für Bildung und Forschung ERA-NET OXYstress Humapathogene Pilze unter Sauerstoff-Stress: Adaptation an Sauerstoffmangel und reaktive Sauerstoffspecies und die Folgen für die Interaktion mit dem Wirt und die Therapie Thorsten Heinekamp Bundesministerium für Bildung und Forschung Zwanzig20 – Partnerschaft für Innovation InfectControl 2020 – Neue Antiinfektionsstrategien – Wissenschaft • Gesellschaft • Wirtschaft; Strategievorhaben Axel Brakhage Bundesministerium für Bildung und Forschung Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) Project: Inprot – Identifizierung von C. albicans und A. fumigatus Proteinantigenen für die Pilzsepsisdiagnostik und Untersuchung der A. fumigatus Stressantwort Olaf Kniemeyer Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Aspergillus fumigatus – Analysis of the interaction of the human pathogenic fungus Aspergillus fumigatus with immune effector cells by functional genomics Axel Brakhage Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Interactions of the fungal pathogen Aspergillus fumigatus with human macrophages Axel Brakhage Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Functional analysis of major transcriptional regulators in pathogenic dermatophytes Axel Brakhage Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Redox regulation, development and hyphal growth in Aspergillus nidulans Axel Brakhage, Peter Hortschansky Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Soil amoeba interactions with filamentous fungi as driving forces for pathogenicity Falk Hillmann Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project Z02: Methoden zur Analyse der zeitlichen und räumlichen Änderung der Proteome bei der Interaktion pilzlicher Pathogene mit dem Wirt Olaf Kniemeyer Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project Z01: Projektmanagement Axel Brakhage European Union Framework Programm 7 European Re-integration Grants (ERG) CellWallPath – Identification of new molecules able to bypass the cell wall compensatory pathways in the pathogenic fungus Aspergillus fumigatus Vito Valiante European Union Framework Programm 7 Marie Curie Initial Training Network ARIADNE – Signaling circuitry controlling fungal virulence: identification and characterization of conserved and specific fungal virulence genes as common antifungal targets Axel Brakhage, Thorsten Heinekamp European Union Framework Programm 7 Marie Curie Initial Training Network Quantfung – Quantitative Biology for Fungal Secondary Metabolite Producers Axel Brakhage Freistaat Thüringen ProExzellenz Initiative NanoConSens – Nanocontainer und nanostrukturierte Trägermaterialien für Sensorik und Wirkstofftransport Axel Brakhage, Christian Hertweck Freistaat Thüringen ProExzellenz Initiative MikroInter – Characterisation of receptors of the human-pathogenic fungus Aspergillus fumigatus – How does the fungus communicate with the environment and the host Axel Brakhage Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013 77 Freistaat Thüringen Thüringer Aufbaubank Geschäftsstelle InfectControl 2020 Axel Brakhage Charité, Universitätsmedizin Berlin Prof. Dr. Alexander Scheffold German Israeli Foundation The hypoxic response in the pathogenic mold Aspergillus fumigatus and its relevance to disease Olaf Kniemeyer Leibniz-Gemeinschaft Leibniz-Wettbewerb, Förderlinie 3: Vernetzung Kryostress – Anpassungsmechanismen der Zelle an Tiefstemperaturen (KAIT) Olaf Kniemeyer Selected publications Scharf DH, Groll M, Habel A, Heinekamp T, Hertweck C, Brakhage AA, Huber EM (2014) Flavoenzyme-catalyzed formation of disulfide bonds in natural products. Angew Chem Int Ed 53, 2221-2224. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis. A four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges in fungal ETP toxins. Angew Chem Int Ed 52, 11092-11095. Duke University, Durham, NC, USA Prof. Dr. Rytas Vilgalys Dr. Andrej Gryganskyi Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Armilla, Granada, Spain Dr. Olga Genilloud Friedrich Schiller University, Jena, Germany Prof. Dr. Ulrich S. Schubert Prof. Dr. Stefan Schuster Konrad Grützmann Prof. Dr. Jürgen Popp Dr. Petra Rösch Ute Müncheberg PD Dr. Frank Peters Dr. Akos Kovacs Henkel AG & Co KGaA, Düsseldorf, Germany Dr. Mirko Weide Max-Planck-Institut für chemische Ökologie Jena Prof. Dr. Wilhelm Boland Yamuna Sahadevan Innsbruck Medical University, Innsbruck, Austria Prof. Dr. Hubertus Haas Brakhage AA (2013) Regulation of fungal secondary metabolism. Nat Rev Microbiol 11, 21-32. Institut Pasteur, Frankreich, Paris Prof. Dr. Jean-Paul Latgé Bacher P, Kniemeyer O, Schönbrunn A, Sawitzki B, Assenmacher M, Rietschel E, Steinbach A, Cornely OA, Brakhage AA, Thiel A, Scheffold A (2013) Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. Mucosal Immunol 7, 916-928. Institute of Animal and Invertebrate Genetics, Czech Academy of Sciences Dr. Katerina Fliegerová König CC, Scherlach K, Schroeckh V, Horn F, Nietzsche S, Brakhage AA, Hertweck C (2013) Bacterium induces cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus. ChemBioChem 14, 938-942. Montana State University, Bozeman, USA Prof. Dr. Robert A.Cramer Philipps University Marburg, Germany Prof. Dr. Shu-Ming Li Royal Botanic Gardens Kew, UK Dr. Paul M. Kirk University Hospital Jena, Germany Dr. Sandor Nietzsche Collaborations Albert Ludwigs University Freiburg, Germany Prof. Dr. Michael Müller University of Florida, Gainsville, FL, USA Dr. Gerald L. Benny BASF, Ludwigshafen, Germany Dr. Lutz Petzke Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherland Prof. Dr. Sybren de Hoog Dr. Benjamin Stielow Dr. Gerard Verkley Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Servicio de Micologia, Madrid, Spain Dr. Emilia Mellado 78 University of California Los Angeles, USA Prof. Dr. Scott F. Filler University of Manchester, UK Dr. Elaine Bignell Dr. Michael Bromley Dr. Paul Bowyer University of Missouri-Kansas City, USA Prof. Dr. Ted White Technical University Munich, Germany Prof. Dr. Michael Groll HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 79 Department of Cell and Molecular Biology Research in the Department of Cell and Molecular Biology is devoted to the flow of molecular information during host-pathogen interactions. As model hosts we are using human cell lines, mice and chick embryos in ovo, and the pathogens of our main interest are Chlamydiales and pathogenic fungi. Within this framework, we aim at the elucidation of how infections proceed in living organisms (imaging) and how infected organs react on a molecular level (e.g. comparative genomics, transcriptomics and interactomics). Imaging is performed by means of our latest generation positron emission tomography-computed tomography (PET-CT) instrument that provides co-registered images, ie. it combines the high spatial reso- lution and anatomical detail of CT with the molecular, quantifiable images obtained by PET. Comparative genomics, transcriptomics and interactomics involve massive parallel sequencing. To broaden information-solving abilities we have set out to adopt and to develop highly advanced micro- and nanosytems, which allow the simultaneous handling of multiple samples within sets of different biomolecules under nearly identical experimental conditions. At present we are focussing on multicolour hyperspectral imaging of biomolecules on solid body surfaces and in single living infected cells. I N T R O D U C T I O N | E I N LE I T U N G Die Abteilung Zell- und Molekularbiologie beschäftigt sich mit den molekularen Grundlagen von Wirt-Pathogen-Interaktionen. Als Modellwirte untersuchen wir menschliche Zellen, Mäuse und Hühnerembryonen im Ei. Die Pathogene, die für uns eine zentrale Rolle spielen, sind Chlamydien und pathogene Pilze. Head: Prof. Dr. Hans Peter Saluz Im Rahmen der biologischen Projekte erforschen wir Infektionsabläufe in lebenden Organismen (Imaging) und die Reaktion infizierter Organe auf molekularer Ebene (mittels komparative Genomics, Transcriptomics und Interactomics). Imaging führen wir vor allem mit einem modernen kombinierten PET-CT (Positron Emission Tomography-Computed Tomography)-Instrument durch, das die hohe räumliche Auflösung und anato- 80 mischen Details des CT mit molekular quantifizierbaren Bildern des PET kombiniert. Sowohl die Genom-, Transkriptom - als auch die Interaktomanalysen involvieren Hochdurchsatzsequenzierung. Um die verschiedenen wirts-spezifischen Antworten wirkungsvoll erfassen zu können, entwickeln wir hochmoderne Mikro- und Nanosysteme. Mit ihnen können jeweils mehrere tausend Proben aus einer Reihe unterschiedlicher Biomoleküle gleichzeitig und unter nahezu identischen Versuchsbedingungen untersucht werden. Zur Zeit beschäftigen wir uns mit Vielfarben-hyperspektralem Imaging von Biomolekülen auf Festkörperoberflächen und in einzelnen infizierten, lebenden Zellen. Unsere Mikro- und Nanosysteme eignen sich auch zur Anwendung in anderen Abtei- HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology The micro- and nanosystems are also ideally suited for the application in other departments of our institute, as well as within the entire Beutenberg Campus in Jena, where research interests in different fields, such as physics, chemistry, and biology meet. Combined with the automation of techniques, these systems will also provide effective tools for the rapid realisation of products and instruments - an important aspect in the context of the “BioRegio” Jena network. To expand optimum access to the most advanced scientific know-how and technical equipment we also cooperate with several institutions and industries, locally and inter- lungen unseres Institutes, sowie auf dem gesamten Beutenberg Campus in Jena, wo die Forschungsinteressen aus so verschiedenen Gebieten wie Physik, Chemie und Biologie in einzigartiger Weise aufeinander treffen. Außerdem liefern diese Systeme, kombiniert mit der Möglichkeit zur Automatisierung, eine Voraussetzung für die effiziente Realisierung von Produkten und Instrumenten, für die „BioRegio“ Jena ein nicht unwesentlicher Aspekt. Um eine optimale Erweiterung durch neueste wissenschaftliche Erkenntnisse und technische Mittel zu haben, kooperieren wir mit mehreren lokalen und internationalen Instituten und Firmen. Die Erfahrungen und Kenntnisse, die wir aus unseren Arbeiten gewinnen, erlauben es uns, Studenten nationally. Experience and knowledge gained from our projects allow us to teach and confront students theoretically and practically with modern aspects of basic and applied research. During the last two years, several bachelor, master, diploma and doctoral students from our department have graduated successfully. Within our technological framework, we have had some real success related to rapid heat block thermo cycling of small samples. The ability to rapidly amplify nucleic acids has been particularly important for pathogen detection in diagnostic applications, as well as in life science research (e.g. library theoretisch und praktisch mit modernsten Aspekten der Grundlagenforschung und angewandten Wissenschaft zu konfrontieren. So hatten wir in den letzten zwei Jahren wiederum mehrere Bachelor- und Masterstudenten, Diplomanden und Doktoranden in unserer Abteilung, die ihre Studien mit Erfolg abschließen konnten. Was unsere technischen Projekte anbetrifft, konnten wir sehr gute Erfolge auf dem Gebiet der ultraschnellen PCR verzeichnen. Die Möglichkeit, Nukleinsäuren sehr rasch und präzise amplifizieren zu können, war vor allem für den Nachweis von Pathogenen bei diagnostischen Anwendungen, aber auch für Forschungs- (z. B. für die Konstruktion von Bibliotheken) und Industrieanwendungen wichtig (z. B. für Pathogennachweise in ex- Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 81 constructions) and industrial applications (e.g. pathogen detection in exported marine organisms). Additional developments concerning in vivo ChIPSeq are currently providing novel and unexpected information on regulatory protein-DNA interactions in promoter, intron, and exon regions of the human genome. Finally, our home-made molecular hyper spectral imager (two dimensional) and our hyper spectral microscope (one dimensional) coupled with multivariate data analysis provide powerful new tools for understanding complex biological and biomedical samples and infection studies in single cells. portierten marinen Organismen). Andererseits ergaben die Interaktomanalysen neuartige und unerwartete Informationen über die Interaktionen von regulatorischen Proteinen mit DNA in allen Promoter-, Intron- und Exonregionen des menschlichen Genoms. Schließlich ermöglicht das selbstgebaute hyperspektrale Imaging-Instrument (zweidimensional) und unser hyperspektrales Mikroskop (eindimensional), kombiniert mit multivariater Datenanalyse, neue Möglichkeiten zum Verständnis von biologischer und biomedizinischer Materialien und Infektionsstudien in einzelnen Zellen. 82 HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology Scientific Projects 1 The primary transcriptomes of infectious and noninfectious chlamydial particles Group Leader: Gerhard Schöfl Chlamydiae are obligate intracellular Gramnegative bacteria parasitising a diverse array of eukaryotic hosts ranging from amoebas to humans. They are responsible for a wide spectrum of medical conditions in mammals and birds. All members of the family Chlamyidaceae share a unique biphasic life-cycle: Infectious elementary bodies (EBs) attach to the host cell and are internalised by endocytosis. The DNA of EBs is condensated by histone-like proteins, which renders EBs small and dense, and effectively suppresses most transcriptional activity. Once enveloped by the host cell, EBs develop into metabolically active, but non-infectuous reticulate bodies (RBs). The RBs proliferate by cell division, and, after several cycles of reproduction, redifferentiate into EBs. The host cell containing these EBs bursts and new cells can be infected. EBs are slightly larger than RBs, have a granular cytoplasm and diffuse nucleic acid, which makes them considerable less dense than the EBs. The various species and strains within Chlamydiaceae are ecologically separated by differential host preferences, host specificities, virulence, and tissue tropism. Investigations into the molecular and genetic bases of these differentiating factors have been hampered by the fact that these bacteria cannot be cultivated outside of their host cells and their targeted genetic manipulation is difficult. Therefore, approaches such as comparative genomics, microarray studies, and genomescale proteomics have been employed to elucidate genes or genomic regions likely responsible for differences in host range and virulence. However, while these studies reveal suggestive patterns of divergence between species they don’t convey information about the relative importance of diverging genes nor about the roles of regulatory RNAs during different stages of the chlamydial life cycle. To address this question we initiated a project to monitor the primary and processed transcriptomes of chlamydial EBs and RBs at various stages of the chlamydial life cycle. Our initial target species are the highly virulent host-generalist C. psittaci, the less virulent host-specialist C. abortus und and the chlamydia-like Waddlia chondrophila as an outgroup. A basic requirement for investigating differential patterns of gene expression in EBs and RBs was to develop a method for a clean mechanical separation of eukaryotic and prokaryotic material, as well as the separation of purification of the two life-cycle stages. Therefore, at a first stage, we established a protocol for the purification of infectuous EBs and metabolically active RBs utilising the differences in the density of these bodies. A separation of eukaryotic and prokaryotic material, as well as EBs and RBs was achieved by ultracentrifugation through a discontinuous gradient of Visipaque® 320, based on the estimated densities of EBs and RBs. The purity of the EB and RB preparations was verified by infection tests (Fig. 1A) and electron microscopy (Fig. 1B). Total-RNA extracts were prepared from the EB and RB fractions (Fig. 1C) and subjected to a differential exonuclease treatment, which leads to a depletion of processed transcripts in the treated samples. A comparison of cDNA libraries generated from untreated versus exonuclease-treated samples will allow a global mapping of transcriptional start sites, the determination of promoter regions and the definition of operon and suboperon structures at whole-genome Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 83 Figure 1 (A): Representative visual fields of infected HEp-2 cells with RB and EB fractions obtained by the purification protocol. Magnification 400x, bar 20 µm. (B) Electron micrographs of RB and EB fractions. RBs and EBs are tagged by white and black arrows respectively. Bar 500 nm. (C) Gel-like image of total RNA obtained from the input material and the RB and EB fractions. Prominent eukaryotic (28S, 18S) and prokaryotic (23S, 16S) rRNAs were labelled. level. It will also allow the global detection of small regulatory RNAs and thus reveal the whole complexity of gene regulation during an infection time-course of Chlamydia. 2 Identification of antigens in calves induced by Chlamydia psittaci infection Group Leader: Hans Peter Saluz (in cooperation with Konrad Sachse, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena The obligate intracellular bacterium Chlamydia psittaci shows a unique life cycle consisting of infectious extracellular elementary bodies (EBs) and metabolically active noninfectious intracellular reticulate bodies (RBs). Because of the ability of C. psittaci to enter host cells, the combined action of cellular and humoral immune response is often 84 needed to combat its infection. To study chlamydia-host interactions in calf we applied modified in vivo-induced antigen technology (IVIAT; Fig. 2) to C. psittaci (DC15 strain). IVIAT is an in vivo high throughput immunoscreening technique capable of identifying immunogenic antigens that are upregulated or specifically expressed upon infection. The aim of this study is the identification of antigens to gain insights into the virulence of these pathogenic organisms. Upon infection of host cells, many chlamydial genes are expressed. We used IVIAT for the identification of immunogenic bacterial proteins. Several chlamydial antigenic determinants, being expressed during calf infection, have been identified and predicted to be involved in cell wall synthesis/structures, transport, metabolism, metal acquisition, virulence, regulatory and hypothetical functions. Both humoral and cell mediated immunity are important for the protection against chlamydial infection and HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology Sera preparation + Y Y Y Y Y Y Y Y Y Generate Protein Expression Library Y Figure 2 Modified in vivo-induced antigen technology (IVIAT). …AGTATAGCA… its clearance, thus IVIAT can point out the antibody-mediated immune response. 3 Protein-protein interactions during infection and cellular stress Group Leader: Frank Hänel (in cooperation with Shamci Monajembashi, Leibniz Institute for Age Research – Fritz Lipmann Institute, Jena) Association of Chlamydia psittaci inclusion membrane protein IncB with host protein Snapin to interact with the host microtubule network All members of the family Chlamydiaceae are obligate intracellular parasites that display a biphasic developmental cycle. The elementary body (EB) form of the bacteria attaches and invades host-epithelial cells. The nascent phagocytic compartment is rapidly modified by Chlamydia-derived proteins to generate a parasitophorous vacuole termed an inclusion. Within the inclusion EBs differentiate into non-infectious but metabolically active reticulate bodies (RB), which proliferate within the expanding inclusion, giving rise to 1000 or more progeny per host cell. The developmental cycle ends after 2 - 3 days depending on the strain, when RBs transform back into EBs and are released into the extracellular medium by cell lysis and/or extrusion to initiate new rounds of infection. During this unique biphasic developmental cycle replicating bacteria acquire energy and biosynthetic precursors from the infected cell. Furthermore, Chlamydiae modulate cellular functions such as apoptotic programs and immune response (reviewed by Bastidas et al. 2013). Studies on inhibitors of bacterial protein synthesis suggest that modulation of the host cell functions requires the activity of chlamydial proteins. All Chlamydiaceae ssp. possess genes encod- Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 85 References Bastidas RJ, Elwell CA, Engel JN, Valdivia RH (2013) Chlamydial intracellular survival strategies. Cold Spring Harb Perspect Med 3, a010265. Peters J, Wilson DP, Myers G, Timms P, Bavoil PM (2007) Type III secretion à la Chlamydia. Trends Microbiol 15, 241-251. Borth N, Litsche K, Franke C, Sachse K, Saluz HP, Hänel F (2011) Functional interaction between type III-secreted protein IncA of Chlamydophila psittaci and human G3BP1. PLoS One 6, e16692. ing core components of a Type III Secretion (TTS) apparatus, a protein transport system used by Gram-negative bacteria to translocate proteins into the cytoplasm of the host cell. Therefore, it is commonly accepted that chlamydial effector proteins are targeted by the TTS to the inclusion membrane and that their interaction with host proteins cause the modulation of host cell functions (Peters et al. 2007). Interactions of chlamydial effector proteins with host proteins seem to play a role at all stages of chlamydial developmental cycle – from adhesion and internalisation of EBs till their exit from the host cell. In C. pneumoniae invasin protein Pmp21 recruits the EGF receptor for host cell entry. Other chlamydial proteins playing a role in invasion are TARP and CT694. The well conserved among all Chlamydia TARP is bacterial nucleator of actin. An important group of chlamydial infector proteins are the Inc (inclusion) proteins which share a common secondary structural feature of a bilobed hydrophobic domain. While this hydrophobic domain enables the anchoring of the proteins in the inclusion membrane the cytoplasmic tail is responsible for interaction with host proteins. IncD of C. trachomatis interacts with the lipid transfer proteins CERT and CT229 from C. trachomatis binds to the GTPase Rab4 – both interactions may affect trafficking processes of the host cell. One of the first discovered protein-protein interactions was the association of C. trachomatis IncG with the mammalian 14-3-3 protein which contributes to circumvention of host cell apoptosis similar to the interaction of TARP with the human adaptor protein SHC1. Finally, C. trachomatis Inc protein CT228 recruits elements of the myosin phosphatase pathway to regulate release mechanisms (reviewed by Bastidas et al. 2013). acting host proteins that could indicate functions of this proteins. In a first attempt we found the interaction between Type III-secreted protein IncA of C. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localisation of G3BP1 near the inclusion membrane of C. psittaci-infected Hep2 cells was demonstrated. G3BP1 harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3´-untranslated region of human c-myc mRNA. Notably, infection of Hep-2 cells with C. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesise that lowering the host cell c-Myc protein concentration may be part of a strategy employed by C. psittaci to avoid apoptosis and scale down host cell proliferation (Borth et al. 2011). Furthermore we could show that IncB in early inclusions of C. psittaci interacts und co-localises with components of the dynein complex. It was known that within the first few hours of infection, endocytosed C. trachomatis EBs are transferred to a perinuclear location corresponding to the microtubule-organizing centre (MTOC) of the host cell using the tubulin-dynein system. Like C. trachomatis, C. psittaci also utilises dynein motor proteins for optimal early development. The interplay of IncB of C. psittaci with dynein components is the first allusion to the nature of chlamydial proteins involved in this transport process. While the present knowledge on interactions of chlamydial effector proteins with host proteins almost entirely refers to the human pathogens C. trachomatis and C. pneumoniae, we focused on the IncA and IncB proteins (Fig. 3) of the zoonotic agent C. psittaci using yeast two-hybrid screens to search for inter- 86 HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology Figure 3 IncB of C. psittaci localises to the inclusion membrane. HEp-2 cells were infected with C. psittaci at a MOI of 3, and at 24 h post infection, the cells were fixed and immunolabeled with anti-IncB (green), counterstained with DAPI (blue, nuclei), and viewed under a confocal laser-scanning microscope. IncB shows a typical inclusion membrane staining. Bar: 20 µm. 4 Interaction of the multifunctional signal protein TopBP1 with the helicase RecQL4 Group Leader: Frank Hänel (in cooperation with Peter Hortschansky, Department of Molecular and Applied Microbiology, HKI, and Shamci Monajembashi, Frank Grosse, Matthias Görlach and Helmut Pospiech, Leibniz Institute for Age Research – Fritz Lipmann Institute, Jena). TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by yeast two-hybrid experiments. As TopBP1 binding partner we identified the helicase RecQL4 and confirmed the physical interaction by GST pull-down assays, co-immuno- precipitations and by surface plasmon resonance binding assays. RecQ-like DNA helicases form a ubiquitous protein family that plays a pivotal role in the maintenance of genome stability from bacteria to man. Five RecQ-like DNA helicases have been identified in the human cell, mutations of three of which are associated with autosomal, recessive human disorders that display various symptoms of genomic instability and premature ageing. Mutations in the RecQL4 gene are associated with the Rothmund–Thomson (RTS), RAPADILINO and Baller-Gerold syndromes. Numerous studies have indicated a role for RecQL4 helicase at the crossroads of DNA replication, recombination and DNA damage response. The amino-terminus of RecQL4 possesses limited homology to the essential yeast replication factor Sld2. In yeast, the binding of Sld2 to Dpb11 (the yeast orthologue of TopBP1) constitutes a critical regulatory step dur- Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 87 References Ohlenschläger O, Kuhnert A, Schneider A, Haumann S, Bellstedt P, Keller H, Saluz HP, Hortschansky P, Hänel F, Grosse F, Görlach M, Pospiech H (2012) The N-terminus of the human RECQL4 helicase is a homeodomainlike DNA interaction motif. Nucleic Acids Res 40, 8309-8324. ing initiation of DNA replication, and Xenopus laevis RecQL4 (XRecQL4) has also been reported to bind to XCut5, the frog orthologue of Dpb11. The amino-terminal ~55 amino acids of human RecQL4 display the highest sequence homology to the N-terminus of Sld2, and this region is conserved in metazoans. This led us to speculate that this part of the protein constitutes an autonomous subdomain of the RecQL4 N-terminal region. To address this assumption, we assessed binding of the human Dbp11 homologue TopBP1 and of DNA to the N-terminal 54 residues of RecQL4 (RecQL4_N54). In addition, we determined its solution structure via NMR spectroscopy. In essence, RecQL4_N54 represents the minimal region for the interaction with TopBP1, binds Y-shaped and double-stranded (ds)DNA with no apparent sequence specificity and adopts a fold highly similar to homeodomains (Ohlenschläger et al. 2012). 5 Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells Group Leaders: Gerhard Schöfl, Hans Peter Saluz (in cooperation with the Leibniz Center for Tropical Marine Ecology (ZMT), Bremen; Leibniz Institute for Baltic Sea Research Warnemünde; University Rostock; NTT, and the Indonesian Ministry for Marine Affairs and Fisheries) In 2012 we launched a project within the SPICE III framework that aims at the taxonomic and functional characterisation of the microbial gut communities in economically and alimentary relevant marine organisms in Indonesian coastal areas under pollutant and non-pollutant conditions. The microbiome includes bacterial, fungal, protistan, and viral species, many of which may pose threats to human and animal health, and/or impact sustainability of food production. It is estimated that about 98 % of the prokaryotic species cannot be cultivated under laboratory conditions, and consequently are not 88 easily detectable. By employing a culture-independent metagenomic approach, we aim to generate taxonomic profiles and snapshots of metagenome gene content for faecal samples collected in different coastal areas and under different environmental conditions. At present, the main goals are to (i) establish the bioinformatical infrastructure necessary to handle and analyse metagenomic data generated by a next-generation sequencing platform, and (ii) to demonstrate, whether and if yes, what kind of pathogenic microorganisms can be found in food fish derived from Jakarta bay. The most commonly used next-generation sequencing platforms (454/Roche pyrosequencing and Illumina/Solexa) have now been extensively applied to metagenome samples. While the 454/Roche technology produces average read-lengths of ~600 pb (with a relatively high error rate), a typical run will yield only ~500 Mbp. Illumina HiSeq2000 instruments on the other hand will produce ~60 Gbp per channel with, however, drastically reduced read-lengths (only up to 150 bp). The trade-off between information loss due to short read-lengths and information loss due to lower yield is alleviated by the recent introduction of the Illumina MiSeq technology. With read-lengths of 2x250 bp (paired-end libraries) and a total output of up to 15 Gbp, the most current MiSeq technology greatly surpasses 454 technology in terms of yield at only a marginal loss of information due to read-length reduction. This method has not yet been used extensively for metagenomics, but first comparisons with established methodologies highlight its promise. Therefore we chose to evaluate metagenomic MiSeq sequencing within the framework of this SPICE III project. Initially, a total of 24 faecal samples from three species of local food fish, Epinephelus fuscoguttatus (brown-marbled grouper), E. sexfasciatus (six-striped grouper), and Atule mate (yellow-fin scad) were obtained from Jakarta Bay (north-eastern Java, polluted) and Cilacap (south-western Java, HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology unpolluted). To generate a comprehensive taxonomic profile, paired-end 16S rRNA community sequencing for all samples was performed on the Illumina MiSeq platform using conserved bacterial/archeal primers for the variable V4 region of the 16S gene. To generate functional profiles, whole genome metagenomic sequencing was performed on total DNA extracted from 12 E. fuscoguttatus samples. For the 16S amplicon sequencing, all 24 samples were multiplexed on a single MiSeq channel, yielding approx. 15.2 m 250 bp paired-end reads, of which 8.9 m could be mapped to one of 530 operational taxonomic units (OTUs) inferred using the uparse OTU clustering method. Total DNA fragments from 12 samples were sequenced on 2 MiSeq channels, yielding a total of 20.6 m and 20.9 m paired-end reads, respectively. The development of a classification pipeline optimised for whole-genome DNA derived MiSeq reads is currently ongoing. Preliminary analyses of the taxonomic composition of the gut microbiomes suggests some habitat effects, possibly attributable to pollution levels, as well as strong betweenspecific variability, with, e.g. E. fuscoguttatus microbiomes being dominated by gamma-proteobacteria, while the dominating taxa in E. sexfasciatus and Atule mate were members of the classes beta-proteobacteria and clostridia, and alpha-proteobacteria and beta-proteobacteria, respectively. With the establishment of an analysis pipeline a larger-scale comparison of the compositions of microbial communities harvested from fish and shellfish exposed to the relatively polluted conditions of Jakarta Bay with control samples from less polluted areas will be conducted to elucidate the influence of environmental conditions on intestinal microbiomes. A special interest will be qualitative and quantitative differences in the pathogenic or potentially pathogenic gut microbiota. 6 Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells Group Leaders: Hans Peter Saluz (in cooperation with Axel A. Brakhage, Department of Molecular and Applied Microbiology, HKI) Host cell death is a critical component of innate immunity and often determines the progression and outcome of infections. The opportunistic human pathogen Aspergillus fumigatus can manipulate the immune system either by inducing or inhibiting host cell apoptosis dependent on its distinct morphological form. Previous studies (Volling et al. 2011) showed that conidia of Aspergillus ssp. inhibit apoptosis of macrophages induced via the intrinsic (staurosporine) and extrinsic (Fas ligand) pathway. Hence, mitochondrial cytochrome c release and caspase activation were prevented. It was further found that the anti-apoptotic effect depends on both host cell de novo protein synthesis and phagocytosis of conidia by macrophages. For the first time, it could be shown that the fungal dihydroxynaphthalene (DHN) melanin is a crucial component to inhibit macrophage apoptosis which may contribute to dissemination of the fungus. Apoptosis inhibition even works with the isolated DHN melanin and also with the corresponding synthetic melanin. These findings have been further investigated these findings in apoptosis-induced human lung A549 epithelial cells. Aspergillus fumigatus enters the human body through inhalation of airborne conidia, thus making close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of A. fumigatus conidia in the human host. Therefore, by using uracilauxotrophic mutant strains, one has been able to investigate the interaction of the lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, un- Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 89 like professional phagocytes, the study has shown that the presence of conidial DHN melanin enhances the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus are able to survive within epithelial cells. This is due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects cannot be observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia are able to inhibit both the intrinsic and extrinsic apoptotic pathway in the lung epithelial cells even for longer periods. The anti-apoptotic effect is not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, act anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells. 7 Positron emission tomography/ computed tomography (PET/CT) for depicting infection, bone metabolism and inflammation: reduction, refinement, and replacement of animal experiments Group Leader: Hans Peter Saluz (in cooperation with Bernhard Hube, Department of Microbial Pathogenicity Mechanisms, HKI; Marc Thilo Figge, Research Group Applied Systems Biology, HKI; Konrad Sachse, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, and Thomas Kamradt and Ingo Irmler, Institute for Immunology, University Hospital Jena) After establishing molecular imaging techniques by combining positron emission tomography (PET) with computed tomography 90 (CT) we started investigating the pathogenicity of microbial infections, bone growth and inflammations using established and new animal model systems. Because PET/CT is a non-invasive imaging method, stress on laboratory animals is heavily reduced (refinement). A reduction of animal experiments by using PET/CT is achieved because the same animal can be subjected to repeated measurements and moreover, it can act as its own control. Finally, by introducing the embryonated chicken egg as an alternative animal model, we were able to replace rodents with bird embryos, especially well suited for in vivo experiments in the context of infections with C. psittaci, the original hosts of which are birds. 8 Automated segmentation and object classification of CT images: Application to in vivo molecular imaging of avian embryos Group Leader: Hans Peter Saluz Although chick embryogenesis has been studied extensively, there has been growing interest in the investigation of skeletogenesis. In addition to improved poultry health and minimised economic loss, a better understanding of skeletal abnormalities can also have implications for human medicine. True in vivo studies require non-invasive imaging techniques such as high-resolution µCT. However, the manual analysis of acquired images is both time consuming and subjective. For these reasons we have developed a system for automated image segmentation that entails object-based image analysis followed by the classification of the extracted image objects. For image segmentation, a rule set was developed using Definiens image analysis software. The classification engine was implemented using the WEKA machine learning tool. Our system reduces analysis time and observer bias while maintaining high accuracy (Fig. 4). Applying the system to the quantification of long bone growth has allowed us to present the first HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology Figure 4 Graphical representation of the complete automated image segmentation procedure. (From: Heidrich et al. 2013) true in ovo data for bone length growth recorded in the same chick embryos. The procedures developed represent an innovative approach for the automated segmentation, classification, quantification, and visualization of µCT images (Fig. 5). µCT offers the possibility of performing longitudinal studies and thereby provides unique insights into the morphogenesis and embryogenesis of live chick embryos. 9 Insights into bone metabolism of avian embryos in ovo via 3d and 4d 18 F-fluoride Positron Emission Tomography Group Leader: Hans Peter Saluz (in cooperation with Thomas Opfermann, Institute of Nuclear Medicine, University Hospital Jena) Although the chick embryo is a well-known economical and widely applied in vivo model system for e.g., bone development studies, it is surprising that no studies concerning the application of 18F-fluoride microPET to bone metabolism were reported so far. Reasons for this lack might be motion artifacts and the lack of convenient tracer injection tools and sites. We therefore resolved the above problems by a combination of embryo in ovo anesthesia, microPET imaging, followed by special computational processing. By these means we developed a convenient way of visualising three- and four- dimensional features of bone metabolism in living chick embryos. Thereby the application of 18 F-fluoride microPET facilitates repeat measurements, highly reproducible and motionartifact-free skeletal imaging, and provides quantitative measurements of in ovo metabolic activities in the bones of developing chicks. During micro-PET measurement, a radio tracer was injected intravascularly using a custom-made catheter system, allowing us to additionally investigate early time Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 References Heidrich A, Schmidt J, Zimmermann J, Saluz HP(2013) Automated Segmentation and Object Classification of CT Images: Application to In Vivo Molecular Imaging of Avian Embryos. Int J Biomed Imaging, 508474. 91 Figure 5 Segmentation and classification of micro CT images of Group 1 chick embryos from d 13 to d19 . The bone names and their corresponding colors are presented. Image artifacts are circled in green. (From: Heidrich et al. 2013) points in tracer kinetics and uptake. Our results clearly show that bone metabolism in living chick embryos can be reproducibly studied and quantified in ovo, even for multiple tracer injections over a longer time period. The use of dynamic 18F-fluoride microPET imaging made it possible to visualise and analyse even small bone structures with excellent quality. Moreover, as our data are comparable to data from corresponding rodent experiments, the use of embryonated chicken eggs is a convenient and economical alternative to other animal models. 10 PET/CT image analysis in experimental arthritis Group Leader: Hans Peter Saluz (in cooperation with Ingo Irmler and Thomas Kamradt, Institute for Immunology, University Hospital Jena, and Marc Thilo Figge, Research Group Applied Systems Biology, HKI) 92 Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and mainly characterised by joint inflammation, bone erosion and deformation. Up to now there exists no cure and the causes of RA are also not resolved in detail. Early detection of the disease can improve the treatment success and is important to evaluate the effectiveness of new drugs. The gold standard for exploring experimental arthritis in murine models is histopathological investigation, meaning that animals have to be sacrificed to assess joint inflammation and bone destruction. In contrast, imaging with positron emission tomography-computed tomography (PET/CT) is minimal invasive and therefore a promising approach to investigate arthritic progresses in murine models. This multimodal imaging technique offers the opportunity to explore experimental arthritis in vivo and in longitudinal studies. Besides this the number of animals needed for preclinical studies can be reduced in a large amount. In this HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology Figure 6 PET/CT imaging in experimental arthritis and high-resolution surface reconstruction. (A) 18F-fluoride PET and CT image fusion at day 28 after G6PI immunization. Inflamed joints show a high tracer uptake. (B) High-resolution surface reconstruction of the zoomed region in A. (C) Highlighting of bone destruction. Blue colour denotes smooth regions, green colour denotes rough and eroded regions of metatarsal bones. subproject the usability of the PET tracer 18Ffluorodeoxyglucose (18F-FDG) for quantification of joint inflammation in a model of glucose-6-phosphate isomerase (G6PI)-induced murine arthritis could already been shown. The current research covers the application of 18F-fluoride as a tracer for bone metabolism as well as a more extensive quantification of bone erosion in the paws based on CT images. CT offers a higher spatial resolution than PET imaging and therefore may allow better discrimination of different stages in arthritic progress, but also earlier detection of the disease based on symptoms like bone erosion and malformation. New computer aided approaches for (semi-)automated image analysis are under development in a close cooperation with the HKI research group Applied Systems Biology. Two different concepts are pursued within these studies. The first one is based on measurements of cortical bone thickness, as arthritic mice show a loss of cortical bone substance dur- ing progress of the disease. Additionally, the second approach uses high-resolution surface reconstruction of metatarsal bones to quantify bone erosion based on surface roughness (Fig. 6). The combination of functional imaging via PET, giving insights into bone metabolism, and high-resolution anatomical imaging via CT may give the opportunity to improve diagnostics and also treatment testing in murine models, towards RA in humans. Department of Cell and Molecular Biology HKI Research Report 2012 / 2013 References Irmler IM, Opfermann T, Gebhardt P, Gajda M, Brauer R, Saluz HP, Kamradt T (2010) In vivo molecular imaging of experimental joint inflammation by combined (18)F-FDG positron emission tomography and computed tomography. Arthritis Res Ther 12, R203. 93 Group members Dr. Kusuma Jitsaeng Ubon Ratchathani University, Thailand 10/2012–11/2012 Head Prof. Dr. Hans Peter Saluz Phone +49 3641 532-1200 Fax +49 3641 532-0805 [email protected] Dr. Brent Sørensen Max Planck Institute for Chemical Ecology, Jena, Germany 04/2013–06/2013 Secretary Svetlana Bauer Dr. Ingo Irmler Friedrich Schiller University Jena, Germany Since 03/2012 Scientists Dr. Anne Bleicher (until 11/2013) Dr. Frank Hänel Dr. Peter Gebhardt Dr. Hans Krügel (until 10/2012 ) Dr. Jens Müller (since 11/2013) Dr. Gerhard Schöfl Dr. Brent Sørensen Dr. Alexander Tretiakov External funding Freistaat Thüringen Thüringer Aufbaubank Integriertes Multiplex-Genotyping-System basierend auf einem hyperspektralen Vielfarben-Scanner zur schnellen qualitativen und quantitativen Sequenzanalyse von SNPs und DNA-Polymorphismen auf Microarrayoberflächen Hans Peter Saluz Ph.D. Students Shayista Amin Thomas Beder (since 11/2013) Selina Böcker Alexander Heidrich (until 06/2013) Bianca Hoffmann Julia Kästner Anja Kuhnert (until 02/2012) Katrin Litsche (until 03/2012) Sara Mohebbi Philipp Sehnert Arndt Steube Anja Voigt Lydia Würbach (until 04/2012) Bundesministerium für Bildung und Forschung Verbundprojekt Zoonotische Chlamydien – Wechselwirkung von zoonotischen Chlamydien mit ihren Wirtszellen Hans Peter Saluz, Frank Hänel Bundesministerium für Bildung und Forschung Resistom – Antibiotika-Resistom in der Lebensmittelkette Hans Krügel Diploma / Bachelor / Master Students Jochen Bick (until 05/2013) Thomas Beder (10/2012–10/2013) Anna Dettling Sabine Kupis Martin Schneider (since 10/2012) Philipp Sehnert (until 07/2012) Martin Thamm (until 03/2013) Ding Wang Christina Wichmann Bundesministerium für Bildung und Forschung PET-CT II – In-vivo-Quantifizierung von Inflammation und Destruktion in murinen Arthritismodellen mittels PET/CT als Beitrag zur Reduktion der Zahl der in Forschung und Entwicklung verwendeten Versuchstiere Hans Peter Saluz Bundesministerium für Bildung und Forschung SPICE III – Wissenschaftlich-technische Zusammenarbeit mit Indonesien: MABICO - Einfluss von Meeresverschmutzung auf Biodiversität und den Lebensunterhalt von Küstenbewohnern; Mikrobielle Biodiversität Hans Peter Saluz Research Assistants Claudia Franke Grit Mrotzek Vera Mittenzwei-Klujewa (until 09/2012) Yvonne Hupfer (until 03/2012) Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Comparative genomics of host / chlamydiales interactions Hans Peter Saluz Trainees Karthika Annamalai (07/2012–05/2013) Sarah Behr Alexander Korn (02/2013–03/2013) Juliane Kießling Sabine Kupis Anna Lisa Mitter Abdul Muslihudeen Hanna Noack (until 04/2012) Dominique Prätzsch Mareike Scheven Sebastian Schneider (until 03/2012) Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Pathogen/host communication: dual RNA expression dynamics to investigate differential patterns of virulence Hans Peter Saluz Klimina KM, Kjasova DCh, Poluektova EU, Krügel H, Leuschner Y, Saluz HP, Danilenko VN (2013) Identification and characterization of toxin-antitoxin systems in strains of Lactobacillus rhamnosus isolated from humans. Anaerobe 22, 82-89. Bleicher A, Schöfl G, Rodicio MD, Saluz HP (2013) The plasmidome of a Salmonella enterica serovar Derby isolated from pork meat. Plasmid 69, 202-210. Groot AT, Staudacher H, Barthel A, Inglis O, Schöfl G, Santangelo RG, Gebauer-Jung S, Vogel H, Emerson J, Schal C, Heckel DG, Gould F (2013) One quantitative trait locus for intra- and interspecific ariations in a sex pheromone. Mol Ecol 22, 10651080. Rassmann A, Martin U, Saluz HP, Peter S, Munder T, Henke A (2013) Identification of gene expression profiles in HeLa cells and HepG2 cells infected with Coxsackievirus B3. J Virol Methods 187, 190-194. Collaborations Food GmbH Jena, Germany Dr. Michael Grün Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena Dr. Konrad Sachse University Hospital Jena Dr. Thomas Opfermann Gesellschaft zur Förderung von Medizin-, Bio- und Umwelttechnologien e.V. (GMBU), Jena, Germany Dr. Emanuel Gutmann Dipl.-Ing. Florian Erfurth Gondol Research Institute for Mariculture, Indonesia Dr. Haryanti Leibniz Center for Marine Tropical Ecology (ZMT), Bremen, Germany Dr. Claudia Schultz Dr. Sebastian Ferse Leibniz Institute for Age Research, Fritz-Lipmann-Institut, Jena, Germany Prof. Frank Große Max Planck Institute for Chemical Ecology, Jena, Germany Dr. Ales Svatos Research Ministry of Marine Affairs and Fisheries, Jakarta, Indonesia Dr. Ketut Sugama Universtiy of Applied Sciences, Jena, Germany Prof. Dr. Thomas Munder University of Rostock, Germany Prof. Dr. Harry Palm Selected publications Visiting Scientists Dr. Haryanti Gondol Research Institute for Mariculture, Indonesia 11/2012–12/2012 11/2013 94 Heidrich A, Schmidt J, Zimmermann J, Saluz HP (2013) Automated segmentation and object classification of CT images: application to in vivo molecular imagin of avian embryos. Int J Biomed Imaging 2013, 508474. HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology Research Group Applied Systems Biology HKI Research Report 2012 / 2013 95 Research Group Applied Systems Biology The focus of the research group Applied Systems Biology (ASB) is on the quantitative analysis and mathematical modelling of microscopy image data. Image-based Systems Biology represents an essential connective link in joint studies of experiment and theory and complements bioinformatics analysis of “omics”-data by addressing the dynamical, functional and morphological aspects of biological processes. Image-based Systems Biology includes the following three main aspects: (i) acquisition and automated analysis of image data for high-content and high-throughput screening, (ii) quantitative description of biological processes by appropriate characteristic measures, (iii) con- struction of image-derived spatiotemporal models and predictive computer simulations. Applying the Image-based Systems Biology approach is a unique feature of the research group ASB at the HKI which collaborates with experimentalists at the HKI and beyond on both of the institute’s research priorities: natural product research and infection biology of human-pathogenic fungi. Microscopy data are provided by the Research Group Fungal Septomics, the Department Microbial Pathogenicity Mechanisms and the Bio Pilot Plant. Horn. External collaborations exist with Prof. Dr. J. Stein (Lymphocyte Trafficking and Activation) at the Theodor Kocher I N T R O D U C T I O N | E I N LE I T U N G Head: Prof. Dr. Marc Thilo Figge Der Schwerpunkt der Forschungsgruppe Angewandte Systembiologie (ASB) liegt auf der quantitativen Analyse und mathematischen Modellierung von Mikroskopie-Bilddaten. Die Bildbasierte Systembiologie ist ein wesentliches Bindeglied in gemeinsamen Studien von Experiment und Theorie und die Berücksichtigung von dynamischen, funktionellen und morphologischen Aspekten biologischer Prozesse ist ein zur bioinformatorischen Analyse von „omics“-Daten komplementärer Ansatz. Bildbasierte Systembiologie umfasst die folgenden drei Hauptaspekte: (i) Erstellung von Bilddaten und deren automatisierte Analyse für ein high-content- und high-troughput-screening, (ii) quantitative Beschreibung biologischer Prozesse durch geeignete charakteristische Maße, (iii) Generierung von Bild-basierten 96 Raum-Zeit-Modellen und prädiktiven Computersimulationen. Die Anwendung der Bildbasierten Systembiologie ist ein Alleinstellungsmerkmal der Forschungsgruppe ASB am HKI. Die theoretischen Studien werden in Zusammenarbeit mit Experimentatoren am HKI und darüber hinaus durchgeführt und umfassen beide Forschungsschwerpunkte des Instituts: die Naturstoff-Forschung und die Infektionsbiologie human-pathogener Pilze. Mikroskopie-Daten werden erstellt in der Gruppe Fungal Septomics, in der Abteilung Mikrobielle Pathogenitätsmechanismen und im Biotechnikum. Externe Kooperationen sind etabliert mit Prof. Dr. J. Stein (Transport und Aktivierung von Lymphozyten) am Theodor-Kocher-Institut in Bern, Prof. Dr. A. Hauser (Im- HKI Research Report 2012 / 2013 Research Group Applied Systems Biology Institute in Bern, Prof. Dr. A. Hauser (Immunodynamics) at the Leibniz Institute German Rheumatism Research Centre in Berlin and Prof. Dr. M. Gunzer (Experimental Immunology and Imaging) at the University Duisburg-Essen. In the research group ASB, Image-based Systems Biology is realized with regard to all three aforementioned aspects: (i) Fully automated image analysis is performed by the postdoctoral researchers Dr. Franziska Mech and Dr. Carl Svensson as well as the PhD student Susanne Brandes. The implementation ranges from the use of the commercial software Definiens Developer XD® mundynamik) am Leibniz-Institut Deutsches Rheuma-Forschungszentrum in Berlin und Prof. Dr. M. Gunzer (Experimentelle Immunologie und Bildgebung) an der Universität Duisburg-Essen. In der Forschungsgruppe ASB wird die Bild-basierte Systembiologie in Bezug auf alle drei zuvor genannten Aspekte realisiert: (i) Die vollautomatische Analyse von Bilddaten wird von den Postdoktoranden Dr. Franziska Mech und Dr. Carl Svensson sowie von der Doktorandin Susanne Brandes bewerkstelligt. Die Implementierung der Algorithmen reicht von der Programmierung im Rahmen der kommerziellen Software Definiens Developer XD® und Imaris® zu selbst geschriebenen Codes in den Sprachen C++ und Python mit modernen Methoden aus dem and Imaris® to self-written code in C++ and Python languages using state-of-the-art machine learning methods. (ii) Mathematical analysis of the image-derived raw data to quantify biological processes is realized by the PhD student Zeinab Mokhtari. (iii) Integration of the quantified spatio-temporal information into models – to generate testable predictions and hypotheses by computer simulations – is realized by the PhD students Johannes Pollmächer and Teresa Lehnert through applying ordinary differential equations, state-based models or agent-based models – depending on the properties of the biological process under consideration. Bereich des maschinellen Lernens. (ii) Die Anwendung von mathematischen Konzepten zur Herleitung charakteristischer Größen für die Quantifizierung von biologischen Prozessen, die in den Rohdaten aus der Bildanalyse enthalten sind, wird von der Doktorandin Zeinab Mokhtari realisiert. (iii) Die Integration der quantitativ erfassten raum-zeitlichen Information in Modelle, die zur Generierung von überprüfbaren Voraussagen und Hypothesen auf dem Computer simuliert werden können, wird von den Doktoranden Johannes Pollmächer und Teresa Lehnert realisiert. Abhängig von den Eigenschaften des betrachteten biologischen Prozesses reichen die Modellierungsansätze von gewöhnlichen Differentialgleichungen über Zustand-basierte Modelle bis hin zu Agenten-basierten Modellen. Research Group Applied Systems Biology HKI Research Report 2012 / 2013 97 In joint experimental and theoretical studies, we investigate phagocytosis assays for alveolar macrophages versus Aspergillus fumigatus or Lichtheimia, as well as invasion assays for Candida albicans on oral epithelial cells, and a whole blood model to quantify innate effector mechanisms and immune evasion by C. albicans in human blood. In addition, we develop image processing algorithms for recognition and sorting of Actinobacteria in picoliter-droplets in real time. The development of tracking algorithms goes hand in hand with the identification of mathematical measures for the characterization and classification of cell tracks from image sequences. In gemeinsamen experimentellen und theoretischen Studien untersuchen wir Phagozytose-Assays für Alveolarmakrophagen und Aspergillus fumigatus oder Lichtheimia, sowie einen Invasionsassay für Candida albicans auf oralen Epithelzellen und ein Vollblut-Modell zur Quantifizierung von angeborenen Effektor-Mechanismen und der Immunevasion von C. albicans im menschlichen Blut. Darüber hinaus entwickeln wir Bildverarbeitungsalgorithmen zur Erkennung und Sortierung von Actinobacteria in Pikoliter-Tröpfchen in Echtzeit. Die Entwicklung von Tracking-Algorithmen geht Hand in Hand mit der Identifizierung mathematischer Maße, mit denen Zell-Tracks aus Bildsequenzen charakterisiert und klassifiziert werden können. 98 HKI Research Report 2012 / 2013 Research Group Applied Systems Biology Scientific Projects Figure 1 (A) Overview of segmentation and subsequent classification of C. albicans cells by the automated image analysis. (a) Original image. (b) Application of the maximum intensity transformation. (c) Recognition of the region of interest (white outline) as a C. albicans cell. (d) The filamentous cell was classified into a yeast cell (red outline) and its two hyphae (green outline). (e) Hyphae were further partitioned into invaded (blue) and non-invaded (pink) segments. (B) Original and analysed images for six time points. Hyphae and yeast segmentation is shown in comparison with the original images for each time point. 1 Interaction between host cells and human pathogenic fungi: Quantification of microscopic image data for different confrontation assays Group Leaders: Franziska Mech, Marc Thilo Figge In collaboration with the Department Molecular and Applied Microbiology (A. Brakhage), automated analysis of microscopic image data was previously established for phagocytosis assays of murine alveolar macrophages and Aspergillus fumigatus conidia (Mech et al. 2011). This ongoing collaboration is now focused on the quantitative image analysis of phagocytosis assay with Lichtheimia species. Futhermore, in cooperation with the Department Microbial Pathogenicity Mechanisms (B. Hube), the automated analysis of image data was applied to invasion assays for Candida albicans and human epithelial cells. This required an adaptation of the exist- ing algorithm for spherical image objects to recognize the morphologically changed yeast cells forming hyphae and invading the epithelium. A selection of typical image data is presented in Figure 1. Based on the automated image analysis, the kinetics of hyphal growth and of epithelial invasion were quantitatively described by mathematical models consisting of coupled differential equations. This analysis revealed that the initiation of hyphae formation represents an ultimate commitment to invasive growth, which suggests that the yeast to hypha transition in vivo must be under exquisitely tight negative regulation to avoid the transition from commensal to pathogen and hyphal invasion of the epithelium (Mech et al. 2012, Mech et al. 2013). Research Group Applied Systems Biology HKI Research Report 2012 / 2013 References Coelho FM, Natale D, Soriano SF, Hons M, Swoger J, Mayer J, Danuser R, Scandella E, Pieczyk M, Zerwes HG, Junt T, Sailer AW, Ludewig B, Sharpe J, Figge MT, Stein J (2013) Naïve B cell trafficking is shaped by local chemokine availability and LFA-1 independent stromal interactions. Blood 121, 4101-4109. Figge MT, Reichert AS, MeyerHermann M, Osiewacz HD (2012) Deceleration of fusion–fission cycles improves mitochondrial quality control during aging. PLoS Computational Biology 8, e1002576. Figge MT, Osiewacz HD, Reichert AS (2013) Quality control of mitochondria during aging: Is there a good and a bad side of mitochondrial dynamics? Bioessays 35, 314-322. 99 Figure 2 Schematic representation of the state-based model. Circular symbols depict different states of the model, i.e. the green circle represents extracellular alive C. albicans (CAE), the red circle indicates extracellular killed C. albicans (CKE), the black circles symbolise resistant C. albicans that are alive (CAR) or killed (CKR), orange circles represent states of monocytes (Mi,j) with i alive and j killed C. albicans and the blue circles depict different states of granulocytes (Gi,j). The arrows between states represent transitions studied in the virtual whole-blood infection model. References Guthke R, Linde J, Mech F, Figge MT (2012) Systems biology of microbial infection. Frontiers in Microbiology 3, 328. Horn F, Heinekamp T, Kniemeyer O, Pollmächer J, Valiante V, Brakhage AA (2012) Systems biology of fungal infection. Frontiers in Microbiology 3, 108. Mech F, Thywißen A, Guthke R, Brakhage AA, Figge MT (2011) Automated image analysis of the host-pathogen interaction between phagocytes and Aspergillus fumigatus. PLoS ONE 6, e19591. Mech F, Figge MT (2012) Image-based systems biology: A quantitative approach to elucidate the kinetics of fungal morphologies and virulence. Proceedings of the German Conference on Bioinformatics 2012, HighlightPaper. 100 2 Mathematical modelling: From strategies of the immune response to intracellular infection processes Group Leaders: Johannes Pollmächer, Teresa Lehnert, Marc Thilo Figge Modelling of the immune response to human-pathogenic fungi Computer simulations are a valuable tool to investigate strategies of the immune response against fungal infections within mathematical models (Guthke et al. 2012). In the research group ASB, agent-based models are developed that describe each biological object within a system as an individual agent, which moves in space and time and interacts with other agents according to defined rules. This approach allows to integrate the information obtained from image data analysis, e.g. on the migration behaviour of cells, into computer simulations (Horn et al. 2012). This was realized in a compara- tive study of virtual phagocytosis assays for neutrophils versus A. fumigatus conidia to investigate the phagocytosis efficiency as a function of the migration behaviour of neutrophils (Tokarski et al. 2012). We currently develop a software package for agent-based modelling that will allow testing of hypotheses about the immune response in complex and dynamic structures, such as alveoli in the presence of respiration. Such structures are not accessible for investigation under physiological conditions and represent an ideal field for application of virtual infection models. Furthermore, in collaboration with the Research Group Fungal Septomics (O. Kurzai) a virtual infection model for infection assays of whole blood with C. albicans is developed. Using the state-based model depicted in Figure 2, in combination with a Metropolis Monte Carlo algorithm to quantitatively determine the optimal model parameters, we investigate innate effector HKI Research Report 2012 / 2013 Research Group Applied Systems Biology Figure 3 Schematic representation of antibody feedback during a germinal centre reaction. Circulating high-affinity antibodies (red) that are dynamically binding to antigen presented on follicular dendritic cells reduce its availability for B lymphocytes and by that increase the selection pressure for retrieval of antibodies with even higher binding affinities. mechanisms and immune evasion by C. albicans in human blood. the product and the regulator of the germinal centre reaction. Modelling antibody affinity maturation in germinal centres Studies on the antibody affinity maturation in germinal centres pose a prime example for the fruitful exchange between experimental and theoretical research. In such a joint study with scientists from Geneva (CH), Birmingham (UK) and Braunschweig (G), we recently proved that germinal centres can communicate with each other through the direct exchange of high-affinity antibodies (Zhang et al. 2013). Computer simulations of agent-based models did not only predict an increased selection pressure and thus the production of even higher affinity antibodies, but also the termination of a germinal centre reaction, if no further improvement of the antibody affinity was possible. Thus, as is schematically shown in Figure 3, antibodies play the central role of being Modelling of mitochondrial network dynamics – a view of aging as intracellular infection process In cooperation with experimentalists from Goethe University Frankfurt, the spread of molecular damage in the mitochondrial network of a cell was investigated by a statebased model. The loss of mitochondrial functionality contributes to the formation of degenerative processes, which are thought to be responsible for neurodegenerative diseases and to be a cause of aging. The dynamics of the mitochondrial network plays a crucial role for the bio-energetic quality control. However, the model developed in the research group ASB reveals that mitochondrial dynamics is a double-edged sword: too frequent fusion and fission processes can highly increase the probability of molecular damage spread and degeneration of the intra- Research Group Applied Systems Biology HKI Research Report 2012 / 2013 References Mech F, Wilson D, Lehnert T, Hube B, Figge MT (2013) Epithelial invasion outcompetes hypha development during Candida albicans infection as revealed by an image-based systems biology approach. Cytometry Part A, DOI: 10.1002/cyto.a.22418. Mokhtari Z, Mech F, Zitzmann C, Hasenberg M, Gunzer M, Figge MT (2013) Automated characterization and parameter-free classification of cell tracks based on local migration behavior. PLoS One 8: e80808. Tokarski C, Hummert S, Mech F, Figge MT, Germerodt S, Schroeter A, Schuster S (2012) Agent-based modelling approach of immune defense against spores of opportunistic human pathogenic fungi. Frontiers in Microbiology 3, 129. 101 Figure 4 Three phases for triggered imaging and sorting. Droplet detection by the photodiode and subsequent triggered imaging occurs in phase I. During phase II, image retrieval and analysis is executed. Based on the outcome of the image analysis, a sorting pulse is generated and transmitted through the electrodes in phase III, changing the directionality of the droplets. References Zang E, Brandes S, Tovar M, Martin K, Mech F, Horbert P, Henkel T, Figge MT, Roth M (2013) Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets. Lab on a Chip 13, 3707-3713. Zhang Y, Meyer-Hermann M, Geroge L, Figge MT, Khan M, Goodall M, Young SP, Reynolds A, Falciani F, Waisman A, Notley CA, Ehrenstein MR, Kosco-Vilbois M, Toellner KM (2013) Germinal center B cells govern their own fate via antibody feedback. The Journal of Experimental Medicine 210, 457-464. 102 cellular mitochondrial network, ultimately resulting into aging of cells (Figge et al. 2012, Figge et al. 2013). 3 Image processing for recognition and sorting of Actinobacteria in pL-droplets in real time Group Leaders: Susanne Brandes, Marc Thilo Figge The subject of this cooperation with the Bio Pilot Plant (U. Horn, M. Roth) is the development of an automated image analysis procedure for microfluidic application. The aims are (i) to detect the growth of actinobacteria in microfluidic droplets with pico-liter (pL) volumes and (ii) to sort the droplets depending on the content of actinobacteria in real time. This was successfully achieved by a setup that is depicted in Figure 4 and yielded the high-throughput cultivation of more than half a million of cultures per hour and subsequent sorting of unlabelled droplets in a real-time analysis of triggered images with a frequency above 100 Hz (Zang and Brandes et al. 2013). The technique has a high potential for screening of unknown or difficult to cultivate actinobacteria and thus for the discovery of novel natural products. 4 Characterisation and classification of cell migration from image sequences Group Leaders: Zeinab Mokhtari, Susanne Brandes, Marc Thilo Figge The characterisation and classification of cell migration generally requires the analysis of individual cell tracks relative to their surroundings, because useful knowledge about the migration behaviour can be lost in the process of averaging over cell populations HKI Research Report 2012 / 2013 Research Group Applied Systems Biology Group members Head Prof. Dr. Marc Thilo Figge Phone +49 3641 532-1416 Fax +49 3641 532-2416 [email protected] Scientists Dr. Carl Svensson Anna Medyukhina (since 11/2013) Dr. Zoltán Cseresnyés (since 10/2013) Dr. Franziska Mech (until 09/2013) Ph.D. Students Susanne Brandes Bianca Hoffmann Kaswara Kraibooj (since 09/2013) Teresa Lehnert Zeinab Mokhtari Johannes Pollmächer Sandra Timme (since 11/2013) Diploma / Bachelor / Master Students Sandra Timme (until 11/2013) Bertram Vogel Carolin Zitzmann (until 12/2013) Research Assistants Sören Totzauer (since 07/2013) Vladimir Shelest External funding Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Development of a multi-scale simulator to model host-pathogen interactions Marc Thilo Figge (Coelho et al. 2013). The research group ASB develops algorithms for the automated cell tracking as well as methods for the systematic and quantitative characterization of cell tracks and the classification of cell populations (Mokhtari et al. 2013). The aim is to arrive at a comprehensive tool for the extraction and analysis of cell tracks in image data. Deutsche Forschungsgemeinschaft JIMI – ein Netzwerk für intravitale Mikroskopie: Gezielte Projektbetreuung von der Mikroskopie in der Zelle bis zum Zelltracking in lebenden Organismen Marc Thilo Figge Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project B04: Bilddatenanalyse und Agenten-basierte Modellierung der Wechselwirkung zwischen Immunzellen und humanpathogenen Pilzen in Raum und Zeit Marc Thilo Figge Selected publications Mech F, Wilson D, Lehnert T, Hube B, Figge MT (2014) Epithelial invasion outcompetes hypha development during Candida albicans infection as revealed by an image-based systems biology approach. Cytometry Part A 85, 126-139. Mokhtari Z, Mech F, Zitzmann C, Hasenberg M, Gunzer M and Figge MT (2013) Automated characterization and parameter-free classification of cell tracks based on local migration behavior. PLoS ONE 8, e80808. Zang E*, Brandes S*, Tovar M, Martin K, Mech F, Horbert P, Henkel T, Figge MT, Roth M (2013) Realtime image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets. Lab Chip 13, 3707-3713. *Authors contributed equally. Research Group Applied Systems Biology HKI Research Report 2012 / 2013 103 Coelho FM, Natale D, Soriano SF, Hons M, Swoger J, Mayer J, Danuser R, Scandella E, Pieczyk M, Zerwes HG, Junt T, Sailer AW, Ludewig B, Sharpe J, Figge MT, Stein J (2013) Naïve B cell trafficking is shaped by local chemokine availability and LFA1-independent stromal interactions. Blood 121, 4101-4109. Zhang Y, Meyer-Hermann M, Geroge L, Figge MT, Khan M, Goodall M, Young SP, Reynolds A, Falciani F, Waisman A, Notley CA, Ehrenstein MR, KoscoVilbois M, Toellner KM (2013) Germinal center B cells govern their own fate via antibody feedback. J Exp Med 210, 457-464. Collaborations Buchmann Institute for Molecular Life Sciences, Frankfurt, Germany Prof. Dr. Andreas Reichert Frankfurt Institute for Advanced Studies (FIAS), Frankfurt, Germany Dr. Jörg Lücke Friedrich Schiller University, Jena, Germany Prof. Dr. Stefan Schuster German Rheumatism Research Centre (DRFZ), Berlin, Germany Dr. Raluca Niesner Prof. Dr. Anja Hauser Goethe University Frankfurt, Germany Prof. Dr. Heinz Osiewacz Helmholtz Centre for Infection Research, Braunschweig, Germany Prof. Dr. Michael Meyer-Hermann University of Birmingham, UK Dr. Kai Tollner University of Duisburg-Essen, Germany Prof. Dr. Matthias Gunzer Theodor Kocher Institut, Bern, Switzerland Prof. Dr. Jens Stein 104 HKI Research Report 2012 / 2013 Research Group Applied Systems Biology Research Group Fungal Septomics HKI Research Report 2012 / 2013 105 Research Group Fungal Septomics Fungal Septomics investigates the infection biology of invasive fungal infections (IFI), focusing on the interaction between fungal pathogens and the human host. The group is located at the Center for Innovation Competence Septomics and forms an independent associated research group at the HKI. Septomics is a faculty-spanning centre of the Friedrich-Schiller-University and scientifically interconnected with the Department of Anesthesiology and Intensive Care Medicine, the Institute for Medical Microbiology and the HKI. Within Septomics, the fields of pathogen and host biology in sepsis are approached complementary and linked to clinical practice. Numbers of IFI have constantly increased over the last decades and a steadily growing number of patients must be considered at risk for IFI. The human pathogenic yeast Candida albicans and the filamentous fungus Aspergillus fumigatus are by far the most important causes of life-threatening mycoses in Europe, whereas Cryptococcus neoformans plays a major role in Africa. Sepsis - an uncontrolled immune reaction to infection affecting the whole body - can be both a consequence of and a risk factor for IFI. Within Fungal Septomics, we work with infection models for a range of primary human immune cells, including neutrophilic granulocytes, monocytes, monocyte derived den- I N T R O D U C T I O N | E I N LE I T U N G Die Forschungsgruppe Fungal Septomics untersucht die Infektionsbiologie invasiver Pilzinfektionen und legt dabei den Schwerpunkt auf die Interaktion zwischen den pathogenen Pilzen und dem menschlichen Wirt. Die Forschungsgruppe gehört zum Zentrum für Innovationskompetenz (ZIK) Septomics und bildet eine unabhängige assoziierte Forschungsgruppe am HKI. Septomics ist ein fakultätsübergreifendes Zentrum der Friedrich-Schiller-Universität und wird wissenschaftlich getragen von der Klinik für Anästhesiologie und Intensivmedizin, dem Institut für Medizinische Mikrobiologie und dem HKI. Head: Prof. Dr. Oliver Kurzai Im Septomics Forschungszentrum wird die Biologie von Erregern und Wirt im Rahmen der Sepsis in komplementärer Weise bearbeitet und mit der klinischen Praxis dieser Infektion verknüpft. Die Zahl invasiver Pilz- 106 infektionen hat in den letzten Jahrzehnten konstant zugenommen und eine ständig wachsende Zahl von Patienten besitzt ein hohes Risiko für invasive Pilzinfektionen. Die humanpathogene Hefe Candida albicans und der Schimmelpilz Aspergillus fumigatus sind mit Abstand die wichtigsten Erreger lebensbedrohlicher Mykosen in Europa. In Afrika ist zusätzlich insbesondere Cryptococcus neoformans relevant. Sepsis – eine unkontrollierte Immunreaktion auf eine invasive Infektion, die den gesamten Körper betrifft – kann sowohl eine Folge als auch ein Risikofaktor invasiver Pilzinfektionen sein. In der Arbeitsgruppe Fungal Septomics haben wir Infektionsmodelle für ein breites Spektrum primärer humaner Immunzellen, darunter neutrophile Granulozyten, Monozyten, Monozyten-generierte dendritische Zellen und natürliche Killerzel- HKI Research Report 2012 / 2013 Research Group Fungal Septomics dritic cells and natural killer (NK-) cells. For this purpose, a large cohort of healthy blood donors as well as several protocols for isolation and cultivation of primary human immune cells have been established. Our work addresses aspects of fungal virulence biology that determine the human host response to the pathogen. A major example is filamentation of C. albicans which is closely related to tissue invasion and innate immune activation. A whole blood infection model is used to analyse host-pathogen interaction in a complex environment that closely mimics early stages of in vivo interaction. In addition, Fungal Septomics is analysing interindividual differences in human susceptibil- ity to fungal pathogens. For this we look at genetic diversity and correlate genetic polymorphisms with functional consequences in immune responses to fungal pathogens. A major effort is the coordination of the AspIRS international multi-centre study on the genetic risk for invasive aspergillosis in stem cell transplant recipients. len etabliert. Um diese Modelle bearbeiten zu können, wurde eine große Kohorte von gesunden Blutspendern rekrutiert und verschiedene Protokolle für die Isolierung und Kultivierung primärer humaner Immunzellen etabliert. Unsere Arbeit adressiert verschiedene Aspekte der Pathobiologie von Pilzen, die einen Einfluß auf die Interaktion mit dem menschlichen Immunsystem haben. Ein wichtiges Beispiel hierfür ist die Filamentierung von C. albicans, die eine zentrale Rolle bei der Gewebsinvasion und der Aktivierung des angeborenen Immunsystems spielt. Wir verwenden ein Vollblutinfektionsmodell, um die pathogene Interaktion in einem komplexen Umfeld, das eng mit der in vivo Situation verwandt ist, zu untersuchen. invasive Pilzinfektionen. Zu diesem Zweck untersuchen wir die genetische Diversität bei Patienten und korrelieren genetische Polymorphismen mit funktionellen Aspekten der Immunantwort auf humanpathogene Pilze. Ein wesentliches Standbein dieses Arbeitsfelds ist die Koordinierung der internationalen Multicenterstudie AspIRS (Aspergillosis Intrinsic Risk Stratification), die das genetische Risiko für invasive Aspergillose bei stammzelltransplantierten Patienten adressiert. Zusätzlich bearbeitet Fungal Septomics die genetischen Grundlagen für interindividuelle Unterschiede in der Suszeptibilität für The resulting data will lead to a better understanding of molecular mechanisms in IFI. Furthermore they build a basis for translational projects that should result in the development of diagnostic tests which will help to improve clinical management of patients at risk for or affected by IFI. Die erzielten Ergebnisse sollen neben einem verbesserten Verständnis der Pathophysiologie invasiver Pilzinfektionen auch die Entwicklung neuer diagnostischer Verfahren im klinischen Management von Risikopatienten ermöglichen. Research Group Fungal Septomics HKI Research Report 2012 / 2013 107 Scientific Projects 1 Adaptation of fungal pathogens to host niches Virulence of C. albicans is directly related to the ability of this yeast to respond to changes in environmental conditions with a morphological switch between the yeast form and filamentous growth. In our group we work on several aspects of filamentation in C. albicans. To determine the effects of filamentation on the interaction with human host cells, we identified a group of genes that are differentially regulated under all conditions which favour filamentous growth, termed core filamentation response. By correlationbased network modelling of the transcriptional response to different defined external stimuli for morphogenesis (cooperation with the Research Group Systems Biology / Bioinformatics, R. Guthke) we identified a set of eight genes with highly correlated expression patterns (Fig. 1; Martin et al. 2013). The validity of network modelling was confirmed using datasets of advanced complexity that describe the transcriptional response of C. albicans during epithelial invasion as well as comparing our results with other previously published transcriptome studies. As filamentation is mainly triggered by environmental cues we have studied the role of CO2 as an environmental signal for C. albicans in cooperation with the group of Fritz Mühlschlegel, University of Kent (GB). High levels of CO2 are linked to regulation of filamentation in C. albicans. On a molecular level CO2 adaptation involves activation of the multi-sensor adenylyl cyclase (AC), and additionally an AC-independent CO2 sensing pathway. The central transcriptional regulator of the latter, Rca1p, was recently identified by Fritz Mühlschlegel in collaboration with Fungal Septomics. Unravelling of this alternate cascade, which is conserved in yeasts, is currently in progress. For this we make use of the genomic resources available for the closely re- 108 lated model yeast Saccharomyces cerevisiae, including completed knock-out libraries, possibility of synthetic-lethal screens, highthroughput transformation and multiple reporter systems (Cottier et al. 2012, 2013). Integrated knowledge on the initial triggers for filamentation and the resulting changes in gene expression is currently used in ongoing projects to extent our knowledge on the impact of this central virulence trait on pathogen – host interaction. 2 Innate immune recognition of fungal pathogens The contact between specialised immune effector cells and invading pathogens is of central importance in invasive infections. Activation of innate immunity can result both in the successful elimination of invading pathogens and in systemic hyperinflammation and sepsis. Therefore a major focus in the group are studies on the activation of innate immune cells by fungal pathogens. The preferred model organism is C. albicans. Whenever possible, comparative analyses with the second major fungal pathogen in Europe, A. fumigatus are performed in cooperation with the Department Molecular and Applied Microbiology (A. Brakhage) and Jürgen Löffler, University of Würzburg. Furthermore, we have recently established infection models for C. neoformans, thus enabling us to perform comparative analyses for the major invasive fungal pathogens. A major focus is put on polymorphonuclear neutrophils (PMN), which play a central role in antifungal immunity. In addition, the role of monocytes, dendritic cells and NK cells is studied. PMN activation is mainly triggered by filamentous forms of C. albicans and results in targeted migration and uptake of the fun- HKI Research Report 2012 / 2013 Research Group Fungal Septomics Figure 1 Defining a core filamentation response (CFR) in Candida albicans. Candida albicans wild type was grown for a total of 12h at 37°C and in M199 medium with a pH8. (A) This environment triggered the transition from unicellular yeasts to long and branching filaments as shown by microscopy. It was therefore possible to distinguish between germ tube formation and hyphal elongation. (B) From these cells, total RNA was isolated and used for whole- genome expression analysis with DNA- microarrays. Out of these data, the transcriptional landscape was defined by correlation- based networks. (C) Independent from the external stimulus, distinct networks of up- regulated genes which were specific for germ tube formation or hyphal elongation could be identified. Combination of these two networks allowed the modelling of a core filamentation network which was restricted to the eight genes which were up- regulated during all stages of filamentous growth in C. albicans. gus. We have established live cell imaging systems for primary human PMN to further analyse these activation patterns. For analysis of video microcopy we established a close cooperation with the Research Group Applied Systems Biology (M. T. Figge) who develop automated analysis tools for live cell imaging. In addition, comparative studies for PMN activation by C. albicans and C. glabrata are performed in cooperation with the Department Microbial Pathogenicity Mechanisms (B. Hube). NK cells are innate lymphocytes with potent cytotoxic activity. In cooperation with the group of Jürgen Löffler activity of NK cells has been demonstrated against the fungal pathogen A. fumigatus. Other groups have shown activity against C. neoformans, but little was known about their role in invasive candidiasis. Primary human NK cells were isolated from buffy coats, primed with a cytokine cocktail and used for confrontation assays with C. albicans. Interaction was monitored and quantified using live cell imaging, confocal microscopy, flow cytometry and Luminex/ELISA. Cytokine-primed primary human NK cells recognised C. albicans, resulting in degranulation and secretion of GM-CSF, IFN-g and TNF-a. Activation of NK cells is triggered by actin-dependent phagocytosis of C. albicans. Although antifungal activity could be detected and attributed to secreted perforin, NK cells were unable to inhibit filamentation of C. albicans. Human PMN potently counteracted the pro-inflammatory reaction of NK cells by preventing a direct contact between Research Group Fungal Septomics HKI Research Report 2012 / 2013 109 Figure 2 NK cells completely engulf C. albicans. To confirm internalisation of C. albicans by human NKC we performed differential staining of extracellular and intracellular fungal cells. Extracellular C. albicans cells were stained with Calcofluor-White before permeabilisation (blue). After permeabilisation, both intra- and extracellular C. albicans were stained using a specific antibody (red). Immune cells were identified as NKC by CD56-positivity (orange) and staining of the actin cytoskeleton (green) further confirmed an embedding of the fungal cells within the NKC. Pictures are representative for cells from three independent experiments using cells from different donors. NK cells and the fungal pathogen. Activation of human PMN was enhanced in the presence of NK cells. Our results clearly assign a function to NK cells in C. albicans infection, which involves both direct pro-inflammatory activation and modulation of PMN activity. For the first time, phagocytosis of a pathogen is shown to contribute to NK cells activation. 3 In/ex vivo infection models All in vivo studies are performed in cooperation with the Research Group Microbial Immunology (I. Jacobsen) using the expertise of the HKI infection models group. To back up in vitro data of the infection model with NK cells and C. albicans a mouse model was established by generating NK cell deficient mice using an anti-NK1.1 antibody. Compared to wild type mice NK cell deficient mice showed prolonged survival and lower pro-inflammatory cytokine levels in blood 110 upon infection with a lethal dose of C. albicans. These data indicate a pro-inflammatory role of NK cells in mice during C. albicans infection. In cooperation with the Research Groups Microbial Biochemistry and Physiology (M. Brock) and Microbial Immunology, we have used the Septomics infrastructure to establish in vivo imaging in murine models of systemic candidiasis. We developed a novel real-time non-invasive bioluminescence imaging technique which allows monitoring of infection progression and determination of fungal burden in vivo in mice. In addition to murine infection models, we work with a human whole blood model to address immune activation in response to fungal pathogens in a situation close to in vivo. While many parameters of host-pathogen interaction are accessible to direct experimental quantification in the whole-blood infection assay, others are not (Fig. 3). To overcome these limitations, in cooperation with the Research Group Applied Systems Biology we generated a virtual HKI Research Report 2012 / 2013 Research Group Fungal Septomics Figure 3 C. albicans predominantly associates with PMN. Representative blood smears of C. albicans-infected blood after 30 min (A), 60 min (B) and 240 min (C) demonstrate association of C. albicans with PMN and continuous filamentation of extracellular fungi. infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modelling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on a priori unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of PMN, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidial activity. Both mechanisms together account for almost 98 % of C. albicans killing. Independent of filamentation and not linked to exhaustion of inactivation of innate immune cells a fraction of C. albicans cells escaped phagocytosis and remained extracellular which may account for the high proportion of dissemination in C. albicans bloodstream infection (Hünniger, Lehnert et al. 2014). Furthermore, in cooperation with Niels Riedemann (InflaRx GmbH, Jena) and the Department Infection Biology (P. Zipfel) we have used the human whole blood model to analyse the impact of C5a inhibition on the immune response against C. albicans. We have found that early PMN activation and effector function against C. albicans clearly rely on C5a – a highly potent pro-inflammatory product, which is generated right upon activation of the complement cascade. 4 Genetic risk factors for fungal infection Fungal Septomics coordinates the AspIRS (Aspergillosis – intrinsic risk stratification) study which is the first systematic approach to identify genetic markers associated with invasive aspergillosis (IA). For this purpose, genome-wide screening for single nucleotide polymorphisms (SNPs) in allogeneic stem cell Research Group Fungal Septomics HKI Research Report 2012 / 2013 111 transplant (SCT) recipients, a well-defined high-risk cohort of patients, is performed in a multi-center approach, including 16 leading clinical partners from Europe and the US. Within AspIRS an unprecedented cohort of >500 cases of IA and >900 control SCT patients has been collected. The resulting data will (i) increase our understanding of the genetic background for IA, (ii) allow the design of diagnostic tools to estimate the intrinsic risk of individual patients and (iii) open new ways to analyse pathogenesis and immune response in IA. In addition to genome-wide analyses, we have used AspIRS samples for confirmation of prior work from our cooperation partner A. Carvalho (Perugia, Italy), who has studied the long pentraxin 3 (PTX3) in IA. PTX3 has a nonredundant role in antifungal immunity as it promotes the control of A. fumigatus infection in vivo. The contribution of SNPs in PTX3 in the development of IA is unknown. We screened an initial cohort of 229 recipients of hematopoietic stem cell transplants and their donors for PTX3 SNPs modifying the risk for developing IA. The analysis was validated in a large multi-center study involving 107 cases of IA and 223 matched controls. The functional consequences of PTX3 SNPs were investigated in vitro and in lung specimens from SCT recipients. A donor homozygous haplotype (h2/h2) in PTX3 increased the risk of infection in the discovery (adjusted hazard ratio, 3.15; P=0.002) and validation (adjusted odds ratio, 2.14; P=0.01) studies and was associated with defective expression of PTX3 in transplant recipients. Functionally, PTX3 deficiency in h2/h2 PMN, presumably due to impaired mRNA stability, led to impaired phagocytosis and clearance of the fungus. Genetic deficiency of PTX3 affects the antifungal capacity of PMN and contributes to risk of IA in SCT recipients (Cunha et al. 2014). 112 HKI Research Report 2012 / 2013 Research Group Fungal Septomics Group members Deutsche Forschungsgemeinschaft DFG KU1540/3-1 Project: CO2 Adaptation in Candida glabrata und ihre Rolle in der Pathogen-Wirt-Interaktion Oliver Kurzai Head Prof. Dr. Oliver Kurzai Phone +49 3641 532-1347 Fax +49 3641 532-2347 [email protected] Deutsche Forschungsgemeinschaft SFB/TR 124 FungiNet Project: Intrinsic Modulation of Neutrophil antifungal Activity against Candida albicans Oliver Kurzai Secretary Silke Pfohl Administrative Manager Dr. Katrin Haupt Selected publications Scientists Dr. Michael Böhringer Dr. Daniela Hellwig (since 02/2013) Dr. Kerstin Hünniger Dr. Ronny Martin Dr. Jessica Voigt (since 05/2013) Dr. Michael Weber (since 06/2013) Dr. Hanna Windecker (until 01/2013) InflaRx GmbH Prof. Dr. Niels Riedemann Prof. Dr. Ren-Feng Guo University Hospital Jena Prof. Dr. Dagmar Barz Prof. Dr. Michael Bauer PD Dr. Ralf Claus Prof. Dr. Michael Kiehntopf Prof. Dr. Hortense Slevogt Leibniz Institute for Age Research – Fritz-Lipmann-Institute, Jena PD Dr. Matthias Platzer Voigt J, Hünniger K, Bouzani M, Jacobsen I, Barz D, Hube B, Löffler J, Kurzai O (2014) Phagocytosis of Candida albicans by human NK Cells results in a proinflammatory response J Inf Dis 209, 616-626. Martin R, Albrecht-Eckardt D, Brunke S, Hube B, Hünniger K, Kurzai O (2013) A core filamentation response network in Candida albicans is restricted to eight genes. PLoS One 8, e58613. Diploma / Bachelor / Master Students Laura Bauer (since 10/2013) Fabian Essig (03/2013–10/2013) Antje Häder Johanna Huyke (since 02/2013) Toni Kaulfuß (until 03/2013) Judith Piegsa (04/2013–08/2013) Ann-Katrin Schmidt (04/2013–08/2013) Steffi Spielberg (10/2013) Cunha C, Aversa F, Lacerda JF, Busca A, Kurzai O, Grube M, Löffler J, Maertens JA, Bell AS, Inforzato A, Barbati E, Almeida B, Santos e Sousa P, Barbui A, Potenza L, Caira M, Rodrigues F, Salvatori G, Pagano L, Luppi M, Mantovani A, Velardi A, Romani L, Carvalho A. (2014) Genetic PTX3 deficiency and aspergillosis in stem-cell transplantation. N Engl J Med (accepted for publication) Research Assistents Cindy Reichmann Collaborations Trainees Josephine Dietrich (07/2013–09/2013) External funding Bundesministerium für Bildung und Forschung Zentren für Innovationskompetenz – Unternehmen Region Zentrum für Innovationskompetenz Septomics – Nachwuchsgruppe Fungal Septomics Oliver Kurzai Bundesministerium für Bildung und Forschung Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) Project: VisIPLI – Visualisierung der Immunzellaktivierung durch Sepsispathogene durch Life-Cell Imaging Oliver Kurzai Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: The interaction of Candida glabrata with human neutrophils Oliver Kurzai AspIRS Study Group (Medizinische Klinik und Poliklinik II, University Hospital Würzburg, Microarray Facility, University Hospital Tübingen, Bundesinstitut für Arneimittelforschung, Bonn, University Hospital Dresden, Jena, Regensburg, Köln, Mannheim, Allgemeines Krankenhaus Wien, Karolinska Institute, Stockholm, Trinity College & Saint James Hospital, Dublin, University of Leuven, Universitätsklinikum Jena, Abteilung Hämatologie und Internistische Onkologie, University Hospital of Wales, Cardiff, Medizinische Universität Innsbruck, University of Perugia, University Hospital of Leiden, Fred Hutchinson Cancer Research Center, Seattle) Julius Maximilians University, Würzburg, Germany Prof. Dr. Jürgen Löffler Prof. Dr. Hermann Einsele University of Bonn, Germany PD Dr.Johannes Schumacher University of Kent, Canterbury, UK Prof. Dr. Fritz Mühlschlegel Radboud University Nijmegen Medical Center, Nijmegen, Netherlands Prof. Dr. Mihai G. Netea University of Perugia Dr. Agostinho Carvalho Prof. Dr. Luigina Romani Research Group Fungal Septomics HKI Research Report 2012 / 2013 113 114 HKI Research Report 2012 / 2013 Research Group Microbial Immunology HKI Research Report 2012 / 2013 115 Research Group Microbial Immunology The independent Research Group Microbial Immunology, established in August 2013, derives from a research group that was formerly integrated in the Department Microbial Pathogenicity Mechanisms. The group investigates pathogenesis and host-pathogen interactions of human pathogenic fungi with a focus on Candida albicans. We aim to elucidate which mechanisms contribute to the outcome of infection by using microbiology, cell biology, biochemistry, infection biology and immunology approaches in combination with complex infection models. We are especially interested in how Candida albicans interacts with the host’s immune system in disseminated infections and which attributes contribute to either protection or disease. In this context, projects address the role of fungal morphology in the development of disease and fungal factors that modulate the host immune response. Recently, we began a project that extends our work to the mechanisms that facilitate translocation of C. albicans from the intestinal tract, which is an essential step in the development of disseminated candidiasis. Further projects address the pathogenesis of infection with less common fungal pathogens, Aspergillus terreus and Lichtheimia species. In 2012 to 2013, our research was supported I N T R O D U C T I O N | E I N LE I T U N G Die unabhängige Forschungsgruppe Mikrobielle Immunologie ist im August 2013 aus einer Arbeitsgruppe innerhalb der Abteilung Mikrobielle Pathogenitätsmechanismen hervorgegangen. Die Forschungsgruppe untersucht die Pathogenese und Wirt-Erreger-Interaktionen humanpathogener Pilze, insbesondere Candida albicans. Um Mechanismen zu identifizieren und zu charakterisieren, die den Infektionsverlauf maßgeblich bestimmen, setzen wir mikrobiologische, zellbiologische, infektionsbiologische und immunologische Methoden ein. Unsere besondere Expertise liegt dabei im Bereich komplexer Infektionsmodelle. Head: Ilse D. Jacobsen, PhD Der Forschungsschwerpunkt der Gruppe Mikrobielle Immunologie liegt in der Un- 116 tersuchung der Interaktion von C. albicans mit dem Immunsystem des Wirtes während disseminierter Infektionen, da diese Interaktionen sowohl zur Protektion als auch zum Fortschreiten der Erkrankung beitragen können. Dabei interessieren uns insbesondere die Rolle der Pilzmorphologie bei der Entstehung von Erkrankungen und Pilzfaktoren, die die Immunantwort des Wirtes modulieren können. Neben disseminierten Infektionen adressieren wir dabei seit kurzem auch die Translokation von C. albicans aus dem Darm, die den initialen Schritt in der Entstehung der disseminierten Candidiose darstellt. Weitere Projekte beschäftigen sich mit der Pathogenese seltener Pilzinfektionen, ausgelöst durch Aspergillus terreus und Lichtheimia-Arten. HKI Research Report 2012 / 2013 Research Group Microbial Immunology by the Jena School for Microbial Communication (JSMC), the Deutsche Forschungsgemeinschaft (DFG), the Studienstiftung des Deutschen Volkes and the Federal Ministry of Education and Research (BMBF). Projects are integrated into the Center for Sepsis Control and Care (CSCC; joined project with MPM) and the DFG Collaborative Research Center / Transregio 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet”. Unsere Forschung wurde in den Jahren 2012 und 2013 finanziell von der Jena School for Microbial Communication (JSMC), der Deutschen Forschungsgemeinschaft (DFG), der Studienstiftung des Deutschen Volkes und dem Bundesministeriums für Bildung und Forschung (BMBF) unterstützt. Wir sind mit unseren Projekten an dem Center for Sepsis Control and Care (CSCC, gemeinsames Projekt mit der Abteilung MPM) und dem DFG Sonderforschungsbereich / Transregio 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet” beteiligt. Research Group Microbial Immunology HKI Research Report 2012 / 2013 117 Scientific Projects 1 The role of morphology for fungal pathogenicity Group Leader: Ilse D. Jacobsen The switch between the spherical yeast and the filamentous hyphal growth form of Candida albicans is a two-step process including the initial formation of germ tubes, subsequent elongation and maintenance of hyphal growth. A complex network of regulatory factors controls the morphological state. 1.1 The role of filamentation in the pathogenesis of candidiasis The ability of C. albicans to switch between yeast and hyphal growth forms is believed to be crucial for virulence during systemic infections and sepsis. In fact, all growth forms, yeast, pseudohyphae and true hyphae are found during systemic infection. In general, mutants locked either in the yeast or hyphal phase are often strongly attenuated in virulence. Although it is clear that hyphal formation is essential for tissue invasion in vitro, the role of filamentation in the pathogenesis of systemic candidiasis remains unknown. However, we found that the filament-deficient mutant eed1∆ is able to cause mortality in the absence of hyphae. This recent finding challenges the long-standing hypothesis that morphogenesis is essential for disseminated candidiasis. We aim to clarify the role of hyphal formation for pathogenesis of disseminated C. albicans infection. Therefore, conditional knock-out or overexpression mutants of EED1 and UME6, regulatory factors supporting the maintenance of hyphal growth, and NRG1, a negative regulator of yeast-tohypha transition, have been constructed using a tetracycline-regulable expression system (Fig. 1). With the help of these strains, the effect of morphology on virulence and immune response is studied in different infection models. Further, in vitro studies with immune cells, endothelial and epithelial 118 cells are performed to understand the role of morphology in the interaction with distinct host cell types and the contribution of morphology to direct vs. immune-mediated tissue damage. This combination of in vitro and in vivo approaches will allow us to dissect the different mechanisms that contribute to the development of disease and will provide insight into the role of filamentation in pathogenesis. 1.2 Eed1 – a regulator of hyphal maintenance The C. albicans EED1 gene plays a crucial role in hyphal extension and maintenance of filamentous growth. Mutants lacking the EED1 gene (eed1∆) do not form true hyphae but only pseudohyphae before switching back into an elongated yeast cell-like growth form (Martin et al. 2011). We observed that this phenotype depends on the inoculum size: eed1∆ cells seeded at moderate cell densities formed shorter filaments than cells seeded at low cell densities. Using filamentation studies we were able to show that this effect is due to the quorum sensing molecule farnesol to which eed1∆ mutant cells are significantly more sensitive than wild type cells. Since it has been described that farnesol especially acts on the cAMP/PKA signalling pathway to inhibit filamentation, the association between EED1 and cAMP signalling is currently under investigation by testing the effect of db-cAMP on cells and constructing overexpression or deletion strains of several factors within this signalling pathway in an eed1∆ mutant background. To provide further insights into the role of Eed1p within the cellular machinery, His-tagged protein versions have been constructed to analyse the protein localisation. Eed1 C-terminal peptides were produced using a heterologous E. coli expression system which are tested in ongoing experiments for interaction with DNA and proteins. In collaboration with Vito Valiante (Department MAM) another expres- HKI Research Report 2012 / 2013 Research Group Microbial Immunology Figure 1 Morphology of C. albicans wild type and regulable mutant strains in vivo. Calcofluor white stained C. albicans cells from murine liver tissue 24 h after intraperitoneal infection. – Dox: Absence of doxycycline, the respective target genes are expressed. + Dox: Doxycyclin added to the drinking water, gene expression is repressed. Repression of Nrg1, a transcriptional repressor of hyphal formation, results in hyperfilamentous growth while the presence of doxycyclin allows NRG1 expression resulting in yeast growth. Doxycyclin represses expression of the positive regulators EED1 or UME6, thus cells grow dominantly in yeast or pseudohyphal-like form. In the absence of doxycyclin EED1 and UME6, respectively, are expressed, resulting in production of true hyphae. sion system involving the yeast Saccharomyces cerevisiae is used to generate full length Eed1 protein for further studies. These experiments will provide valuable information about the possible role of EED1 in C. albicans tolerance to farnesol and contribute to the elucidation of its molecular function in morphogenesis. The projects are supported by the DFG and Studienstiftung des Deutschen Volkes (joined project with MPM). 2 Molecular mechanisms of Candida sepsis Group Leader: Betty Hebecker During systemic infections, C. albicans can infect almost all organs; however, the host response and the ability to clear C. albicans cells is organ-specific. While C. albicans is gradually cleared from liver and spleen, infection progresses in the kidneys due to a delayed, but prolonged influx of neutrophils and is accompanied by a strong cytokine response. The mechanisms underlying these organ-specific differential host responses are not known. We hypothesised that distinct fungal genes elicit an organ-specific host response, which is either protective or non-protective, without directly affecting host cell damage. To identify C. albicans factors or activities which contribute to protective or non-protective host responses, we use large scale host response screening (Fig. 2). Therefore, screening protocols for several host cell lines including hepatocytes, renal, oral and intestinal epithelial cells, and macrophages were established. So far, 1180 defined fungal mutants were screened for their potential to damage renal, oral and intestinal epithelial cells and to induce the expression of Il-6 Research Group Microbial Immunology HKI Research Report 2012 / 2013 119 Figure 2 Molecular mechanisms of Candida sepsis: Project outline. (1) Organ-specific outcome of systemic infection with C. albicans. Approaches to identify fungal factors involved in the organ-specific host responses and outcome: (2) Large-scale host response screening of C. albicans mutants. Mutants with significantly altered induction of cytokines without changes in growth rates, filamentation and cytotoxicity are selected for further analysis. (3) Identification of C. albicans genes that are differentially expressed in different organs. C. albicans RNA is isolated from infected organs using an optimized protocol to perform microarray analysis. and Il-8 in these host cell lines. From this mutant collection 37 knock out mutants could be identified which showed a significantly altered induction of cytokines without changes in growth rates, filamentation and cytotoxicity. These candidate genes can be categorized in three classes: (a) mutants with known reduced virulence potential, which demonstrate the suitability of this approach, (b) mutants causing reduced cytokine induction in all analysed cell lines and (c) mutants which have a host cell type specific phenotype indicating organ specific factors. These genes and their role during systemic C. albicans infection will be further investigated in detail. Another approach to identify niche-specific fungal genes is in vivo transcriptional profiling of C. albicans from different organs. In vivo transcriptional profiling is technically challenging due to low amounts of fungal RNA in the organs. Therefore, we established 120 an efficient RNA isolation protocol, which includes centrifugation and washing steps to separate host and fungal RNA. Preliminary tests using this isolation protocol applied to mouse organs spiked with C. albicans cells allowed the reproducible isolation of good quality RNA in sufficient amounts to perform microarray analysis. We could show that this optimised RNA isolation protocol has no impact on gene expression by analysing the expression of heat shock genes before and after spiking. Analysis of organ samples from infected mice is currently under way. The combination of these two approaches will allow us to detect genes which are both infection-associated and niche-specific and which contribute to organ-specific pathogenesis. The project is performed in close collaboration with the Department MPM and supported by the CSCC (Project D1.15: MomCanSep). HKI Research Report 2012 / 2013 Research Group Microbial Immunology A . te rreus 5 d p. inf. Figure 3 Persistence of A. terreus conidia in immunocompetent mice. Histological sections of murine lung, 5 days after infection with A. terreus SBUG844, PAS-stained. Left panel: arrow indicates immune cell infiltrates; right panel: arrows indicate A. terreus conidia stained in pink; scale bars represent 100µm (left panel) and 10µm (right panel). 3 Pathogenesis of aspergillosis caused by Aspergillus terreus Group Leader: Ilse D. Jacobsen Aspergillus terreus is an emerging life-threatening agent of invasive aspergillosis. In collaboration with Matthias Brock (Research Group Microbial Biochemistry and Physiology) and financially supported by the DFG, two murine infection models were developed to study A. terreus mediated disease. Leukopenic mice showed a similar outcome of infection in comparison to A. fumigatus, though a 102 to 103 fold higher infectious dose was required for 100 % lethality. In surviving animals conidia were found to persist in pulmonary tissue. Contrasting an A. fumigatus infection, a fatty liver degeneration appeared in all animals infected with A. terreus, suggesting production of secondary toxic metabolites. Persistence and subclinical liver damage are unique features of A. terreus invasive aspergillosis in mice (Slesiona et al. 2012a). The murine data implied that the initial disease establishment might be fundamentally different between A. terreus and A. fumigatus. Therefore, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages, one of the initial phagocytes facing inhaled conidia in the lung. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to a higher exposure of β-1,3-glucan and galactomannan on the conidial surface, as confirmed by reduced phagocytosis rates when blocking dectin-1 and the mannose-receptor. Once phagocytosed A. terreus did not inhibit acidification of phagolysosomes, but remained viable, as inert conidia both in vitro and in immunocompetent mice, contrasting A. fumigatus (Fig. 3). The inability of A. terreus to germinate and pierce macrophages resulted in significantly Research Group Microbial Immunology HKI Research Report 2012 / 2013 121 A B lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by bafilomycin, a v-ATPase blocker, increased A. terreus germination rates and cytotoxicity. In summary A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts (Slesiona et al. 2012b). mycosis in Europe, virulence and pathogenesis of this genus has not been addressed in detail yet. Only three of the five species of the genus Lichtheimia have been found to be involved in mucormycosis. Using an alternative infection model based on chicken embryos to screen the virulence potential of the different Lichtheimia species we showed that the three clinically relevant Lichtheimia species, L. ramosa, L. corymbifera and L. ornata, have a higher virulence potential than the species that have only been isolated from the environment. Furthermore, we observed differences in virulence between strains of the same species, independent of their origin (environmental vs. clinical) or phylogenetic position within the species (Schwartze et al. 2012). Figure 4 Lung pathology after infection with L. corymbifera. (A) HE-stained overview of an infected murine lung showing alterations typical for mucormycosis. (B) PASstained area indicated by the box in (A). Arrows indicate fungal hyphae (stained in pink) within the thrombus. H: hemorrhage, T: thrombosis. References Martin R, Moran GP, Jacobsen ID, Heyken A, Domey J, Sullivan DJ, Kurzai O, Hube B (2011) The Candida albicans-Specific Gene EED1 Encodes a Key Regulator of Hyphal Extension. PLoS One 6, e18394. Slesiona S, Ibrahim-Granet O, Olias P, Brock M, Jacobsen ID (2012) Murine infection models for Aspergillus terreus pulmonary aspergillosis reveal long-term persistence of conidia and liver degeneration. J Infect Dis 205, 1268-1277. Slesiona S, Gressler M, Mihlan M, Zaehle C, Schaller M, Barz D, Hube B, Jacobsen ID, Brock M (2012) Persistence versus Escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages. PLoS ONE 7, e31223. Schwartze VU, Hoffmann K, Nyilasi I, Papp T, Vágvölgyi C, de Hoog S, Voigt K, Jacobsen ID (2012) Lichtheimia Species Exhibit Differences in Virulence Potential. PLoS One 7, e40908. 122 4 Pathogenesis of Lichtheimia infections Group Leader: Ilse D. Jacobsen Although Lichtheimia species represent the second to third most common cause of mucor- We selected a L. corymbifera strain of typical virulence in the chicken embryo model to establish murine pulmonary infection models (Fig. 4). Clinical disease developed only in HKI Research Report 2012 / 2013 Research Group Microbial Immunology Group members Head PD Ilse D. Jacobsen, PhD Phone +49 3641 532-1223 Fax +49 3641 532-2223 [email protected] Scientists Dr. Betty Hebecker (together with MPM) Ph.D. Students Christine Dunker (since 06/2013) Tony Pawlik (since 04/2013, together with MPM) Melanie Polke Diploma / Bachelor / Master Students Marcel Sprenger (since 10/2013) Research Assistents Birgit Weber Visiting Scientists Barbara Schaarschmidt University Hospital Jena 03/2013-05/2013 Josephine Fischer Laboratory of Molecular Stem Cell Biology, Westfälische Wilhelms-Universität Münster 11/2013 External funding immunocompromised animals. Acute disease was characterized by respiratory symptoms and extensive necrosis and hemorrhage in the lung. Histology revealed abundant mycelia, angioinvasion and thrombosis. Dissemination from the lung to other internal organs, especially the kidneys, was observed in all animals that became moribund ≥ 6 days post infection. Similar to the lung, abundant mycelia, angioinvasion and thrombosis characterised alterations in the various internal organs. Thus, the pathological alterations in the murine model accurately resemble disease in human patients. We are currently employing this model to analyse whether fungal strains differ in virulence and to determine the immune response to infection. This project is performed in close collaboration with Kerstin Voigt, JMRC. Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Mechanisms of Candida albicans colonization and translocation Ilse Jacobsen Deutsche Forschungsgemeinschaft MorphPath – The Role of morphogenesis in the pathogenesis of systemic candidiasis Ilse Jacobsen Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project C05: Mechanismen der gastrointestinalen Kolonisierung und der Translokation von Candida albicans durch die Darmschleimhaut Ilse Jacobsen Selected publications Lüttich A, Brunke S, Hube B, Jacobsen ID (2013) Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney. PLoS One 8, e64482. Schwartze VU, Hoffmann K, Nyilasi I, Papp T, Vágvölgyi C, de Hoog S, Voigt K, Jacobsen ID (2012) Lichtheimia species exhibit differences in virulence potential. PLoS One 7, e40908. Slesiona S, Ibrahim-Granet O, Olias P, Brock M, Jacobsen ID (2012) Murine infection models for Aspergillus terreus pulmonary Aspergillosis reveal long-term persistence of conidia and liver degeneration. J Infect Dis 205, 1268-1277. Research Group Microbial Immunology HKI Research Report 2012 / 2013 123 Slesiona S, Gressler M, Mihlan M, Zaehle C, Schaller M, Barz D, Hube B, Jacobsen ID, Brock M (2012) Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages. PLoS One 7, e31223. Jacobsen ID, Wilson D, Wächtler B, Brunke S, Naglik JR, Hube B (2012) Candida albicans dimorphism as a therapeutic target. Expert Rev Anti Infect Ther 10, 85-93. Collaborations Max von Pettenkoffer-Institut, LudwigMaximilians-Universität, Munich, Germany Prof. Dr. Frank Ebel Laboratory of Molecular Stem Cell Biology, Medizinische Fakultät, Westfälische WilhelmsUniversität, Münster, Germany Prof. Dr. Frank Rosenbauer Leibniz Institute for Age Research, Fritz-Lipmann-Institute, Jena, Germany Prof. Dr. Falk Weih University Hospital Jena, Germany Prof. Dr. Michael Bauer VIB Department of Molecular Microbiology, K. U. Leuven, Laboratory of Molecular Cell Biology, Belgium Prof. Patrick van Dijck ZIK Septomics, Research Group Host Septomics, Jena, Germany Prof. Dr. Hortense Slevogt 124 HKI Research Report 2012 / 2013 013 Research Group Microbial Immunology Research Group Systems Biology and Bioinformatics HKI Research Report 2012 / 2013 125 Research Group Systems Biology and Bioinformatics The Research Group Systems Biology and Bioinformatics (SBI) is dedicated to the integrated computational analysis of genome, transcriptome, proteome and metabolome as well as microbiological and clinical data. Data analysis comprises the management of heterogeneous large-scale experimental data in a data warehouse, pre-processing, discovery of the main features and patterns by data mining techniques and – finally – prediction of experimentally testable hypotheses. Hypotheses of interest concern molecular interactions between host and pathogen, or between transcription factors and their target genes within the host or the pathogen or the fungus that produces an natural product. The hypotheses have been embedded in predictive mathematical and computational models that represent both prior knowledge available from experts or extracted from biomolecular databases or by text mining in research papers and the pattern found by the computational analysis of experimentally measured data. We developed and applied novel bioinformatic tools that identify the structure and parameter values of network models of gene regulation and signalling. In addition, we developed and applied bioinformatic tools of genome sequence analysis to predict protein coding genes, transcription factor binding sites and secondary metabolite synthesis gene clusters. Fungi are known to produce pharmaceutically important natural products (secondary metabolites, SMs) synthesised by polyketide I N T R O D U C T I O N | E I N LE I T U N G Die Forschungsgruppe Systembiologie und Bioinformatik (SBI) befasst sich mit der bioinformatischen Analyse von genomweiten Hochdurchsatzdaten, wie Genom-, Transkriptom- und Proteom-Daten in Verbindung mit mikrobiologischen und klinischen Daten. Die Datenanalyse umfasst das Management von umfangreichen heterogenen experimentellen Daten in einem „Data Warehouse“, die Vorverarbeitung, die Entdeckung von Mustern in den Daten, sowie schließlich die Vorhersage von experimentell prüfbaren Hypothesen. Interessante Hypothesen betreffen vor allem die Wirt-Pathogen-Interaktion, aber auch molekulare Wechselwirkungen, darunter Transkriptionsfaktor-Zielgen-Beziehungen innerhalb der Wirt- oder der Erregerzelle oder in einem Pilz, der einen Wirkstoff produziert. Die Hypothesen sind Head: Prof. Dr. Reinhard Guthke 126 in mathematischen Modellen formuliert, die einerseits das Vorwissen und andererseits die entdeckten Muster in den experimentell gemessenen Daten widerspiegeln. Wir haben neue bioinformatische Werkzeuge entwickelt und angewandt, die der Identifikation der Struktur und Parameterwerte von Netzwerkmodellen der Genregulation und von Signalwegen dienen. Ferner haben wir bioinformatischen Werkzeuge für die Genomsequenzanalyse entwickelt, die der Vorhersage von Genen, Transkriptionsfaktor-Bindestellen und von Genclustern der SekundärmetabolitSynthese dienen. Pilze sind bekannt als Produzenten von pharmazeutisch wichtigen Naturstoffen (Sekundärmetabolite, SM), die mittels Polyketidsynthetasen (PKS) und Nicht-ribosomalen Peptidsynthetasen synthetisiert werden. Zahlreiche „kryptische“ HKI Research Report 2012 / 2013 Research Group Systems Biology and Bioinformatics synthases (PKS) and nonribosomal peptide synthetases (NRPS). Analyses of sequenced fungal genomes have revealed numerous examples of ‘cryptic’ biosynthetic gene clusters, which can potentially be responsible for the production of the novel SMs. Our work is devoted to the genome-driven detection of new complex natural products in selected fungi and bacteria. Next Generation Sequencing techniques paved the way for advanced genomic and transcriptomic studies, in particular for infection research. We demonstrated how to model the host-pathogen interaction by simultaneous monitoring of gene expression profiles from both the pathogen and the host using RNA-seq technique. In addition, using Biosynthese-Gencluster wurden mittels bioinformatischer Untersuchungen sequenzierter Pilzgenomen gefunden. Unsere Forschung zielt auf die Genom-getriebene Entdeckung neuer komplexer Naturstoffe in ausgewählten Pilzen und Bakterien. Die großen Fortschritte bei der Etablierung neuer Seqenziertechniken (sogenanntes Next Generation Sequencing, z. B. RNA-seq) haben neue Wege für Genom- und Transkriptom-Studien, die besonders wertvoll für die Infektionsforschung sind, eröffnet. Wir konnten zeigen, wie die Wirt-Pathogen-Interaktion mittels eines Netzwerkmodells beschrieben werden kann auf der Grundlage von simultan (mittels RNA-seq) gemessenen Genexressionsprofilen. Außerdem konnten wir mittels RNA-seq-Datenanalyse Pilzgeno- RNA-seq analysis, we established and applied a pipeline for structural fungal genome annotation and improved the genome annotation e.g. for A. fumigatus and C. glabrata. Thus, we found that the transcriptional potential of A. fumigatus was underestimated in the past. The research was done within collaborative networks, such as the graduate schools JSMC and ILRS. The research within the DFG’s Priority Programme SPP1160 ‘Colonisation and Infection by Human-Pathogenic Fungi’ and the newly established Collaborative Research Centre/Transregio 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet” were focused on infections by fungal pathogens, in particular A. fumigatus und C. albicans. me neu annotieren bzw. für Aspergillus fumigatus and Candida glabrata die strukturelle Genomannotation verbessern. Diese Forschungen wurden in interdisziplinärer Zusammenarbeit in Forschungsverbünden wie den Graduiertenschulen JSMC and ILRS durchgeführt. Im DFG-Schwerpunktprogramm SPP1160 „Colonisation and Infection by Human-Pathogenic Fungi“ und im erst kürzlich etablierten Sonderforschungsbereich / Transregio 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet” wurden die Forschungen auf die Infektion mit human-pathogenen Pilzen, insbesondere A. fumigatus und C. albicans konzentriert. Research Group Systems Biology and Bioinformatics HKI Research Report 2012 / 2013 127 Scientific Projects 1 Systems Biology of Fungal Infections Project Leader: Jörg Linde Systems biology of fungal infection comprises all levels of interactions between the pathogen and the host. Here, we aim at describing and analysing the confrontation of the host with fungal pathogens as well as the confrontation of fungal cells with host immune cells of antifungal drugs. We intend to model and to understand the interactions of the host, in particular the immune system of humans, with components of pathogens. Our analysis and models are mainly based on transcriptome data but do also integrate other data as well as current literature knowledge. With help of global transcriptome sequencing, we identified chlamydospore specific markers in Candida albicans and Candida dubliniensis. With the help of microarray expression data we studied the interaction between the soil-derived bacterium Streptomyces rapamycinicus and the human pathogenic fungus Aspergillus fumigatus. Here we found that this interaction leads to the activation of an otherwise silent polyketide synthase gene cluster. For sepsis, the quick identification of the pathogen is crucial to increase the patient’s chances of survival. Currently we are using supervised learning algorithms to analyse the transcriptomic response of human cells to reliably and quickly determine whether the patient is infected by bacteria or fungi. Networks of host-pathogen interactions During the last decade, the group SBI has facilitated transcriptome data to predict mathematical network models which predict regulatory interactions in infection-related conditions. 128 In collaboration with H. Haas (Medical University Innsbruck) and the Department MAM, we reconstructed gene regulatory networks that model the regulation of iron homoeostasis in A. fumigatus (Linde et al. 2012). The network inference was performed by analysis of gene expression profiles as well as soft integration of already known regulatory interactions. The inferred model predicts new interactions between transcription factors and target genes, in particular the activation of the gene expression of hapX by the transcriptional regulator SrbA. This interaction was experimentally verified by northern blots by the collaboration partners. In cooperation with the Department MPM, we predicted regulatory networks of C. albicans under nitrogen starvation as well as after feeding pre-starved cell with proteins (BSA) or arginine. We learnt that C. albicans adopts a particular nitrogen response network, defined by sets of specific gene-gene connections, for each of the environmental condition. In collaboration with the Department MAM, we predicted regulatory networks of A. fumigatus when treated with the antifungal drug Caspofungin. Emergence and spreading of antifungal drug resistance is a growing clinical concern. The RNA-seq based expression data suggests that mpkA and sakA signalling plays an important role for the drug resistance. In order to focus the network modelling on human cells confronted with fungal pathogens we collaborate with the Research Group Fungal Septomics. Here, we predicted a gene regulatory network of MAPK- and NFkBsignalling pathway of epithelial cells infected by C. albicans (Fig. 1). In the future, we will augment this study and analyse these HKI Research Report 2012 / 2013 Research Group Systems Biology and Bioinformatics Candida albicans 97% PTGS2 26% 100% 87% NRAS RIPK1 99% 90% DUSP16 46% DUSP6 91% 94% 63% 99% DUSP1 90% 87% 65% 100% 70% 80% DDIT3 GDF15 99% 86% FLNB GADD45B 88% 91% 90% 99% 58% GADD45A 100% 86% BMP2 MAP3K11 58% 98% 100% FOS 12% 66% 100% DUSP5 50% 76% JUN 100% 49% NFKB1/IA 67% 97% 96% 72% 20% 9% CXCL2 EDN1 35% TRIM25 TICAM1 100% 38% 66% 74% 41% 69% HBEGF 60% CCL20 56% 64% 80% Figure 1 Network model focused on MAPKand NFkB Pathways. The simulation of the network describes the response of intestinal epithelial cells to C. albicans infection. 24 gene nodes (grey boxes indicate transcript decay or autoregulation). Inferred connections are coloured green if prior knowledge was integrated and black without prior knowledge. Edge percentage labels give the frequency of occurrence in the robustness test procedure (from Schulze, 2013). HSPA1A 80% networks of the human immune system confronted with different fungal pathogens. In principal, a molecular model of infection has to comprise interacting molecular networks of both the host and the pathogen. Gene expression analysis by Next Generation Sequencing offers the possibility to monitor the transcriptomic behaviour of host and pathogen simultaneously (Dual RNAseq) and thus, provide data to infer the interaction network of both. A pioneering study for such an integrated systems analysis was done in collaboration with K. Kuchler and coworkers (Medical University of Vienna) and the Department MPM. With the help of our modelling approach we were able to predict a small-scale host-pathogen interaction network (Fig. 2). Improving structural gene prediction of pathogenic fungi by RNA-seq The correct prediction of the localisation of genes in genomes is the most important basis for transcriptomics as well as small-scale experiments, such as knockouts. With the help of RNA-seq data, we are able to identify expressed regions in the genome; which have so far not been indicated as genes. Thus, we can improve the structural genome annotation. In collaborative work with the Department MAM the transcriptome of the wild type and the mpkA mutant of A. fumigatus was analysed using paired-end RNA-seq data and data from four different microarray platforms in comparison with proteome data (Müller et al. 2012). The mutant was investigated to study the regulatory role of the mitogen activated protein kinase MpkA, which has an important function in passing on external cell wall signals. It was demonstrated that by deactivating this kinase, many genes in the pri- Research Group Systems Biology and Bioinformatics HKI Research Report 2012 / 2013 129 Infec&on SOD5 ZPF36 DDR48 HAP3 ALS3 Figure 2 Gene regulatory network inferred from dual RNA-seq data from murin dendritic cells infected with C. albicans. (from Tierney, Linde et al. 2012) References Linde J, Hortschansky P, Fazius E, Brakhage AA, Guthke R, Haas H (2012) Regulatory interactions for iron homeostasis in Aspergillus fumigatus inferred by a Systems Biology approach. BMC Syst Biol 6, 6. Müller S, Baldin C, Groth M, Guthke R, Kniemeyer O, Brakhage AA, Valiante V (2012) Comparison of transcriptome technologies in the pathogenic fungus Aspergillus fumigatus reveals novel insights into the genome and MpkA dependent gene expression. BMC Genomics 13, 519. Wolf T, Shelest V, Shelest E (2013): Motif-based method for genomewide prediction of eukaryotic gene clusters. 2nd International Workshop on Pattern Recognition in Proteomics, Structural Biology and Bioinformatics. In: ICIAP 2013 Workshops, Lecture Notes in Computer Science (LNCS) 8158, Petrosino A, Maddalena L, Pala P (eds), pp. 389–398, Springer-Verlag, Berlin, Heidelberg. 130 CITED2 PTX3 MTA2 FRE10 LIF C. albicans M. musculus mary and secondary metabolism are consequently regulated. In 2013, we implemented a pipeline for structural genome annotation which can be used for re-annotation as well as completely new annotation. This pipeline integrates eleven different tools for structural genome annotation. It has been used for re-annotation of A. fumigatus, C. glabrata as well as de novo annotation of the basal fungal pathogens Lichtheimia corymbifera and Lichtheimia ramosa. 2 RGS1 Genome mining, transcription factors and secondary metabolites in fungi Project Leader: Ekaterina Shelest For prediction of SM genes’ functionality it is essential to know which genes belong to the cluster besides the backbone enzymes. To this end, we have developed a novel non-sim- ilarity based method for the prediction of SM gene clusters in fungal genomes. The motif density method (MDM) is based on the statistical over-representation of binding sites for cluster-specific regulators (Wolf et al. 2013). Importantly, MDM not only allows to reliably predict the SM clusters but also provides information about the potential regulators of the cluster by description of their binding sites. The methods of genome mining and comparative genomics allow to predict the approximate product structure based on the genes’ similarity and/or their domain structure. However, in fungi the amino acid selectivity can be hardly deduced from the adenylation domain sequence and remains a challenging problem. By methods of comparative analysis we showed that the signature for the amino acid selectivity can be found for aromatic amino acids, but not for aliphatic ones; moreover, in PKS-NRPS hybrids the amino HKI Research Report 2012 / 2013 Research Group Systems Biology and Bioinformatics acid selectivity is additionally influenced by the condensation (C) domain (Boettger et al. 2012). We could also infer a non-canonical function of C-terminal C domains in truncated PKS–NRPS homologues. Moreover, sequence and phylogenetic analyses revealed an evolutionary imprint of the PKS–NRPS domains, which reflect the evolutionary history of the entire megasynthase. Transcription factors (TFs) are in the other focus of our research. Families of TFs are not distributed evenly across the fungal kingdom. Combining the information about the expansions and losses of the TF families in different genomes with the specific biological traits of the species, we obtain a valuable source of insights into the evolutionary history and functional importance of particular features, in which the TFs are involved. This is of special value when we discuss such features as virulence of pathogenic fungi, or specific traits of symbionts, adaptation to a particular niche, etc. 3 Gene regulatory network inference Project Leader: Reinhard Guthke Systems biology aims to model living systems to uncover the interaction of molecules, cells, tissues, organs and organisms by dynamic networks. The research of the SBI group has a longstanding tradition in the field of gene expression data-based network modelling of gene regulation and signalling. In 2012 and 2013, we published three novel tools for network modelling. First, Vlaic et al. (2012) presented the ExTILAR approach that combines the advantages of the regression-based inference algorithm TILAR (Transcription factor binding sites Integrating Least Angle Regression) with the advantages of dynamic network modelling based on ordinary differential equations (ODE). TILAR is able to infer large scale networks, whereas ODE-based models allow process simulation of e.g. in silico knockdown experiments. Initially, the novel tool ExTILAR was applied for dynamic modelling of the TF network regulating the adaptation of murine hepatocytes to changed nutrition. Second, NetGenerator 2.0, a new version of the in-house ODE-based network inference tool was developed, assessed and published (Weber et al. 2013a). The tool has many new features, such as to process experimental data of multiple experiments, to model the influence of multiple external signals and to softly integrate prior knowledge. It was applied for various tasks, in particular to model the gene regulation of the fungi A. fumigatus and C. albicans (see above: Networks of hostpathogen interactions). For the first time, we studied the influence of four different stimuli on synovial fibroblast of rheumatoid arthritis patients. In addition, we modelled the dynamic influence of microRNA on the differentiation of human mesenchymal stem cells (Weber et al. 2013b). Third, GRN2SBLM is the name of the third tool developed by the SBI group (Vlaic et al. 2013). It is dedicated to the automated encoding and annotation of inferred gene regulatory networks complying with SBML, the international standard for the storage, exchange and use of quantitative models by the systems biology markup language. By constructing correlation-based difference networks of gene regulation and signalling, we identified those signalling pathways that are most vigorously changed by impaired glycolysis in the roundworm Caenorhabditis elegans (Priebe et al. 2013). Thus, we found genes and pathways that are potentially involved in the extension of life span of this model organism in ageing research. Research Group Systems Biology and Bioinformatics HKI Research Report 2012 / 2013 References Boettger D, Bergmann H, Kuehn B, Shelest E, Hertweck C (2012) Evolutionary imprint of catalytic domains in fungal PKS-NRPS hybrids. ChemBioChem 13, 2363-2373. Vlaic S, Schmidt-Heck W, Matz-Soja M, Marbach E, Linde J, Meyer-Baese A, Zellmer S, Guthke R, Gebhardt R (2012) The extended TILAR approach: a novel tool for dynamic modeling of the transcription factor network regulating the adaption to in vitro cultivation of murine hepatocytes. BMC Syst Biol 6, 147. Weber M, Henkel SG, Vlaic S, Guthke R, van Zoelen EJ, Driesch D (2013) Inference of dynamical gene-regulatory networks based on time-resolved multi-stimuli multi-experiment data applying NetGenerator V2.0. BMC Syst Biol 7, 1. Weber M, Sotoca AM, Kupfer P, Guthke R, van Zoelen EJ (2013) Dynamic modelling of microRNA regulation during mesenchymal stem cell differentiation. BMC Syst Biol 7, 124. Vlaic S, Hoffmann B, Kupfer P, Weber M, Drager A (2013) GRN2SBML: automated encoding and annotation of inferred gene regulatory networks complying with SBML. Bioinformatics 29, 2216-2217. Priebe S, Menzel U, Zarse K, Groth M, Platzer M, Ristow M, Guthke R (2013) Extension of life span by impaired glucose metabolism in Caenorhabditis elegans is accompanied by structural rearrangements of the transcriptomic network. PLoS One 8, e77776. Tierney L, Linde J, Müller S, Brunke S, Molina JC, Hube B, Schock U, Guthke R, Kuchler K (2012) An interspecies regulatory network inferred from simultaneous RNA-seq of Candida albicans invading innate immune cells. Front Microbiol 3, 85. 131 Group members External funding Head Prof. Dr. Reinhard Guthke Phone +49 3641 532-1083 Fax +49 3641 532-0803 [email protected] Bundesministerium für Bildung und Forschung GERONTOSYS II: Rolle von oxidativen Stress in der Alterung und therapeutische Implikationen Reinhard Guthke Bundesministerium für Bildung und Forschung Jenaer Centrum für die Systembiologie des Alterns – JenAge: Systembiologie von mildem Stress beim gesunden Altern ein Multi-Spezies-Ansatz Reinhard Guthke Scientists Dr. Ekaterina Shelest Dr. Uwe Menzel Wolfgang Schmidt-Heck Dr. Jörg Linde Eugen Fazius (until 10/2012) Dr. Fabian Horn (since 09/2012) Dr. Sebastian Müller (06/2012–10/2012) Jonas Schaer (since 02/2012) Philipp Kämpfer (since 10/2012) Janine Freitag (since 11/2012) Bundesministerium für Bildung und Forschung ERASysBio – LINCONET Project: Modellierung des genregulatorischen Netzwerks für die Linien-spezifische Differenzierung in menschlichen mesenchymalen Stammzellen: Identifikation von potenziellen Zielmolekülen für die anabole Therapie von Gewebserkrankungen Reinhard Guthke Ph.D. Students Robert Altwasser Fabian Horn (until 08/2012) Peter Kupfer Sebastian Müller (until 05/2012) Steffen Priebe (until 06/2013) Sylvie Schulze (since 12/2013) Sebastian Vlaic Michael Weber Thoms Wolf (since 04/2012) Andreas Dix (since 06/2012) Müller S, Baldin C, Groth M, Guthke R, Kniemeyer O, Brakhage AA, Valiante V (2012) Comparison of transcriptome technologies in the pathogenic fungus Aspergillus fumigatus reveals novel insights in genome structure and MpkA-dependent gene expression. BMC Genomics 13, 519. Vlaic S, Schmidt-Heck W, Matz-Soja M, Marbach E, Linde J, Meyer-Baese A, Zellmer S, Guthke R, Gebhardt R (2012) The Extended TILAR Approach: A novel tool for dynamic modeling of the transcription factor network regulating the adaption to in vitro cultivation of murine hepatocytes. BMC Syst Biol 6, 147. Collaborations Bundesministerium für Bildung und Forschung Kompetenznetz Die Virtuelle Leber Reinhard Guthke Diploma / Bachelor / Master Students Jonathan Adam Martin Bens Thomas Brockmöller Andreas Dix Janine Freitag Bianca Hoffmann Phillip Kämpfer Sylvie Schulze Thomas Wolf Maximilian Fichtner Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Network Model Fungi – Systems biology of human pathogenic fungi: Modeling transcriptional networks of virulence Reinhard Guthke Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Transcriptome data analysis and modeling of Aspergillus fumigatus – phagocytes interaction Reinhard Guthke Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project B03: Prädiktive Modellierung von WirtPathogen Interaktionen durch Rekonstruktion genregulatorischer Netzwerke Reinhard Guthke, Ekaterina Shelest Research Assistants Sylvie Schulze Carolin Zitzmann (until 08/2012) Vladimir Shelest Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project INF: Integrierte Datenbank für Experimentaldaten (Data Warehouse OmniFung) Reinhard Guthke Trainees Sylvie Schulze (since 10/2012) Christian Kreisel (since 10/2012) European Union Framework Programme 7: Marie Curie Action International Research Staff Exchange Scheme KRAB-ZNF – KRAB zinc finger gene biology in evolution and disease Reinhard Guthke Visiting Scientists Himanshu Manchanda Friedrich Schiller University Jena since 02/2012 Laetitia Muszkieta Institut Pasteur, Laboratoire des Aspergillus, Paris 05/2013 José Muñoz Universidad del Rosario, Bogotá, Medellin, Colombia 06/2012 Linde J, Hortschansky P, Fazius E, Brakhage AA, Guthke R, Haas H (2012) Regulatory interactions for iron homoeostatis in Aspergillus fumigatus inferred by a Systems Biology approach. BMC Syst Biol 6, 6. BioControl Jena GmbH, Jena, Germany Sebastian Henkel Dominik Driesch Florida State University, USA Prof. Dr. Anke Meyer Baese Innsbruck Medical University, Innsbruck, Austria Prof. Dr. Hubertus Haas Medical University of Vienna, Austria Prof. Dr. Karl Kuchler Radboud University Nijmegen, Nijmegen, The Netherlands Prof. Dr. Joop van Zoelen University Hospital Jena, Germany Prof. Dr. Raimund Kinne Prof. Dr. Thomas Kamradt Prof. Dr. Utz Settmacher University of Birmingham, U.K. Prof. Dr. Francesco Falciani University of Leipzig, Germany Prof. Dr. Rolf Gebhard University of Rostock, Germany Prof. Dr. Hans-Jürgen Thiesen Tongji University, Shanghai, China Prof. Dr. Cao Zhiwei Technical University München, Germany Prof. Dr. Andreas Kremling Selected publications Weber M, Henkel SG, Vlaic S, Guthke R, van Zoelen EJ, Driesch D (2013) Inference of dynamical gene-regulatory networks based on time-resolved multi-stimuli multi-experiment data applying the NetGenerator algorithm V 2.0. BMC Syst Biol 7, 1. Vlaic S, Hoffmann B, Kupfer P, Weber M, Dräger A (2013) GRN2SBML: Automated encoding and annotation of inferred gene regulatory networks complying with SBML. Bioinformatics 29, 2216-2217. 132 HKI Research Report 2012 / 2013 Research Group Systems Biology and Bioinformatics Junior Research Group Secondary Metabolism of Predatory Bacteria HKI Research Report 2012 / 2013 133 Junior Research Group Secondary Metabolism of Predatory Bacteria Considering the quick spread of bacterial resistance against antibiotics and the increasing emergence of highly invasive pathogens, the discovery of new antiinfective drugs is urgently needed. Natural products are structurally highly diverse compounds that have been selected during evolution for their affinity to biological targets. Therefore, natural products are ideal candidates for drug discovery. Genomic analyses illuminated the huge proficiency of microorganisms for natural product biosynthesis. Moreover, it became clear that the potential of these organisms for the production of antibiotics is far from being exhausted (Nett 2014). Bacteria and fungi that have as yet been ignored in drug discovery programs are of particular interest in this context, as they provide fresh opportunities for the identification of bioactive molecules. The research program of the Junior Research Group Secondary Metabolism of Predatory Bacteria (SRB) is centred on the exploitation of such neglected microbial resources, focusing mainly on two bacterial groups: •Predatory bacteria, such as Cupriavidus necator or Herpetosiphon aurantiacus, can feed on other microbes under conditions when nutrients become scarce. Available genome sequences show that preda- I N T R O D U C T I O N | E I N LE I T U N G Aufgrund der raschen Ausbreitung von Antibiotika-resistenten Bakterien und dem zunehmenden Auftreten von hochinvasiven Krankheitserregern, besteht ein dringender Bedarf neue antimikrobielle Wirkstoffe zu identifizieren. Naturstoffe umfassen strukturell höchst unterschiedliche Verbindungen, die im Verlauf der Evolution auf ihre Affinität zu biologischen Zielstrukturen selektiert worden sind. Aus diesem Grund sind Naturstoffe ideale Kandidaten bei der Suche nach neuen Wirkstoffen. Genomische Analysen haben das enorme Leistungsniveau von Mikroorganismen für die Biosynthese von Naturstoffen offengelegt. Zudem stellte sich heraus, dass das Potential dieser Organismen zur Produktion von Antibiotika bei weitem nicht ausgeschöpft wurde (Nett 2014). Von Head: Dr. Markus Nett 134 besonderem Interesse sind dabei Bakterien und Pilze, die bislang in der Wirkstoffsuche ignoriert wurden, da sie neue Chancen für die Findung bioaktiver Moleküle bieten. Das Forschungsprogramm der Nachwuchsgruppe Sekundärmetabolismus räuberischer Bakterien (SRB) hat die Nutzbarmachung dieser brachliegenden mikrobiellen Ressourcen zum Ziel. Im Fokus des Interesses stehen dabei zwei bakterielle Gruppen: •Räuberische Bakterien, wie Cupriavidus necator oder Herpetosiphon aurantiacus, können im Falle eines Nahrungsmangels Jagd auf andere Mikroorganismen machen. Verfügbare Genomsequenzen zeigen, dass viele räuberische Bakterien spezifische HKI Research Report 2012 / 2013 Junior Research Group Secondary Metabolism of Predatory Bacteria tory bacteria possess specific biosynthetic pathways that are not found in other organisms. Furthermore, there is growing evidence that many of the secreted natural products contribute to the predatory activity, e.g., in the form of signalling molecules or antibiotics, which promote the killing of the prey. From a pharmaceutical perspective, predatory bacteria are hence a promising source for finding new medicinal drugs. lanacearum, demonstrated that the expression of several biosynthesis genes responds to virulence-associated regulators, assuming a role of natural products in the infection process (Schneider et al. 2009). In the context of drug discovery, secondary metabolites from plant pathogenic bacteria can be valuable lead structures for the development of antibiotics or anticancer agents, as exemplified by micacocidin and rhizoxin. References Nett M (2014) Genome mining – concept and strategies for natural product discovery. Prog Chem Org Nat Prod, in press. •Plant pathogenic bacteria are, with few exceptions, barely characterised with regard to their chemistry. Recent studies, e. g., in the tomato pathogen Ralstonia so- Stoffwechselwege für die Biosynthese von Naturstoffen besitzen, die sich nicht in anderen Produzentengruppen finden. Zudem gibt es vermehrt Hinweise, dass die sezernierten Naturstoffe zur räuberischen Lebensweise beitragen, z. B. in Form von Signalstoffen oder antimikrobiellen Verbindungen, die das Töten der Beute begünstigen. Aus pharmazeutischer Sicht sind räuberische Bakterien damit eine vielversprechende Quelle für die Entdeckung neuer Wirkstoffe. arum, haben gezeigt, dass die Expression zahlreicher Biosynthesegene durch Virulenz-assoziierte Regulatoren kontrolliert wird, was eine Funktion von Naturstoffen im Rahmen des Infektionsprozesses nahe legt (Schneider et al. 2009). Für die Nutzung von pflanzenpathogenen Bakterien als Wirkstoffquelle spricht, dass diese Produzentengruppe bereits einige sehr vielversprechende Leitstrukturen für Antibiotika und Zytostatika geliefert hat. Beispiele sind das Micacocidin und das Rhizoxin. •Pflanzenpathogene Bakterien sind von wenigen Ausnahmen abgesehen bislang nur unzureichend chemisch charakterisiert. Neuere Untersuchungen, u.a. in dem Tomaten-Schaderreger Ralstonia solanace- Junior Research Group Secondary Metabolism of Predatory Bacteria HKI Research Report 2012 / 2013 135 Scientific Projects 1 Natural products from the predatory bacterium Cupriavidus necator and related Cupriavidus species Anecdotal evidence suggests a correlation between the predatory activity of Cupriavidus necator and natural product biosynthesis. In the presence of prey organisms, C. necator secretes a copper-binding peptide, which might be used to deliver toxic amounts of the transition metal to the prey cells. Moreover, the same peptide was also proposed to play an important role in the interaction with the actinomycete Agromyces ramosus. The latter is itself a microbial predator and spreads via the formation of mycelia. Once a mycelial contact is established with potential prey, the actinomycete starts to secrete lytic enzymes in order to feed on these organisms. Interestingly, A amosus was also found to attack C. necator, but in this particular case, the assaulted cells struck back. The counterattack coincided with the release of the aforementioned copper-binding molecule and led to a complete eradication of the A. ramosus mycelium. Notwithstanding its biological significance, the identity of the copper-binding peptide was not resolved. The objective of the junior research group is thus to find this potential antibiotic and to determine its structure. Using a genome mining strategy, a gene cluster was identified in the genome of the C. necator strain H16 that could likely code for the biosynthesis of the molecule sought. The candidate locus contained the genes for the nonribosomal assembly of a peptide and it also included genes involved in the formation of bidentate ligand groups, which would be required for the coordination of metal ions. To retrieve the metabolic product of the cluster, one of the core biosynthesis genes was disrupted through insertional mutagenesis. Comparative profiling of mutant and 136 wildtype strain revealed several peaks that were completely absent in the mutant (Fig. 1). The corresponding compounds were isolated and the planar structure of the main metabolite, cupriachelin, was elucidated by NMR spectroscopy. The stereochemistry of this lipopeptide was solved following chemical derivatisation. A model for the biosynthesis of cupriachelin was subsequently deduced from bioinformatic analyses and the biochemical characterisation of adenylation domain specificity (Kreutzer et al. 2012a). Since the predatory activity of C. necator cannot be detected in established assays, the group also developed a new predation test system. The latter relies on the co-cultivation of predatory bacteria with prey organisms producing distinctive pigments and the subsequent enumeration of the individual populations at defined time points. Using the new assay, the predatory activity of C. necator could be confirmed. However, cupriachelindeficient mutants of C. necator H16 were not affected in their capability to feed on prey bacteria, indicating that cupriachelin is not essential for predation. This result was further corroborated by comparative genomics, which revealed that the cupriachelin biosynthesis genes are restricted to C. necator strains originating from freshwater environments. Subsequent studies showed a preference of cupriachelin for the chelation of ferric iron. The resulting complex is prone to photolytic degradation, which comes along with the release of ferrous iron. This finding suggests an ecological role for cupriachelin in supplying ferrous iron to planktonic communities in exchange against organic carbon (Kreutzer et al. 2012a). In a follow-up study, a cupriachelin-like gene cluster was discovered in the genome of the bacterium Cupriavidus taiwanensis LMG19424. The structure of the encoded lipo- HKI Research Report 2012 / 2013 Junior Research Group Secondary Metabolism of Predatory Bacteria Figure 1 Structure of cupriachelin (A). Metabolic profiles of the C. necator H16 wildtype (profile a) and a cupriachelin-deficient mutant strain (profile b). The metabolites 1-3 represent cupriachelin congeners, which possess differing acyl chains (B). peptide could be predicted with sufficient accuracy from the molecular assembly line to enable an NMR-assisted isolation. This study hence represents the first example of a genome mining attempt that relied on the readout of distinctive NMR resonances (Kreutzer et al. 2012b). Similar to cupriachelin, the isolated taiwachelin was shown to undergo a photoreductive dissociation, when an aqueous solution of its ferric iron complex was exposed to sunlight. 2 Natural products from the predatory bacterium Herpetosiphon aurantiacus The genome of the predatory bacterium Herpetosiphon aurantiacus harbours a plethora of genes that are predicted to be involved in natural product assembly. Although a total of 20 biosynthetic gene clusters had been identified in a preceding study (Kiss et al. 2011), their metabolic products remained elusive, most likely due to cultivation issues of this filamentous microorganism. To retrieve the natural products of H. aurantiacus, we joined forces with Martin Roth (Bio Pilot Plant) to optimise the fermentation conditions. A systematic evaluation of different cultivation parameters in combination with comparative NMR analyses led to the discovery of a new diterpene featuring an octahydro-1H-indenyl skeleton. The isolated compound, herpetopanone, bears structural resemblance to the plant-derived cadinane-type sesquiterpenes, which are completely unprecedented in bacteria. Motivated by this finding, we continued to alter the herpetopanone production medium and to analyse the metabolic effects. In this way, two further natural products could be identified, namely the nucleoside antibiotic futalosine and the nonribosomal peptide synthetase-derived niccicin. While the former had been previously described as the intermediate of an alternative mena- Junior Research Group Secondary Metabolism of Predatory Bacteria HKI Research Report 2012 / 2013 137 Figure 2 Structurally diverse natural products from the predatory bacterium Herpetosiphon aurantiacus. References Schneider P, Jacobs JM, Neres J, Aldrich CC, Allen C, Nett M, Hoffmeister D (2009) The global virulence regulators VsrAD and PhcA control secondary metabolism in the plant pathogen Ralstonia solanacearum. ChemBioChem 10, 2730-2732. Kreutzer MF, Kage H, Nett M (2012a) Structure and biosynthetic assembly of cupriachelin, a photoreactive siderophore from the bioplastic producer Cupriavidus necator H16. J Am Chem Soc 134, 5415-5422. Kreutzer MF, Nett M (2012b) Genomics-driven discovery of taiwachelin, a lipopeptide siderophore from Cupriavidus taiwanensis. Org Biomol Chem 10, 9338-9343. 138 quinone pathway, niccicin represents a new compound (Fig. 2). Efforts are now underway to determine the absolute stereochemistry of herpetopanone and niccicin, as well as their biological effects. In addition, we could verify that H. aurantiacus possesses pathways to specific biosynthetic building blocks, such as the non-proteinogenic amino acid 4-hydroxyphenylglycine, which is known as a constituent of glycopeptide antibiotics. In collaboration with the Research Group Systems Biology and Bioinformatics (Reinhard Guthke), we confirmed that the respective 4-hydroxyphenylglycine pathway in H. aurantiacus was not acquired by horizontal gene transfer. According to a phylogenetic reconstruction, it arose by intraspecies gene duplication and concomitant, mutation-induced neofunctionalization (Kastner et al. 2012). 3 Natural Products from the plant pathogenic bacterium Ralstonia solanacearum Although Mycoplasma pneumoniae is actually considered the leading cause of community-acquired pneumonia, the treatment of its infections is still restricted to few antibiotics. In recent years, a dramatic resistance development was observed for this bacterium, underscoring the desperate need for new effective drugs. Micacocidin is a thiazoline-containing natural product that exerts strong and selective inhibitory effects in the nanomolar range against M. pneumoniae. In the previous reporting period, we discovered the micacocidin biosynthesis gene cluster in the plant pathogenic bacterium Ralstonia solanacearum and, together with the group of Hans Peter Saluz (Department CMB), a siderophore function was demonstrated for this molecule in cellular uptake studies (Kreutzer et al. 2011). Subsequent testing re- HKI Research Report 2012 / 2013 Junior Research Group Secondary Metabolism of Predatory Bacteria Figure 3 Modular biosynthesis enzymes for the production of micacocidin. Domain architecture of the micacocidin (Mic) assembly line. Domains that likely account for the structural differences to yersiniabactin (Ybt) are highlighted in blue (A). Chemical structures of micacocidin and yersiniabactin (B). vealed that the antibiotic effect of micacocidin is not linked to its metal-chelating properties. An iron(III)-loaded complex exhibited the same level of activity as the free ligand. Aiming at the engineering of new analogs for structure-activity relationship studies, we then set out to unravel the biosynthesis of micacocidin. The chemical structure of micacocidin is reminiscent of the natural product yersiniabactin, which is also reflected in a largely conserved enzymatic assembly line (Fig. 3). Notwithstanding the close relatedness of the two molecules, the minor variations apparently have important consequences in terms of antibiotic activity, as yersiniabactin is a much weaker antimycoplasma drug than micacocidin. The molecular basis for the subtle, yet biologically significant, structural differences were not clear. In particular, the origin of the distinctive pentylphenol moiety in micacocidin was puzzling, and it was thus interrogated by a combination of stable isotope feeding experiments, reconstitution of biosynthetic steps, and targeted inactivation of catalytic domains. These analyses pointed to an iterative type I polyketide synthase (iPKS) as the key enzyme in the construction of the aromatic residue. The iPKS was shown to generate a linear tetraketide intermediate from acyl carrier protein-tethered hexanoic acid by three consecutive, decarboxylative Claisen condensations with malonyl-coenzyme A. The final conversion into the pentylphenol building block, however, depends on a ketoreductase domain within the enzyme (Fig. 4), as demonstrated by heterologous expression in E. coli and subsequent sitedirected mutagenesis experiments (Kage et al. 2013). It should be noted that the characterized iPKS from R. solanacearum is totally unprecedented in bacteria. Not only is the iteratively acting enzyme integrated into a multimodular assembly line, but it seems mechanistically also related to partially re- Junior Research Group Secondary Metabolism of Predatory Bacteria HKI Research Report 2012 / 2013 References Kiss H, Nett M, Domin N, Martin K, Maresca JA, Copeland A, Lapidus A, Lucas S, Berry KW, Glavina Del Rio T, Dalin E, Tice H, Pitluck S, Richardson P, Bruce D, Goodwin L, Han C, Detter JC, Schmutz J, Brettin T, Land M, Hauser L, Kyrpides NC, Ivanova N, Goker M, Woyke T, Klenk HP, Bryant DA (2011) Complete genome sequence of the filamentous gliding predatory bacterium Herpetosiphon aurantiacus type strain (114-95(T)). Stand Genomic Sci 5, 356-370. Kastner S, Müller S, Natesan L, König GM, Guthke R, Nett M (2012) 4-Hydroxyphenylglycine biosynthesis in Herpetosiphon aurantiacus: a case of gene duplication and catalytic divergence. Arch Microbiol 194, 557-566. 139 Figure 4 Initial steps in micacocidin assembly leading to the formation of the 6-pentylsalicylate starter unit (A). A mutation in the KR domain of the iPKS results in a premature offload of a shunt product due to lactonisation (B). References Kreutzer MF, Kage H, Gebhardt P, Wackler B, Saluz HP, Hoffmeister D, Nett M (2011) Biosynthesis of a complex yersiniabactin-like natural product via the mic locus in phytopathogen Ralstonia solanacearum. Appl Environ Microbiol 77, 6117-6124. Kage H, Kreutzer MF, Wackler B, Hoffmeister D, Nett M (2013) An iterative type I polyketide synthase initiates the biosynthesis of the antimycoplasma agent micacocidin. Chem Biol 20, 764-771. Kreutzer MF, Kage H, Herrmann J, Pauly J, Hermenau R, Müller R, Hoffmeister D, Nett M (2014) Precursor-directed biosynthesis of micacocidin derivatives with activity against Mycoplasma pneumoniae. Org Biomol Chem 12, 113-118. 140 ducing polyketide synthases from fungi, which utilise programmed keto reduction to direct the biosynthesis toward a defined, aromatic product. The possibility to independently reconstitute the function of this enzyme and to heterologously produce 6-pentylsalicylate may ultimately prove useful in the field of combinatorial biosynthesis. We now plan to introduce clickable groups into micacocidin by directed feeding and genetic engineering, respectively, to set the stage for target fishing studies. Together with the group of Dirk Hoffmeister (PB), we then probed the substrate specificity of the hexanoic acid-loading enzyme. A biochemical analysis revealed an extended plasticity, which was exploited for the generation of new micacocidin derivatives by precursor-directed biosynthesis (Kreutzer et al. 2014). The engineered compounds were profiled against M. peumoniae in collaboration with the group of Rolf Müller (Helmholtz Institute for Pharmaceutical Research Saarbrücken), yielding valuable insights with regard to the structural changes that are tolerable in terms of antibiotic activity. HKI Research Report 2012 / 2013 Junior Research Group Secondary Metabolism of Predatory Bacteria Group members Selected publications Head Dr. Markus Nett Phone +49 3641 532-1297 Fax +49 3641 532-2297 [email protected] Kage H, Kreutzer MF, Wackler B, Hoffmeister D, Nett M (2013) An iterative type I polyketide synthase initiates the biosynthesis of the antimycoplasma agent micacocidin. Chem Biol 20, 764-771. Scientists Dr. Hirokazu Kage Dr. Soledad Vela Gurovic (until 03/2012) Ph.D. Students Nicole Domin (until 06/2013) Martin Kreutzer (unitl 11/2013) Xinli Pan (since 10/2013) Sebastian Schieferdecker Ivana Seccareccia Kreutzer MF, Kage H, Nett M (2012) Structure and biosynthetic assembly of cupriachelin, a photoreactive siderophore from the bioplastic producer Cupriavidus necator H16. J Am Chem Soc 134, 5415-5422. Diploma / Bachelor / Master Students Juliane Korp (since 07/2013) Colette Kurth (since 09/2013) Tom Lauterbach (since 10/2013) Bastien Schnell (02/2013–07/2013) Research Assistants Julia Greßler (until 09/2012) Trainees Ron Hermenau (03/2013–03/2013) Juliane Korp (04/2012–07/2012) Colette Kurth (04/2013–05/2013) Franziska Müller von Klingspor (09/2013–09/2013) Sebastian Peil (07/2013–08/2013) Martin Peschel (03/2013–03/2013) Christiane Ring (08/2013–08/2013) Marko Rodewald (09/2013–10/2013) Sophie Seng (02/2013–03/2013) Collaborations University of Potsdam, Germany Prof. Dr. Elke Dittmann Scripps Institution of Oceanography and the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, USA Prof. Dr. Bradley S. Moore Helmholtz-Institut für Pharmazeutische Forschung Saarland (HIPS), Helmholtz-Zentrum für Infektionsforschung (HZI), Institute of Pharmaceutical Biology, University of Saarland, Germany Prof. Dr. Rolf Müller University Hospital Jena, Germany PD Dr. Michaela Schmidtke External funding Bundesministerium für Bildung und Forschung DiNaMid – Genom-basierte Findung neuer antimikrobieller Naturstoffe in mikrofluidischen Chips; Identifizierung neuer Stoffwechselwege und Naturstoff-Isolierung Markus Nett, Martin Roth Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Molecular basis fror predatory interactions of Cupriavidus necator wich ist prey bacteria Markus Nett Junior Research Group Secondary Metabolism of Predatory Bacteria HKI Research Report 2012 / 2013 141 142 HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013 143 Cross-sectional Unit Bio Pilot Plant The research of the HKI Bio Pilot Plant aims at the development and optimisation of biotechnological processes of natural product producers, including strain development and optimisation. The Bio Pilot Plant offers opportunities for the cultivation of a wide spectrum of microorganisms (bacteria, lower and higher fungi, genetically modified organisms, safety group 1 and 2) from flask cultures via laboratory bioreactor to pilot scale fermenter of 2.5 m³ that are unique in the German academic area. All necessary purification processes and the appropriate technical equipment for well-performed purifications of natural products, proteins and biopolymers are available. The development of analytical methods is mainly focused on process-related parameters and the analysis of synthesised products. We are experienced in the cultivation of microorganisms in the chemostat, under anaerobic fermentation conditions, and at high cell densities. The establishment of a microfluidic platform for ultra-high-throughput screening of antimicrobial natural products and enzymes is an example for the development of innovative technologies. The Bio Pilot Plant closely collaborates with the departments and junior research groups of the HKI, institutes of the Friedrich Schiller University, and the Ernst Abbe University of Applied Sciences. I N T R O D U C T I O N | E I N LE I T U N G Die Forschungsarbeiten des Biotechnikum sind auf die Entwicklung und Optimierung bio technologischer Prozesse von Naturstoff-Produzenten einschließlich der Stamm optimierung fokussiert. Das Biotechnikum bietet im deutschsprachigen, akademischen Bereich einzigartige Möglichkeiten, ein weites Spektrum von Mikroorganismen (Bakterien, niedere und höhere Pilze, gentechnisch veränderte Organismen der Risikogruppen 1 und 2) vom Schüttelkolben bis zum Pilotmaßstab von 2,5 m³ Arbeitsvolumen zu kultivieren. Für die dabei gebildeten Naturstoffe, Proteine und Biopolymere stehen alle erforderlichen Aufreinigungsverfahren und die dazu benötigten technischen Aus rüstungen zur Verfügung, um diese bis zur Reinsubstanz zu isolieren. Die Entwicklung Head: Dr. Uwe Horn 144 analytischer Methoden konzentriert sich besonders auf prozessbegleitende Parameter und auf den Nachweis der synthetisierten Produkte. Außerdem haben wir umfangreiche Erfahrungen bei der Kultivierung von Mikroorganismen im Chemostaten, unter anaeroben Fermentationsbedingungen, sowie der Hochzelldichtefermentation. Die Etablierung einer mikrofluidischen Plattform für die Ultrahochdurchsatz-Suche nach antimikrobiellen Naturstoffen und Enzymen ist ein Beispiel für die Entwicklung innovativer Technologien. Das Biotechnikum arbeitet intensiv mit den Forschungsabteilungen und Nachwuchsgruppen des HKI, mit Instituten der Friedrich-Schiller-Universität Jena sowie der Ernst-Abbe-Fachhochschule Jena zusammen. HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant Scientific Projects aminoglycosides β-‐lactams 1940 glycopepAdes 1950 1960 1970 muAlins 1980 1990 2000 innovaAon gap sulpha drugs macrolides quinolones, streptogramins 2010 lipopepAdes oxazolidinones chloramphenicol, tetracyclines Figure 1 Timeline for the discovery of antibiotic substance classes (after Fischbach and Walsh, 2009) 1 Picoliter droplet-based search for natural products – the future of biotechnological screening Group Leaders: Emerson Zang, Martin Roth Antibiotic screening: the past Nature proved to be a rich source of active compounds in the past. Starting with the discovery and commercialisation of penicillin in 1928, an impressive variety of antimicrobial substances was unveiled from soil and marine habitats until the early 1960s. However, after this golden era of antibiotic discovery, researchers increasingly struggled to find new substances as a result of permanent rediscovery of already known producers (Fig. 1). Simultaneously, first evidence of antibiotic-resistant pathogens was reported, aggravating the demand for new substances in the decades to follow. With the dawn of molecular biological methods for target-identification in pathogens, the focus of screening efforts shifted from phenotypical screening to target-based screening approaches. At the same time, natural product libraries were replaced by combinatorial libraries of synthetic compounds. Despite extensive screening programmes, the yield of such techniques was disappointing. The diversity and biological relevance of chemical libraries was found to be inferior to the natural compound portfolio. Moreover, hits that were active in vitro often suffered from unspecificity or insufficient trans-membrane mobility. Antibiotic screening: the future To overcome the crisis in antibiotic research, leading experts postulate the return to phenotypic screening of natural products – the hitherto most successful approach in antibiotic research. However, new cultivation and screening methods are required for significantly increased sample throughput and to provide new means of de-replication. Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013 145 Figure 2 Screening for antibiotic compounds with emulsionbased microfluidics. (1) Droplet generation from spore suspension by flow focusing. (2) Droplet incubation for propagation of Actinobacteria. (3) Dosing of reporter cells. (4) Droplet incubation for propagation of reporter cells. (5) Droplet interrogation and sorting. In 2003, droplet-based microfluidics was used at the HKI for the first time to cultivate soil-borne bacteria by Martin et al. On the basis of this seminal work, a nanoliterdroplet-based screening concept was developed, relying on in-droplet isolation, cultivation and antibiotic activity assessment of Actinobacteria derived from soil samples. The assay was implemented using so-called “micro-segmented” flow, where droplets are continuously kept in serial trains, separated by carrier oil. It could be shown that cultivation of Actinobateria and other organisms on that platform is feasible, as well as the detection of miniminal inhibitory concentrations for various antibiotics. Yet, micro-segmented flow suffered from frequent unintended fusion of droplets, so that complex assay regimes as the intended antibiotic screening were not realizable. As a consequence, we focused on emulsionbased microfluidics in 2013 (Fig. 2), a droplet- 146 Figure 3 Microcultures of green fluorescent Streptomyces lividans in picoliter droplets. based technique that has several advantages over previously applied micro-segmented flow: Droplets are stabilised by surfactant molecules in the carrier oil, so that unintended droplet fusion is avoided. The increased droplet robustness furthermore allows bulk incubation of droplets, which can be exploited for a massive increase in throughput, easily achieving more than 106 samples per day. To make emulsion-based microfluidics available at the Bio Pilot Plant, compliant microfluidic chips were designed in-house and manufactured externally at LionixBV (NL). Apart from droplet generation by flow focusing, the chips allow dosing and sorting of re-injected droplets by integrated electrodes, which are needed to specifically destabilise the surfactant-loaded droplet interface. These operations also require electronic periphery (function generators, voltage amplifiers), and its commissioning was successfully accomplished in 2013. HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant Figure 4 Triggered imaging of droplets. ADC: A/D-converter, ST: Schmitttrigger pL-droplets as high-throughput cultivation platform Having this platform at hand, emulsionbased growth of Actinobacteria was first investigated. To this end, spore suspensions of diverse actinobacterial species were employed as aqueous phase for droplet generation, and the resulting droplet populations were incubated off-chip in handcrafted vials. After one day of incubation, in-droplet germination of spores was noticed and development of small filamentous structures was observed. With prolonged incubation, the filaments grew to dense microcultures (Fig. 3) and the occupied droplets showed shrinkage as a result of metabolic activity concomitant with an osmotic mismatch to non-occupied droplets. In extreme cases, the droplet interface was deformed by growing microcultures. However, filament penetration of the droplet interface was rarely observed, so that the droplet integrity as an isolated cultivation vessel was maintained. Nevertheless, the unequally distributed shrinkage of occupied droplets led to increased polydispersity of the droplet population, impeding subsequent unit operations as dosing and sorting. Image-based droplet sorting To avoid above-mentioned polydispersity during prolonged incubation, image-based droplet sorting (IDS) for growth-dependent fractionation of droplets was developed (Fig. 4): Spore-loaded droplets are incubated until germination of spores and formation of microcultures occurs. The droplets are reinjected into a microfluidic chip and separately imaged using a brightness-dependent trigger signal. Each image is subjected to an online “Difference of Gaussians” analysis developed by the Research Group Applied Systems Biology, enabling detection of droplet boundaries and enclosed filamentous structures within milliseconds. Depending on the presence or absence of filamentous structures, droplets are sorted into either one of Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013 147 no/little mycelium droplet generation incubation reinjection image-based droplet sorting dense mycelium antimicrobial activity testing Figure 5 Screening with image-based droplet sorting for droplet population maintenance and enrichment of slowgrowing actinobacteria. two efferent channels. Using this approach, growth-dependent droplet sorting at 100 Hz was demonstrated. The technique will not only enable reduction of droplet population polydispersities. More importantly, IDS will allow fractionation of unknown Actinobacteria according to their speed of growth, finally achieving enrichment of slow-growing and rare species, being producers of hitherto undiscovered compounds with high probability (Fig. 5). In summary, emulsion-based microfluidics was successfully established as a cultivation platform for Actinobacteria, enabling a culture throughput of far more than 106 samples per day. By the implementation of image-based droplet sorting, a powerful tool for droplet maintenance and enrichment of slow-growing Actinobacteria was developed (Zang et al. 2013). Yet, the proof-of-concept for antibiotic screening in pL-droplet-emulsions must still be provided, but important 148 steps to master this demanding challenge have been taken. 2 Stress response in Escherichia coli bioprocesses related to rare amino acids Group Leaders: Michael Biermann, Uwe Horn Escherichia coli is a commonly used host in industrial applications for the production of pharmaceutical proteins. Because of its high volumetric productivity the fed-batch fermentation under high cell density conditions is one of the most widely used process strategies for the expression of such proteins. Although the fermentation processes are very well-developed today, they demand very high standards on the used host-vector-systems and bioreactors. Particularly in industrial large-scale bioreactors inhomogeneities may occur causing substrate gra- HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant dients and local limitations of dissolved oxygen. The resulting stress situations can lead to instabilities of the process and can have negative effects on the protein yield and quality. Concerning the protein quality, a phenomenon studied intensely in the recent years is the formation and misincorporation of non-canonical amino acids such as norvaline and norleucine into recombinant proteins. This can result in modified variants of the final product. According to the guidelines of regulatory health authorities such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), all of these modified variants need intensive analytical characterisation and are therefore highly undesirable in biopharmaceutical production. A cooperation project of the Bio Pilot Plant with the WACKER Biotech GmbH Jena aimed at the development of new fermentation strategies to prevent the misincorporation of noncanonical amino acids. As analytical basis for the project, a novel ultra-high performance liquid chromatography technique was developed allowing for simultaneous analysis of norvaline, norleucine and 15 standard amino acids (Biermann et al. 2013a). The main focus of the project was the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. We analysed the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with special focus on non-canonical amino acids. Current physiological studies revealed strong evidence for the connection of glucose overflow metabolism and pyruvate accumulation to the occurrence of norvaline in wildtype E. coli fermentations. The trace elements molybdenum, nickel and selenium form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids. During fed-batch cultivation with provoked oxygen limitation and glucose excess, norleucine and norvaline were accumulated only in the absence of molybdenum, nickel and selenium. In contrast, trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional FHL metalloprotein complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration. In conclusion, the formation of norleucine and norvaline by recombinant E. coli at cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low (Biermann et al. 2013b). 3 Life science meets physics - Microbial adhesion and biofilm formation on biomaterials Group Leader: Claudia Lüdecke, Martin Roth (in cooperation with Klaus D. Jandt, Otto Schott Institute of Materials Research, Friedrich Schiller University Jena) Modern medicine increasingly uses biomaterials for implant purposes, e. g., to restore human body functions. Infections related to these biomaterials are most frequently associated with microbial colonisation of the indwelling medical devices. Understanding mechanisms of bacterial adhesion and biofilm formation is thus a crucial prerequisite to develop strategies for the prevention of biomaterial-associated infections. Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013 149 Figure 6 In vitro test system for investigation of microbial adhesion and biofilm formation at constant and reproducible conditions. The experimental setup includes a continuous culture (chemostat), a biofilm reactor (non-constant-depth film fermenter; nCDFF), microscopy and image analysis. (from Lüdecke et al. 2014) A new standard testing system for bacterial adhesion and biofilm formation Various investigations were made to influence bacterial adhesion and biofilm formation by materials’ surface properties, such as surface topography and morphology or chemistry and wettability of the biomaterials. However, these studies are hardly comparable and reproducible since there are no standard tests available for adequately studying microbial adhesion and biofilm formation and all studies differ in experimental conditions and parameters. The first aim of this highly interdisciplinary project was, therefore, to develop an in vitro testing system for the investigation of microbial adhesion and biofilm formation on biomaterials surfaces at constant and reproducible conditions. To realise the new testing system, a biofilm reactor was coupled to a continuous culture (chemostat) of Escherichia coli. For evaluation of the system, biofilms were cultivated in several repeated experimental runs on titanium surfaces for up to 9 days (Fig. 6). Using a continuous culture for inoculation of the biofilm reactor provided intact and viable microbial cells for adhesion and biofilm cultivation compared to the most often used Figure 7 Adhesion and biofilm formation of Escherichia coli on titanium surfaces after 24 hours (A), 2 days (B), 3 days (C) and 6 days (D), respectively, cultivated with the new in vitro testing system. (from Lüdecke et al. 2014) 150 HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant Figure 8 PhD student Claudia Lüdecke investigates the adhesion of Escherichia coli on nanorough titanium surfaces using a scanning electron microscope at the Otto Schott Institute of Materials Research (OSIM), Friedrich Schiller University, Jena. (Photo by Jan-Peter Kasper, FSU Jena) microtiter well plate based approaches where cells are weakened by centrifugation and resuspension procedures are used. The cultivated biofilms showed equal and reproducible growth with a typical four-stagesdevelopment including initial bacterial adhesion, micro colony formation and early and late maturation (Fig. 7). Inoculation of the biofilm reactor with a continuous culture minimised variations between different experimental runs. The new in vitro testing system thus allows the investigation of microbial adhesion and reproducible cultivation of biofilms at constant experimental conditions. The system will support developing and adequate testing of biomaterial’s surface modifications for prevention of biomaterial-associated infections (Lüdecke et al. 2014). Microbial adhesion on nanorough titanium surfaces It is well known that surface topographical features of biomaterials in the micrometer range strongly affect the adhesion of microorganisms and subsequent biofilm formation. To date, the impact of materials surface topography on the nanometer range was much less investigated and fundamental mechanisms have not been identified. The second aim of this project was to identify fundamental mechanisms involved in bacterial adhesion on nanorough titanium surfaces. The adhesion of the pathogenic microorganisms Staphylococcus aureus and Escherichia coli most often involved in biomaterial-associated infections was investigated on titanium surfaces with different nanoroughness using the new in vitro testing system (Fig. 8). Titanium surface coverage by E. coli and S. aureus was decreased by approximately Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013 151 50% with increasing surface nanoroughness from 2 to 6 nm. Two main mechanisms were identified to be most probably responsible for the reduced number of adhered bacteria with increasing titanium surface nanoroughness: With increasing roughness, the size of titanium grains at the surface increased and, accordingly, the relative number of surface grains decreased. For first contact of bacterial cells with the titanium surface (early adhesion), fewer contact points due to the lower number of surface grains were available with increasing roughness resulting in a reduced number of adhered cells. A scanning electron microscope equipped with a focused ion beam (FIB-SEM) provided direct insight into the interface between bacterial cell and nanorough titanium surface. After 3 h of adhesion (early adhesion), bacterial cells were attached to the nanorough surfaces only at few points. After 9 h of adhesion (late adhesion), bacterial cells produced extracellular polymeric substances (e.g. carbohydrates) and were in complete contact with the titanium surface. With increasing titanium surface roughness, the specific surface area available for contact between the bacterial cell and the titanium decreased resulting also in a reduced number of adhered cells (Fig. 9). In this project it was shown that surface roughness even on the very low nanometer scale influences bacterial adhesion behavior. These findings can give new momentum to biomaterials research and will support the development of biomaterials surfaces with anti-infectious surface properties (Lüdecke et al. 2013). This interdisciplinary project, a cooperation between the HKI Bio Pilot Plant and the Otto Schott Institute of Materials Research (OSIM) at the Friedrich Schiller University, Jena, is supported with a PhD fellowship awarded by the Graduate School “Jena School for Microbial Communication (JSMC)”. The successful collaboration will be continued in a second JSMC PhD project. 152 4 Production of microbial biomass and natural products for research projects of HKI groups and external academic partners Group Leaders: Uwe Horn, Martin Roth A large number of microbial products from different microorganisms were produced for projects of research groups at HKI. 620 fermentations at the 1- to 7-litre-laboratory-scale and 155 fermentations at the 10 to 500-litrepilot-scale were performed within 2012-2013 including preparation of inoculum cultures, fermentation and monitoring of the fermentations by data acquisition and analysing samples. The following downstream processing procedures have been carried out: separation of supernatant and biomass, fractionation of supernatant by preparative HPLC, capture of natural products by adsorption on synthetic resins or by solvent extraction, and concentration and freeze drying of the extracts. In 2012-2013, products from about 5,500 litres fermentation broth were prepared. Selected projects Isolation and structural elucidation of bioactive natural products and genetics of biosynthetic pathways (Department of Biomolecular Chemistry) 41 pilot scale fermentations (30 – 300 L) with different Streptomyces and Burkholderia strains and mutants, and with Aspergillus oryzae were performed. Downstream processing, especially solvent extraction and preparative HPLC, was done to isolate the natural products. Discovery of new drugs and metabolites (Junior Research Group Secondary Metabolism of Predatory Bacteria) Predatory and phytopathogenic bacteria were cultivated in pilot scale fermentations (30 – 75 L) in order to discover and to isolate new natural products. HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant Figure 9 Staphylococcus aureus cells adhered to nanorough titanium (after 3 h of adhesion (A, C) and after 9 h of adhesion (B, D)) cross-sectioned with a focused ion beam. Arrows indicate the adhesion points between microbial cell and titanium surface. With increasing time, the cells are in complete contact to the surface. Genome mining in clostridia for natural product discovery (Department of Biomolecular Chemistry) More than 400 fermentations with strictly anaerobic Clostridium species were performed in lab-scale bioreactors (1 – 7 L) using a variety of media for the isolation of novel natural products (Fig. 10). Molecular biology of natural products (Department of Molecular and Applied Microbiology) 40 pilot scale fermentations (30 L) of Aspergillus fumigatus mutants were performed to enable the elucidation of the biosynthesis of the toxin gliotoxin. Analysis of the Aspergillus fumigatus response to low atmospheric oxygen levels (Department of Molecular and Applied Microbiology) 31 laboratory fermentations in the 7-L-scale were performed to investigate the dynamic changes of the A. fumigatus transcriptome and proteome in hypoxia utilising an oxygen-controlled bioreactor system. Identification of protein antigens in the secretome of Candida albicans for the diagnosis of fungal sepsis (Department of Molecular and Applied Microbiology) This projcet aims at characterising the secretome of the two morphotypes of C. albicans, yeast cell and hypha. The cultivation of both cell types in 10-litre-fermenters and the sample preparation were established and improved in order to obtain sufficient amounts of the proteins for the secretome analyses, because C. albicans secretes only low protein concentrations in contrast to saprophytic fungi. By using this approach it was possible to identify 43 proteins excreted by the yeast cell type, and 114 proteins secreted by the hypha type. Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013 153 Figure 10 Anaerobic cultivation of clostridia in 1 liter bioreactors (BIOSTAT Qplus, Sartorius Stedim). Use of heavy metal resistant streptomycetes in bioremediation (Friedrich Schiller University Jena, Institute of Microbiology, Microbial Communication) Pilot scale fermentation (30 – 300 L) of heavy metal resistant Streptomyces strains and preparation of biomass for bioremediation studies at the former uranium mining site in Eastern Thuringia. 5 Development of high performance bioprocesses in cooperation with academic and industrial partners Group Leaders: Bettina Bardl, Klaus-Dieter Menzel, Gundela Peschel, Martin Roth The development of integrated fermentation and downstream processes for the production of antibiotics, enzymes, toxins and biopolymers in one of the main tasks of the Bio Pilot Plant. Our research is aimed at GMP- 154 compliant processes in order to transfer them to GMP production facilities. Integrated bioprocesses for the following products were developed or improved: Corallopyronin A (in cooperation with the University Hospital Bonn (A. Hörauf), the Institute of Pharmaceutical Biology, University Bonn (G. König), funded by the German Center for Infection Research (DZIF)) Corallopyronin A (CorA) is an antibiotic from the myxobacterium Corallococcus coralloides that inhibits the bacterial DNA dependent RNA polymerase. CorA is effective against Gram-positive bacteria, e.g. Staphylococcus aureus, but has recently been shown to also be effective against the intracellular Gramnegative bacteria Wolbachia spp., endosymbionts in filarial worms. However, it is ineffective against Mycobacterium spp. The mode of action (MoA) of CorA is different to that HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant of rifampicin. Thus CorA is effective against multi-resistant S. aureus. The MoA also allows the use of CorA against global cases of filaria infections in which rifampicin would be indicated, but is not used due to tuberculosis co-endemicity in order to avoid selecting rifampicin resistant tuberculosis. For preclinical trials CorA is needed in gram amounts. We improved the culture media and developed a GMP-compliant fermentation and purification process. The process was scaled-up to the 500-litre-pilot-scale. Gram quantities of pure CorA can now be produced and provided for preclincal trials. Alpha-amanitin (in collaboration with Heidelberg Pharma GmbH, Ladenburg) A general objective in oncology drug discovery and development is to achieve tumour cell eradication, while maintaining at the same time a high level of tolerability to healthy cells and tissues. Antibody-drug conjugates (ADCs) combine the well-established principle of targeted antibodies (safety and tolerability) with the effectiveness of potent toxins (anti-tumour efficacy). They consist of a monoclonal antibody conjugated to toxins by chemical linkers. The function of the antibody part of the ADC molecule is to ‘guide’ the toxin specifically to the target tumour cells, and to allow healthy cells to be left untouched. Current opinion however is, that tumour relapse and tumour metastases may be related to ‘dormant or quiescent tumour cells’ that are insensitive to most – if not all - currently approved drugs for the treatment of cancer. This explains the tremendous demand for toxins and coupling techniques that result in new ADCs with entirely new properties, in particularly to meet the challenges in treating tumour metastases and preventing tumour relapses. In this environment, the Heidelberg Pharma GmbH (HDP) concentrates on combining the targeting properties of antibodies with the effectiveness of the ultra-potent basidiomycete toxin alpha-amanitin, a specific inhibi- tor of RNA polymerase II. Amanitin differs from other toxins used in today’s ADC therapeutics by its unique intracellular target and its systemic and proteolytic stability. ADCs with amanitin (AMA-ACs) have the potential to treat multiresistant tumours and ‘dormant or quiescent’ tumour cells, which are regarded as a main reason for metastasis and resistance. The availability of sufficient amounts of alpha-amanitin is a prerequisite for the development of AMA-ACs and clinical trials to test their efficiency. Therefore it was the aim of a project funded by the BMBF in the programme ‘KMU-innovativ’ to develop a biotechnological process for the production of alpha-amanitin. A saprophytic basidiomycete was selected as production strain. The developed fermentation and downstream process was scaledup to the 500-litre-pilot-scale. The complete production process is conforming to GMP standards. The transfer of the production process to a GMP certified industrial partner of HDP is in preparation in order to provide the toxin for the production of AMA-ACs for clinical trials. Purified alpha-amanitin is produced in gram quantities at HKI and delivered to HDP for the development of antibody-amanitin conjugates. Polyhydroxyalkanoates In a continued collaboration with Tepha Medical Devices Inc., Lexington, USA we develop and improve fermentation processes for the production of polyhydroxyalkanoates with different specific properties using recombinant E. coli strains. These biocompatible, absorbable polyesters are used at Tepha for developmental work addressing a variety of products for medical applications, e. g. surgical monofilament sutures and meshes, surgical films, composite meshes, absorbable stents and stent coatings, absorbable microspheres and versatile drug release matrices. Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013 155 Group members External funding Collaborations Head Dr. Uwe Horn Phone +49 3641 532-1500 Fax +49 3641 532-2500 [email protected] Bundesministerium für Bildung und Forschung DiNaMid – Genom-basierte Findung neuer antimikrobieller Naturstoffe in mikrofluidischen Chips; Identifizierung neuer Stoffwechselwege und Naturstoff-Isolierung Markus Nett, Martin Roth B.R.A.I.N. AG, Zwingenberg, Germany Dr. Klaus Liebeton Scientists Dr. Bettina Bardl Christine Hoffmeier Uwe Knüpfer Karin Martin Klaus-Dieter Menzel Dr. Jörg-Hermann Ozegowski Dr. Gundela Peschel Dr. Martin Roth Bundesministerium für Bildung und Forschung Deutsches Zentrum für Infektionsforschung New natural compounds – Neue Naturstoffe Martin Roth, Uwe Horn Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Exploiting the metagenome of microbial communities on a microfluidic platform Uwe Horn Ph.D. Students Heike Arnold Magdalena Bereza Michael Biermann (until 09/2013) Alexander Funk Jana Funk Christian Haupt Liesa Heinrich Claudia Lüdecke Lisa Mahler Miguel Tovar Thomas Weber Emerson Zang Selected publications Biermann M, Linnemann J, Knüpfer U, Vollstädt S, Bardl B, Seidel G, Horn U (2013) Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation. Microbial Cell Factories 12, 116-124. Diploma / Bachelor / Master Students Thomas Krauße Annelie Pohle Marco Säuberlich (08/2012–03/2013) Barbara Schaarschmidt Astrid Pflieger (04/2012) Sebastian Tennstedt (04/2012) Miguel Tovar (03/2012–09/2012) Research Assistents Patrick Berthel (until 10/2013) Michael Cyrulies Dirk Femerling Christian Heiden (until 10/2012) Gudrun Krauter Karin Perlet Renate Presselt (until 05/2012) Jan Schönemann Armin Siering Matthias Steinacker Thomas Weber (since10/2012) Karsten Willing Trainees Laura Geilman (06/2012) Verena Hammer (09/2012) Kwon Jisoo (03/2013–04/2013) Michael Meyer (11/2012) Astrid Pflieger (03/2012–04/2012) Stephan Riesenberg (03/2012) Pimolpong Sriyapai (07/2013–09/2013) Sebastian Tennstedt (03/2012–04/2012) Tobias Voigt (02/2012-03/2012) 156 Zang E, Brandes S, Tovar M, Martin K, Mech F, Horbert P, Henkel T, Figge MT, Roth M (2013) Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets. Lab Chip 13, 3707–3713. Friedrich Schiller University Jena, Germany Prof. Dr. Erika Kothe Prof. Dr. Klaus D. Jandt PD Dr. Jörg Bossert Heidelberg Pharma GmbH, Ladenburg, Germany Dr. Jan Anderl Dr. Marcel Linssen Institut für Bioprozess- und Analysenmesstechnik, Heilbad Heiligenstadt, Germany Dr. Gunter Gastrock Leibniz Institute for Photonic Technologies, Jena, Germany Dr. Thomas Henkel Merck KgaA, Darmstadt, Germany Dr. Björn Hock Dr. Lars Toleikis Dr. Andreas Stein Andrè, Kiesewetter Miltenyi Biotec GmbH Teterow, Germany Dr. Bernd Schröder OFS Online Fluid Sensoric GmbH, Ronneburg, Germany Wilried Schellbach Lüdecke C, Bossert J, Roth M, Jandt K D (2013) Physical vapor deposited titanium thin films for biomedical applications: Reproducibility of nanoscale surface roughness and microbial adhesion properties. Applied Surface Science 280, 578-589. Sanofi-Aventis Deutschland GmbH, Frankfurt/M., Germany Dr. Luigi Toti Biermann M, Bardl B, Vollstaedt S, Linnemann J, Knuepfer U, Seidel G, Horn U (2013) Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach. Amino Acids 44, 1225–1231. Tepha Medical Devices, Inc., Lexington, USA Dr. David Martin Andrew Joiner Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage A A, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis: A four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges. Angew Chem Int Ed 52, 1109211095. Technical University Ilmenau, Germany Prof. Dr. J. Michael Köhler University Hospital Bonn, Germany Prof. Dr. Achim Hörauf University of Bonn, Germany Prof. Dr. Gabriele König Wacker Biotech GmbH, Jena, Germany Dr. Guido Seidel HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant International Leibniz Research School for Microbial and Biomolecular Interactions HKI Research Report 2012 / 2013 157 International Leibniz Research School for Microbial and Biomolecular Interactions 158 Graduate training at HKI is organised within the “International Leibniz Research School for Microbial and Biomolecular Interactions” (ILRS Jena). ILRS Jena was established in 2005 and initially financed by the competitively granted means of the Joint Initiative for Research and Innovation. Since 2008, the graduate school has been included in the HKI budget. It is headed by the HKI in close collaboration with the Friedrich Schiller University Jena (FSU) and the Max Planck Institute for Chemical Ecology (MPI CE). ate school and umbrella organisation for microbiological research in Jena funded by the Excellence Initiative of the German Research Foundation. The close network of research activities with a focus on microbial interactions and communication creates an environment with excellent research condidtion. The doctoral researchers benefit from the scientific exchange and extended choice of courses offered by the close network with other graduate schools in Jena and the Jena Graduate Academy. Since 2006, ILRS is a member of the Jena School for Microbial Communication, a gradu- In 2012 and 2013, 10 ILRS doctoral researchers successfully graduated and moved on to Die strukturierte Doktorandenausbildung am HKI findet im Rahmen der „International Leibniz Research School for Microbial and Biomolecular Interactions“ (ILRS Jena) statt. Die ILRS Jena wurde nach Bewilligung des Antrages im Pakt für Forschung und Innovation der Leibniz-Gemeinschaft im Herbst 2005 ins Leben gerufen und ist seit 2008 im Haushalt des HKI verstetigt. Neben dem HKI sind die Friedrich-Schiller-Universität Jena (FSU) und das Max Planck Institut für chemische Ökologie (MPI CE) beteiligt. Communication (JSMC). Die enge Vernetzung der Forschungsaktivitäten auf dem Gebiet der Mikrobiologie mit Schwerpunkt auf dem Aspekt Wechselwirkung und Kommunikation von Mikroorganismen schafft die Voraussetzungen für herausragende wissenschaftliche Leistungen. Die Doktoranden profitieren vom wissenschaftlichen Austausch und erweiterten Kursangebot durch die enge Zusammenarbeit der ILRS mit anderen Graduiertenschulen und der Graduiertenakademie Jena. Seit 2006 ist die ILRS Mitglied der mit Mitteln der Exzellenz-Initiative geförderten Dachorganisation Jena School for Microbial Im Zeitraum von 2012 bis 2013 schlossen 10 Doktoranden ihre Promotion erfolgreich ab und nahmen verantwortungsvolle Tätig- HKI Research Report 2012 / 2013 International Leibniz Research School responsible jobs in industry or academia. The first joint recruitment of ILRS and JSMC was carried out in 2012 and attracted close to 1500 applicants from 82 countries. Currently, 38 doctoral researchers of eleven different nationalities are working towards their PhD degree within ILRS. Prof. Dr. Marc Thilo Figge (systems biology) and Prof. Dr. Matthias Gunzer (infection biology of human pathogenic fungi) joined the faculty of ILRS in 2012. Prof. Gabriele Diekert and Prof. Bernhard Hube. The doctoral researchers are represented by Juliane Fischer and Felix Mingo who succeeded Daniel Schwenk and Sarbani Sarkar as representatives. Prof. Peter F. Zipfel has been the Speaker of ILRS since 2008; the other members of the Steering Committee are Prof. Ian T. Baldwin, keiten in der akademischen oder industriellen Forschung auf. 2012 wurde erstmals ein Auswahlverfahren gemeinsam mit der JSMC durchgeführt. Auf die ausgeschriebenen Stellen bewarben sich knapp 1500 Studenten aus 82 Ländern. Derzeit arbeiten in der ILRS 38 Promovierende aus elf verschiedenen Nationalitäten an ihrer Promotion. Die Fakultät der ILRS wurde 2012 durch die Aufnahme von Prof. Dr. Marc Thilo Figge im Bereich Systembiologie und durch Prof. Matthias Gunzer im Bereich Infektionsbiologie humanpathogener Pilze verstärkt. Prof. Ian T. Baldwin, Prof. Gabriele Diekert und Prof. Bernhard Hube. Die Doktoranden werden durch Juliane Fischer und Felix Mingo vertreten, die Ende 2013 die bisherigen Sprecher (Daniel Schwenk und Sarbani Sarkar) ablösten. Sprecher der ILRS ist seit 2008 Prof. Peter F. Zipfel, die stellvertretenden Sprecher sind International Leibniz Research School HKI Research Report 2012 / 2013 159 160 Scientific Topics In their natural habitat, microorganisms do not occur in isolation but live in close association with other organisms of different species and developmental stages. Their interactions can take many forms, ranging from mutual symbiosis to parasitic interactions. How these complex, multiorganismic networks are regulated by microbial communication is still poorly understood. The ILRS PhD projects therefore aim to gain insight into the microbial and biomolecular interactions underlying the communication processes among microorganisms in diverse habitats with interdisciplinary approaches, combining methods of microbiology, natural product chemistry, chemical ecology, bioinformatics and systems biology. Structured PhD Training The implementation of a curriculum during the course of the PhD programme is an integral part of the structured PhD training. This curriculum consists of advanced methods courses offered by the ILRS Faculty Members. Special soft skill courses such as “Scientific Writing”, “Poster and Oral Presentations”, “Research Funding”, as well as Public Relations Activities and German and English Language courses complement the training on a non-scientific level. In addition, there are regular ILRS group seminars and an annual international symposium. All means of education follow a program of study specifically developed for ILRS. Thus, an efficient qualification of the jun-ior researchers within a tight time frame is ensured. Wissenschaftliche Themenschwerpunkte Mikroorganismen kommen in der Natur in der Regel nicht als Einzelzellen vor, sondern leben in den verschiedensten Habitaten im Verbund mit anderen Organismen, z. B. in mutualistischen Symbiosen oder aber in parasitären Interaktionen. In der Umwelt stehen Mikroorganismen in Wechselwirkung mit vielen anderen Mikroorganismen (Bakterien / Bakterien, Bakterien/Pilze, Pilze/ Pilze) und auch mit höheren Organismen wie Pflanzen und Tieren. Die an dieser Interaktion beteiligten Kommunikationsmechanismen sind weitgehend unverstanden. Die Forschungsprojekte der ILRS versuchen daher, die zugrunde liegenden mikrobiellen und biomolekularen Interaktionen mit Methoden der Mikrobiologie, Naturstoffchemie, chemischen Ökologie, Bioinformatik und Systembiologie zu entschlüsseln. Strukturierte Doktorandenausbildung und Lehre Wesentlicher Bestandteil der strukturierten Doktorandenausbildung ist die Implementierung von Lehrveranstaltungen in die Dissertationszeit. Dazu wird ein vielfältiges Angebot an Methodenpraktika durch die Mitglieder der ILRS Fakulät angeboten. Spezielle soft skill-Kurse, wie „Scientific Writing“, „Poster and Oral Presentations“, „Research Funding“, sowie “Public Relations Activities“ und Sprachkurse in Deutsch und Englisch bieten Qualifizierungsmöglichkeiten über die rein fachliche Weiterbildung hinaus. Außerdem finden regelmäßig Gruppenseminare und jährlich ein internationales Symposium statt. Alle ausbildungsrelevanten Maßnahmen werden von den Doktoranden entsprechend einem für die ILRS entwickelten Program of Study absolviert. Dies sichert eine effiziente Qualifikation des wissenschaftlichen Nachwuchses in einem stringenten Zeitschema. HKI Research Report 2012 / 2013 International Leibniz Research School PhD Student Title of the PhD Project Supervisor Van Thi Luu Mutualistic microbial associates of Nicotiana attenuata Ian T. Baldwin Arne Weinhold Microbial Interactions relevant for the fitness of Nicotiana attenuata in the native environment Ian T. Baldwin Pol Alonso Melolontha hippocastani gut specialized bacterial community: function and structure elucidation Wilhelm Boland Huijuan Guo Isolation, purification, and structural elucidation of active compounds from the tissue of insect Wilhelm Boland Juliane Fischer Post-translational modifications and secondary metabolism of Aspergillus nidulans Axel A. Brakhage Christoph Heddergott Secretome analysis / pathogenicity mechanisms of Arthroderma benhamiae Axel A. Brakhage Andreas Thywißen Molecular mechanisms of the interaction between Aspergillus fumigatus and alveolar macrophages Axel A. Brakhage Shruthi Ramachandra Host pathogen interactions of human-pathogenic yeast Matthias Brock / Bernhard Hube Nayla Jbeily Ceramide formation in host response to bacterial and fungal infection and development of organ failure Ralf A. Claus / Konrad Reinhart Felix Mingo The role of Desulfitobacterium spp. in the global network of O-demethylation in soil Gabriele Diekert Anita Mac Nelly Expression control and biosynthesis of dehalogenating enzymes from anaerobic soil bacteria in response to the interaction with aerobic halogenating fungi Gabriele Diekert Continuation Ú International Leibniz Research School HKI Research Report 2012 / 2013 161 PhD Student Title of the PhD Project Supervisor Susanne Brandes Automated analysis of dynamic properties in biological systems from image data Marc Thilo Figge Pegah Seddigh Identifying the pulmonary phagocytic network of mice infected with Aspergillus fumigatus Matthias Gunzer Fabian Horn Prediction of gene regulatory networks involved in the differentiation, secondary metabolism and cross talk of Aspergillus nidulans Reinhard Guthke Himanshu Manchanda Optimization of the influenza neuraminidase inhibitor therapy by use of secondary bacterial infections Reinhard Guthke Sebastian Müller Integrated genome-wide data analysis by ensemble learning methods to understand infection processes Reinhard Guthke Thomas Wolf Bioinformatic investigation and prediction of gene regulatory elements in pathogenic fungi Reinhard Guthke Swantje Behnken Genome mining of Gram-positive bacteria for secondary metabolites Christian Hertweck Katharina Graupner Molecular basis of bacterial-mushroom interactions Christian Hertweck Srividhya Sundaram Elucidation of complex biosynthetic pathways in microorganisms Christian Hertweck Alexander Funk Regulation of polyketide synthase gene clusters in Aspergillus nidulans Uwe Horn Daniel Braga de Lima Investigation of polyketide biosynthesis in agaricomycetes Uwe Horn / Dirk Hoffmeister Daniel Schwenk Natural products from an unidentified homobasidiomycete which control wood-deteriorating microorganisms Uwe Horn / Dirk Hoffmeister Franziska Gerwien Intracellular survival of Candida glabrata in phagocytes Bernhard Hube Sarah Höfs Candida albicans – The pathway to epithelial damage Bernhard Hube Francois Mayer Identification and characterization of infection-associated genes in Candida albicans Bernhard Hube Volha Skrahina The role of micronutrients during fungal infections Bernhard Hube Kristin Kroll The hypoxic (low-oxygen) response of the pathogenic mould Aspergillus fumigatus and its relevance to pathogenicity Olaf Kniemeyer Elena Shekhova Investigation of the stress response system of A. fumigatus by redox proteomics Olaf Kniemeyer 162 HKI Research Report 2012 / 2013 International Leibniz Research School PhD Student Title of the PhD Project Supervisor Reyna Murry Phosphatidylinositol (PI) signaling in basidiomycete S. commune Erika Kothe Dominik Senftleben A surface hydrophobin in ectomycorrhiza interaction Erika Kothe Wei Li The entrainment of the circadian clock by light-dark and temperature cycles: Functional analysis of components of involved signaling pathways Maria Mittag Karthik Mohan An insertional mutagenesis approach to understand temperature entrainment of the circadian clock in Chlamydomonas reinhardtii Maria Mittag Christine Lembke Induced resistance of phytoplankton against algicidal bacteria Georg Pohnert Michaela Mauß Unraveling the (bio)chemical processes involved in phytoplankton-virus interactions Georg Pohnert Shayista Amin Host Pathogen interactions, Aspergillus fumigatus and PET/CT Technology Hans Peter Saluz Sara Mohebbi Measuring interacellular pH during the apoptosis induction and inhibition regarding the Aspergillus fumigatus infection Hans Peter Saluz Denise Buhlmann Functional characterization of the soluble complement receptor type II (CR2/CD21) in infection Christine Skerka Tina Enghardt Complement evasion of human pathogenic microorganisms Christine Skerka Sandra Gusewski Assessing DNA-binding specificity of MADS-domain transcription factors Günter Theißen Khushboo Jetha Interactions and regulatory networking of MADS-domain transcription factors in model plants (Arabidopsis thaliana) Günter Theißen Isabell Kopka Complement regulation in kidney cells Gunter Wolf Qian Chen The Role of complement in autoimmune disease Peter F. Zipfel Hannes Eberhardt The role of CFHR proteins in human autoimmune diseases Peter F. Zipfel Selina Icke Functional characterizations of variant forms of the human complement regulator CFHR1 Peter F. Zipfel Justus Linden Immunevasion of the human pathogenic yeast Candida albicans Peter F. Zipfel Christiane Pilz Complement evasion strategies of Candida albicans Peter F. Zipfel Sarbani Sarkar Complement escape mechanisms of Streptococcus pneumoniae Peter F. Zipfel International Leibniz Research School HKI Research Report 2012 / 2013 163 164 HKI Research Report 2012 / 2013 Jena School for Microbial Communication HKI Research Report 2012 / 2013 165 Jena School for Microbial Communication 166 The Jena School of Microbial Communication (JSMC) was founded in September 2006 as an umbrella organization for three pre-existing Research Training Groups, the “International Leibniz Research School for Microbial and Biomolecular Interactions” (ILRS) of the HKI, the “International Max Planck Research School“ (IMPRS) of the Max Planck Institute for Chemical Ecology (MPI CE) and the DFG Research Training Group GRK 1257 “Alteration and Element Mobility at Microbe-Mineral Interfaces” of the Friedrich Schiller University. Axel Brakhage, director of the HKI, founded the JSMC as its main coordinator together with Wilhelm Boland, Director of the Department of Bioorganic Chemistry at the MPI-CE, and Erika Kothe, coordinator of the Research Training Group GRK 1257. They act as the Executive Board together with the JSMC management team and two doctoral representatives. In November 2007, the JSMC has been awarded funding by the DFG excellence initiative as the only project in Thuringia, and has been able to secure continued funding for another five years in November 2012 after a highly competitive selection process. Within its structured and interdisciplinary PhD programme, JSMC efficiently educates doctoral researchers to become internationally competitive and versatile independent scientists, aiming at a more detailed and holistic view on microbial communication in all its facets. This only becomes feasible through the inter- Die “Jena School for Microbial Communication” (JSMC) wurde im September 2006 als Dachorganisation der bestehenden strukturierten Doktorandenausbildungen „International Leibniz Research School for Microbial and Biomolecular Interactions“ (ILRS) des HKI, der „International Max Planck Research School“ (IMPRS) des Max-Planck-Instituts für chemische Ökologie (MPI CE) sowie des DFG-Graduiertenkollegs 1257 “Alteration and Element Mobility at Microbe-Mineral Interfaces” (GRK 1257) der Friedrich-Schiller-Universität Jena gegründet. HKI-Direktor Axel Brakhage zeichnete sich damals gemeinsam mit Wilhelm Boland, Direktor der Abteilung „Bioorganische Chemie“ des MPI-CE, und Erika Kothe, Sprecherin des GRK 1257, für die Gründung der JSMC verantwortlich und fungiert seitdem als deren Sprecher und Koordinator, mit W. Boland und E. Kothe als Stellvertretern. Im November 2007 wurde die JSMC als einziges Thüringer Projekt im Rahmen der DFG Exzellenzinitiative bewilligt und finanziert, im November 2012 folgte dann nach einem hochkompetitiven Auswahlverfahren die erfolgreiche Bewilligung zu einer Fortsetzungsfinanzierung für weitere 5 Jahre. Im Zentrum der JSMC steht die strukturierte und fächerübergreifende Ausbildung von Doktoranden zu interdisziplinären und international konkurrenzfähigen Wissenschaftlern, deren fachliche Ausrichtung zu einem detaillierten und holistisch Verständnis der mikrobiellen Kommunikation führen soll. Dies kann erst durch die fachübergreifende Integration der Disziplinen Biologie, Chemie, Medizin, Geologie, Physik und Systembiologie erreicht werden und führt auf diese Weise alle in Jena lokalisierten mikro- HKI Research Report 2012 / 2013 Jena School for Microbial Communication disciplinary integration of biology, chemistry, medicine, geology, physics and systems biology and as such connects all research groups in Jena working on microbiological topics. JSMC doctoral researchers complete an ambitious training programme, which familiarises them with state-of-the-art methods and research concepts while simultaneously providing them with vital transferable skills, an indispensable feature for their later career paths. The most important elements of doctoral training at JSMC are: •Cross-disciplinary PhD research projects situated in the field of tension between basic research and industrial application. biologisch arbeitenden Gruppen zusammen. Die JSMC-Doktoranden absolvieren ein ambitioniertes Lehrprogramm, in welchem sie transdisziplinär mit den neuesten Methoden und Forschungskonzepten vertraut gemacht werden. Wichtigste Elemente dieser Doktoranden-Ausbildung sind: •ein fächerübergreifender Ansatz der Forschungsprojekte, angesiedelt im Spannungsfeld zwischen Grundlagenforschung bis hin zur industriellen Anwendung •frühe Internationalisierung und Netzwerkbildung der Doktoranden durch Konferenzteilnahmen, Auslandsaufenthalte und Kooperationsprojekte •umfangreiche Ausbildung in sogenannten „Transferable Skills“, wie z. B. wissen- •Promotion of research stays abroad, as well as of participation in international conferences and in collaborative projects, to give doctoral researchers early international experience and to enhance their networking skills. •Comprehensive training in ‘transferable skills’, e.g. scientific publication and presentation, acquiring research funds, etc. •Close supervision by a team of mentors to ensure that the students achieve the goals of their PhD projects within three years. schaftliches Publizieren und Präsentieren, Einwerben von Fördergeldern durch gezielte Anträge, etc. •intensive Betreuung durch Mentoren-Teams für eine effiziente Umsetzung der Ziele der Promotionsprojekte innerhalb von 3 Jahren. Die erbrachten Leistungen werden in einem Credit Point System erfasst und zertifiziert. Damit werden hochqualifizierte Nachwuchswissenschaftler herangebildet, die in Jena ein wissenschaftlich äußerst kreatives Netzwerk bilden welches international wettbewerbsfähige und richtungsweisende Forschungsleistungen hervorbringt. 2012 – 2013 verteidigten 31 der insgesamt über 130 JSMC Doktoranden Ihre Doktorarbeit erfolgreich. Auf die offenen Promo- Jena School for Microbial Communication HKI Research Report 2012 / 2013 167 Credit Points are awarded for each achievement and component of the JSMC PhD training, ensuring the conversion to highly qualified young academics, who build an extremely creative scientific network in Jena conducting internationally competitive and seminal research on microbial communication. In 2012 – 2013, 31 of currently over 130 JSMC doctoral students successfully defended their theses. Over 7000 applicants from around 90 different nations worldwide competed in total for available PhD positions, 24 of which were ultimately selected after a rigorous selection procedure and multiple recruiting events as new JSMC PhD students. Dr. Ákos T. Kovács tionsstellen der JSMC bewarben sich insgesamt über 7000 Kandidaten aus rund 90 verschiedenen Nationen, von denen nach rigorosen Selektionsverfahren und mehrtägigen Rekrutierungstreffen schließlich die 24 am besten geeigneten in die JSMC als neue Doktoranden aufgenommen wurden. Dr. Ákos T. Kovács wurde als neuer JSMC Junior Gruppenleiter mit Schwerpunkt „terrestrische Biofilme“ ausgewählt, und ist mit seiner Forschungsgruppe im neu renovierten JSMC Gebäude in der Neugasse 23 tätig welches 168 has been selected as new JSMC junior research group leader, working on terrestrial biofilms at the renovated building in Neugasse 23, which now also houses the JSMC management. Katja Präfcke succeeded Ulrike Schleier as JSMC management assistant in November 2012, and Dr. Jan Büllesbach succeeded Dr. Carsten Thoms as scientific manager of the JSMC in November 2013. Silvio Waschina, Zerrin Üzüm, Alessio Garrone and Juliane Fischer succeeded the previous JSMC doctoral student speakers Markus Bohnert, Martin Föge and Daniel Schwenk in June 2013. nun auch die JSMC Geschäftsführung beherbergt. Katja Präfcke löste Ulrike Schleier als Assistentin der JSMC Geschäftsführung im November 2012 ab, und Dr. Jan Büllesbach trat im November 2013 die Nachfolge von Dr. Carsten Thoms als wissenschaftlicher Geschäftsführer der JSMC an. Silvio Waschina, Zerrin Üzüm, Alessio Garrone und Juliane Fischer übernahmen im Juni 2013 die Funktion als JSMC Doktorandensprecher von Ihren Vorgängern Markus Bohnert, Martin Föge and Daniel Schwenk. HKI Research Report 2012 / 2013 Jena School for Microbial Communication Cross-sectional Unit Jena Microbial Resource Collection HKI Research Report 2012 / 2013 169 Cross-sectional Unit Jena Microbial Resource Collection The Jena Microbial Resource Collection (JMRC) was established in October 2010 as a joint institution of the HKI and the Friedrich-Schiller-University Jena and represents a cross-sectional unit within the HKI. The JMRC manages a collection of living microorganisms comprising about 15,000 fungal and 30,000 bacterial strains. This collection functions as a microbial diversity bank in which the strains serve as microbial resources for academic and industrial research into natural products and infection biology. As regards research, the staff of the JMRC headed by Kerstin Voigt put their focus on pathogenesis, host-pathogen interactions and biodiversity of zygomycetes. Hereby, the main focus is on the evolution of pathogenicity in human pathogenic zygomycetes, namely species of the genera Lichtheimia, Rhizopus and Conidiobolus. Our aim is to decode the mechanisms of infections using evolutionary, cell and infection biology and physiology in combination with Omics-approaches and complex infection models. I N T R O D U C T I O N | E I N LE I T U N G Head: PD Dr. Kerstin Voigt Die Jena Microbial Resource Collection (JMRC) wurde im Oktober 2010 als gemeinsame Einrichtung des HKI und der Friedrich-Schiller-Universität Jena gegründet und repräsentiert eine Querschnittseinrichtung innerhalb des HKI. Die JMRC unterhält eine Sammlung von lebenden Mikroorganismen, die aus 15.000 Pilzstämmen sowie 30.000 Bakterienstämmen besteht. Diese Sammlung fungiert als mikrobielle Diversitätsbank, bei der die Stämme als mikrobielle Ressourcen für die akademische und industrielle Forschung an Naturstoffen und Infektionsbiologie dienen. In der Forschung 170 widmen sich die Mitarbeiter der JMRC unter der Leitung von PD Dr. Kerstin Voigt der Pathogenese, der Wirt-Pathogen Interaktion und der Biodiversität von Zygomyceten. Der Fokus liegt hierbei auf der Evolution der Pathogenität human-pathogener Zygomyceten, namentlich Arten der Gattungen Lichtheimia, Rhizopus und Conidiobolus. Unser Ziel ist es, die Mechanismen der Infektion mit Hilfe von evolutions-, zell- und infektionsbiologischen sowie physiologischen Ansätzen in Kombination mit Omics-Verfahren und komplexen Infektionsmodellen zu entschlüsseln. HKI Research Report 2012 / 2013 Cross-sectional Unit Jena Microbial Resource Collection We are especially interested in the interaction of Lichtheimia with the host’s immune system in disseminated infections in order to unravel the pathogenicity mechanisms and to define targets for prevention or protection against the disease. In this context, projects address the microscopic observation of the interaction of Lichtheimia with murine and human phagocytes (macrophages), the determination of the genome, transcriptome and proteome of Lichtheimia corymbifera, the establishment of a mouse model and the species recognition of Lichtheimia and Rhizopus, which are the main causative agents of zygo- mycoses, based on molecular systematics approaches to aid rapid diagnosis in comparative conjunction to the diversity of zygomycetes. Wir sind im Besonderen an der Interaktion von Lichtheimia mit dem Immunsystem des Wirts in disseminierenden Infektionen interessiert, um die Pathogenitätsmechanismen im Hinblick auf die Definition von Targets für die Prävention von und Schutz gegen Infektionen mit Zygomyceten zu untersuchen. In diesem Kontext befassen sich unsere Projekte mit der mikroskopischen Beobachtung des Zusammenspiels von Lichtheimia mit murinen und humanen Phagozyten (Makrophagen), der Entschlüsselung des Genoms, Transkriptoms und Proteoms von Lichtheimia corymbifera, der Etablierung eines Maus-Modells sowie der Artbildung inner- halb der beiden Haupterreger von Mucormykosen Lichtheimia und Rhizopus mit Hilfe von molekular-systematischen Ansätzen mit dem Ziel der schnellen Diagnose in vergleichender Konjunktion mit der Diversität von Zygomyceten. In 2012 to 2013, our research was supported by the Jena School for Microbial Communication (JSMC), the Friedrich Schiller University Jena and the Deutsche Forschungsgemeinschaft (DFG). Projects are integrated into the DFG Collaborative Research Center / Transregio CRC124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet”. Die Forschungsarbeit der JMRC wurde in den Jahren 2012 und 2013 durch die Jena School for Microbial Communication (JSMC), die Friedrich-Schiller-Universität Jena und die Deutsche Forschungsgemeinschaft (DFG) unterstützt. Die JMRC ist am SFB /TR 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet” beteiligt. Cross-sectional Unit Jena Microbial Resource Collection HKI Research Report 2012 / 2013 171 Scientific Projects Figure 1 Schematic diagram of the phagocytosis assay. 1 The interaction of the human pathogenic zygomycete Lichtheimia with macrophages Group Leader: Kerstin Voigt The number of cases of zygomycosis has increased over the past few years. Lichtheimia species are the 2nd and 3rd most common causative agents for zygomycosis in Europe and USA respectively. The main route of infection is via the respiratory tract where alveolar macrophages (AM) act as the immune system’s first line of defense. A literature review was carried out focusing on the interaction of pathogenic zygomycetes with innate immune cells in order to determine the current knowledge of the interplay at the cellular level between both counterparts. In order to assess the phagocytic efficiency of murine alveolar macrophages (MH-S) (provided by Hans-Martin Dahse, Department Infection Biology), co-incubation of MH-S 172 and two strains of L. corymbifera; FSU 9682 (fully virulent) and 10164 (attenuated) was carried out for an hour with MOI of 5 and fluorescence images were taken for the calculation Phagocytic index (PI). Three conditions; resting, pre-swollen, and opsonised spores were taken for the analysis (Fig. 1). The major findings were: In contrast to the other conditions, FSU 9682 and 10161 showed significant difference in PI in opsonisation. Quantification of C3b (a by-product of C3 which plays a central role in complement system) was followed on two strains via Fluoresence-Activiated Cell Sorting (FACS). Interestingly, complement component C3b was more abundant on FSU 9682 than that of FSU 10164 despite of the similarity in their sizes. These data show for the first time the PI of L. corymbifera by MH-S cells which was never measured previously. We also demonstrated that L. corymbifera binds to main components of the complement system. HKI Research Report 2012 / 2013 Cross-sectional Unit Jena Microbial Resource Collection In addition to the manual analysis, automated image analysis was also carried out in collaboration with Kaswara Kraibooj and Thilo Figge (Research Group Applied Systems Biology). As a perspective we aim to study the interaction of AM and L. corymbifera in more detail at the transcriptomic level using RNA-seq analyses. Various methods such as investigation of binding to complement regulators like factor H, killing assay and transcriptomic analysis of L. corymbifera will be used to extend the current knowledge of pathogensis of L. corymbifera. For transcriptomic analysis, we will be using primary macrophages provided by Kathleen Wagner and Marie von Lilienfeld-Toal from Associated Group Infections in Hematology/Oncology (IHO). This project is funded by the JSMC and performed in close collaboration with Axel A. Brakhage (MAM) and Peter F. Zipfel (IB). 2 De novo sequenced genome from Lichtheimia corymbifera, an ancient human pathogenic basal lineage fungus causing mucormycoses and its functional genomics in the context of evolution of pathogenicity of zygomycetes Group Leaders: Kerstin Voigt, Axel Brakhage Mucoralean fungi are able to cause severe infections in animals and humans. Although these mucormycoses are regarded as uncommon infections the number of cases increased during the last decades. The majority of mucormycotic infections is caused by Rhizopus, Mucor or Lichtheimia species, comprising 70 to 80 % of all clinical cases of zygomycoses. While the genera Rhizopus and Mucor are closely related mucoralean fungi, Lichtheimia represents a more basal group of mucoralean fungi which also includes other pathogenic species such as Rhizomucor pusillus. In addition, the three genera show differences in growth kinetics and morphology (e.g. dimorphism), thermotolerance and resistance to antifungals. The genomes of Rhizopus oryzae and Mucor circinelloides were already sequenced, whereas no genomic data of more basal mucoralean pathogens was available. Here we present first insights into the genome L. corymbifera. Based on 454 and Illumina data contigs with a total length of 33.6 MB were assembled. To aid prediction of protein-coding genes, RNAseq analyses were performed for three different growth conditions (standard growth, hypoxia, iron depletion). The use of different conditions should ensure a higher number of expressed genes, thereby allowing evidencebased gene predictions for many gene models. Prediction of protein-coding genes was performed using AUGUSTUS, resulting in 12,379 predicted genes. The L. corymbifera genome provides first insights into the basal mucoralean pathogens and will contribute to the understanding of these pathogens at the level of genome architecture and its evolution. L. corymbifera represents the most basal mucoralean pathogen sequenced so far. Comparison of the L. corymbifera genome to other mucoralean genomes revealed that less than 40 % of the gene families are shared with all other mucoralean genomes representing 53.7 % of L. corymbifera genes. Conserved regions, in terms of gene order, between mucoralean genomes were examined and a total of 230 regions with a minimum of 3 conserved genes of L. corymbifera were found that were present in at least one of the other genomes. These regions covered only 7.6 % of the L. corymbifera genome reflecting the high dissimilarity between the mucoralean genomes. Comparable to the situation in R. oryzae a high amount of duplicated genes was found in the L. corymbifera genome. In addition, several Lichtheimia-specific gene expansions were found including transcription factors and transporters. Interestingly, gene expansions often overlapped with tandem duplica- Cross-sectional Unit Jena Microbial Resource Collection HKI Research Report 2012 / 2013 173 tions of the genes. Thus retention of tandem duplicated genes could be an explanation for the high incidence of mostly speciesspecific gene duplications in mucoralean genomes. Expression analyses of duplicated genes revealed that the different copies of the genes were differently regulated in many cases. Thus, gene duplication seems to result in functionally diverse genes. Functional genomics will be performed using the genomes of R. oryzae and M. circinelloides, both causing mucormycoses in immunocompromised patients in conjunction with the genome of Condiobolus coronatus, which causes entomophthoromycosis in immuncompetent patients. The genome of C. coronatus was determined in collaboration with the Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI, Matthias Platzer). This project is in part funded by FungiNet and performed in close collaboration with Olaf Kniemeyer and Axel A. Brakhage (MAM). 3 Development of a pulmonary mouse infection model for Lichtheimia spp. Group Leaders: Kerstin Voigt, Ilse D. Jacobsen Although the number of mucormycosis cases has increased during the last decades, little is known about the pathogenesis of mucoralean fungi. While Rhizopus oryzae represents the most-common cause of mucormycosis worldwide, the frequency of Lichtheimia infections differs between geographic regions. Lichtheimia species represent the secondmost common cause of mucormycosis in Europe but play only a minor role in the USA. The primary route of infection is believed to be the inhalation of spores, and pulmonary infection is one of the most-common presentations. Patients with diabetes mellitus and immunocompromised patients following transplantation represent major risk groups. Although different infection models have been used to study Lichtheimia infections, a 174 standardised model has not been established yet. Mostly spores of Lichtheimia were applied via intravenous infection which does not represent the natural route of infection. Based on intranasal infection models for R. oryzae, a model for L. corymbifera was developed and used to test the effect of different risk factors. No clinical disease was observed after infection with L. corymbifera spores in healthy mice. However, immunosuppression with cortisone acetate resulted in development of disease and mortality. The infection was associated with massive tissue destruction in lungs and the kidneys. Additionally, also dissemination into liver, spleen and the brain was observed. Viable spores were found to be present in lungs of immunocompetent and immunocompromised surviving animals 28 days post infection. Although described as a major risk factor for mucormycoses, diabetic mice were not susceptible for L. corymbifera infection. Thus, Lichtheimia species seem to differ from other mucoralean pathogens, e.g. R. oryzae in risk factors. 4 Species recognition and diagnostics of human pathogenic zygomycetes Group Leaders: Grit Walther, Kerstin Voigt Taxonomy and diagnostics of the Rhizopus arrhizus (syn. R. oryzae) species complex Rhizopus arrhizus (syn. R. oryzae) causes by far the most mucormycoses worldwide. On the other hand the species has been used for centuries for the production of fermented food in Asia. Recently, molecular phylogenetic analyses based on four markers revealed the existence of two phylogenetic species, R. arrhizus s. s. and R. delemar differing by the production of lactic acid or fumaric-malic acid, respectively. The phylogenetic species R. arrhizus s. s. contains two copies of Lactate dehydrogenase, LDHA and LDHB, while R. delemar has only a single copy of this HKI Research Report 2012 / 2013 Cross-sectional Unit Jena Microbial Resource Collection gene (LDHB). However, the genetic distance between R. arrhizus s. s. and R. delemar is extremely low compared to distances among other species in the genus Rhizopus. In addition, the formation of zygospores between strains of R. arrhizus s. s. and R. delemar as an indicatior for sexual reproduction have been reported. In cooperation with Somayeh Dolatabadi and Sybren de Hoog from the CBSKNAW Fungal Biodiverstiy Centre (Utrecht, The Netherlands) we currently try to answer the questions if R. arrhizus s. s. and R. delemar should be treated as single or as separate species and if they differ in their pathogenic potential. Therefore we study a set of about 80 strains belonging two both putative species and originating from different sources (clinical, environmental, food). We performed a comprehensive molecular phylogenetic study based on five different markers, AFLPs and MALDI-TOF MS analyses. In order to assess biological species boundaries we performed mating tests. Preliminary results revealed that two out of five phylogenetic gene trees show polyphylies of R. arrhizus and R. delemar within the R. oryzae complex suggesting a single species. Currently the sequencing is repeated in order to exclude that this deviating position of the strains in the trees is only caused by the confusion of strain numbers. Mating tests were successful only in two pairings, one of which represents a pairing between R. arrhizus sensu stricto and R. delemar, a fact that is furthermore supporting the hypothesis of a single species. other genera in the fungal kingdom. Recent molecular phylogenetic studies based only on a small number of strains revealed that the morphologically defined species Syncephalastrum racemosum consists of at least two distinct groups that might represent independent species. In cooperation with numerous partners we are currently studying the Syncephalastrum racemosum species complex molecular phylogenetically, morphologically, physiologically and by mating tests for biological species recognition. Central questions are: how many species to this complex? Do they differ in their pathogenic potential and in their susceptibility for antifungals? And how can they be diagnosed? Preliminary results of the molecular phylogenetic study revealed a surprisingly high biodiversity. To date we recognized five phylogenetic species that differ neither in their morphology nor in their susceptibility profiles. Mating tests to recognise the biological species boundaries were still not successful. The project is performed in close collaboration with the National Reference Center for Invasive Mycoses (NRZMyk) and is funded by grant of the DFG to Dr. Grit Walther. References Grützmann K1, Szafranski K, Pohl M, Voigt K, Petzold A, Schuster S (2014) Fungal alternative splicing is associated with multicellular complexity and virulence: a genome-wide multispecies study. DNA Res 21, 27-39. Schwartze VU, Hoffmann K, Nyilasi I, Papp T, Vágvölgyi C, de Hoog S, Voigt K, Jacobsen ID (2012) Lichtheimia species exhibit differences in virulence potential. PLoS One 7, e40908. Schrödl W, Heydel T, Schwartze VU, Hoffmann K, Walther G, AlastrueyIzquierdo A, Rodriguez-Tudela JL, Olias P, Jacobsen ID, de Hoog GS, Voigt K (2012) Direct analysis and identification of opportunistic Lichtheimia species by Matrix Assisted Laser Desorption Ionization (MALDI) - Time-Of-Flight (TOF) analyzermediated mass spectrometry. J Clin Microbiol 50, 419-427. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W; Bolchacova E, Voigt K and the Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 109, 6241-6246. Hoffmann K, Pawłowska J, Walther G, Wrzosek M, Hoog GS de, Benny GL, Kirk PM, Voigt K (2013) The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies. Persoonia 30, 57–76. Taxonomy and diagnostics of the Syncephalastrum racemosum species complex Syncephalastrum racemosum is a thermotolerant saprotropic fungus belonging to the order Mucorales with a main distribution in the tropics and subtropics. It is able to cause infections in humans with impaired immunity but these mucormycoses are rare and restricted to its main distribution area. The species is also of interest because it possesses two distinctly deviating types of ITS sequences which are known only from two Cross-sectional Unit Jena Microbial Resource Collection HKI Research Report 2012 / 2013 175 Group members Collaborations Head PD Dr. Kerstin Voigt Phone +49 3641 532-1395 Fax +49 3641 532-2395 [email protected] Friedrich Schiller University Jena, Germany Prof. Dr. Stefan Schuster Prof. Dr. Erika Kothe Ph.D. Students Volker Schwartze Hea Reung Park Leibniz Institute for Age Research – Fritz Lipmann Institute, Jena, Germany Max Planck Institute for Chemical Ecology, Jena Freie Universität Berlin, Germany Research Assistents Christiane Weigel Caroline Semm Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany External funding Centre for Genomic Regulation (CRG), Barcelona, Spain Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project Z01: Die Rolle der Regulation putativer Pathogenitäts-assoziierter Gene in der Virulenz des humanpathogenen Mucormykose-Erregers Lichtheimia corymbifera: Vergleichende RNAseq-Expressionsanalyse von attenuiertem versus virulentem Stamm Kerstin Voigt Universitat Pompeu Fabra (UPF), Barcelona, Spain Centre Nacional d'Anàlisi Genòmica (CNAG), Barcelona, Spain Laboratório Nacional de Computação Científica (LNCC), Rio de Janeiro, Brazil Ruhr University Bochum, Germany Selected publications Medical University Innsbruck, Austria Schwartze VU, Winter S, Shelest E, MarcetHouben M, Horn F, Wehner S, Linde J, Valiante V, Sammeth M, Riege K, Nowrousian M, Kaerger K, Jacobsen ID, Marz M, Brakhage AA, Gabaldón T, Böcker S, Voigt K (2014) Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: an evolutionary genomics analysis in the ancient terrestrial Mucorales (Mucoromycotina). PLoS Genetics, in press. Institut Pasteur, Paris, France Schoch CL, Robbertse B, Robert V, …, Voigt K, …, Federhen S (2014) Finding needles in haystacks: linking scientific names, reference specimens. Database (Oxford), 1-21. Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Braunschweig, Germany University of Szeged, Hungary Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech Republic Botanische Staatssammlungen München, Germany CBS Utrecht, The Netherlands Hoffmann K, Pawlowska J, Walther G, Wrzosek M, de Hoog GS, Benny GL, Kirk PM, Voigt K (2013) The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies. Persoonia 30: 57–76. Federal Rural University of Pernambuco – Academic Unit of Serra Talhada, Pernambuco, Brazil NIH/NLM/NCBI Bethesda, Maryland, USA Schrödl W, Heydel T, Schwartze VU, Hoffmann K, Walther G, Alastruey-Izquierdo A, RodriguezTudela JL, Olias P, Jacobsen ID, de Hoog GS, Voigt K (2012) Direct analysis and identification of opportunistic Lichtheimia species by Matrix Assisted Laser Desorption Ionization (MALDI) Time-Of-Flight (TOF) analyzer-mediated mass spectrometry. J Clin Microbiol 50, 419-427. Royal Botanic Gardens Kew, Mycology section, UK University of Gainsville Florida, USA Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W; Bolchacova E, Voigt K and the Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci USA 109, 6241-6246. 176 HKI Research Report 2012 / 2013 Cross-sectional Unit Jena Microbial Resource Collection Collaborative Research Centre / Transregio 124 FungiNet HKI Research Report 2012 / 2013 177 Collaborative Research Centre / Transregio 124 – FungiNet 178 Pathogenic fungi and their human host: Networks of interaction The Collaborative Research Centre (CRC) / Transregio 124 “FungiNet” started its work, funded by the German Research Foundation (DFG) in October 2013. It brings together natural scientists, clinicians and bioinformaticians from Jena and Würzburg, who will work in 18 different research projects. They join forces to study the two major pathogenic fungi Candida albicans and Aspergillus fumigatus and their interaction with the human host. The aim of FungiNet is to apply a sytems biological approach to decipher the complex pathobiology of the two human-pathogenic fungi and the response of the host’s immune system. This approach will uncover novel regulatory and metabolic pathways that are important in host-pathogen interactions and may help to redefine the basic concepts of virulence and pathogenicity of human-pathogenic fungi. The aims of FungiNet are: •to identify pathogenic determinants specific for A. fumigatus or C. albicans •to investigate the roles of epithelial barriers, the innate and adaptive immune system for the pathogenesis of fungal infections Pathogene Pilze und ihr menschlicher Wirt: Netzwerke der Interaktion Der SFB / Transregio 124 „FungiNet“, der von der Deutschen Forschungsgemeinschaft (DFG) gefördert wird, nahm im Oktober 2013 seine Arbeit auf. In 18 Teilprojekten arbeiten Naturwissenschaftler, Mediziner und Bioinformatiker aus Jena und Würzburg zusammen daran, die beiden bedeutenden human-pathogenen Pilze Aspergillus fumigatus und Candida albicans zu untersuchen und die Interaktionen mit dem menschlichen Wirt zu entschlüsseln. Gemeinsames Ziel ist es, die komplexen Mechanismen der Pil- zinfektionen zu verstehen und mittels eines systembiologischen Ansatzes ein „virtuelles Infektionsmodell” zu generieren, das als Ausgangspunkt zur Entwicklung effektiver Präventions- und Therapiestrategien dient. FungiNet will •Spezifische Pathogenitätsfaktoren für A. fumigatus und C. albicans identifizieren •Die Rolle von Gewebeschranken sowie des angeborenen und adaptiven Immunsystems bei der Pathogenese pilzlicher Infektionen untersuchen •Die komplexen Interaktionsmechanismen während des Infektionsprozesses HKI Research Report 2012 / 2013 Collaborative Research Centre / Transregio 124 – FungiNet •to elucidate the complex mechanisms of interactions during the infection process and to develop virtual models and tools, which can describe these interplays •to use the gained knowledge for the development of therapeutic approaches •Candida albicans: from commensal to pathogen. Additionally, the CRC comprises central projects which provide services to the involved scientists. Axel Brakhage (HKI) is the speaker of FungiNet. FungiNet is divided in three interlinked research areas, namely •Aspergillus fumigatus: from environmental microorganisms to pathogen, •Bioinformatics / Computational systems biology of infection; creation of a „virtual infection model“ entschlüsseln und virtuelle Modelle und Werkzeuge entwickeln, die dieses Zusammenspiel beschreiben •Das erworbene Wissen dafür einsetzen, neue Therapiestrategien für Pilzinfektionen zu entwickeln •Candida albicans: vom Kommensalen zum Pathogen Außerdem beinhaltet der SFB / Transregio noch Zentralprojekte, die Services für die beteiligten Wissenschaftler liefern. Axel Brakhage ist der Sprecher des SFB / Transregio. Dazu kommen in FungiNet drei miteinander verknüpfte Ansätze zum Einsatz: •Aspergillus fumigatus: vom allgegenwärtigen Mikroorgansimus zum Pathogen •Bioinformatik / Systembiologie der Infektion: Erstellung eines „virtuellen Infektionsmodells” Collaborative Research Centre / Transregio 124 – FungiNet HKI Research Report 2012 / 2013 179 180 HKI Research Report 2012 / 2013 Associated Group Infections in Hematology and Oncology HKI Research Report 2012 / 2013 181 Associated Group Infections in Hematology and Oncology The Associated Group Infections in Hematology and Oncology (IHO) was founded in summer 2013 with the appointment of Marie von Lilienfeld-Toal and consists of clinical scientists based at the University Hospital Jena and of natural scientists in the laboratory based within the HKI. Our research interest comprises all aspects of infectious complications in patients with malignancies with a special focus on atypical infections like invasive fungal infections. We form a link between the clinical departments and institutes of the medical faculty on one side and the HKI on the other side which enables interdisciplinary research of clinically relevant questions (Fig. 1). Infections are the leading cause of treatment-related mortality in patients with malignant diseases. This is commonly believed to be due to a profound immunosuppression, which makes patients susceptible to opportunistic infections. Good examples for such opportunistic infections are invasive fungal infections, which almost never occur in healthy individuals and which are I N T R O D U C T I O N | E I N LE I T U N G Head: Prof. Dr. Marie von Lilienfeld-Toal 182 Die assoziierte Gruppe Infektionen in der Hämatologie und Onkologie (IHO) entstand im Sommer 2013 mit der Berufung von Marie von Lilienfeld-Toal und besteht aus wissenschaftlich tätigen Ärzten im Universitätsklinikum Jena-Lobeda sowie Naturwissenschaftlern in den Laborräumen im HKI. Wir beschäftigen uns mit allen Gesichtspunkten von Infektionen bei Patienten mit malignen Systemerkrankungen mit einem speziellen Schwerpunkt im Bereich atypischer Infektionen wie zum Beispiel invasiver Pilzerkrankungen. Mit dem Standort Universitätsklinikum Jena-Lobeda auf der einen Seite und dem Standort Hans-Knöll-Institut auf der anderen Seite bilden wir eine Verbindung zwischen klinischen und theoretischen Instituten und ermöglichen so die interdisziplinäre Bearbeitung klinisch relevanter Fragestellungen (Fig. 1). Infektionen sind die wichtigste Ursache für therapieassoziierte Mortalität bei Patienten mit Krebserkrankungen. Man geht davon aus, dass dies vor allem der ausgeprägten Immunsuppression, die die Patienten für opportunistische Infektionen prädisponiert, geschuldet ist. Invasive Pilzinfektionen sind HKI Research Report 2012 / 2013 Associated Group Infections in Hematology and Oncology exceptionally difficult to diagnose. In addition, treatment of invasive fungal infections is often toxic and not very successful. For these reasons, invasive fungal infections in patients with hematological malignancies have a very high mortality rate. Besides opportunistic infections, cancer patients are at risk of common infections, which often take a more severe clinical course than in healthy individuals. This is believed to be the case for infections with respiratory viruses like influenza or adenovirus. We aim at understanding the pathogenesis and clini- ein gutes Beispiel für solche opportunistischen Infektionen, da sie bei gesunden Menschen fast nie vorkommen. Sie sind schwierig zu diagnostizieren und erfordern eine relativ toxische Therapie, die oft nicht ausreichend wirkt. Aus diesen Gründen sind invasive Pilzinfektionen mit einer hohen Mortalitätsrate assoziiert. Neben dem Risiko für opportunistische Infektionen besteht außerdem das Risiko für „normale“ Infektionen, die bei Krebspatienten oft schwerer verlaufen als bei Gesunden. Infektionen mit respiratorischen Viren wie Influenza oder Adenovirus sind Beispiele, für die schwerere Verläufe bei cal course of some of these infections with the goal to improve diagnostics and clinical treatment of these patients. In 2013 our research was supported by the Federal Ministry of Education and Research (BMBF) within the Center for Sepsis Control and Care (CSCC) and by the Interdisciplinary Centre for Clinical Research (IZKF) of the Medical Faculty of Friedrich Schiller University Jena. Krebspatienten beschrieben werden. Unser Ziel ist es, das Verständnis für die Entstehung dieser Infektionen und ihren klinischen Verlauf zu vertiefen, um so die Diagnostik und Therapie verbessern zu können. 2013 erhielt unsere Gruppe finanzielle Unterstützung vom Bundesministerium für Bildung und Forschung (BMBF) innerhalb des Verbundes Center for Sepsis Care and Control (CSCC)und vom Interdisziplinären Zentrum für Klinische Forschung (IZKF) der medizinischen Fakultät der Friedrich-Schiller-Universität Jena. Associated Group Infections in Hematology and Oncology HKI Research Report 2012 / 2013 183 Scientific Projects IHO takes part in a number of ongoing multicentre clinical trials which will not be discussed in detail. The following projects have been developed over the last six months within our group in Jena. 1 Establishment of registry of infections in patients with hematological malignancies Group Leader: Tobias Rachow Prior research has been partly based on a large registry of clinical cases from the university hospital at Bonn (Schwab et al. 2014, Hahn-Ast et al. 2010). We are now in the process of establishing a registry of infections in patients with hematological malignancies treated in the University Hospital Jena. A retrospective documentation of the years 2012 and 2013 is the foundation for the prospective documentation of the following years. We document the clinical courses of patients receiving myelosuppressive chemotherapy for acute leukaemia with a special focus on all aspects of infectious complications including the respective diagnostic and therapeutic interventions. We will enlarge the registry to include additional types of malignant diseases apart from leukaemia in the future. In particular, we focus on the following aspects: a) Efficacy of antimicrobial prophylaxis and therapy Guidelines on the management of infectious complications in cancer patients are mostly based on the results of prospective clinical trials. However, for better comparability these trials include only very selected patient populations. Thus, the results often do not reflect clinical reality as they cannot 184 be transferred from the homogenous trial population (often only one disease included, no co-morbidities, median age low, very specified co-medication allowed) to the more heterogeneous patient population (different diseases, many co-morbidities, median age higher, large variety of co-medication) inhabiting the wards. It is therefore necessary to acquire knowledge about the clinical course and the treatment efficacy of infections in clinical reality by descriptive analysis of patient cohorts (Schwab et al. 2014). In cooperation with the University Hospital Bonn (Karin Mayer), we conducted a first analysis of 286 patients with acute myeloid leukaemia with regard to the efficacy of two different regimens of antibiotic prophylaxis and found, in contrast to published trials, an equivalent efficacy of the newer drugs (fluorochinolones) and the older drug (cotrimoxazole). Current efforts will lead to enlargement of this patient cohort and additional aspects like empirical antimicrobial treatment in the first or second line will be analysed. b) Incidence, risk factors and clinical course of invasive fungal infections As part of the registry, patients with suspected or diagnosed invasive fungal infections are also documented. Diagnosis of Aspergillus infection in these patients is based on the detection of the galactomannan-antigen specific for Aspergillus spp. in the serum or in bronchial-lavage fluid. Importantly, for these patients we collect left-over serum samples from test tubes collected for detection of the Aspergillus antigen. Over the past four months, more than 400 samples have been collected and stored. This allows the testing of novel diagnostic tools on serum with known results in the conventional diagnostic methods. Here, we collaborate in particular with the Department Molecular and Applied HKI Research Report 2012 / 2013 Associated Group Infections in Hematology and Oncology IHO Infec?ons in Haematology and Oncology Respiratory Viruses in Healthy Donors Sample Storage Hans Knöll Ins*tute Associated Research Group IHO University Hospital Jena Research Group IHO Sample and Data Collec*on PCR Registry Infec?ons in Haematology and Oncology Sample Storage Clinical Trials C. diff, CMV, ESBL Immune response against Aspergillus Cell Culture, Flow Cytometry, Cytokine Response Infec*on Biology Microbiology. In addition, we are able to provide fresh samples of patients with proven fungal disease or with specified immune defects to other research groups, in particular to Fungal Septomics and the Jena Microbial Resource Collection (JMRC). c) Epidemiology, diagnosis and treatment of atypical mycobacteria Infections with atypical mycobacteria such as Mycobacterium avium intracellulare are rare events in patients with hematological malignancies. Nonetheless, they do occur and little is known about their relevance in terms of morbidity and mortality. Most treatment recommendations have been deduced from patients with HIV as these patients are believed to be similarly immunosuppressed. However, it has been recognised that patients with hematological malignancies might differ in their susceptibility to infection and in their clinical course, but there are still not enough epidemiological data to draw any conclusions. We set out to document patient records with proven or suspected infection with atypical mycobacteria and describe the severity of the infection as well as the response to treatment (if administered). This project is being conducted in collaboration with B. Hermann from the Institute of Medical Microbiology, University Hospital Jena. Associated Group Infections in Hematology and Oncology HKI Research Report 2012 / 2013 Figure 1 Depiction of the Research Group IHO and its collaborations within the HKI. 185 References Schwab KS, Hahn-Ast C, Heinz WJ, Germing U, Egerer G, Glasmacher A, Leyendecker C, Marklein G, Nellessen CM, Brossart P, von Lilienfeld-Toal M (2014) Tigecycline in febrile neutropenic patients with haematological malignancies: a retrospective case documentation in four university hospitals. Infection 42, 97-104. Hahn-Ast C, Glasmacher A, Mückter S, Schmitz A, Kraemer A, Marklein G, Brossart P, von Lilienfeld-Toal M (2010) Overall survival and fungal infection-related mortality in patients with invasive fungal infection and neutropenia after myelosuppressive chemotherapy in a tertiary care centre from 1995 to 2006. J Antimicrob Chemother 65, 761-768. von Lilienfeld-Toal M, Frank S, Leyendecker C, Feyler S, Jarmin S, Morgan R, Glasmacher A, Märten A, Schmidt-Wolf IG, Brossart P, Cook G (2010) Reduced immune effector cell NKG2D expression and increased levels of soluble NKG2D ligands in multiple myeloma may not be causally linked. Cancer Immunol Immunothe. 59, 829-839 2 Immune response against Aspergillus infections in patients with hematological malignancies (IMPACT), CSCC Group Leader: Marie von Lilienfeld-Toal Group Leader: Kathleen Wagner Patients with hematological malignancies are believed to be susceptible to invasive fungal infections mainly because of their therapy-induced neutropenia (i.e. low number of immune cells). However, it is likely that they also exhibit a functional defect of the immune response, as other patient populations with low cell numbers (for example patients receiving treatment with interferon) do not show the same risk and because of observed defects in the tumour immune response as described by us and others (von LilienfeldToal et al. 2010). In this project, we analyse the innate immune response to Aspergillus fumigatus in healthy subjects compared to patients with interferon-therapy and patients with acute myeloid leukaemia prior to, during and after regeneration of chemotherapyinduced neutropenia. Immune cells from the peripheral blood are characterised by flowcytometry and cytokine release to determine the activation profile (pro-inflammatory versus anti-inflammatory). In addition, freshly sampled immune cells and germinating conidia of Aspergillus spp. are co-incubated to investigate the interaction between immune cells and conidia with regard to phagocytosis, viability and activation of immune cells. This project is funded by the BMBF/Center for Sepsis Control and Care (CSCC) and the experiments are performed in close collaboration with the Departments Molecular and Applied Microbiology, Infection Biology and ZIK Fungal Septomics. 186 3 Respiratory viruses in healthy individuals – Prospective surveillance with a multiplex PCR Recently, infections with respiratory viruses such as respiratory syncytial virus or influenza A have received much attention. This is partly because of the 2009 pandemic, but may also be due to the fact that refined diagnostic techniques like multiplex PCR are increasingly used to examine nasopharyngeal aspirates or swabs. With these nucleic-acid tests, viruses are now detected which have no clearly defined role such as metapneumovirus, bocavirus or parainfluenzavirus. However, case reports describe potentially fatal courses of cancer patients with pneumonia and evidence of metapneumovirus or parainfluenzavirus, which are then assumed to be causally linked. As there are no data whatsoever on the prevalence of these newly described viruses in healthy individuals, it is impossible to draw any firm conclusions on these findings. We have started a prospective diagnostic study to determine the prevalence and relevance of 21 respiratory pathogens in healthy individuals. The study started in November 2013 and has included 85 individuals so far. PCR measurements take place in our laboratory with a commercial multiplex PCR kit. We plan to recruit altogether 225 individuals over the course of two winters to be able to detect a prevalence of 10 % in the whole population in a statistically valid fashion. The study is conducted under the patronage of the Infectious Disease Working Party of the German Society for Haematology and Oncology (AGIHO) and is funded in part by the Interdisciplinary Centre for Clinical Research (IZKF) of the Medical Faculty, FSU Jena. HKI Research Report 2012 / 2013 Associated Group Infections in Hematology and Oncology Group members Head Prof. Dr. Marie von Lilienfeld-Toal Phone +49 3641 532-1720 Fax +49 3641 532-0815 [email protected] Scientists Dr. Kathleen Wagner Medical Doctors Dr. Beate Hermann Dr. Tobias Rachow Neumann S, Krause SW, Maschmeyer G, Schiel X, von Lilienfeld-Toal M, Infectious Diseases Working Party (AGIHO), German Society of Hematology and Oncology (DGHO) (2013) Primary prophylaxis of bacterial infections and Pneumocystis jirovecii pneumonia in patients with hematological malignancies and solid tumors: guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO). Ann Hematol 92, 433-442. Fleischhacker M, Schulz S, Jöhrens K, von Lilienfeld-Toal M, Held T, Fietze E, Schewe C, Petersen I, Ruhnke M (2012) Diagnosis of chronic disseminated candidosis from liver biopsies by a novel PCR in patients with haematological malignancies. Clin Microbiol Infect 18, 1010-1016. MD. Students Jana Kalkreuth Julia Kurrek Stephanie Kurze Katharina Meckel Verena Schlüter Maria Schuwirth Diploma / Bachelor / Master Students Franziska Wittig Mayer K, Kapelle M, Kaeferstein A, Weßling M, Bekeredjian-Ding I, Leutner C, von LilienfeldToal M, Brossart P, Wolf D (2013) Successful management of Candida krusei monoarthritis after allo-SCT. Bone Marrow Transplant 48, 1585-1586. Research Assistants Pia Konowski Collaborations External funding Bundesministerium für Bildung und Forschung Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) Project: IMPACT: Immune response against mould species in patients with acute myeloid leukaemia Marie von Lilienfeld-Toal Universitätsklinikm Bonn, Germany Prof. Dr. Georg Baumgarten, Dr. Karin Mayer Universitätsklinikum Jena, Germany Prof. Dr. Frank Brunkhorst PD Dr. Michael Kiehntopf Prof. Dr. Wolfgang Pfister Prof. Dr. Matthias Pletz Selected publications Schwab KS, Hahn-Ast C, Heinz WJ, Germing U, Egerer G, Glasmacher A, Leyendecker C, Marklein G, Nellessen CM, Brossart P, von Lilienfeld-Toal M (2014) Tigecycline in febrile neutropenic patients with haematological malignancies: a retrospective case documentation in four university hospitals. Infection 42, 97-104. Epub 2013 Aug 25. Lo-Coco F, Avvisati G, Vignetti M, Thiede C, Orlando SM, Iacobelli S, Ferrara F, Fazi P, Cicconi L, Di Bona E, Specchia G, Sica S, Divona M, Levis A, Fiedler W, Cerqui E, Breccia M, Fioritoni G, Salih HR, Cazzola M, Melillo L, Carella AM, Brandts CH, Morra E, von Lilienfeld-Toal M, Hertenstein B, Wattad M, Lübbert M, Hänel M, Schmitz N, Link H, Kropp MG, Rambaldi A, La Nasa G, Luppi M, Ciceri F, Finizio O, Venditti A, Fabbiano F, Döhner K, Sauer M, Ganser A, Amadori S, Mandelli F, Döhner H, Ehninger G, Schlenk RF, Platzbecker U; Gruppo Italiano Malattie Ematologiche dell'Adulto; German-Austrian Acute Myeloid Leukemia Study Group; Study Alliance Leukemia (2013) Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med 11, 111-121. Associated Group Infections in Hematology and Oncology HKI Research Report 2012 / 2013 187 188 HKI Research Report 2012 / 2013 Associated Group Microbial Biochemistry and Physiology HKI Research Report 2012 / 2013 189 Associated Group Microbial Biochemistry and Physiology The Associated Group Microbial Biochemistry and Physiology (MBP) investigates fungal nutrition during pathogenesis, the effect of environmental conditions on secondary metabolite production and the real-time visualisation of fungal infections by bioluminescence imaging. Currently, we focus our research on the filamentous ascomycetes Aspergillus fumigatus and Aspergillus terreus and the dimorphic yeast Candida albicans. way in A. fumigatus, Aspergillus nidulans and Saccharomyces cerevisiae. Here, a special interest was given to the isomerisation reaction between homocitrate and homoisocitrate that is required for α-aminoadipate synthesis, which is, in some fungi, the precursor for penicillins and cephalosporins. Furthermore, α-oxidation of fatty acids was studied in C. albicans to investigate its importance during infection. In terms of nutrient acquisition during infection, we elucidated the synthesis of the amino acid lysine via the α-aminoadipate path- A. terreus is a potent source of secondary metabolites, since its genome contains a great variety of secondary metabolite gene I N T R O D U C T I O N | E I N LE I T U N G Die Asoziierte Gruppe Mikrobielle Biochemie und Physiologie beschäftigt sich maßgeblich mit der Nährstoff-Verwertung pathogener Pilze im Infektionsprozess, der Bedeutung der Sekundärstoff-Produktion in Abhängigkeit von Umweltbedingungen und der Echtzeit-Visualisierung von Pilzinfektionen am lebenden Versuchstier. Hierbei stehen insbesondere die filamentösen Pilze Aspergillus fumigatus und Aspergillus terreus, sowie die dimorphe Hefe Candida albicans im Zentrum der Studien. Head: Dr. habil. Matthias Brock Im Rahmen der Nährstoff-Verwertung wurde die Synthese der Aminosäure Lysin anhand von A. fumigatus, Aspergillus nidulans und Saccharomyces cerevisiae über den 190 α-Aminoadipatweg näher untersucht. Hierbei stand die Isomerisierung von Homocitrat zu Homoisocitrat im Vordergrund, die für den Vorläufer α-Aminoadipat essentiell ist. Dieser Vorläufer wird von einigen Pilzen gleichzeitig für die Synthese von Penicillinen und Cephalosporinen benötigt. Des Weiteren wurden Untersuchungen zur Fettsäure-Verwertung von Candida albicans angestellt, um den Einfluss dieser Nährstoffquelle auf den Infektionsprozess zu beleuchten. A. terreus ist eine bedeutende Quelle für Sekundärstoffe, da das Genom zahlreiche Gencluster für solche Metabolite besitzt. Hierbei ist A. terreus besonders für die Produktion des HMG-CoA Reduktase-Inhibi- HKI Research Report 2012 / 2013 Associated Group Microbial Biochemistry and Physiology clusters. Due to its ability to produce the HMG-CoA reductase inhibitor lovastation, this fungus is currently used in white biotechnology. However, A. terreus is also found as a pathogen of humans and is, similar to A. fumigatus, able to provoke life-threatening invasive aspergillosis. However, in contrast to A. fumigatus, invasive aspergillosis caused by A. terreus is frequently associated with dissemination. Additionally, due to an intrinsic Amphotericin B resistance, one of the most important first-line drugs to combat fungal infections is not available. Therefore, we established infection models to compare invasive aspergillosis caused by both fungi and are currently investigating the requirement of secondary metabolite production for environmental competition of A. terreus. tors Lovastatin bekannt und wird zur biotechnologischen Produktion dieses Statins eingesetzt. Daneben ist A. terreus aber in der Lage, lebensbedrohliche invasive Aspergillosen hervorzurufen, die insbesondere durch den verwandten Vertreter A. fumigatus bekannt sind. Im Gegensatz zu A. fumigatus sind Infektionen mit A. terreus häufig von einer Disseminierung der Infektion begleitet. Zusätzlich zeigt A. terreus eine intrinsische Resistenz gegenüber dem bedeutenden Antimykotikum Amphotericin B, was die Behandlung zusätzlich erschwert. Daher wurden Infektionsmodelle etabliert, die es erlauben, den Infektionsverlauf über A. fumigatus und A. terreus zu vergleichen. Weiterhin wird zurzeit der Einfluss der Se- kundärstoff-Produktion auf die Kompetition von A. terreus mit der Umwelt studiert. Last but not least, bioluminescence imaging has been optimised to monitor onset and progression of invasive fungal infections under in vivo conditions. This technique has now been applied to study the effect of immunosuppression on disease development and to elucidate the in vivo efficacy of antifungal drugs. This technique allows the visualisation of temporal and spatial progression of Außerdem wurde die Methode des Biolumineszenz-Imaging optimiert, um Beginn und Verlauf invasiver Pilzinfektionen unter in vivo-Bedingungen zu untersuchen. Diese Technik wurde angewendet, um den Effekt von Immunsuppressionen auf den Krankheitsverlauf zu studieren und die Wirksamkeit antifungaler Therapien im lebenden Tier zu dokumentieren. Da sowohl der zeitliche als auch der räumliche Verlauf von Pilzinfektionen sichtbar gemacht werden kann, ist es mit dieser Technik möglich, die Strategien zur Therapie von Pilzinfektionen zu verbessern. Associated Group Microbial Biochemistry and Physiology HKI Research Report 2012 / 2013 191 fungal infections and can, thus, help to identify new therapeutic strategies against fungal infections. In 2012 to 2013, our research was supported by the International Leibniz Research School (ILRS) in a joint project with the department of Microbial Pathogenicity Mechanisms, the Deutsche Forschungsgemeinschaft (DFG) and the DFG Collaborative Research Center / Transregio CRC124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet”. In 2012 und 2013 wurden die Forschungsarbeiten durch ein gemeinsames Projekt mit der Abteilung Mikrobielle Pathogenitätsmechanismen im Rahmen der “International Leibniz Research School” (ILRS), durch Einzelförderung der Deutschen Forschungsgemeinschaft (DFG), dem DFG SFB/TR 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet” gefördert. 192 HKI Research Report 2012 / 2013 Associated Group Microbial Biochemistry and Physiology Scientific Projects 1 Nutrients and fungal pathogenesis Nutrient acquisition is important for fungal pathogens, as for all microorganisms, to proliferate within the host. In recent studies we focused on the synthesis of the amino acid lysine, which is an essential amino acid for humans, but is synthesised via the α-aminoadipate pathway in fungi. Since this pathway is also important for production of the antibiotics Penicillin and Cephalosporin, a main focus was given to reactions leading to the common precursor α-aminoadipate. In addition, fatty acids are very abundant in the host environment, since they are major components of membranes and used as energy storage compounds in the form of lipids. Literature on the impact of lipid metabolism on pathogenesis of C. albicans and enzymes involved in this metabolic path has remained confusing and was, therefore, revisited in our investigations. 1) Synthesis of the amino acid lysine Fungi synthesise lysine via the α-aminoadipate pathway and the precursor α-aminoadipate is additionally used by some fungi to produce beta-lactam antibiotics. Despite the great importance of α-aminoadipate production, not all reactions leading to this intermediate have been studied in detail. Thus, we concentrated on the isomerisation between homocitrate and homoisocitrate, a reaction that is similar to the aconitase reaction in the citrate cycle. However, while the citrate cycle only requires a single enzyme for this isomerisation, two enzymes are essentially required in the α-aminoadipate pathway. Unexpectedly, we discovered that the citrate cycle aconitase contributes to the α-aminoadipate pathway by performing the dehydration of homocitrate to homoaconitate. Subsequently, the rehydration of homoaconitate to homoisocitrate is performed by a pa- thway specific homoaconitase. The requirement for two enzymes in this isomerisation is due to the extended aliphatic chain-length in homocitrate compared citrate (Fig. 1). Isomerisation requires a 180° rotation of the substrate, which is characterised by a citrate and isocitrate binding mode in aconitases. The additional methylene-group in homocitrate prevents correct orientation of the substrate in the isocitrate mode. Thus, aconitase dehydrates homocitrate in the citrate mode, releases homoaconitate that is subsequently hydrated by homoaconitase in the isocitrate mode. Thus, the catabolic enzyme aconitase is directly linked with the anabolism of lysine. However, a second unexpected observation resulted from a difference between filamentous fungi and yeasts. Although both species contain two aconitase isoenzymes, the aconitase B is inactive in filamentous fungi, but has specifically adapted to serve for the α-aminoadipate pathway in yeasts by displaying high affinity for homocitrate but not citrate. This allows fermenting yeasts to separate anabolic lysine synthesis from the catabolic citrate cycle, which is dispensable for yeasts on fermentable nutrient sources. In contrast, filamentous fungi rely on an active citrate cycle since they are not able to ferment. Thus, differences in the life-style of fungi have manifested in the adaptation of isoenzymes to specific metabolic needs. While these discoveries shed new light on the fungal synthesis of lysine, they are also important for optimisation of β-lactam antibiotics production in biotechnological processes due to the tight link of cata- and anabolism in α-aminoadipate synthesis. 2) Fatty acid oxidation in Candida albicans: The role of thiolases Genes involved in β-oxidation are highly upregulated during the interaction of C. albicans with immune cells. The final reaction of α-oxidation is the release of acetyl-CoA units Associated Group Microbial Biochemistry and Physiology HKI Research Report 2012 / 2013 193 Figure 1 Scheme of substrate conversion by aconitase and homoaconitase. In the citrate cycle aconitase binds citrate in the “citrate mode” and performs a dehydration to aconitate. After a substrate flip and binding of aconitate in the isocitrate mode water is added by aconitase to yield isocitrate. Homocitrate can also bind to the citrate cycle aconitase, but binding is limited to the citrate mode. Homoaconitate resulting from the dehydration cannot be stabilised in the isocitrate mode due to the extended aliphatic chain. Therefore, rehydration in the isocitrate mode requires homoaconitase, which, in turn, is not able to bind the substrate in the citrate mode. from the oxidised fatty acid via a β-ketothiolase. While filamentous fungi degrade fatty acids in mitochondria and peroxisomes, yeasts only posses a peroxisomal path. Nevertheless, the dimorphic yeast C. albicans contains three isoenzymes of β-ketothiolases, whereas the yeast S. cerevisiae only contains a single enzyme. Therefore, we analysed the contribution of each isoenzyme to fatty acid degradation and virulence. Two of the three isoenzymes in C. albicans (Pot1 and Fox3) showed close phylogenetic relation to the single thiolase from S. cerevisiae, whereas the third enzyme (Pot13) was only distantly related and clustered with mitochondrial thiolases from filamentous fungi. Analyses of single, double and triple deletion mutants revealed that Pot1 is the major thiolase in C. albicans and generally required for utilisation of fatty acids. Interestingly, while Pot13 did not contribute to fatty acid utilisation – which is in agreement with a lacking mitochondrial β-oxidation cycle ‑ Fox3 was 194 required for wild-type growth at elevated temperatures. Thus, Fox3 seems to have adapted to support growth of C. albicans when growing as a commensal or pathogen inside the mammalian host. However, analyses of the virulence revealed that none of the β-ketothiolases was essential for virulence in a hen’s egg infection model. Although this does not exclude an importance of β-oxidation in the commensal state of the fungus, it supports the assumption that other nutrients such as proteins and/or glucose may serve as the primary nutrients during infection. 2 Pathogenesis of A. terreus infections A. terreus is an important source of natural products, but also an emerging cause of life-threatening invasive aspergillosis. Thus, in collaboration with Ilse Jacobsen, Research Group Microbial Immunology and Chri- HKI Research Report 2012 / 2013 Associated Group Microbial Biochemistry and Physiology A Lung Histology B Tissue homogenate Figure 2 Aspergillus terreus persistence in infected lung tissues. (A) Lung histology (PAS staining) of mice infected with A. terreus conidia reveals persisting conidia (indicated by arrows). (B) Plating of tissue homogenates yields a dense lawn of A. terreus colonies, indicating that conidia remained in a viable state. stian Hertweck, Department Biomolecular Chemistry, we are investigating virulence of A. terreus in comparison to A. fumigatus and analyse natural products specifically produced by A. terreus. Although leukopenic mice showed a similar outcome of infection in comparison to A. fumigatus, a 102 to 103 fold higher infectious dose was required for 100% lethality. Interestingly, in corticosteroid treated animals, all mice revealed an initial acute infection, whereby approximately 50% of infected mice survived and showed no signs of infection at the end of the observation period. However, the surviving animals revealed a persistence of ungerminated, but viable conidia in the lung tissue (Fig. 2). Furthermore, all mice that succumbed to infection and regardless the immunosuppression regimen showed a fatty liver degeneration, suggesting production of toxic secondary metabolites. Persistence of A. terreus in mice implied a fundamental difference between A. terreus and A. fumigatus infections that was assumed to derive from differences in the interaction with primary immune cells. A comparative study between both fungi revealed a substantially higher exposure of the surface antigens beta-1,3-glucan and galactomannan on A. terreus conidia, which was responsible for fast phagocytosis by macrophages. Additionally, despite acidification of phagolysosomes, A. terreus conidia were not inactivated, which contrasts A. fumigatus conidia that prevent phagocytosis and rapidly escape by germination. A major difference between both species derived from the lack of a DHN-melanin derivative in A. terreus conidia, which inhibits phagolysosome acidification in other Aspergillus species. Accordingly, recombinant production of the respective polyketide in A. terreus inhibited acidification, enhanced escape from macrophages and increased virulence. Further studies now focus on the identification of the pigment produced by A. terreus conidia and secondary metabolites provoking liver da- Associated Group Microbial Biochemistry and Physiology HKI Research Report 2012 / 2013 195 Figure 3 Bioluminescence of Aspergillus fumigatus reporter strains. (A) Dark-field photograph of an A. fumigatus colony expressing a codon-optimised luciferase. (B) In vivo imaging of a mouse infected with the bioluminescent A. fumigatus reporter strain. Photons emitted by the reporter strain from the infected lung are detected by a highly sensitive CCD camera and visualised in the overlay image. mage in infected mice to elucidate different pathogenicity mechanisms among Aspergillus species. 3 Monitoring of therapy efficacy by bioluminescence imaging Bioluminescence imaging (BLI) allows the visualisation of infections in living animals by collecting photons from the site of infection that are emitted by a luminescent pathogen. In our studies, we optimised A. fumigatus reporter strains (Fig. 3) with the aim to monitor drug efficacy under in vivo conditions. Intranasal infection of leukopenic mice with the reporter strain resulted in rapid increase in the pulmonary luminescence signal accompanied with 100 % mortality. Amphotericin B (AmB) treatment was effective to reduce mortality, but imaging implied a low efficacy of AmB in the sinus region, which led to lethal infections in some animals. In 196 contrast, Anidulafungin (Anid) was not sufficient to prevent pulmonary aspergillosis, but no signal increase was observed from the sinus. Thus, a combination of AmB and Anid was applied, which resulted in 100 % survival of mice with a control of infection in both, sinus and pulmonary region. Thus, bioavailabilty of AmB and Anid may differ in sinus and lung, which results in a synergistic therapeutic effect when given in parallel. Thus, BLI enables to visualise disease progression in temporal and spatial resolution and allows to follow the efficacy of antifungal drugs at diverse body sites. This results in an increased resolution of the outcome of therapy approaches by simultaneously reducing the number of animals in such investigations. HKI Research Report 2012 / 2013 Associated Group Microbial Biochemistry and Physiology Group members Collaborations Head Dr. habil. Matthias Brock Phone +49 3641 532-1710 Fax +49 3641 532-0809 [email protected] Goethe University Frankfurt, Frankfurt/Main, Germany Prof. Dr. Thomas Lehrnbecher Scientists Dr. Felicitas Schöbel (until 10/2012) Ph.D. Students Markus Gressler Christian Otzen Shruthi Ramachandra (until 09/2013) Amélie Savers (since 10/2013) Institute Pasteur, Paris, France Dr. Oumaima Ibrahim-Granet Dr. Grégory Jouvion Max Planck Institute for Chemical Ecology, Jena, Germany Dr. Almuth Hammerbacher Research Institute of Vall d´Hebron Hospital, Barcelona, Spain Dr. Joan Gavaldà Diploma / Bachelor / Master Students Katrin Emmelmann (11/2012–01/2013) St George's University of London, UK Dr. Kai Hilpert Research Assistants Daniela Hildebrandt Universidade Federal de Goiás, Goiás, Brazil Prof. Dr. Maristela Pereira Prof. Dr. Célia Maria de Almeida Soares External funding University of Leipzig, Germany Prof. Dr. Gottfried Alber Deutsche Forschungsgemeinschaft Aspergillus terreus as a cause of invasive aspergillosis: Impact of secondary metabolites on pathogenesis Matthias Brock Deutsche Forschungsgemeinschaft SFB/TR FungiNet Project A03: In vivo Bildgebung der Ausbreitung und immunologischen Kontrolle von Aspergillus fumigatus und Candida albicans Infektionen Matthias Brock Selected publications Otzen C, Müller S, Jacobsen ID, Brock M (2013) Phylogenetic and phenotypic characterisation of the 3-ketoacyl-CoA thiolase gene family from the opportunistic human pathogenic fungus Candida albicans. FEMS Yeast Res 13, 553-564. Galiger C*, Brock M*, Jouvion G, Savers A, Parlato M, Ibrahim-Granet O (2013) Assessment of efficacy of antifungals against Aspergillus fumigatus: the value of real-time bioluminescence imaging. Antimicrob Agents Chemother 57, 3046-3059. (*equal contribution) Fazius F, Zaehle C, Brock M (2013) Lysine biosynthesis in microbes: relevance as drug target and prospects for β-lactam antibiotic production. Appl Microbiol Biotechnol 97: 3763-3772 Associated Group Microbial Biochemistry and Physiology HKI Research Report 2012 / 2013 197 198 HKI Research Report 2012 / 2013 Associated Group Pharmaceutical Microbiology HKI Research Report 2012 / 2013 199 Associated Group Pharmaceutical Microbiology The Associated Group Pharmaceutical Microbiology is affiliated with the Pharmaceutical Institute of the Friedrich Schiller University and, at the same time, associated with the HKI. The department thus represents another excellent example for the HKI’s close interaction with the university. As a group focusing on pharmaceutical research our objective is to isolate and analyse microbial small molecule natural products as potential new pharmaceuticals or as chemical signals in microbial communication processes. Along with this endeavour, we elucidate the biochemical and genetic basis of natural product biosyntheses. A particular emphasis is placed on basidiomycete secondary metabolism, however, our projects also include bacterial small molecules. As a university-affiliated group, the Department Pharmaceutical Microbiology actively engages in teaching within the curriculum of the Pharmaceutical Sciences. Students can elect to fulfil the requirements for diploma theses or laboratory internships in the department and gain hands-on experience in natural product research early on. I N T R O D U C T I O N | E I N LE I T U N G Die assoziierte Gruppe Pharmazeutische Mikrobiologie ist Teil des Institutes für Pharmazie der Friedrich-Schiller-Universität Jena, jedoch gleichzeitig dem HKI assoziiert. Sie stellt damit ein weiteres Beispiel für die enge Verzahnung des HKI mit der universitären Forschung dar. Als eine pharmazeutisch ausgerichtete Gruppe betreibt sie Forschung über Naturstoffe mit dem Ziel der Isolation und Analyse von kleinen mikrobiellen Sekundärmetaboliten als potentielle neue Wirkstoffe oder Informationsträger für mikrobielle Kommunikation. Ein Schwerpunkt ist die Erforschung der genetischen und biochemischen Grundlagen, auf denen die jeweiligen zellulären Bio- Head: Prof. Dr. Dirk Hoffmeister 200 synthesewege dieser Sekundärmetabolite aufbauen. Hauptsächlich werden Ständerpilze (Basidiomyceten) untersucht, jedoch ist auch der bakterielle Stoffwechsel durch ein Projekt in der Gruppe repräsentiert. Als universitäre Gruppe ist die assoziierte Gruppe Pharmazeutische Mikrobiologie auch aktiv in Lehraufgaben zur Ausbildung von Apothekern eingebunden. Durch die Vergabe von Praktikums- und Diplomarbeiten sowie Plätzen für Wahlpflichtpraktika gemäß der Approbationsordnung für Apotheker kann der pharmazeutische Nachwuchs auf diese Weise frühzeitig an Naturstoff-Forschung herangeführt werden. HKI Research Report 2012 / 2013 Associated Group Pharmaceutical Microbiology Scientific Projects SidE A T C GreA A T TE LnaA/ LnbA 1 A T C Figure 1 Domain layout and products of the peptide synthetase SidE and peptide synthetase-like enzymes GreA and LnaA/LnbA. Enzyme domain abbreviations are A: adenylation; T: thiolation; C: condensation; TE: thioesterase; R: reductase. A T R Multi-domain enzymes in fungal natural product biosyntheses Fungal peptide synthetases and related enzymes A major focus of our scholarly work lies on the non-ribosomal peptide synthetases (NRPSs), i.e., multi-domain enzymes that catalyse the assembly of complex natural products from a large variety of monomeric building blocks. Through their inherent logic of self-activating their substrates via specific adenylation, NRPSs are not limited to the usual proteinogenic L-amino acids. The cumulative genomic sequence data shows that fungi usually encode numerous NPRS biosynthetic pathways. However, the majority of their products is still unknown. If it was possible to accurately predict substrate specificities of NRPSs from their primary amino acid sequence, we could anticipate product structures and, ultimately, fungal secondary metabolomes from genomic data in silico. This would help accelerate discovery of drugs and other potentially useful compounds. Currently, such predictions are, to a degree, possible but by far not as accurate as for their bacterial counterparts. Still, progress toward deciphering and refining such specificities has been made, and the results by the Department Pharmaceutical Microbiology contributed to this objective. The Aspergillus fumigatus bimodular NRPS SidE (Fig. 1) is encoded by a gene that was previously shown to be strongly induced during initiation of murine lung infection. Using heterologously produced full-length SidE, fumarylalanine formation was observed in vitro, thereby disproving the hypothesis that SidE was related to siderophore formation. Therefore, SidE allows, for the first time, insight how NRPSs select fumaric acid, i.e., a dicarboxylic non-amino acid substrate. Furthermore, fumarylalanine was also identified in A. fumigatus culture supernatants Associated Group Pharmaceutical Microbiology HKI Research Report 2012 / 2013 201 Figure 2 Structure of dehydroarmillylorsellinate (left) and ArmB-catalysed cross-coupling during melleolide biosynthesis (right). under inducing conditions, unless sidE was genetically inactivated. This project was accomplished in close collaboration with Prof. Hubertus Haas (Innsbruck Medical University). Our current hypothesis includes that fumarylalanine production could result in local immunosuppression, so the results may help better understand the processes behind infection by A. fumigatus. The basidiomycete Suillus grevillei (“larch bolete”) encodes the gene greA for a monomodular NRPS-like biosynthesis enzyme that lacks a condensation (C) domain but is composed of three domains for adenylation, thiolation, and thioesterase (Fig. 1). GreA was biochemically characterized in vitro and shown to possess atromentin synthetase activity. Specificity-conferring amino acid residues were identified which showed an unprecedented combination specific for aromatic α-keto acid substrates. 202 In cooperation with Prof. Nancy Keller’s group at the University of Wisconsin - Madison, a second class of C-domainless NRPS-like enzyme has recently been found relevant for the biosynthesis of small natural products. These enzymes, LnaA and LnbA of Aspergillus flavus, feature an adenylation, thiolation, and reductase domain arrangement (Fig. 1). Therefore, they resemble α-aminoadipic acid reductases, a unique class of fungal enzymes that serve fungal L-lysine biosynthesis, i.e., primary metabolism. LnaA and LnbA are functionally redundant and govern the production of piperazines and morpholines via a reductive mechanism whose details are not yet understood. Consistent with the chemical structure of the final product, in vitro work demonstrated that L-tyrosine is the preferred substrate of either enzyme. A double mutant (deletion and silencing, respectively, of both lnaA and lnbA) showed an altered phenotype as the strain was defec- HKI Research Report 2012 / 2013 Associated Group Pharmaceutical Microbiology tive in the formation of sclerotia. This result therefore suggests that natural products also play a role for the producer itself as its own small molecules help it complete its life cycle properly. Fungal polyketide synthases – Large-scale genome sequencing efforts (see also section 3) have helped anticipate the capacity of basidiomycetes to synthesize polyketides. A phylogenetic analysis of 111 type I polyketide synthase genes from 35 sequenced basidiomycete genomes was carried out. The resulting phylogeny suggests that all main types of fungal iterative PKS had already evolved before the Ascomycota and Basidiomycota diverged during evolution. We expect this study supports genomic mining projects aimed at discovering structurally diverse mushroom-derived polyketides. In cooperation with Prof. Barbara Howlett’s group at the University of Melbourne, Australia, the fungus Leptosphaeria maculans, a serious pathogen on canola, was investigated regarding its capacity to produce the phytotoxin phomenoic acid. It was shown that the polyketide synthase PKS2 catalyses the biosynthesis of this long-chain aliphatic carboxylic acid. 2 Melleolide natural products from Armillaria species Many species within the basidiomycete genus Armillaria (“honey mushroom”) are known as notorious butt and root rot agents and globally present as pathogens both in managed and unmanaged hardwood and conifer forests. Armillaria species produce melleolides (e.g., dehydroarmillylorsellinate, Fig. 2) which are composed of a sesquiterpenoid protoilludene whose alcohol functionality at position 5 engages in ester bond formation with orsellinic acid. This tetraketide is a common microbial metabolite, and protoilludenes are frequently found in basidiomycetes. However, the esterification of these two building blocks into a more complex metabolite is a unique biosynthetic capacity of Armillaria. Further, a permutation-like biosynthetic combination of methylation, chlorination, and oxidation/reduction generates in excess of 50 melleolide analogs which make them one of the most diverse groups of fungal natural products and an attractive model to study structure-activity-relationships. An Armillaria mellea non-reducing polyketide synthase, termed ArmB, was identified as orsellinic acid synthase. Further, cross-coupling activity of the ArmB thioesterase domain, i.e., esterification of orsellinic acid with various alcohols, among them a tricyclic Armillaria sesquiterpene alcohol, was shown (Fig. 2). Therefore, the biosynthesis of the melleolide melledonol was reconstituted in vitro. The pharmaceutical aspect of research on melleolides stems from their antifungal activity and their inhibitory activities against human cell lines. Five new melleolides were isolated and their structure elucidated. Using these new and a series of known melleolides, the ∆2,4 -double bond in the protoilludene moiety was identified as a key structural feature for antifungal activity, but turned out irrelevant for cytotoxicity against human cells. Further, treatment with an antifungally active melleolide impacts upon transcription of Aspergillus nidulans natural product genes. Transcriptomic studies were accomplished in close cooperation with the Department of Molecular and Applied Microbiology (A. Brakhage). After melleolide treatment, the most intriguing transcriptional up-regulation pertained to genes in a silent cluster which represents a portion of an extended genetic locus that had previously been recognized by HKI members as responsive to the presence and physical contact of streptomycetes. Therefore, our findings add to the notion of this Aspergillus locus as critical for intermicrobial communication. This study was integrated into the Jena School of Microbial Communication (JSMC). Associated Group Pharmaceutical Microbiology HKI Research Report 2012 / 2013 203 3 Basidiomycete genome projects The associated group Pharmaceutical Microbiology has been involved in various multi-PI, international consortia to annotate the genomic sequences of basidiomycetes whose DNA was sequenced at the Joint Genome Institute (JGI) in Walnut Creek, CA, USA. The group provided expertise to annotate genes that encode biosynthetic capacity for natural products in the respective species and to modify gene models, if necessary. In the 2012/2013 biennium, the group primarily focused on i) the white-rot fungus Ceriporiopsis subvermispora, which is a model basidiomycete for selective ligninolysis, as this fungus readily depolymerizes lignin with relatively little cellulose degradation; ii) the tree pathogen Heterobasidion annosum, the causative agent of root rot and a cosmopolitan pathogen in coniferous forests; iii) and Phlebiopsis gigantea, which degrades all components of plant cell walls and is uniquely able to colonize freshly exposed conifer sapwood. Spore suspensions of this fungus are marketed as a biological pesticide to control root rot. The genomic sequences of the former two species, including the contributions regarding the natural products, have been published. A two-fold benefit results from this work. Regarding the goals of the HKI and discovery of new compounds, such projects help identify species which encode a particularly diverse secondary metabolism or which harbour unusual biosynthesis genes. These species then can be given priority to search for new biochemistry and unprecedented chemical compounds and bioactivities. Seen from a more ecological angle, this work contributes to the focus area “microbial communication”, as 204 interorganismal interactions are frequently mediated by small molecule natural products. The careful genetic analysis provides us with a map to the secondary metabolome of a species. 4 Secondary metabolism of Ralstonia solanacearum Ralstonia solanacearum is a Gram-negative bacterium and notorious plant pathogen as it causes a lethal wilt disease. The host range of this organism is remarkably broad and includes plants used for staple food production, such as banana and potato, and species of significant commercial value, including tomato, eggplant, and tobacco. Previous studies identified the global regulatory proteins VsrAD and PhcA as central for both virulence and for biosynthesis of ralfuranones, a small family of phenylpyruvate-derived aryl-substituted furanones. The ralfuranone synthetase RalA was characterised biochemically and several sulfur-containing derivatives were isolated, among them the methyl thioether-containing ralfuranone D. The project included stable isotope feeding, headspace analyses of volatile compounds, gene inactivation, as well as bioinformatic analyses of the ralfuranone biosynthesis. A non-enzymatic one-step mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid was established. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate, was isolated and characterized by NMR (Fig. 3). The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione. These results may point to the actual biological role of ralfuranones in interference with glutathione-mediated signalling in host plant cells. These results, again, may contribute to the focus area “microbial communication”. HKI Research Report 2012 / 2013 Associated Group Pharmaceutical Microbiology γ α α Figure 3 Mechanism of methylthioether formation during ralfuranone D biosynthesis. This work was accomplished in cooperation with i) a partner group at the University of Wisconsin-Madison (transcriptome analysis), ii) the Max Planck Institute for Chemical Ecology in Jena (headspace analysis) and iii) HKI’s Junior Research Group Secondary Metabolism of Predatory Bacteria (NMR). In return, the latter group was supported with biochemical experiments and in vitro enzyme characterisations during their research on micacocidin, i.e., a thiazoline-containing antibiotic with significant activity against Mycoplasma pneumoniae. Associated Group Pharmaceutical Microbiology HKI Research Report 2012 / 2013 205 Group members Kalb D, Lackner G, Hoffmeister D (2013) Fungal peptide synthetases – an update on functions and specificity signatures. Fungal Biol Rev 27, 43-50. Head Prof. Dr. Dirk Hoffmeister Phone +49 3641 949-850 Fax +49 3641 949-852 [email protected] Steinchen W, Lackner G, Yasmin S, Schrettl M, Dahse HM, Haas H, Hoffmeister D (2013) Bimodular peptide synthetase SidE produces fumarylalanine in the human pathogen Aspergillus fumigatus. Appl Environ Microbiol 79, 6670-6676. Scientists Dr. Gerald Lackner Ph.D. Students Markus Bohnert Jana Braesel Daniel Braga de Lima (since 05/2013) Daniel Kalb Julia Pauly Daniel Schwenk Collaborations Max-Planck-Institute for Chemical Ecology, Jena, Germany Dr. Jeanine Linz Diploma / Bachelor / Master Students Philip Brandt (since 10/2013) Matthias Böhm (since 10/2013) Wieland Steinchen (until 05/2013) Jonas Wick (until 11/2013) Research Assistants Julia Greßler Elke Hänert (until 01/13) External funding Deutsche Forschungsgemeinschaft Jena School for Microbial Communication Project: Chemical ecology of the honey mushroom Armillaria mellea: Investigations on the mode of action of melleolide antibiotics Dirk Hoffmeister Medical University Innsbruck, Austria Prof. Dr. Hubertus Haas The University of Melbourne, Australia Prof. Dr. Barbara Howlett Dr. Candace Elliott University of Wisconsin, Madison, WI, USA Prof. Dr. Caitilyn Allen Prof. Dr. Nancy Keller United States Department of Agriculture – Forest Products Laboratory, Madison, WI, USA Dr. Daniel Cullen Selected publications Forseth RR, Amaike SA, Schwenk D, Affeldt KJ, Hoffmeister D, Schroeder FC, Keller NP (2013) Homologous non-canonical NRPS gene clusters mediate redundant small-molecule biosynthesis and sclerotia formation in Aspergillus flavus. Angew Chem Intl Ed 52, 1590-1594. Lackner G, Bohnert M, Wick J, Hoffmeister D (2013) Assembly of melleolide antibiotics involves a polyketide synthase with cross-coupling activity. Chem Biol 20, 1101-1106. Pauly J, Spiteller D, Linz J, Jacobs JM, Allen C, Nett M, Hoffmeister D (2013) Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum. ChemBioChem 14, 2169-2178. 206 HKI Research Report 2012 / 2013 Associated Group Pharmaceutical Microbiology Appendix HKI Research Report 2012 / 2013 207 Appendix Peer Reviewed Articles 2012 Originalarbeiten 2012 Department of Biomolecular Chemistry Behnken S, Hertweck C (2012) Cryptic polyketide synthase genes in non-pathogenic Clostridium spp. PLoS One 7, e29609. Behnken S, Lincke T, Kloss F, Ishida K, Hertweck C (2012) Antiterminator-mediated unveiling of cryptic polythioamides in an anaerobic bacterium. Angew Chem Int Ed 51, 2425-2428. Böttger D, Bergmann H, Kuehn B, Shelest E, Hertweck C (2012) Evolutionary imprint of catalytic domains in fungal PKS-NRPS hybrids. ChemBioChem 13, 23632373. Bretschneider T, Zocher G, Unger M, Scherlach K, Stehle T, Hertweck C (2012) A ketosynthase homolog uses malonyl units to form esters in cervimycin biosynthesis. Nat Chem Biol 8, 154-161. Busch B, Ueberschaar N, Sugimoto Y, Hertweck C (2012) Interchenar retrotransfer of aureothin intermediates in an iterative polyketide synthase module. J Am Chem Soc 134, 12382-12385. Ding L, Dahse HM, Hertweck C (2012) Cytotoxic alkaloids from Fusarium incarnatum associated with the mangrove tree Aegiceras corniculatum. J Nat Prod 75, 617621. Ding L, Maier A, Fiebig HH, Lin WH, Peschel G, Hertweck C (2012) Kandenols A-E, eudesmenes from an endophytic Streptomyces sp. of the mangrove tree Kandelia candel. J Nat Prod 75, 2223-2227. Ding L, Ndejouong Ble S, Maier A, Fiebig HH, Hertweck C (2012) Elaiomycins D-F, antimicrobial and cytotoxic azoxides from Streptomyces sp. strain HKI0708. J Nat Prod 75, 1729-1734. Ding L, Peschel G, Hertweck C (2012) Biosynthesis of archetypal plant self-defensive oxylipins by an endophytic fungus residing in mangrove embryos. ChemBioChem 13, 2661-2664. 208 Franke J, Ishida K, Hertweck C (2012) Genomics-driven discovery of burkholderic acid, a noncanonical, cryptic polyketide from human pathogenic Burkholderia species. Angew Chem Int Ed 51, 11611-11615. Brakhage AA, Hertweck C, Horn U (2012) Differential expression of silent polyketide biosynthesis gene clusters in chemostat cultures of Aspergillus nidulans. J Biotechnol 160, 64-71. Graupner K, Scherlach K, Bretschneider T, Lackner G, Roth M, Groß H, Hertweck C (2012) Imaging mass spectrometry and genome mining reveal highly antifungal virulence factor of mushroom soft rot pathogen. Angew Chem Int Ed 51, 13173-13177. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Roth M, Brakhage AA, Hertweck C (2012) Epidithiol formation by an unprecedented twin carbon-sulfur lyase in the gliotoxin pathway. Angew Chem Int Ed 51, 10064-10068. Henrot M, Richter ME, Maddaluno J, Hertweck C, De Paolis M (2012) Convergent asymmetric synthesis of (+)-aureothin employing an oxygenase-mediated resolution step. Angew Chem Int Ed 51, 95879591. Ishida K, Lincke T, Hertweck C (2012) Assembly and absolute configuration of short-lived polyketides from Burkholderia thailandensis. Angew Chem Int Ed 51, 5470-5474. Jensen K, Niederkruger H, Zimmermann K, Vagstad AL, Moldenhauer J, Brendel N, Frank S, Poplau P, Kohlhaas C, Townsend CA, Oldiges M, Hertweck C, Piel J (2012) Polyketide proofreading by an acyltransferase-like enzyme. Chem Biol 19, 329-339. Lupo A, Coyne S, Berendonk TU (2012) Origin and evolution of antibiotic resistance: the common mechanisms of emergence and spread in water bodies. Front Microbiol 3, 18. 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Junior Research Group Secondary Metabolism of Predatory Bacteria Kastner S, Müller S, Natesan L, König GM, Guthke R, Nett M (2012) 4-Hydroxyphenylglycine biosynthesis in Herpetosiphon aurantiacus: a case of gene duplication and catalytic divergence. Arch Microbiol 194, 557-566. Kreutzer MF, Kage H, Nett M (2012) Structure and biosynthetic assembly of cupriachelin, a photoreactive siderophore from the bioplastic producer Cupriavidus necator H16. J Am Chem Soc 134, 5415-5422. Kreutzer MF, Nett M (2012) Genomics-driven discovery of taiwachelin, a lipopeptide siderophore from Cupriavidus taiwanensis. Org Biomol Chem 10, 9338-9343. Junior Research Group Cellular Immunobiology This group ceased in 2012. Head: Dr. Mihály Józsi Castiblanco-Valencìa MM, Fraga TR, Silva LB, Monaris D, Abreu PA, Strobel S, Józsi M, Isaac L, Barbosa AS (2012) Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP. J Infect Dis 205, 995-1004. Appendix HKI Research Report 2012 / 2013 Hebecker M, Józsi M (2012) Factor H-related protein 4 activates complement by serving as a platform for the assembly of alternative pathway C3 convertase via its interaction with C3b protein. J Biol Chem 287, 19528-19536. Kopp A, Hebecker M, Svobodová E, Józsi M (2012) Factor H: A complement regulator in health and disease, and a mediator of cellular interactions. Biomolecules 2, 46-75. Kopp A, Strobel S, Tortajada A, Rodríguez de Córdoba S, SánchezCorral P, Prohászka Z, LópezTrascasa, Józsi M (2012) Atypical hemolytic uremic syndrome-associated variants and autoantibodies impair binding of factor H and factor H-related protein 1 to pentraxin 3. J Immunol 189, 1858-1867. Krmar RT, Skattum L, Barany P, Józsi M (2012) Screening for antifactor B autoantibody in a patient with acute renal injury due to dense deposit disease. Clin Nephrol 77, 85-86. Nozal P, Strobel S, Ibernon M, López D, Sánchez-Corral P, Rodríguez de Córdoba S, Józsi M, LópezTrascasa M (2012) Anti-factor H antibody affecting factor H cofactor activity in a patient with dense deposit disease. Clin Kidney J 5, 133-136. Svobodová E, Staib P, Losse J, Hennicke F, Barz D, Józsi M (2012) Differential interaction of the two related fungal species Candida albicans and Candida dubliniensis with human neutrophils. J Immunol 189, 2502-2511. Junior Research Group Fundamental Molecular Biology of Pathogenic Fungi This group ceased in 2012. Head: Dr. habil. Peter Staib Hof H, Eigner U, Maier T, Staib P (2012) Differentiation of Candida dubliniensis from Candida albicans by means of MALDI-TOF mass spectrometry. Clin Lab 58, 927-931. Svobodová E, Staib P, Losse J, Hennicke F, Barz D, Józsi M (2012) Differential interaction of the two related fungal species Candida albicans and Candida dubliniensis with human neutrophils. J Immunol 189, 2502-2511. Bio Pilot Plant Ding L, Maier A, Fiebig HH, Lin WH, Peschel G, Hertweck C (2012) Kandenols A-E, eudesmenes from an endophytic Streptomyces sp. of 211 the mangrove tree Kandelia candel. J Nat Prod 75, 2223-2227. Ding L, Peschel G, Hertweck C (2012) Biosynthesis of archetypal plant self-defensive oxylipins by an endophytic fungus residing in mangrove embryos. ChemBioChem 13, 2661-2664. Gläser SP, Galatis H, Martin K, Kämpfer P (2012) Luteolibacter cuticulihirudinis sp. nov., isolated from Hirudo medicinalis. Antonie Van Leeuwenhoek 102, 319-324. Graupner K, Scherlach K, Bretschneider T, Lackner G, Roth M, Gross H, Hertweck C (2012) Imaging mass spectrometry and genome mining reveal highly antifungal virulence factor of mushroom soft rot pathogen. Angew Chem Int Ed 51, 13173-13177. Kämpfer P, Arun AB, Young CC, Rekha PD, Martin K, Busse HJ, Chen WM (2012) Microbulbifer taiwanensis sp. nov., isolated from coastal soil. Int J Syst Evol Microbiol 62, 2485-2489. Kämpfer P, Falsen E, Lodders N, Martin K, Kassmannhuber J, Busse HJ (2012) Paenibacillus chartarius sp. nov., isolated from a paper mill. Int J Syst Evol Microbiol 62, 13421347. Kämpfer P, Lai WA, Arun AB, Young CC, Rekha PD, Martin K, Busse HJ, Chen WM (2012) Paracoccus rhizosphaerae sp. nov., isolated from the rhizosphere of the plant Crossostephium chinense (L.) Makino (Seremban). Int J Syst Evol Microbiol 62, 2750-2756. Kämpfer P, Lodders N, Grün-Wollny I, Martin K, Busse HJ (2012) Nocardia grenadensis sp. nov., isolated from sand of the Caribbean Sea. Int J Syst Evol Microbiol 62, 693-697. Kämpfer P, Lodders N, Martin K, Avendano-Herrera R (2012) Flavobacterium chilense sp. nov. and Flavobacterium araucananum sp. nov., isolated from farmed salmonid fish. Int J Syst Evol Microbiol 62, 1402-1408. Kämpfer P, Lodders N, Martin K, Falsen E (2012) Massilia oculi sp. nov., isolated from a human clinical specimen. Int J Syst Evol Microbiol 62, 364-369. Kämpfer P, Schäfer J, Lodders N, Martin K (2012) Microbacterium murale sp. nov., isolated from an indoor wall. Int J Syst Evol Microbiol 62, 2669-2673. 212 Kämpfer P, Wellner S, Lohse K, Martin K, Lodders N (2012) Duganella phyllosphaerae sp. nov., isolated from the leaf surface of Trifolium repens and proposal to reclassify Duganella violaceinigra into a novel genus as Pseudoduganella violceinigra gen. nov., comb. nov. Syst Appl Microbiol 35, 19-23. Martinez-Ramirez JA, Voigt K, Peters FT (2012) Studies on the metabolism of five model drugs by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS analysis. Anal Bioanal Chem 404, 1339-1359. Möbius N, Ross C, Scherlach K, Rohm B, Roth M, Hertweck C (2012) Biosynthesis of the respiratory toxin bongkrekic acid in the pathogenic bacterium Burkholderia gladioli. Chem Biol 19, 1164-1174. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Fungal Barcoding Consortium (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 109, 6241-6246. Morgado I, Wieligmann K, Bereza M, Ronicke R, Meinhardt K, Annamalai K, Baumann M, Wacker J, Hortschansky P, Malesevic M, Parthier C, Mawrin C, SchieneFischer C, Reymann KG, Stubbs MT, Balbach J, Gorlach M, Horn U, Fändrich M (2012) Molecular basis of beta-amyloid oligomer recognition with a conformational antibody fragment. Proc Natl Acad Sci USA 109, 12503-12508. Schrödl W, Heydel T, Schwartze VU, Hoffmann K, Große-Herrenthey A, Walther G, AlastrueyIzquierdo A, Rodriguez-Tudela JL, Olias P, Jacobsen ID, de Hoog GS, Voigt K (2012) Direct analysis and identification of pathogenic Lichtheimia species by matrix-assisted laser desorption ionization-time of flight analyzer-mediated mass spectrometry. J Clin Microbiol 50, 419-427. Sarkar A, Funk AN, Scherlach K, Horn F, Schroeckh V, Chankhamjon P, Westermann M, Roth M, Brakhage AA, Hertweck C, Horn U (2012) Differential expression of silent polyketide biosynthesis gene clusters in chemostat cultures of Aspergillus nidulans. J Biotechnol 160, 64-71. Schwartze VU, Hoffmann K, Nyilasi I, Papp T, Vagvolgyi C, de Hoog S, Voigt K, Jacobsen ID (2012) Lichtheimia species exhibit differences in virulence potential. PLoS One 7, e40908. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Roth M, Brakhage AA, Hertweck C (2012) Epidithiol formation by an unprecedented twin carbon-sulfur lyase in the gliotoxin pathway. Angew Chem Int Ed 51, 10064-10068. Jena Microbial Resource Collection Choi JS, Gräser Y, Walther G, Peano A, Symoens F, de Hoog S (2012) Microsporum mirabile and its teleomorph Arthroderma mirabile, a new dermatophyte species in the M. cookei clade. Med Mycol 50, 161-169. Ebersberger I, de Matos Simoes R, Kupczok A, Gube M, Kothe E, Voigt K, von Haeseler A (2012) A consistent phylogenetic backbone for the fungi. Mol Biol Evol 29, 1319-1334. Gryganskyi AP, Humber RA, Smith ME, Miadlikowska J, Wu S, Voigt K, Walther G, Anishchenko IM, Vilgalys R (2012) Molecular phylogeny of the Entomophthoromycota. Mol Phylogenet Evol 65, 682-694. Vitale RG, de Hoog GS, Schwarz P, Dannaoui E, Deng S, Machouart M, Voigt K, van de Sande WW, Dolatabadi S, Meis JF, Walther G (2012) Antifungal susceptibility and phylogeny of opportunistic members of the order mucorales. J Clin Microbiol 50, 66-75. Associated Group Microbial Biochemistry and Physiology Brock M (2012) Application of bioluminescence imaging for in vivo monitoring of fungal infections. Int J Microbiol 2012, 956794. Citiulo F, Jacobsen ID, Miramón P, Schild L, Brunke S, Zipfel P, Brock M, Hube B, Wilson D (2012) Candida albicans scavenges host zinc via Pra1 during endothelial invasion. PLoS Pathog 8, e1002777. Fazius F, Shelest E, Gebhardt P, Brock M (2012) The fungal α-aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate. Mol Microbiol 86, 1508-1530. Fekkar A, Pionneau C, Brossas JY, Marinach-Patrice C, Snounou G, Brock M, Ibrahim-Granet O, HKI Research Report 2012 / 2013 Appendix Mazier D (2012) DIGE enables the detection of a putative serum biomarker of fungal origin in a mouse model of invasive aspergillosis. J Proteomics 75, 2536-2549. Jouvion G, Brock M, Droin-Bergere S, Ibrahim-Granet O (2012) Duality of liver and kidney lesions after systemic infection of immunosuppressed and immunocompetent mice with Aspergillus fumigatus. Virulence 3, 43-50. Slesiona S, Greßler M, Mihlan M, Zaehle C, Schaller M, Barz D, Hube B, Jacobsen ID, Brock M (2012) Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages. PLoS One 7, e31223. Slesiona S, Ibrahim-Granet O, Olias P, Brock M, Jacobsen ID (2012) Murine infection models for Aspergillus terreus pulmonary aspergillosis reveal long-term persistence of conidia and liver degeneration. J Infect Dis 205, 1268-1277. Research Group Pharmaceutical Microbiology Fernandez-Fueyo E, Ruiz-Duenas FJ, Ferreira P, Floudas D, Hibbett DS, Canessa P, Larrondo LF, James TY, Seelenfreund D, Lobos S, Polanco R, Tello M, Honda Y, Watanabe T, Watanabe T, Ryu JS, Kubicek CP, Schmoll M, Gaskell J, Hammel KE, St John FJ, Vanden Wymelenberg A, Sabat G, Splinter BonDurant S, Syed K, Yadav JS, Doddapaneni H, Subramanian V, Lavin JL, Oguiza JA, Perez G, Pisabarro AG, Ramirez L, Santoyo F, Master E, Coutinho PM, Henrissat B, Lombard V, Magnuson JK, Kues U, Hori C, Igarashi K, Samejima M, Held BW, Barry KW, LaButti KM, Lapidus A, Lindquist EA, Lucas SM, Riley R, Salamov AA, Hoffmeister D, Schwenk D, Hadar Y, Yarden O, de Vries RP, Wiebenga A, Stenlid J, Eastwood D, Grigoriev IV, Berka RM, Blanchette RA, Kersten P, Martinez AT, Vicuna R, Cullen D (2012) Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis. Proc Natl Acad Sci U S A 109, 5458-5463. Lackner G, Misiek M, Braesel J, Hoffmeister D (2012) Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases. Fungal Genet Biol 49, 996-1003. Misiek M, Hoffmeister D (2012) Sesquiterpene aryl ester natural products in North American Armillaria species. Mycol Prog 11, 7-15. Olson A, Aerts A, Asiegbu F, Belbahri L, Bouzid O, Broberg A, Canbäck B, Coutinho PM, Cullen D, Dalman K, Deflorio G, van Diepen LT, Dunand C, Duplessis S, Durling M, Gonthier P, Grimwood J, Fossdal CG, Hansson D, Henrissat B, Hietala A, Himmelstrand K, Hoffmeister D, Högberg N, James TY, Karlsson M, Kohler A, Kües U, Lee YH, Lin YC, Lind M, Lindquist E, Lombard V, Lucas S, Lundén K, Morin E, Murat C, Park J, Raffaello T, Rouzé P, Salamov A, Schmutz J, Solheim H, Ståhlberg J, Vélëz H, de Vries RP, Wiebenga A, Woodward S, Yakovlev I, Garbelotto M, Martin F, Grigoriev IV, Stenlid J (2012) Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen. New Phytol 194, 1001-1013. Wackler B, Lackner G, Chooi YH, Hoffmeister D (2012) Characterization of the Suillus grevillei quinone synthetase GreA supports a nonribosomal code for aromatic alpha-keto acids. ChemBioChem 13, 1798-1804. Peer Reviewed Articles 2013 Originalarbeiten 2013 Department of Biomolecular Chemistry Baunach M, Ding L, Bruhn T, Bringmann G, Hertweck C (2013) Regiodivergent N-C and N-N aryl coupling reactions of indoloterpenes and cycloether formation mediated by a single bacterial flavoenzyme. Angew Chem Int Ed 52, 9040-9043. Bretschneider T, Heim JB, Heine D, Winkler R, Busch B, Kusebauch B, Stehle T, Zocher G, Hertweck C (2013) Vinylogous chain branching catalysed by a dedicated polyketide synthase module. Nature 502, 124-128. Busch B, Ueberschaar N, Behnken S, Sugimoto Y, Werneburg M, Traitcheva N, He J, Hertweck C (2013) Multifactorial control of iteration events in a modular polyketide assembly line. Angew Chem Int Ed 52, 5285-5289. Coyne S, Chizzali C, Khalil MN, Litomska A, Richter K, Beerhues L, Hertweck C (2013) Biosynthesis of the antimetabolite 6-thioguanine in Erwinia amylovora plays a key role in fire blight pathogenesis. Angew Chem Int Ed 52, 10564-10568. pathway in the pathogenic fungus Aspergillus fumigatus. ChemBioChem 14, 938-942. Fazius F, Zähle C, Brock M (2013) Lysine biosynthesis in microbes: relevance as drug target and prospects for beta-lactam antibiotics production. Appl Microbiol Biotechnol 97, 3763-3772. Letzel AC, Pidot SJ, Hertweck C (2013) A genomic approach to the cryptic secondary metabolome of the anaerobic world. Nat Prod Rep 30, 392-428. Franke J, Ishida K, Ishida-Ito M, Hertweck C (2013) Nitro versus hydroxamate in siderophores of pathogenic bacteria: effect of missing hydroxylamine protection in malleobactin biosynthesis. Angew Chem Int Ed 52, 8271-8275. Gatte-Picchi D, Weiz A, Ishida K, Hertweck C, Dittmann E (2014) Functional analysis of environmental DNA-derived microviridins provides new insights into the diversity of the tricyclic peptide family. Appl Environ Microbiol 80, 1380-1387. Epub 2013 Dec 13. Grumbt M, Monod M, Yamada T, Hertweck C, Kunert J, Staib P (2013) Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump. J Invest Dermatol 133, 15501555. Hennicke F, Grumbt M, Lermann U, Ueberschaar N, Palige K, Böttcher B, Jacobsen ID, Staib C, Morschhäuser J, Monod M, Hube B, Hertweck C, Staib P (2013) Factors supporting cysteine tolerance and sulfite production in Candida albicans. Eukaryot Cell 12, 604-613. Hölscher D, Dhakshinamoorthy S, Alexandrov T, Becker M, Bretschneider T, Buerkert A, Crecelius AC, De Waele D, Elsen A, Heckel DG, Heklau H, Hertweck C, Kai M, Knop K, Krafft C, Maddula RK, Matthäus C, Popp J, Schneider B, Schubert US, Sikora RA, Svatoš A, Swennen RL (2014) Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis. Proc Natl Acad Sci USA 111, 105-110. Epub 2013 Dec 9. Kloss F, Pidot S, Goerls H, Friedrich T, Hertweck C (2013) Formation of a dinuclear copper(I) complex from the clostridium-derived antibiotic closthioamide. Angew Chem Int Ed 52, 10745-10748. König CC, Scherlach K, Schroeckh V, Horn F, Nietzsche S, Brakhage AA, Hertweck C (2013) Bacterium induces cryptic meroterpenoid Mayeku PW, Hassanali A, Kiremire BT, Odalo JO, Hertweck C (2013) Anti-bacterial activities and phytochemical screening of extracts of different parts of Thallictrum rhynchocarpum. Afri J Tradit Complement Altern Med, 10, 341-344. Meyer F, Ueberschaar N, Dahse HM, Hertweck C (2013) Synthesis and biological evaluation of hydrazidomycin analogues. Bioorg Med Chem Lett 23, 6043-6045. Meyer F, Ueberschaar N, Hertweck C (2013) Concise total synthesis of hydrazidomycin A, a rare hydrazide metabolite of Eur J Org Chem 2013, 4242-4244. Nützmann HW, Fischer J, Scherlach K, Hertweck C, Brakhage AA (2013) Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans. Appl Environ Microbiol 79, 6102-6109. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis: a four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges. Angew Chem Int Ed 52, 11092-11095. Scherlach K, Lackner G, Graupner K, Pidot S, Bretschneider T, Hertweck C (2013) Biosynthesis and mass spectrometric imaging of tolaasin, the virulence factor of brown blotch mushroom disease. ChemBioChem 14, 24392443. Ueberschaar N, Dahse HM, Bretschneider T, Hertweck C (2013) Rational design of an apoptosis-inducing photoreactive DNA intercalator. Angew Chem Int Ed 52, 6185-6189. Ueberschaar N, Xu Z, Scherlach K, Metsä-Ketelä M, Bretschneider T, Dahse HM, Görls H, Hertweck C (2013) Synthetic remodeling of the chartreusin pathway to tune antiproliferative and antibacterial activities. J Am Chem Soc 135, 17408-17416. Appendix HKI Research Report 2012 / 2013 Department of Infection Biology Ansari M, McKeigue PM, Skerka C, Hayward C, Rudan I, Vitart V, Polasek O, Armbrecht AM, Yates JR, Vatavuk Z, Bencic G, Kolcic I, Oostra BA, Van Duijn CM, Campbell S, Stanton CM, Huffman J, Shu X, Khan JC, Shahid H, Harding SP, Bishop PN, Deary IJ, Moore AT, Dhillon B, Rudan P, Zipfel PF, Sim RB, Hastie ND, Campbell H, Wright AF (2013) Genetic influences on plasma CFH and CFHR1 concentrations and their role in susceptibility to age-related macular degeneration. Hum Mol Genet 22, 4857-4869. Bohnert M, Dahse HM, Gibson DM, Krasnoff SB, Hoffmeister D (2013) The fusarin analog NG-391 impairs nucleic acid formation in K-562 leukemia cells. Phytochemistry Letters 6, 189-192. Caesar JJ, Wallich R, Kraiczy P, Zipfel PF, Lea SM (2013) Further structural insights into the binding of complement factor H by complement regulator-acquiring surface protein 1 (CspA) of Borrelia burgdorferi. Acta Crystallogr Sect F Struct Biol Cryst Commun 69, 629-633. Chen Q, Wiesener M, Eberhardt HU, Hartmann A, Kirschfink M, Uzonyi B, Amann K, Buettner M, Goodship T, Hugo C, Skerka C, Zipfel PF (2014) Complement deregulation by a CFHR2-CFHR5 hybrid-protein in Dense Deposit Disease. J Clin Invest 124, 145-155. Epub 2013 Dec 16. Eberhardt HU, Buhlmann D, Hortschansky P, Chen Q, Böhm S, Kemper MJ, Wallich R, Hartmann A, Hallström T, Zipfel PF, Skerka C (2013) Human Factor H-Related Protein 2 (CFHR2) regulates complement activation. PLoS One 8, e78617. Hallström T, Siegel C, Mörgelin M, Kraiczy P, Skerka C, Zipfel PF (2013) CspA from Borrelia burgdorferi inhibits the terminal complement pathway. MBio 4, pii e00481-13. Hammerschmidt C, Königs A, Siegel C, Hallström T, Skerka C, Wallich R, Zipfel PF, Kraiczy P (2013) Versatile roles of CspA orthologs in complement inactivation of serumresistant lyme disease spirochetes. Infect Immun 82, 380-392. Epub 2013 Nov 4. Königs A, Hammerschmidt C, Jutras BL, Pogoryelov D, Barthel D, 213 Skerka C, Kugelstadt D, Wallich R, Stevenson B, Zipfel PF, Kraiczy P (2013) BBA70 of Borrelia burgdorferi is a novel plasminogen-binding protein. J Biol Chem 288, 2522925243. Lesher AM, Zhou L, Kimura Y, Sato S, Gullipalli D, Herbert AP, Barlow PN, Eberhardt HU, Skerka C, Zipfel PF, Hamano T, Miwa T, Tung KS, Song WC (2013) Combination of factor H mutation and properdin deficiency causes severe C3 glomerulonephritis. J Am Soc Nephrol 24, 53-65. Luo S, Hoffmann R, Skerka C, Zipfel PF (2013) Glycerol-3-phosphate dehydrogenase 2 is a novel factor H-, factor H-like protein 1-, and plasminogen-binding surface protein of Candida albicans. J Infect Dis 207, 594-603. Macabeo APG, Lopez ADD, Schmidt S, Heilmann J, Dahse HM, Alejandro GJD, Franzblau SG (2013) Antitubercular and cytotoxic constituents from Goniothalamus gitingensis. Rec Nat Prod 8, 41-45. Meyer F, Ueberschaar N, Dahse HM, Hertweck C (2013) Synthesis and biological evaluation of hydrazidomycin analogues. Bioorg Med Chem Lett 23, 6043-6045. Noone D, Waters A, Pluthero FG, Geary DF, Kirschfink M, Zipfel PF, Licht C (2014) Successful treatment of DEAP-HUS with eculizumab. Pediatr Nephrol 29, 841-851. Epub 2013 Nov 20. Poolpol K, Orth-Höller D, Speth C, Zipfel PF, Skerka C, de Cordoba SR, Brockmeyer J, Bielaszewska M, Würzner R (2014) Interaction of Shiga toxin 2 with complement regulators of the factor H protein family. Mol Immunol 58(1), 77-84. Schrader FC, Glinca S, Sattler JM, Dahse HM, Afanador GA, Prigge ST, Lanzer M, Müller AK, Klebe G, Schlitzer M (2013) Novel type II fatty acid biosynthesis (FAS II) inhibitors as multistage antimalarial agents. Chem Med Chem 8, 442-461. Schwab J, Hammerschmidt C, Richter D, Skerka C, Matuschka FR, Wallich R, Zipfel PF, Kraiczy P (2013) Borrelia valaisiana resist complement-mediated killing independently of the recruitment of immune regulators and inactivation of complement components. PLoS One 8, e53659. Simon N, Lasonder E, Scheuermayer M, Kühn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G (2013) Malaria parasites coopt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13, 29-41. Steinchen W, Lackner G, Yasmin S, Schrettl M, Dahse HM, Haas H, Hoffmeister D (2013) Bimodular peptide synthetase SidE produces fumarylalanine in the human pathogen Aspergillus fumigatus. Appl Environ Microbiol 79, 66706676. Ueberschaar N, Dahse HM, Bretschneider T, Hertweck C (2013) Rational design of an apoptosisinducing photoreactive DNA intercalator. Angew Chem Int Ed 52, 6185-6189. Ueberschaar N, Xu Z, Scherlach K, Metsä-Ketelä M, Bretschneider T, Dahse HM, Görls H, Hertweck C (2013) Synthetic remodeling of the chartreusin pathway to tune antiproliferative and antibacterial activities. J Am Chem Soc 135, 17408-17416. Unnewehr H, Rittirsch D, Sarma JV, Zetoune F, Flierl MA, Perl M, Denk S, Weiss M, Schneider ME, Monk PN, Neff T, Mihlan M, Barth H, Gebhard F, Ward PA, Huber-Lang M (2013) Changes and regulation of the C5a receptor on neutrophils during septic shock in humans. J Immunol 190, 4215-4225. Voss S, Hallström T, Saleh M, Burchhardt G, Pribyl T, Singh B, Riesbeck K, Zipfel PF, Hammerschmidt S (2013) The cholinebinding protein PspC of Streptococcus pneumoniae interacts with the C-terminal heparin-binding domain of vitronectin. J Biol Chem 288, 15614-15627. Watson R*, Lindner S*, Bordereau P*, Hunze EM, Tak F, Ngo S, Zipfel PF, Skerka C#, Dragon-Durey MA#,Marchbank KJ# (2014) Standardisation of the factor H autoantibody assay. Immunbiol 219, 9-16. Epub 2013 Jun 26. *shared firstauthors; #shared last authors Weinberger AWA, Eddahabi X, Carstesen D, Zipfel PF, Walter P, Skerka C (2014) Human complement factor H and factor H-like protein1 (FHL-1) are expressed in human retinal pigment epithelial (RPE) cells. Opht Res 51, 59-66. Zipfel PF, Skerka C (2014) Staphylococcus aureus: The multi headed 214 hydra resists and controls human complement response in multiple ways. Int J Med Microbiol 304, 188194. Epub 2013 Dec 1. Department of Microbial Pathogenicity Mechanisms Hennicke F, Grumbt M, Lermann U, Ueberschaar N, Palige K, Böttcher B, Jacobsen ID, Staib C, Morschhäuser J, Monod M, Hube B, Hertweck C, Staib P (2013) Factors supporting cysteine tolerance and sulfite production in Candida albicans. Eukaryot Cell 12, 604-613. Krljanac B1, Weih D, Jacobsen ID, Hu D, Koliesnik I, Reppe K, Witzenrath M, Weih F (2014) NF-κB2/ p100 deficiency impairs immune responses to T-cell-independent type 2 antigens. Eur J Immunol 44, 662-672. Epub 2013 Dec 16. Lüttich A, Brunke S, Hube B, Jacobsen ID (2013) Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney. PLoS One 8, e64482. Martin R, Albrecht-Eckardt D, Brunke S, Hube B, Hünniger K, Kurzai O (2013) A core filamentation response network in Candida albicans is restricted to eight genes. PLoS One 8, e58613. Mayer FL, Wilson D, Hube B (2013) Hsp21 potentiates antifungal drug tolerance in Candida albicans. PLoS One 8, e60417. Mech F, Wilson D, Lehnert T, Hube B, Figge MT (2014) Epithelial invasion outcompetes hypha development during Candida albicans infection as revealed by an imagebased systems biology approach. Cytometry Part A, 85, 126-139. Epub 2013 Nov 20. Otto GP, Ludewig K, Jacobsen ID, Schaarschmidt B, Hube B, Bauer M (2013) Limitation of (1->3)-beta-Dglucan monitoring in major elective surgery involving cardiopulmonary bypass. Crit Care 17, 437. Otzen C, Müller S, Jacobsen ID, Brock M (2013) Phylogenetic and phenotypic characterisation of the 3-ketoacyl-CoA thiolase gene family from the opportunistic human pathogenic fungus Candida albicans. FEMS Yeast Research 13, 553-564. Palige K, Linde J, Martin R, Böttcher B, Citiulo F, Sullivan DJ, Weber J, Staib C, Rupp S, Hube HKI Research Report 2012 / 2013 Appendix B, Morschhäuser J, Staib P (2013) Global transcriptome sequencing identifies chlamydospore specific markers in Candida albicans and Candida dubliniensis. PLoS One 8, e61940. Pietrella D, Pandey N, Gabrielli E, Pericolini E, Perito S, Kasper L, Bistoni F, Cassone A, Hube B, Vecchiarelli A (2013) Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome. Eur J Immunol 43, 679-692. Wagener J, Schneider JJ, Baxmann S, Kalbacher H, Borelli C, Nuding S, Kuchler R, Wehkamp J, Kaeser MD, Mailänder-Sanchez D, Braunsdorf C, Hube B, Schild L, Forssmann WG, Korting HC, Liepke C, Schaller M (2013) A peptide derived from the highly conserved protein GAPDH is involved in tissue protection by different antifungal strategies and epithelial immunomodulation. J Invest Dermatol 133, 144-153. Voigt J, Hünniger K, Bouzani M, Jacobsen ID, Barz D, Hube B, Löffler J, Kurzai O (2014) Human NK cells act as phagocytes against Candida albicans and mount an inflammatory response which modulates neutrophil antifungal activity. J Infect Dis 209, 616-626. Wilson D, Hebecker B, Moyes DL, Miramón P, Jablonowski N, Wisgott S, Allert S, Naglik JR, Hube B (2013) Clotrimazole dampens vaginal inflammation and neutrophil infiltration in response to Candida albicans infection. Antimicrob Agents Chemother 57, 5178-5180. Department of Molecular and Applied Microbiology Alborzinia H, Schmidt-Glenewinkel H, Ilkavets I, Breitkopf-Heinlein K, Cheng X, Hortschansky P, Dooley S, Wolfl S (2013) Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists. J Cell Sci 126, 117-127. Bacher P, Kniemeyer O, Schönbrunn A, Sawitzki B, Assenmacher M, Rietschel E, Steinbach A, Cornely OA, Brakhage AA, Thiel A, Scheffold A (2013) Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. Mucosal Immunol. Epub 2013 Dec 4. Bacher P, Schink C, Teutschbein J, Kniemeyer O, Assenmacher M, Brakhage AA, Scheffold A (2013) Antigen-reactive T cell enrichment for direct, high-resolution analysis of the human naive and memory Th cell repertoire. J Immunol 190, 3967-3976. Beauvais A, Bozza S, Kniemeyer O, Formosa C, Balloy V, Henry C, Roberson RW, Dague E, Chignard M, Brakhage AA, Romani L, Latge JP (2013) Deletion of the alpha-(1,3)glucan synthase genes induces a restructuring of the conidial cell wall responsible for the avirulence of Aspergillus fumigatus. PLoS Pathog 9, e1003716. Bibashi E, de Hoog GS, Pavlidis TE, Symeonidis N, Sakantamis A, Walther G (2013) Wound infection caused by Lichtheimia ramosa due to a car accident. Medical Mycology Case Reports 2, 7-10. Coburger I, Dahms SO, Roeser D, Gührs KH, Hortschansky P, Than ME (2013) Analysis of the overall structure of the multi-domain amyloid precursor protein (APP). PLoS One 8, e81926. Dolatabadi S, Walther G, Gerrits van den Ende AHG, Hoog GS (2013) Diversity and delimitation of Rhizopus microsporus. Fungal Diversity 1-19. Eberhardt HU, Buhlmann D, Hortschansky P, Chen Q, Böhm S, Kemper MJ, Wallich R, Hartmann A, Hallström T, Zipfel PF, Skerka C (2013) Human factor H-related protein 2 (CFHR2) regulates complement activation. PLoS One 8, e78617. Eberhardt U, Beker H, Vesterholt J, Dukik K, Walther G, Vila J, Fernández Brime S (2013) European species of Hebeloma section Theobromina. Fungal Diversity 58, 103-126. Grützmann K, Szafranski K, Pohl M, Voigt K, Petzold A, Schuster S (2014) Fungal alternative splicing is associated with multicellular complexity and virulence: A genome-wide multi-species study. DNA Res 21, 27-39. Gryganskyi AP, Humber RA, Smith ME, Hodge K, Huang B, Voigt K, Vilgalys R (2013) Phylogenetic lineages in entomophthoromycota. Persoonia 30, 94-105. Hoffmann K, Pawlowska J, Walther G, Wrzosek M, de Hoog GS, Benny GL, Kirk PM, Voigt K (2013) The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies. Persoonia 30, 57-76. Knop K, Pavlov GM, Rudolph T, Martin K, Pretzel D, Jahn BO, Scharf DH, Brakhage AA, Makarov V, Möllmann U, Schacher FH, Schubert US (2013) Amphiphilic star-shaped block copolymers as unimolecular drug delivery systems: investigations using a novel fungicide. Soft Matter 9, 715-726. Knop K, Pretzel D, Urbanek A, Rudolph T, Scharf DH, Schallon A, Wagner M, Schubert S, Kiehntopf M, Brakhage AA, Schacher FH, Schubert US (2013) Star-shaped drug carriers for doxorubicin with POEGMA and POEtOxMA brushlike shells: a structural, physical, and biological comparison. Biomacromolecules 14, 2536-2548. König CC, Scherlach K, Schroeckh V, Horn F, Nietzsche S, Brakhage AA, Hertweck C (2013) Bacterium induces cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus. ChemBioChem 14, 938-942. Mischo A, Ohlenschläger O, Hortschansky P, Ramachandran R, Görlach M (2013) Structural insights into a wildtype domain of the oncoprotein E6 and its interaction with a PDZ domain. PLoS One 8, e62584. Münchberg U, Wagner L, Spielberg ET, Voigt K, Rosch P, Popp J (2013) Spatially resolved investigation of the oil composition in single intact hyphae of Mortierella spp. with micro-Raman spectroscopy. Biochim Biophys Acta 1831, 341-349. Muszkieta L, Beauvais A, Pähtz V, Gibbons JG, Anton Leberre V, Beau R, Shibuya K, Rokas A, Francois JM, Kniemeyer O, Brakhage AA, Latge JP (2013) Investigation of Aspergillus fumigatus biofilm formation by various “omics” approaches. Front Microbiol 4, 13. Nützmann HW, Fischer J, Scherlach K, Hertweck C, Brakhage AA (2013) Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans. Appl Environ Microbiol 79, 6102-6109. Pawłowska J, Walther G, Wilk M, Hoog S, Wrzosek M (2013) The use of compensatory base change analysis of ITS2 as a tool in the phylogeny of Mucorales, illustrated by the Mucor circinelloides complex. Organisms Diversity & Evolution 13, 497-502. Sahadevan Y, Richter-Fecken M, Kaerger K, Voigt K, Boland W (2013) Early and late trisporoids differentially regulate β-carotene production and gene transcript levels in the mucoralean fungi Blakeslea trispora and Mucor mucedo. Appl Environ Microbiol 79. 7466-7475. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis: a four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges. Angew Chem Int Ed 52, 11092-11095. Shopova I, Bruns S, Thywißen A, Kniemeyer O, Brakhage AA, Hillmann F (2013) Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus. Front Microbiol 4, 141. Voigt K, Vaas L, Stielow B, de Hoog GS (2013) The zygomycetes in a phylogenetic perspective. Persoonia 30, i–iv. Wagner L, Stielow B, Hoffmann K, Petkovits T, Papp T, Vagvolgyi C, de Hoog GS, Verkley G, Voigt K (2013) A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA. Persoonia 30, 77-93. Walther G, Pawłowska J, Alastruey-Izquierdo A, Wrzosek M, Rodriguez-Tudela JL, Dolatabadi S, Chakrabarti A, de Hoog GS (2013) DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia 30, 11-47. Department of Cell and Molecular Biology Bleicher A, Schöfl G, Rodicio Mdel R, Saluz HP (2013) The plasmidome of a Salmonella enterica serovar Derby isolated from pork meat. Plasmid 69, 202-210. Gebhardt P, Würbach L, Heidrich A, Heinrich L, Walther M, Opfermann T, Sørensen B, Saluz HP (2013) Dynamic behaviour of selected PET tracers in embryonated chicken eggs. Rev Esp Med Nucl Imagen Mol 32, 371-377. Groot AT, Staudacher H, Barthel A, Inglis O, Schöfl G, Santangelo RG, Gebauer-Jung S, Vogel H, Emerson J, Schal C, Heckel DG, Gould F (2013) One quantitative trait locus for intra- and interspecific variation in a sex pheromone. Mol Ecol 22, 1065–1080. Appendix HKI Research Report 2012 / 2013 Heidrich A, Schmidt J, Zimmermann J, Saluz HP (2013) Automated segmentation and object classification of CT images: Application to in vivo molecular imaging of avian embryos. Int J Biomed Imaging 2013, 508474. Klimina KM, Kjasova DK, Poluektova EU, Krügel H, Leuschner Y, Saluz HP, Danilenko VN (2013) Identification and characterization of toxin-antitoxin systems in strains of Lactobacillus rhamnosus isolated from humans. Anaerobe 22, 82-89. Rassmann A, Martin U, Saluz HP, Peter S, Munder T, Henke A (2013) Identification of gene expression profiles in HeLa cells and HepG2 cells infected with Coxsackievirus B3. J Virol Methods 187, 190-194. Research Group Applied Systems Biology Coelho FM, Natale D, Soriano SF, Hons M, Swoger J, Mayer J, Danuser R, Scandella E, Pieczyk M, Zerwes HG, Junt T, Sailer AW, Ludewig B, Sharpe J, Figge MT, Stein JV (2013) Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions. Blood 121, 4101-4109. Figge MT, Osiewacz HD, Reichert AS (2013) Quality control of mitochondria during aging: is there a good and a bad side of mitochondrial dynamics? Bioessays 35, 314-322. Hünniger K, Lehnert T, Bieber K, Martin R, Figge MT, Kurzai O (2014) A virtual infection model quantifies innate effector mechanisms and candida albicans immune escape in human blood. PLoS Comput Biol 10, e1003479. Mech F, Wilson D, Lehnert T, Hube B, Figge MT (2014) Epithelial invasion outcompetes hypha development during Candida albicans infection as revealed by an imagebased systems biology approach. Cytometry Part A, 85, 126-139. Epub 2013 Nov 20. Mokhtari Z, Mech F, Zitzmann C, Hasenberg M, Gunzer M, Figge MT (2013) Automated characterization and parameter-free classification of cell tracks based on local migration behavior. PLos One 8, e80808. Zang E, Brandes S, Tovar M, Martin K, Mech F, Horbert P, Henkel T, Figge MT, Roth M (2013) Real-time image processing for label-free 215 enrichment of Actinobacteria cultivated in picolitre droplets. Lab Chip 13, 3707-3713. markers in Candida albicans and Candida dubliniensis. PLoS One 8, e61940. ducer of telomycin, isolated from soil. Int J Syst Evol Microbiol 63, 3812-3817. regulation is linked to methylation of nicotinamide. Nat Chem Biol 9, 693-700. Zhang Y, Meyer-Hermann M, George LA, Figge MT, Khan M, Goodall M, Young SP, Reynolds A, Falciani F, Waisman A, Notley CA, Ehrenstein MR, Kosco-Vilbois M, Toellner KM (2013) Germinal center B cells govern their own fate via antibody feedback. J Exp Med 210, 457-464. Voigt J, Hünniger K, Bouzani M, Jacobsen ID, Barz D, Hube B, Löffler J, Kurzai O (2014) Human natural killer cells acting as phagocytes against Candida albicans and mounting an inflammatory response that modulates neutrophil antifungal activity. J Inf Dis 209, 616-626. König CC, Scherlach K, Schroeckh V, Horn F, Nietzsche S, Brakhage AA, Hertweck C (2013) Bacterium induces cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus. ChemBioChem 14, 938-942. Schmeisser S, Priebe S, Groth M, Monajembashi S, Hemmerich P, Guthke R, Platzer M, Ristow M (2013) Neuronal ROS signaling rather than AMPK/sirtuin-mediated energy sensing links dietary restriction to lifespan extension. Mol Metab 2, 92-102. Research Group Fungal Septomics Research Group Microbial Immunology Cottier F, Leewattanapasuk W, Kemp LR, Murphy M, Supuran CT, Kurzai O, Mühlschlegel FA (2013) Carbonic anhydrase regulation and CO(2) sensing in the fungal pathogen Candida glabrata involves a novel Rca1p ortholog. Bioorg Med Chem 21, 1549-1554. Hennicke F, Grumbt M, Lermann U, Ueberschaar N, Palige K, Böttcher B, Jacobsen ID, Staib C, Morschhäuser J, Monod M, Hube B, Hertweck C, Staib P (2013) Factors supporting cysteine tolerance and sulfite production in Candida albicans. Eukaryot Cell 12, 604-613. Cunha C, Aversa F, Lacerda JF, Busca A, Kurzai O, Grube M, Löffler J, Maertens J, Bell AS, Inforzato A, Barbati E, Almeida B, Santos e Sousa P, Barbui A, Potenza L, Caira M, Rodrigues F, Salvatori G, Pagano L, Luppi M, Mantovani A, Velardi A, Romani L, Carvalho A (2014) Genetic deficiency of PTX3 and invasive aspergillosis in stem cell transplantation. N Engl J Med 370, 421-432. Krljanac B, Weih D, Jacobsen ID, Hu D, Koliesnik I, Reppe K, Witzenrath M, Weih F (2014) NF-κB2/ p100 deficiency impairs immune responses to T-cell-independent type 2 antigens. Eur J Immunol 44, 662-672. Epub 2013 Dec 16. Hentschel J, Müller U, Doht F, Fischer N, Böer K, Sonnemann J, Hipler C, Hünniger K, Kurzai O, Markert UR, Mainz JG (2014) Influences of nasal lavage collection-, processing- and storage methods on inflammatory markers - Evaluation of a method for non-invasive sampling of epithelial lining fluid in cystic fibrosis and other respiratory diseases. J Immunol Methods 404, 41-51. Hünniger K, Lehnert T, Bieber K, Martin R, Figge MT, Kurzai O (2014) A virtual infection model quantifies innate effector mechanisms and candida albicans immune escape in human blood. PLoS Comput Biol 10, e1003479. Martin R, Albrecht-Eckardt D, Brunke S, Hube B, Hünniger K, Kurzai O (2013) A core filamentation response network in Candida albicans is restricted to eight genes. PLoS One 8, e58613. Palige K, Linde J, Martin R, Böttcher B, Citiulo F, Sullivan DJ, Weber J, Staib C, Rupp S, Hube B, Morschhäuser J, Staib P (2013) Global transcriptome sequencing identifies chlamydospore specific 216 Lüttich A, Brunke S, Hube B, Jacobsen ID (2013) Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney. PLoS One 8, e64482. Otto GP, Ludewig K, Jacobsen ID, Schaarschmidt B, Hube B, Bauer M (2013) Limitation of (1->3)-beta-Dglucan monitoring in major elective surgery involving cardiopulmonary bypass. Crit Care 17, 437. Otzen C, Müller S, Jacobsen ID, Brock M (2013) Phylogenetic and phenotypic characterisation of the 3-ketoacyl-CoA thiolase gene family from the opportunistic human pathogenic fungus Candida albicans. FEMS Yeast Research 13, 553-564. Voigt J, Hünniger K, Bouzani M, Jacobsen ID, Barz D, Hube B, Löffler J, Kurzai O (2014) Human NK cells act as phagocytes against Candida albicans and mount an inflammatory response which modulates neutrophil antifungal activity. J Infect Dis 209, 616-626.) Research Group Systems Biology and Bioinformatics Gurovic MS, Müller S, Domin N, Seccareccia I, Nietzsche S, Martin K, Nett M (2013) Micromonospora schwarzwaldensis sp. nov., a pro- Kramer M, Sponholz C, Slaba M, Wissuwa B, Claus RA, Menzel U, Huse K, Platzer M, Bauer M (2013) Alternative 5’ untranslated regions are involved in expression regulation of human heme oxygenase-1. PLoS ONE 8, e77224. Otzen C, Müller S, Jacobsen I, Brock M (2013): Phylogenetic and phenotypic characterisation of the 3-ketoacyl-CoA thiolase gene family from the opportunistic human pathogenic fungus Candida albicans. FEMS Yeast Research 13, 553-564. Palige K, Linde J, Martin R, Böttcher B, Citiulo F, Sullivan DJ, Weber J, Staib C, Rupp S, Hube B, Morschhäuser J, Staib P (2013) Global transcriptome sequencing identifies chlamydospore specific markers in Candida albicans and Candida dubliniensis. PLoS One 8, e61940. Petzold A, Reichwald K, Groth M, Taudien S, Hartmann N, Priebe S, Shagin D, Englert C, Platzer M (2013) The transcript catalogue of the short-lived fish Nothobranchius furzeri provides insights into agedependent changes of mRNA levels. BMC Genomics 14, 185. Priebe S, Menzel U, Zarse K, Groth M, Platzer M, Ristow M, Guthke R (2013) Extension of life span by impaired glucose metabolism in Caenorhabditis elegans is accompanied by structural rearrangements of the transcriptomic network. PLoS One 8, e77776. Schleicher J, Guthke R, Dahmen U, Dirsch O, Holzhütter HG, Schuster S (2013) A theoretical study of lipid accumulation in the liverimplications for nonalcoholic fatty liver disease. Biochim Biophys Acta 1841, 62-69. Schmeisser K, Mansfeld J, Kuhlow D, Weimer S, Priebe S, Heiland I, Birringer M, Groth M, Segref A, Kanfi Y, Price NL, Schmeisser S, Schuster S, Pfeiffer AF, Guthke R, Platzer M, Hoppe T, Cohen HY, Zarse K, Sinclair DA, Ristow M (2013) Role of sirtuins in lifespan HKI Research Report 2012 / 2013 Appendix Schmeisser S, Schmeisser K, Weimer S, Groth M, Priebe S, Fazius E, Kuhlow D, Pick D, Einax JW, Guthke R, Platzer M, Zarse K, Ristow M (2013) Mitochondrial hormesis links low-dose arsenite exposure to lifespan extension. Aging Cell 12, 508-517. Ullmann-Zeunert L, Stanton MA, Wielsch N, Bartram S, Hummert C, Svatos A, Baldwin IT, Groten K (2013) Quantification of growthdefense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover. Plant J 75, 417-429. Vlaic S, Hoffmann B, Kupfer P, Weber M, Drager A (2013) GRN2SBML: automated encoding and annotation of inferred gene regulatory networks complying with SBML. Bioinformatics 29, 2216-2217. Weber M, Henkel SG, Vlaic S, Guthke R, van Zoelen EJ, Driesch D (2013) Inference of dynamical gene-regulatory networks based on time-resolved multi-stimuli multiexperiment data applying NetGenerator V2.0. BMC Syst Biol 7, 1. Weber M, Sotoca AM, Kupfer P, Guthke R, van Zoelen EJ (2013) Dynamic modelling of microRNA regulation during mesenchymal stem cell differentiation. BMC Syst Biol 7, 124. Junior Research Group Secondary Metabolism of Predatory Bacteria Elliott CE, Callahan DL, Schwenk D, Nett M, Hoffmeister D, Howlett BJ (2013) A gene cluster responsible for biosynthesis of phomenoic acid in the plant pathogenic fungus, Leptosphaeria maculans. Fungal Genet Biol 53, 50-58. Kage H, Kreutzer MF, Wackler B, Hoffmeister D, Nett M (2013) An iterative type I polyketide synthase initiates the biosynthesis of the antimycoplasma agent micacocidin. Chem Biol 20, 764-771. Kersten RD, Lane AL, Nett M, Richter TK, Duggan BM, Dorrestein PC, Moore BS (2013) Bioactivity-guided genome mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica. ChemBioChem 14, 955-962. Kreutzer MF, Kage H, Herrmann J, Pauly J, Hermenau R, Müller R, Hoffmeister D, Nett M (2014) Precursor-directed biosynthesis of micacocidin derivatives with activity against Mycoplasma pneumoniae. Org Biomol Chem 12, 113-118. Pauly J, Spiteller D, Linz J, Jacobs J, Allen C, Nett M, Hoffmeister D (2013) Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum. ChemBioChem 14, 2169-2178. Vela Gurovic MS, Müller S, Domin N, Seccareccia I, Nietzsche S, Martin K, Nett M (2013) Micromonospora schwarzwaldensis sp. nov., a producer of telomycin, isolated from soil. Int J Syst Evol Microbiol 63, 3812-3817. Junior Research Group Cellular Immunobiology This group ceased in 2012. Head: Dr. Mihály Józsi Hebecker M, Alba-Domínguez M, Roumenina LT, Reuter S, Hyvärinen S, Dragon-Durey MA, Jokiranta TS, Sánchez-Corral P, Józsi M (2013) An engineered construct combining complement regulatory and surface-recognition domains represents a minimal-size functional factor H. J Immunol 191, 912-921. Junior Research Group Fundamental Molecular Biology of Pathogenic Fungi This group ceased in 2012. Head: Dr. habil. Peter Staib Grumbt M, Monod M, Yamada T, Hertweck C, Kunert J, Staib P (2013) Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump. J Invest Dermatol 133, 15501555. Hennicke F, Grumbt M, Lermann U, Ueberschaar N, Palige K, Böttcher B, Jacobsen ID, Staib C, Morschhäuser J, Monod M, Hube B, Hertweck C, Staib P (2013) Factors supporting cysteine tolerance and sulfite production in Candida albicans. Eukaryot Cell 12, 604-613. Palige K, Linde J, Martin R, Böttcher B, Citiulo F, Sullivan DJ, Weber J, Staib C, Rupp S, Hube B, Morschhäuser J, Staib P (2013) Global transcriptome sequencing identifies chlamydospore specific markers in Candida albicans and Candida dubliniensis. PLoS One 8, e61940. Symoens F, Jousson O, Packeu A, Fratti M, Staib P, Mignon B, Monod M (2013) The dermatophyte species Arthroderma benhamiae: intraspecies variability and mating behaviour. J Med Microbiol 62, 377-385. Wu H, Downs D, Ghosh K, Ghosh AK, Staib P, Monod M, Tang J (2013) Candida albicans secreted aspartic proteases 4-6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism. FASEB J 27, 2132-2144. Bio Pilot Plant Biermann M, Bardl B, Vollstadt S, Linnemann J, Knupfer U, Seidel G, Horn U (2013) Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach. Amino Acids 44, 1225-1231. Biermann M, Linnemann J, Knüpfer U, Vollstädt S, Bardl B, Seidel G, Horn U (2013) Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation. Microbial Cell Factories 12, 116-124. Cao J, Goldhan J, Martin K, Köhler JM (2013) Investigation of mixture toxicity of widely used drugs caffeine and ampicillin in the presence of an ACE inhibitor on bacterial growth using droplet-based microfluidic technique. Green Process Synth 2, 591-601. Cao J, Kürsten D, Krause K, Kothe E, Martin K, Roth M, Köhler JM (2013) Application of micro-segmented flow for two-dimensional characterization of the combinatorial effect of zinc and copper ions on metal-tolerant Streptomyces strains. Appl Microbiol Biotechnol 97, 8923-8930. Gläser SP, Bolte K, Martin K, Busse HJ, Grossart HP, Kämpfer P, Gläser J (2013) Novosphingobium fuchskuhlense sp. nov., isolated from the north-east basin of Lake Grosse Fuchskuhle. Int J Syst Evol Microbiol 63, 586-592. Gläser SP, Galatis H, Martin K, Kämpfer P (2013) Castellaniella hirudinis sp. nov., isolated from the skin of Hirudo verbana. Int J Syst Evol Microbiol 63, 521-525. Gläser SP, Galatis K, Martin K, Kämpfer P (2013) Flavobacterium cutihirudinis sp. nov., isolated from the skin of the medical leech Hirudo verbana. Int J Syst Evol Microbiol 63, 2841-2847. Gläser SP, Galatis H, Martin K, Kämpfer P (2013) Kaistia hirudinis sp. nov., isolated from the skin of Hirudo verbana. Int J Syst Evol Microbiol 63, 3209-3213. Gläser SP, Galatis H, Martin K, Kämpfer P (2013) Niabella hirudinis and Niabella drilacis sp. nov., isolated from the medicinal leech Hirudo verbana. Int J Syst Evol Microbiol 63, 3487-3493. Gläser SP, Falsen E, Martin K, Kämpfer P (2013) Alicyclobacillus consociatus sp. nov., isolated from a human clinical specimen. Int J Syst Evol Microbiol 63, 3623-3627. Gebhardt P, Würbach L, Heidrich A, Heinrich L, Walther M, Opfermann T, Sørensen B, Saluz HP (2013) Dynamic behaviour of selected PET tracers in embryonated chicken eggs. Rev Esp Med Nucl Imagen Mol 32, 371-377. Gurovic MS, Müller S, Domin N, Seccareccia I, Nietzsche S, Martin K, Nett M (2013) Micromonospora schwarzwaldensis sp. nov., a producer of telomycin, isolated from soil. Int J Syst Evol Microbiol 63, 3812-3817. Kämpfer P, Gläser SP, Schäfer J, Lodders N, Martin K, Schumann P (2013) Ornithinimicrobium murale sp. nov., isolated from an indoor wall colonized by moulds. Int J Syst Evol Microbiol 63, 119-123. Kämpfer P, Martin K, Dott W (2013) Gordonia phosphorivorans sp. nov., isolated from a wastewater bioreactor with phosphorus removal. Int J Syst Evol Microbiol 63, 230-235. Kämpfer P, Wellner S, Lohse K, Lodders N, Martin K (2013) Rhodococcus cerastii sp. nov. and Rhodococcus trifolii sp. nov., two novel species isolated from leaf surfaces. Int J Syst Evol Microbiol 63, 1024-1029. Knop K, Pavlov GM, Rudolph T, Martin K, Pretzel D, Jahn BO, Scharf DH, Brakhage AA, Makarov V, Möllmann U, Schacher FH, Schubert US (2013) Amphiphilic Appendix HKI Research Report 2012 / 2013 star-shaped block copolymers as unimolecular drug delivery systems: investigations using a novel fungicide. Soft Matter 9, 715-726. Lüdecke C, Bossert J, Roth M, Jandt KD (2013) Physical vapor deposited titanium thin films for biomedical applications: Reproducibility of nanoscale surface roughness and microbial adhesion properties. Applied Surface Science 280, 578-589. Lüdecke C, Jandt KD, Siegismund D, Kujau MJ, Zang E, Rettenmayr M, Bossert J, Roth M (2014) Reproducible biofilm cultivation of chemostat-grown Escherichia coli and investigation of bacterial adhesion on biomaterials using a non-constant-depth film fermenter. PLoS ONE 9, e84837. Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis: a four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges. Angew Chem Int Ed 52, 11092-11095. Schütze E, Klose M, Merten D, Nietzsche S, Senftleben D, Roth M, Kothe E (2014) Growth of streptomycetes in soil and their impact on bioremediation. J Hazard Mater 267, 128-135. Schütze E, Miltner A, Nietzsche S, Achtenhagen J, Klose M, Merten D, Greyer M, Roth M, Kästner M, Kothe E (2013) Live and death of streptomycetes in soil - what happens to the biomass? J Plant Nutr Soil Sci 176, 665–673. Wink J, Schumann P, Spöer C, Eisenbarth K, Glaeser SP, Martin K, Kämpfer P (2014) Emended description of Actinoplanes friuliensis and description of Actinoplanes nipponensis sp. nov., antibiotic-producing species of the genus Actinoplanes. Int J Syst Evol Microbiol 64, 599-606. Epub 2013 Oct 24. Zang E, Brandes S, Tovar M, Martin K, Mech F, Horbert P, Henkel T, Figge MT, Roth M (2013) Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets. Lab Chip 13, 3707-3713. Jena Microbial Resource Collection Bibashi E, de Hoog GS, Pavlidis TE, Symeonidis N, Sakantamis A, Walther G (2013) Wound infection 217 caused by Lichtheimia ramosa due to a car accident. Medical Mycology Case Reports 2, 7-10. Dolatabadi S, Walther G, Gerrits van den Ende AHG, Hoog GS (2013) Diversity and delimitation of Rhizopus microsporus. Fungal Diversity 1-19. Eberhardt U, Beker H, Vesterholt J, Dukik K, Walther G, Vila J, Fernández Brime S (2013) European species of Hebeloma section Theobromina. Fungal Diversity 58, 103-126. Grützmann K, Szafranski K, Pohl M, Voigt K, Petzold A, Schuster S (2014) Fungal alternative splicing is associated with multicellular complexity and virulence: A genome-wide multi-species study. DNA Res 21, 27-39. Gryganskyi AP, Humber RA, Smith ME, Hodge K, Huang B, Voigt K, Vilgalys R (2013) Phylogenetic lineages in entomophthoromycota. Persoonia 30, 94-105. Hoffmann K, Pawlowska J, Walther G, Wrzosek M, de Hoog GS, Benny GL, Kirk PM, Voigt K (2013) The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies. Persoonia 30, 57-76. Münchberg U, Wagner L, Spielberg ET, Voigt K, Rosch P, Popp J (2013) Spatially resolved investigation of the oil composition in single intact hyphae of Mortierella spp. with micro-Raman spectroscopy. Biochim Biophys Acta 1831, 341-349. Pawłowska J, Walther G, Wilk M, Hoog S, Wrzosek M (2013) The use of compensatory base change analysis of ITS2 as a tool in the phylogeny of Mucorales, illustrated by the Mucor circinelloides complex. Organisms Diversity & Evolution 13, 497-502. Sahadevan Y, Richter-Fecken M, Kaerger K, Voigt K, Boland W (2013) Early and late trisporoids differentially regulate β-carotene production and gene transcript levels in the mucoralean fungi Blakeslea trispora and Mucor mucedo. Appl Environ Microbiol 79. 7466-7475. Voigt K, Vaas L, Stielow B, de Hoog GS (2013) The zygomycetes in a phylogenetic perspective. Persoonia 30, i–iv. 218 Wagner L, Stielow B, Hoffmann K, Petkovits T, Papp T, Vagvolgyi C, de Hoog GS, Verkley G, Voigt K (2013) A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA. Persoonia 30, 77-93. Walther G, Pawłowska J, Alastruey-Izquierdo A, Wrzosek M, Rodriguez-Tudela JL, Dolatabadi S, Chakrabarti A, de Hoog GS (2013) DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia 30, 11-47. Associated Group Microbial Biochemistry and Physiology Galiger C, Brock M, Jouvion G, Savers A, Parlato M, IbrahimGranet O (2013) Assessment of efficacy of antifungals against Aspergillus fumigatus: value of real-time bioluminescence imaging. Antimicrob Agents Chemother 57, 3046-3059. Otzen C, Müller S, Jacobsen ID, Brock M (2013) Phylogenetic and phenotypic characterisation of the 3-ketoacyl-CoA thiolase gene family from the opportunistic human pathogenic fungus Candida albicans. FEMS Yeast Res 13, 553-564. Associated Group Pharmaceutical Microbiology Bohnert M, Dahse HM, Gibson DM, Krasnoff SB, Hoffmeister D (2013) The fusarin analog NG-391 impairs nucleic acid formation in K-562 leukemia cells. Phytochemistry Letters 6, 189-192. Elliott CE, Callahan DL, Schwenk D, Nett M, Hoffmeister D, Howlett BJ (2013) A gene cluster responsible for biosynthesis of phomenoic acid in the plant pathogenic fungus, Leptosphaeria maculans. Fungal Genet Biol 53, 50-58. Forseth RR, Amaike S, Schwenk D, Affeldt KJ, Hoffmeister D, Schröder FC, Keller NP (2013) Homologous NRPS-like gene clusters mediate redundant small-molecule biosynthesis in Aspergillus flavus. Angew Chem Int Ed 52, 1590-1594. Kage H, Kreutzer MF, Wackler B, Hoffmeister D, Nett M (2013) An iterative type I polyketide synthase initiates the biosynthesis of the antimycoplasma agent micacocidin. Chem Biol 20, 764-771. Kreutzer MF, Kage H, Herrmann J, Pauly J, Hermenau R, Müller R, Hoffmeister D, Nett M (2014) Precursor-directed biosynthesis of micacocidin derivatives with activity against Mycoplasma pneumoniae. Org Biomol Chem 12, 113-118. Böttger D, Hertweck C (2013) Molecular diversity sculpted by fungal PKS-NRPS hybrids. ChemBioChem 14, 28-42. Lackner G, Bohnert M, Wick J, Hoffmeister D (2013) Assembly of melleolide antibiotics involves a polyketide synthase with crosscoupling activity. Chem Biol 20, 1101-1106. Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19, 792-798. Pauly J, Spiteller D, Linz J, Jacobs JM, Allen C, Nett M, Hoffmeister D (2013) Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum. ChemBioChem 14, 2169-2178. Steinchen W, Lackner G, Yasmin S, Schrettl M, Dahse HM, Haas H, Hoffmeister D (2013) The bimodular peptide synthetase SidE produces fumarylalanine in the human pathogen Aspergillus fumigatus. Appl Environ Microbiol 79, 6670-6676. Pidot SJ, Coyne S, Kloss F, Hertweck C (2014) Antibiotics from neglected bacterial sources. Int J Med Microbiol 304, 14-22. Epub 2013 Sep 4. Sasso S, Pohnert G, Lohr M, Mittag M, Hertweck C (2012) Microalgae in the postgenomic era: a blooming reservoir for new natural products. FEMS Microbiol Rev 36, 761-785. Scharf DH, Heinekamp T, Remme N, Hortschansky P, Brakhage AA, Hertweck C (2012) Biosynthesis and function of gliotoxin in Aspergillus fumigatus. Appl Microbiol Biotechnol 93, 467-472. Reviews, Monographs, Book Chapters 2012/2013 Übersichtsarbeiten, Monographien, Beiträge zu Sammelwerken 2012/2013 Scherlach K, Graupner K, Hertweck C (2013) Molecular bacteriafungi interactions: effects on environment, food, and medicine. Annu Rev Microbiol 67, 375-397. Department of Biomolecular Chemistry Department of Infection Biology Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA, Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E, Donadio S, Dorrestein PC, Entian KD, Fischbach MA, Garavelli JS, Goransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Muller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJ, Rebuffat S, Ross RP, Sahl HG, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Sussmuth RD, Tagg JR, Tang GL, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, van der Donk WA (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30, 108-160. Behnken S, Hertweck C (2012) Anaerobic bacteria as producers of antibiotics. Appl Microbiol Biotechnol 96, 61-67. HKI Research Report 2012 / 2013 Appendix Lindner S, Zipfel PF (2012) Atypisches hämolytisch-urämisches Syndrom – Genetik, Atypical hemolytic uremic syndrome – Genetics. In: Dialyse aktuell, 16, 512-522, Georg Thieme Verlag Stuttgart, New York. Luo S, Skerka C, Kurzai O, Zipfel PF (2013) Complement and innate immune evasion strategies of the human pathogenic fungus Candida albicans. Mol Immunol 56, 161-169. Skerka C, Chen Q, FremeauxBacchi V, Roumenina LT (2013) Complement factor H related proteins (CFHRs). Mol Immunol 56, 170-180. Zipfel PF, Hallström T, Riesbeck K (2013) Human complement control and complement evasion by pathogenic microbes - tipping the balance. Mol Immunol 56, 152-160. Zipfel PF, Lauer N (2012) Defective complement action and control defines disease pathology for retinal and renal disorders and provides a basis for new therapeutic approaches. In: Advances in Experimental Medicine and Biology, Lambris JD (ed), Springer. Zipfel PF, Lauer N (2013) Defective complement action and control defines disease pathology for retinal and renal disorders and provides a basis for new therapeutic approaches. Adv Exp Med Biol 734, 173-187. Zipfel PF, Skerka C (2012) Hämolytisch–Urämisches Syndrom und Thrombotisch-Thrombozytopenische Purpura. In: Klinische Immunologie, Peter HH. Pichler WJ Müller-Lander U (eds), Urban & Fischer. Zipfel PF, Skerka C (2012) Thrombotic Microangiopathies: Thrombus Formation due to Common or Related Mechanisms? In: Kidney disorders, Sethi S, Fervenza FC (eds), Springer. Zipfel PF, Skerka C (2012) Complement, Candida, and cytokines: the role of C5a in host response to fungi. Eur J Immunol 42, 822-825. Zipfel PF, Skerka C, Hallström T, Wolf G (2013) 14th European meeting on complement in human disease, Jena, Germany, August 1721, 2013. Mol Immunol 56, 151. Department of Microbial Pathogenicity Mechanisms Brunke S, Hube B (2013) Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol 15, 701-708. Gow NA, Hube B (2012) Importance of the Candida albicans cell wall during commensalism and infection. Curr Opin Microbiol 15, 406-412. Jacobsen ID, Große K, Hube B (2012) Part VI: Responses to infection in vivo: Host response. Title: Embryonated chicken eggs as alternative infection model for pathogenic fungi. In: Methods in Molecular Biology. Host-Fungus Interactions: Volume 845, Methods and Protocols, Brand AC, MacCallum DM (eds), Springer Verlag, 487-496. Jacobsen ID, Wilson D, Wächtler B, Brunke S, Naglik JR, Hube B (2012) Candida albicans dimorphism as a therapeutic target. Expert Rev Anti Infect Ther 10, 85-93. Lüttich A, Brunke S, Hube B (2012) Part V: Responses to infection in vivo: Fungal response. Title: Isolation and amplification of fungal RNA for microarray analysis from host samples. In: Methods in Molecular Biology. Host-Fungus Interactions: Volume 845, Methods and Protocols, Brand AC, MacCallum DM (eds), Springer Verlag, 411-421. Mayer FL, Wilson D, Hube B (2013) Candida albicans pathogenicity mechanisms. Virulence 4, 119-128. Miramón P, Kasper L, Hube B (2013) Thriving within the host: Candida spp. interactions with phagocytic cells. Med Microbiol Immunol 202, 183-195. Wilson D, Citiulo F, Hube B (2012) Pearl review: Zinc exploitation by pathogenic fungi. PLoS Pathog 8, e1003034. Department of Molecular and Applied Microbiology Brakhage AA (2013) Regulation of fungal secondary metabolism. Nat Rev Microbiol 11, 21-32. Fliegerova K, Mrazek J, Voigt K (2012) 1. Gut Fungi: Classification, Evolution, Life Style and Application. In: Fungi: Types, Environmental Impact and Role in Disease. Series: Environmental Health - Physical, Chemical and Biological Factors. Silva AP, Sol M (eds), Nova Science Publisher, USA. Heinekamp T, Thywißen A, Macheleidt J, Keller S, Valiante V, Brakhage AA (2013) Aspergillus fumigatus melanins: interference with the host endocytosis pathway and impact on virulence. Front Microbiol 3, 440. Horn F, Heinekamp T, Kniemeyer O, Pollmächer J, Valiante V, Brakhage AA (2012) Systems biology of fungal infection. Front Microbiol 3, 108. Kroll K, Pähtz V, Kniemeyer O (2014) Elucidating the fungal stress response by proteomics. J Proteomics 97, 151-163. Nützmann HW, Schroeckh V, Brakhage AA (2012) Regulatory cross talk and microbial induction of fungal secondary metabolite gene clusters. Methods Enzymol 517, 325-341. Scharf DH, Brakhage AA (2013) Engineering fungal secondary metabolism: a roadmap to novel compounds. J Biotechnol 163, 179-183. Scharf DH, Heinekamp T, Remme N, Hortschansky P, Brakhage AA, Hertweck C (2012) Biosynthesis and function of gliotoxin in Asper- gillus fumigatus. Appl Microbiol Biotechnol 93, 467-472. Voigt K, de Hoog S, Ho HM, Hoffmann K, Jacobsen ID, Fischer G (2012) International cooperation in zygomycete research. IMA fungus 3, 20-23. Voigt K, Marano AV, Gleason F (2013) Ecological and economical importance of parasitic and zoosporic true Fungi In: The Mycota Vol. XI: Agricultural Applications. 2nd edition. Kempken F (ed), pp. 243-270, Springer Verlag, Berlin, Heidelberg, New York. Voigt K, Shelest E (2013) Genomics to study basal lineage fungal biology: phylogenomics suggest a common origin. In: Mycota XIII, Fungal Genomics, 2. Edition. Werner W, Herold M, Ruffert K, Merkle K, Brakhage AA, Leoni L, Cheson BD (2013) Developmental history: bendamustine yesterday, today, tomorrow. Onkologie 36 Suppl 1, 2-10. Research Group Applied Systems Biology Guthke R, Linde J, Mech F, Figge MT (2012) Systems biology of microbial infection. Front Microbiol 3, 328. Horn F, Heinekamp T, Kniemeyer O, Pollmächer J, Valiante V, Brakhage AA (2012) Systems biology of fungal infection. Front Microbio 3, 108. Mech F, Figge MT (2012) Imagebased systems biology: A quantitative approach to elucidate the kinetics of fungal morphologies and virulence. In: German Conference on Bioinformatics, September 19-22, 2012, Jena, Germany. Proceedings, pp. 9-12. Research Group Fungal Septomics Luo S, Skerka C, Kurzai O, Zipfel PF (2013) Complement and innate immune evasion strategies of the human pathogenic fungus Candida albicans. Mol Immunol 56, 161-169. Willinger B, Kienzl D, Kurzai O (2013) Diagnostics of Fungal Infections. In: Human Fungal Pathogens Vol. XII, Kurzai O (ed), The Mycota, Springer Verlag, Heidelberg. Research Group Systems Biology / Bioinformatics Altwasser R, Guthke R, Vlaic S, Emmett MR, Conrad CA, MeyerBaese A (2013) Model order reduc- Appendix HKI Research Report 2012 / 2013 tion of deterministic and stochastic gene regulatory networks. In: BIOCOMP’12 - 13th Int. Conf. Bioinformatics and Computational Biology, July 16-19, 2012, Las Vegas, USA. Proc. BIOCOMP’13, CSREA Press, USA, pp. 487-496. Altwasser R, Guthke R, Vlaic S, Emmett MR, Conrad CA, MeyerBaese A (2013) Almost sure stability of stochastic gene regulatory networks with mode-dependent interval delays. In: BIOCOMP’12 13th Int. Conf. Bioinformatics and Computational Biology, July 16-19, 2012, Las Vegas, USA. Proc. BIOCOMP’13, CSREA Press, USA, pp. 468-477. Bettenbrock K, Jahreis K, Kremling A, Pfaff M, Rinas U, Schuster S, Guthke R (2012) Das Bakterium Escherichia coli - Ein Modellorganismus für die Systembiologie. sytembiologie.de - Ein Magazin für systembiologische Forschung in Deutschland, 4, 84-86. Guthke R, Linde J, Mech F, Figge MT (2012) Systems biology of microbial infection. Front Microbiol 3, 328. Horn F, Guthke R (2012) Crosshybridizations - corrupting and enhancing microarray probe designs. Biomed Tech (Berl) 57, 268. Horn F, Guthke R (2012) Probe selection for pathway-specific microarray probe design minimizing melting temperature variance. In: Proc Int Conf Bioinf and Biomed ICBB 2012, Madrid, Spain, March 28 - 29, 2012, pp. 301-306. Horn F, Heinekamp T, Kniemeyer O, Pollmächer J, Valiante V, Brakhage AA (2012) Systems biology of fungal infection. Front Microbiol 3, 108. Kottenhagen N, Gramzow L, Horn F, Pohl M, Theißen G (2012) Polyglutamine and polyalanine tracts are enriched in transcription factors of plants. In: Proc German Conf Bioinf - GCB 2012, Jena, September 19 - 22, 2012, pp. 93-107. Kupfer P, Guthke R, Pohlers D, Huber R, Koczan D, Kinne RW (2012) Batch correction of microarray data substantially improves the identification of genes differentially expressed in rheumatoid arthritis and osteoarthritis. BMC Med Genomics 5, 23. Kupfer P, Vlaic S, Huber R, Kinne RW, Guthke R (2013) Different stimuli for inference of gene 219 regulatory network in rheumatoid arthritis. In: International Conference on Bioinformatics Models, Methods and Algorithms, 11-14 February 2013, Barcelona, Spain. Conference Proceedings, pp. 282287. Priebe S, Menzel U (2013) Assignment of orthologous genes by utilization of multiple databases: the orthology package in R. In: International Conference on Bioinformatics Models, Methods and Algorithms, 11-14 February 2013, Barcelona, Spain. Conference Proceedings, pp. 105-110. Socota AM, Weber M, Zoelen EJ (2012) Gene expression regulation underlying osteo-, adipo-, and chondrogenic lineage commitment of human mesenchymal stem cells. In: Daskalaki A: Medical advancements in aging and regenerative technologies: Clinical tools and applications. IGI Global, Vol. 1, pp. 76-94. Voigt K, Shelest E (2013) Genomics to study basal lineage fungal biology: phylogenomics suggest a common origin. In: Mycota XIII, Fungal Genomics, 2. Edition.c Wolf T, Shelest V, Shelest E (2013) Motif-based method for genomewide prediction of eukaryotic gene clusters. 2nd International Workshop on Pattern Recognition in Proteomics, Structural Biology and Bioinformatics. In: ICIAP 2013 Workshops, Lecture Notes in Computer Science (LNCS) 8158, Petrosino A, Maddalena L, Pala P (eds), pp. 389–398, Springer-Verlag, Berlin, Heidelberg. Junior Research Group Cellular Immunobiology This group ceased in 2012. Head: Dr. Mihály Józsi Jena Microbial Resource Collection Fliegerova K, Mrazek J, Voigt K (2012) 1. Gut Fungi: Classification, Evolution, Life Style and Application. In: Fungi: Types, Environmental Impact and Role in Disease. Series: Environmental Health - Physical, Chemical and Biological Factors. Silva AP, Sol M (eds), Nova Science Publisher, USA. Voigt K, de Hoog S, Ho HM, Hoffmann K, Jacobsen ID, Fischer G (2012) International cooperation in zygomycete research. IMA fungus 3, 20-23. Voigt K, Marano AV, Gleason F (2013) Ecological and economical importance of parasitic and zoosporic true Fungi In: The Mycota Vol. XI: Agricultural Applications. 2nd edition. Kempken F (ed), pp. 243-270, Springer Verlag, Berlin, Heidelberg, New York. Voigt K, Shelest E (2013) Genomics to study basal lineage fungal biology: phylogenomics suggest a common origin. In: Mycota XIII, Fungal Genomics, 2. Edition. Associated Group Microbial Biochemistry and Physiology Brock M (2012) Application of bioluminescence imaging for in vivo monitoring of fungal infections. Int J Microbiol 2012, 956794. Fazius F, Zaehle C, Brock M (2013) Lysine biosynthesis in microbes: relevance as drug target and prospects for beta-lactam antibiotics production. Appl Microbiol Biotechnol 97, 3763-3772. Associated Group Pharmaceutical Microbiology Józsi M (2012) Anti-Factor H autoantibodies in kidney disease: Detection, pathogenic role, and relevance for treatment. In: Autoantibodies: Detection, Pathogenicity and Health Implications. Jenkins GE, Hall JI (eds), Hauppauge, Nova Science Publishers, pp.37-58. Junior Research Group Fundamental Molecular Biology of Pathogenic Fungi This group ceased in 2012. Head: Dr. habil. Peter Staib Monod M, Staib P, Borelli C (2013) Candidapepsin (Chapter 33) In: The handbook of proteolytic enzymes. Rawlings ND, Salvesen G (eds), Elsevier Academic Press, Oxford, UK. 220 Kalb D, Lackner G, Hoffmeister D. (2013) Fungal peptide synthetases – an update on functions and specificity signatures. Fungal Biol Rev 27, 43-50. Memberships in Editorial Boards 2012/2013 Mitgliedschaften in Editorial Boards 2012/2013 Axel A Brakhage Applied Environmental Microbiology Applied Microbiology and Biotechnology Current Genetics eLife Eukaryotic Cell Frontiers in Fungi and Their Interactions Frontiers in Microbiology Matthias Brock FEMS Microbiology Letters Pathogens Oliver Kurzai Medical Mycology Case Reports Journal of Basic Microbiology Christine Skerka Journal of Genetic Syndromes and Gene Therapy Kerstin Voigt Frontiers in Evolutionary and Population Genetics Journal of Basic Microbiology MycoKeys Persoonia (Guest editor) Peter F Zipfel Molecular Immunology Frontiers in Molecular Innate Immunity Marc Thilo Figge Cytometry Part A, Special Issue Image-based Systems Biology FIAS Interdisciplinary Science Series, Springer Int. Publishing Frontiers in Computational Physics Frontiers Special Topic Systems Biology of Microbial Infection ISRN Biomathematics Journal of Computational Medicine Lectures at the HKI 2012 Kolloquien am HKI 2012 Reinhard Guthke BMC Systems Biology Frontiers Special Topic Systems Biology of Microbial Infection Eduard Reithmeier Leibniz-Universität Hannover 3D-Object-/Surface-Measurement using a SEM with 4 EverhartThornley Detectors 07.03.2012 Host: Brakhage AA Christian Hertweck Bioorganic Chemistry ChemBioChem Chemistry & Biology Organic and Biomolecular Chemistry The Journal of Antibiotics Kerstin Hoffmann Frontiers in Evolutionary and Population Genetics Uwe Horn Microbiological Research Bernhard Hube BMC Microbiology BMC Research Notes Cellular Microbiology FEMS Yeast Research Frontiers in Fungi and Their Interactions Frontiers in Molecular Innate Immunity mBio Pathogens Ilse Jacobsen Medical Mycology Case Reports PLoS One Olaf Kniemeyer Archives of Microbiology HKI Research Report 2012 / 2013 Appendix Christoph M. Tang Sir William Dunn School of Pathology, Oxford Complement and infection of Neisseria meningitidis 24.01.2012 Host: Zipfel PF Matthias Mörgelin Lund University, Schweden Antimicrobial properties of the extracellular matrix 20.03.2012 Host: Zipfel PF Candace Elliott Faculty of Science School of Botany, Melbourne Unravelling the roles of peptide synthetases and polyketide synthases in the canola pathogen Leptosphaeria maculans 27.03.2012 Host: Hoffmeister D Frank Martin Brunkhorst Universitätsklinikum Jena Die Gefährlichkeit von Krankenhausinfektionen 03.04.2012 Host: Brakhage AA, Stelzner A, Zipfel PF Kai Naumann Leibniz-Institut für Pflanzenbiochemie, Halle Label-free shotgun proteomics as versatile tool in biological research 08.05.2012 Host: Kniemeyer O Hortense Slevogt Universitätsklinikum Jena Danger signals in der Pathogenese der Sepsis 15.05.2012 Host: Brakhage AA, Stelzner A, Zipfel PF Chambers C. Hughes University of California, San Diego The discovery and development of marine microbial natural products as therapeutic leads and cellular probes 29.05.2012 Anja Forche Bowdoin College, Brunswick, ME, USA Increase of genetic and phenotypic diversity as an adaptation mechanism to changing host environments 05.06.2012 Host: Hube B, Brakhage AA Mathias Pletz Universitätsklinikum Jena Antibiotikaresistenzen 17.07.2012 Host: Brakhage AA, Stelzner A, Zipfel PF Pierre Stallforth Harvard Medical School Metabolomic analysis of the social amoeba’s bacterial symbionts reveals a highly adapted mutualistic association 25.09.2012 Host: Habel A Oliver Werz Friedrich-Schiller-Universität Jena Molekulare Mechanismen geschlechts-spezifischer Unterschiede bei entzündlichen Reaktionen 09.10.2012 Host: Brakhage AA, Stelzner A, Zipfel PF Christine Beemelmanns Harvard Medical School Symbiotic and associated bacteria: big players in evolution and rich sources of new natural compounds 16.10.2012 Host: Habel A Lectures at the HKI 2013 Kolloquien am HKI 2013 Thomas Lehrnbecher Universitätsklinikum Frankfurt Immunotherapeutic approaches to invasive fungal infections 05.02.2013 Host: Brock M Gregers Rom Andersen University of Aarhus, Denmark Structural basis for proteolytic cleavage of complement C4 and C5 19.02.2013 Host: Zipfel PF Brandon Jutras University of Kentucky, College of Medicine 20.03.2013 Host: Zipfel PF Richard Pott Leibniz-Universität Hannover Biodiversitätskrise – Das sechste Massensterben auf der Erde? 16.04.2013 Host: Brakhage AA Walter Doerfler Friedrich-Alexander Universität Erlangen-Nürnberg Epigenetics – a different way of looking at genetics 23.04.2013 Host: Brakhage AA, Stelzner A, Zipfel PF Thorsten Heinzel Friedrich-Schiller-Universität Jena Lysine acetylation as a regulatory protein modification 14.05.2013 Host: Brakhage AA, Stelzner A, Zipfel PF Frank Ebel Max von Pettenkofer-Institut, LMU München Woronin bodies - their anchoring at the septal pore and contribution to the stress resistance of Aspergillus fumigatus 23.07.13 Host: Köster-Eiserfunke N Mike Bromley University of Manchester Antifungal resistance and drug discovery 22.08.13 Utz Reichard Universitätsmedizin Göttingen Aspergillus fumigatus and vaccines - is there hope? 05.12.13 Andreas Holzenburg Microscopy and Imaging Center, Texas A&M University Microscopy and Imaging in the Life Sciences 19.12.13 Host: Brakhage AA Scientific Awards 2012 Preise und Auszeichnungen 2012 Böhm, Sascha Posterpreis XXIV International Complement Workshop, Kreta, Griechenland 2012 Bretschneider, Tom Posterpreis Irseer Naturstofftagung 2012 Chankhamjon, Pranatchareeya medac-Forschungspreis medac GmbH, Wedel 2012 Chen, Qian Else Körner-Fresenius Posterpreis 43. Jahrestagung der Gesellschaft für Pädiatrische Nephrologie 2012 Chen, Qian Vortragspreis XXIV International Complement Workshop, Kreta, Griechenland 2012 Eberhardt, Hannes Posterpreis XXIV International Complement Workshop, Kreta, Griechenland 2012 Greßler, Markus Posterpreis European Fungal Genetics Conference, Marburg 2012 Hallström, Teresia Vortragspreis XXIV International Complement Workshop, Kreta, Griechenland 2012 Heinekamp, Thorsten medac-Forschungspreis medac GmbH, Wedel 2012 Horn, Fabian Vortragspreis, 5. ILRS Symposium ILRS 2012 Kasper, Lydia Vortragspreis DFG Schwerpunkt-Meeting 1580, Bonn 2012 Kreutzer, Martin Vortragspreis MiCom JSMC 2012 Kreutzer, Martin Vortragspreis VAAM International Workshop Biology and Chemistry of Antibiotic-Producing Bacteria and Fungi VAAM 2012 Appendix HKI Research Report 2012 / 2013 Letzel, Anne-Catrin Posterpreis, VAAM-Naturstofftagung Liebig-Vereinigung der Gesellschaft deutscher Chemiker 2012 Roth, Martin medac-Forschungspreis medac GmbH, Wedel 2012 Scharf, Daniel medac-Forschungspreis medac GmbH, Wedel 2012 Scherlach, Kirstin medac-Forschungspreis medac GmbH, Wedel 2012 Seccareccia, Ivana Posterpreis MiCom JSMC 2012 Seider, Katja Forschungspreis der DGHM Fachgruppe Eukaryontische Krankeitserreger DGHM 2012 Thywißen, Andreas Posterpreis Keystone Symposium, Santa Fe, USA 2012 Wolf, Thomas Vortragspreis MiCom JSMC 2012 Scientific Awards 2013 Preise und Auszeichnungen 2013 Behnken, Swantje DECHEMA Doktorandenpreis DECHEMA 2013 Brunke, Sascha Vortragspreis FEBS Young Investigator Award 5th FEBS Advanced Lecture Course 2013 Buhlmann, Denise Posterpreis 14th European Meeting on Complement in Human Disease, Jena 2013 Chankhamjon P, Scherlach K, Hertweck C Publikationspreis der DMykG Stiftung der Deutschsprachigen Mykologischen Gesellschaft 2013 221 Chen, Qian Vortragspreis 14th European Meeting on Complement in Human Disease, Jena 2013 Citiulo, Francesco Publikationspreis der DMykG Stiftung der Deutschsprachigen Mykologischen Gesellschaft 2013 Kästner, Julia Young Scientist Presentation Award 2nd European Meeting on Animal Chlamydioses and Zoonotic Implications (EMAC-2), Jena 2013 Kloss F, Pidot S, Goerls H, Friedrich T, Hertweck C Posterpreis des VAAM Workshop Biology of Bacteria Producing Natural Products VAAM 2013 Mayer, Francois Young Investigator Award der DGHM Fachgruppe Eukaryontische Krankeitserreger DGHM 2013 Figge MT Jena, Germany 2012 3rd International Student Conference on Microbial Communication (MiCom 2012) Pollmächer J, Sarkar S Jena, Germany 2012 5th ILRS Symposium Vogler C, Zipfel PF Jena, Germany 2012 11th European Congress on Fungal Genetics (ECFG 11) Brakhage AA Marburg, Germany 2012 11th European Conference on Computational Biology Figge MT Basel, Switzerland 2012 11th International Conference on Artifical Immune Systems Figge MT Taormina, Italy 2012 18th Congress of the International Society for Human and Animal Mycology (ISHAM) 2012 Hube B, Brock M Berlin, Germany 2012 Scharf, Daniel medac Forschungspreis medac GmbH, Wedel 2013 Scharf, Daniel, Chankhamjon P, Scherlach K, Hertweck C Publikationspreis der DMykG Stiftung der Deutschsprachigen Mykologischen Gesellschaft 2013 Scharf, Daniel Promotionspreis Beutenberg Campus e.V. 2013 BMT 2012 – 46 Jahrestagung der Deutschen Gesellschaft für Biomedizinische Technik (section on imaging and pattern recognition) Saluz HP Jena, Germany 2012 German Conference on Bioinformatics 2012 Figge MT, Guthke R Jena, Germany 2012 Scharf, Daniel Promotionspreis VAAM 2013 Ueberschaar N, Dahse H-M, Bretschneider T, Hertweck C Posterpreis, VAAM Workshop Biology of Bacteria Producing Natural Products VAAM 2013 Meetings, Workshops, Symposia 2012/2013 Wissenschaftliche Veranstaltungen 2012/2013 1 International Workshop “Image-based Systems Biology” st 222 Irseer Naturstofftage Hertweck C Irsee, Germany 2012 Marie-Curie ITN ARIADNE Summer School Kniemeyer O et al. Jena, Germany 2012 1st International Conference on Natural Products Brakhage AA Frankfurt, Germany 2013 2nd International Workshop Systems Biology of Microbial Infection, S2B0M1I3 Figge MT, Guthke R Jena, Germany 2013 6th ILRS Symposium Vogler C, Zipfel PF Jena, Germany 2013 14th European Meeting on Complement in Human Disease Zipfel PF, Skerka C, Hallström T et al. Jena, Germany 2013 CBS Workshop Emerging Zygomycetes, a new problem in the clinical lab A workshop of the ECMM/ISHAM Working Group on Zygomycetes Voigt K et al. Utrecht, The Netherlands 2013 FEBS Advanced practical course “State-of-the-art infection models for human pathogenic fungi” Hube B, Jacobsen ID Jena, Germany 2013 Irseer Naturstofftage Hertweck C Irsee, Germany 2013 Jahrestagung der Deutschsprachigen Mykologischen Gesellschaft DMykG Kurzai O Tübingen, Germany 2013 Saluz HP 10/2008 – 01/2012 Teilprojekt 39: Analysis of the interaction of the human pathogenic fungus Aspergilllus fumigatus with immune effector cells by functional genomics Brakhage AA 09/2009 – 12/2012 Teilprojekt 52: The interaction of Candida glabrata with human Neutrophils Kurzai O 11/2010 – 07/2014 Teilprojekt 55: Development of a multi-scale simulator to model host-pathogen interactions Figge MT 10/2011 – 09/2015 Teilprojekt 56: Transcriptome data analysis and modeling of Aspergillus fumigatus - phagocytes interaction Guthke R 08/2011 – 07/2014 Teilprojekt 58: Molecular basis for predatory interactions of Cupriavidus necator wich ist prey bacteria Nett M 10/2011 – 09/2014 Teilprojekt 59: Functional analysis of major transcriptional regulators in pathogenic dermatophytes Brakhage AA 01/2013 – 09/2014 Teilprojekt 67: Bacterial endosymbionts in plant-pathogenic fungi Hertweck C 11/2012 – 10/2015 Participation in Research Networks 2012/2013 Beteiligung an Netzwerken und Verbundprojekten 2012/2013 Teilprojekt 72: Interactions of the fungal pathogen Aspergillus fumigatus with human macrophages Brakhage AA 11/2012 – 10/2015 Deutsche Forschungsgemeinschaft Exzellenzgraduiertenschule Jena School for Microbial Communication Teilprojekt 77: Exploiting the metagenome of microbial communities on a microfluidic platform Horn U 03/2013 – 02/2016 Teilprojekt 6: Molecular Basis of Bacterial-Fungal Symbioses Hertweck C 11/2007 – 10/2012 Teilprojekt 21: MicroCom – Host pathogen interactions of human pathogenic yeast Hube B 10/2008 – 01/2012 Teilprojekt 31: Comparative genomics of host/chlamydiales interactions HKI Research Report 2012 / 2013 Appendix Teilprojekt 78: Infection-associated genes of Candida glabrata Hube B 02/2013 – 02/2016 Teilprojekt 79: Immunevasion of the Gram positive bacterium Streptococcus pneumoniae Zipfel PF 10/2013 – 09/2016 Teilprojekt 83: Pathogen/host communication: dual RNA expression dynamics to investigate differential patterns of virulence Saluz HP 10/2013 – 10/2016 Teilprojekt 87 Shelest E 10/2013 – 09/2016 Teilprojekt 89: Soil amoeba interactions with filamentous fungi as driving forces for pathogenicity Hillmann F 10/2013 – 01/2017 Teilprojekt 92: Mechanisms of Candida albicans colonization and translocation Jacobsen ID 02/2013 – 01/2017 Deutsche Forschungsgemeinschaft Sonderforschungsbereich/ Transregio 124 FungiNet Teilprojekt B03: Prädiktive Modellierung von Wirth-Pathogenen-Interaktionen durch Rekonstruktion genregulatorischer Netzwerke Guthke R, Shelest E 10/2013 – 06/2017 Teilprojekt B04: Bilddatenanalyse und Agenten-basierte Modellierung der Wechselwirkung zwischen Immunzellen und humanpathogenen Pilzen in Raum und Zeit Figge MT 10/2013 – 06/2017 Teilprojekt C01: Charakterisierung molekularer Mechanismen der Translokation von Candida albicans durch Epithelgewebe Hube B 10/2013 – 06/2017 Teilprojekt C04: Mechanismen der Adhäsion und Modulation humaner Immunzellen durch Candida albicans Skerka C 10/2013 – 06/2017 Teilprojekt C05: Mechanismen der gastrointestinalen Kolonisierung und der Translokation von Candida albicans durch die Darmschleimhaut Jacobsen ID 10/2013 – 06/2017 Teilprojekt C06: Einfluss sekretierter Proteine von Candida albicans bei der Immunevasion und der Pathogenität Zipfel PF 10/2013 – 06/2017 Teilprojekt INF: Integrierte Datenbank für Experimentaldaten (Data Warehouse OmniFung) Guthke R 10/2013 – 06/2017 Teilprojekt Z01: Projektmanagement Brakhage AA 10/2013 – 06/2017 Teilprojekt Z01: Die Rolle der Regulation putativer Pathogenitätsassoziierter Gene in der Virulenz des humanpathogenen Mucormykose-Erregers Lichtheimia corymbifera: Vergleichende RNAseq-Expressionsanalyse von attenuiertem versus virulentem Stamm Voigt K 10/2013 – 12/2013 Teilprojekt Z02: Methoden zur Analyse der zeitlichen und räumlichen Änderung der Proteome bei der Interaktion pilzlicher Pathogene mit dem Wirt Kniemeyer O 10/2013 – 06/2017 Deutsche Forschungsgemeinschaft Schwerpunktprogramm 1580: Intracellular Compartments as Places of Pathogen-Host Interaction Survival and proliferation of human pathogenic Candida species within phagocytes Hube B 10/2010 – 08/2014 Forschergruppe FOR 1334: Determinants of Polarized Growth and Development in Filamentous Fungi Redox regulation, development and hyphal growth in Aspergillus nidulans Brakhage AA, Hortschansky P 07/2010 – 06/2013 Europäische Union, 7. Forschungsrahmenprogramm Marie Curie International Research Staff Exchange Scheme KRAB-ZNF: KRAB zinc finger gene biology in evolution and disease Guthke R 07/2011 – 06/2013 Marie Curie Initial Training Network ARIADNE – Signaling circuitry controlling fungal virulence: identification and characterization of conserved and specific fungal virulence genes as common antifungal targets Brakhage AA, Heinekamp T 01/2010 – 12/2013 Marie Curie Initial Training Network Quantfung – Quantitative Biology for Fungal Secondary Metabolite Producers Brakhage AA 10/2013 – 09/2017 Marie Curie Initial Training Network FINSysB – Pathogenomics and Systems Biology of Fungal Infections – An Integrative Approach Hube B 11/2008 – 09/2012 Large-scale Integrating Project EURenOmics – European Consortium for High-Throughput Research in Rare Kidney Diseases WP 05: Complement Disorders Zipfel PF 10/2012 – 10/2016 Bundesministerium für Bildung und Forschung DiNaMid: Genom-basierte Findung neuer antimikrobieller Naturstoffe in mikrofluidischen Chips Teilprojekt: Identifizierung neuer Stoffwechselwege und NaturstoffIsolierung Nett M, Roth M 10/2009 – 12/2012 ERA-NET AspBIOmics Biomarker zur Therapie von invasiver Aspergillosis Kniemeyer O 05/2011 – 10/2014 ERA-Net – CandiCol Grundlagen zur Kolonisation und der Dissemination pathogener Candida-Spezies: Entwicklung früher Diagnose- und Therapieansätze Hube B 05/2011 – 04/2014 ERA-NET OXYstress Humapathogene Pilze unter Sauerstoff-Stress: Adaptation an Sauerstoffmangel und reaktive Sauerstoffspecies und die Folgen für die Interaktion mit dem Wirt und die Therapie Heinekamp T 04/2011 – 09/2014 ERA-NET PathoGenoMics 2 Apergillosis – Die Zellwand als Target zur Verbesserung der antifungalen Therapie der Aspergillose Kniemeyer O 02/2009 – 05/2012 ERASysBio – LINCONET Modellierung des genregulatorischen Netzwerks für die Linienspezifische Differenzierung in menschlichen mesenchymalen Stammzellen: Identifikation von potenziellen Zielmolekülen für die Appendix HKI Research Report 2012 / 2013 anabole Therapie von Gewebserkrankungen Guthke R 03/2010 – 08/2013 GenBioCom: Genombasierte Produktion bioaktiver Verbindungen aus Aktinomyceten Komplexe Polyketide aus Aktinomyceten: Genom-Analyse, Gensynthese und Aktivitätsbestimmung Hertweck C 03/2010 – 07/2013 GERONTOSYS II Rolle von oxidativen Stress in der Alterung und therapeutische Implikationen Guthke R 03/2011 – 05/2014 Integrierte Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) In-house Professorship Molekulare Mechanismen der Candida-Sepsis Hube B, Jacobsen ID Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) Project: Inprot Identifizierung von C. albicans und A. fumigatus Proteinantigenen für die Pilzsepsisdiagnostik und Untersuchung der A. fumigatus Stressantwort Kniemeyer O Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC) Project: Pathogen-Wirt-Interaktion während der Infektion mit Candida albicans und die Entwicklung zur Sepsis Hube B, Brunke S Jenaer Centrum für die Systembiologie des Alterns – JenAge Systembiologie von mildem Stress beim gesunden Altern ein MultiSpezies-Ansatz Guthke R 10/2009 – 09/2014 Kompetenznetz Die Virtuelle Leber Guthke R 04/2020 – 03/2015 PET-CT II In-vivo-Quantifizierung von Inflammation und Destruktion in murinen Arthritismodellen mittels PET/CT als Beitrag zur Reduktion der Zahl der in Forschung und Entwicklung verwendeten Versuchstiere Saluz HP 11/2011 – 10/2014 223 Resistom – Antibiotika-Resistom in der Lebensmittelkette Krügel H 04/2011 – 03/2012 SPICE III – Wissenschaftlichtechnische Zusammenarbeit mit Indonesien MABICO – Einfluss von Meeresverschmutzungauf Biodiversität und den Lebensunterhalt von Küstenbewohnern; Mikrobielle Biodiversität Saluz HP 03/2012 – 02/2015 Technologietransfer Verstetigung der Verwertungskonzepte in den lebenswissenschaftlichen Instituten der WGL – LeibnizInstitut für Naturstoff-Forschung und Infektionsbiologie e.V. – HansKnöll-Institut - (HKI) Ramm M 01/2009 – 03/2012 Verbundvorhaben Medizinische Infektionsgenomik: Wirt-Pathogen-Interaktion: Wirkung sekretierter Proteine von Staphylococcus aureus auf Zellen und Komponenten des Immunsystems Zipfel PF 10/2010 – 12/2013 ZIK Septomics Forschungsgruppe Fungal Septomics: Molekulare Mechanismen in der Pathogenese der Sepsis durch Pilze Brakhage A, Kurzai O 11/2009 – 10/2014 Zoonotische Chlamydien Teilprojekt 4: Wechselwirkung von zoonotischen Chlamydien mit ihren Wirtszellen Saluz HP, Hänel F 0172011 – 04/2014 Zwanzig20 – Partnerschaft für Innovation InfectControl 2020 – Neue Antiinfektionsstrategien – Wissenschaft • Gesellschaft • Wirtschaft; Strategievorhaben Brakhage AA Weitere Verbundprojekte Freistaat Thüringen Integriertes Multiplex Genotyping System basierend auf einem hyperspektralen Vielfarben-Scanner zur schnellen qualitativen und quantitativen Sequenzanalyse von SNPs und DNA-Polymorphismen auf Microarrayoberflächen Saluz HP 10/2009 – 09/2012 Freistaat Thüringen, ProExzellenz NanoConSens – Nanocontainer 224 und nanostrukturierte Trägermaterialien für Sensorik und Wirkstofftransport Brakhage AA, Hertweck C 10/2009 – 09/2012 Freistaat Thüringen, ProExzellenz MikroInter – Characterisation of receptors of the human-pathogenic fungus Aspergillus fumigatus - How does the fungus communicate with the environment and the host Brakhage AA 04/2010 – 03/2013 German-Israeli Foundation The hypoxic response in the pathogenic mold Aspergillus fumigatus and its relevance to disease Kniemeyer O 01/2010 – 06/2013 Leibniz-Gemeinschaft Kryostress – Anpassungsmechanismen der Zelle an Tiefstemperaturen (KAIT) Kniemeyer O 05/2013 – 05/2017 NIH Collaborative Study of Membranoproliferative Glomerulonephritis Type II Zipfel PF 09/2007 – 04/2012 Calls for Appointments 2012/2013 Rufe 2012/2013 Dr. habil. Guthke, Reinhard apl. Professor für Systembiologie an der Friedrich-SchillerUniversität Jena (2011) Prof. Hoffmeister, Dirk Professor für Pharmazeutische Biologie an der Friedrich-SchillerUniversität Jena Ruf an die Universität des Saarlandes, Saarbrücken (2013) – abgewendet – PD Jacobsen, Ilse, PhD Ruf an die Universität Leipzig (2013) – abgewendet – Postdoctoral Lecture Qualifications 2012/2013 Habilitationen 2012/2013 Ilse Jacobsen, PhD Infektionsmodelle zur Untersuchung der Pathogenese von Pilzinfektionen Friedrich-Schiller-Universität Jena 2013 Graduations 2012 Promotionen 2012 Brendel, Nicole Molekularbiologische Untersuchungen und Manipulation der Rhizoxin-Biosynthese in Burkholderia rhizoxinica Friedrich-Schiller-Universität Jena Kusebauch, Björn Biogenese und synthetische Modifizierung des antimitotischen Makrolids Rhizoxin Friedrich-Schiller-Universität Jena Barthel, Diana Die Rolle von Proteasen bei der Immunevasion von pathogenen Mikroorganismen Friedrich-Schiller-Universität Jena Braukmann, Maria Wirt-Pathogen-Wechselwirkungen in Chlamydia psittaci- und Chlamydia abortus-infizierten Hühnerembryonen Friedrich-Schiller-Universität Jena Linde, Jörg Systems biology of human pathogenic fungi: Modelling transcriptional networks of virulence Friedrich-Schiller-Universität Jena Mayer, François Indentification and characterization of novel infection-associated genes in Candida albicans Friedrich-Schiller-Universität Jena Mech, Franziska Image-based Systems Biology of Human-Pathogenic Fungi Friedrich-Schiller-Universität Jena Müller, Sebastian Application of Bayesian statistics in natural product research and infection biology Friedrich-Schiller-Universität Jena Nützmann, Hans-Wilhelm Protein acetyltransferases of Aspergillus nidulans – impact on secondary metabolism and fungal – bacterial interaction Friedrich-Schiller-Universität Jena Bruns, Sandra Interaktion des human-pathogenen Schimmelpilzes Aspergillus fumigatus mit humanen Neutrophil Extracellular Traps: Induktion und Evasion Friedrich-Schiller-Universität Jena Scharf, Daniel Neue Mechanismen der Regulation und Biochemie von Penicillin- und Gliotoxin-Biosynthese in Aspergillus sp. Friedrich-Schiller-Universität Jena Heddergott, Christoph Sekretierte Pathogenitätsfaktoren des Dermatophyten Arthroderma benhamiae Friedrich-Schiller-Universität Jena Seider, Katja Immune evasion as a pathogenicity strategy of Candida glabrata: survival within macrophages Friedrich-Schiller-Universität Jena Horn, Fabian Integrative Analysis of Omics-data for Aspergillus nidulans Friedrich-Schiller-Universität Jena Slesiona, Silvia Etablierung von Infektionsmodellen zur Untersuchung invasiver Aspergillosen durch Aspergillus terreus Freie Universität Icke, Selina Rolle von Akut-Phase-Proteinen und Komplement in entzündlichen Prozessen Friedrich-Schiller-Universität Jena Koch, Tina Evasionsstrategien pathogener Bakterien gegen die angeborene Immunreaktion Friedrich-Schiller-Universität Jena Kopp, Anne Crosstalk between the long pentraxin PTX3 and soluble complement inhibitors Friedrich-Schiller-Universität Jena Kuhnert, Anja Die Rolle des multifunktionellen Proteins TopBP1 bei der Initiation von Replikation und DNA-Schadensantwort Friedrich-Schiller-Universität Jena HKI Research Report 2012 / 2013 Appendix Svobodová, Eliška Interaction of Candida albicans and Candida dubliniensis with cells of the human innate immune system Friedrich-Schiller-Universität Jena Voigt, Anja Comperative genomics of chlamydial pathogens Friedrich-Schiller-Universität Jena Wartenberg, Dirk Identifizierung und Charakterisierung von Effektorproteinen bei Aspergillus fumigatus und Aspergillus nidulans Friedrich-Schiller-Universität Jena Graduations 2013 Promotionen 2013 Behnken, Swantje Genome mining of bacteria leads to closthioamide, the first antibiotic from the anaerobic world Friedrich-Schiller-Universität Jena Bereza, Magdalena Assessment of structural diversity of ß-amyloid with genetically engineered antibody fragments Friedrich-Schiller-Universität Jena Böhm, Sascha Immune evasion proteins from Staphylococcus aureus and therapeutic evaluation of staphylococcal complement-controlling proteins Friedrich-Schiller-Universität Jena Boettger, Daniela Exploring the biosynthetic potential of PKS and NRPS pathways in filamentous fungi Friedrich-Schiller-Universität Jena Bretschneider, Tom In-vitro-Charakterisierung nichtkanonischer Katosynthasen und Imaging-Massenspektrometrie von Naturstoffen Friedrich-Schiller-Universität Jena Döring, Nadia Die Rolle der Plasmaproteine Kallikrein, Plasmin und beta2-Glykoprotein I (ß2GPI) bei der Regulation des alternativen Komplementaktivierungsweges. Friedrich-Schiller-Universität Jena Eberhardt, Hannes The role of CFHR proteins in human autoimmune diseases Friedrich-Schiller-Universität Jena Friedrich, Lydia Etablierung des Hühnerembryonenmodells zur Darstellung von Infektionen mittels Positronenemissionstomographie/Computertomographie (PET/CT) am Beispiel von Chlamydophila psittaci Friedrich-Schiller-Universität Jena Heidrich, Alexander A glance through the shell: establishing the chick embryo as in vivo model system for preclinical molecular imaging by PET and CT Friedrich-Schiller-Universität Jena Kupfer, Peter Dynamic modeling of stimulated cells involved in cartilage and bone degenerative diseases Friedrich-Schiller-Universität Jena Lopez, Crisanto Immunoevasion of pathogenic microbes Friedrich-Schiller-Universität Jena Miramón, Pedro Molecular dissection of the interactions between Candida albicans and polymorphonuclear granulocytes Friedrich-Schiller-Universität Jena Priebe, Steffen Transcriptome analysis of multiple species using novel sequencing technologies Friedrich-Schiller-Universität Jena Thywißen, Andreas Molekulare Mechanismen der Interaktion von Aspergillus fumigatus mit Makrophagen Friedrich-Schiller-Universität Jena Voigt, Jessica The role of NK cells in immuneresponse against Candida albicans Friedrich-Schiller-Universität Jena Weber, Michael Integrative Methods for reconstruction of dynamic networks in chondrogenesis Friedrich-Schiller-Universität Jena Bachelor / Master / Diploma Theses 2012 Bachelor-/ Master-/ Diplomarbeiten 2012 Adam, Jonathan Approaches to detecting boundaries of biosynthetic gene clusters (pathways) in bacterial genomes Friedrich-Schiller-Universität Jena Bartz, Christoph Charakterisierung von Aspergillus fumigatus Gendeletionsmutanten unter hypoxischen Wachstumsbedingungen Fachhochschule Jena Bauer, Laura Konstruktion und Verifizierung von filamentösen und filamentdefizienten Candida albicans Stämmen unter Einsatz des Tetrazyklinregulierbaren Expressionssystems Friedrich-Schiller-Universität Jena Baunach, Martin Aufklärung der Indolterpenbiosynthese in Mangrovenendophyten Friedrich-Schiller-Universität Jena Bens, Martin Analyse der transkriptionellen Antwort von Rattenhepatozyten auf Hepatotoxine Friedrich-Schiller-Universität Jena Bonkowski, Katharina Die Rolle der DNasen von Aspergillus fumigatus beim Stickstoffmetabolismus und der Pathogenese Friedrich-Schiller-Universität Jena Bresan, Stefanie Epidemiologie von Klebsiella ESBLStämmen Friedrich-Schiller-Universität Jena Brockmöller, Thomas Metabolische Rekonstruktion des Malonyl-CoA-Biosynthese-Weges in Aspergillus nidulans Friedrich-Schiller-Universität Jena Buhlmann, Denise Expression und Charakterisierung des humanen löslichen Komplementrezeptors Typ 2 (CR2/CD21) Friedrich-Schiller-Universität Jena Conrad, Theresia Die Bedeutung von Single Nucleotide Polymorphismen (SNPs) der Alarmin-erkennenden Rezeptoren MINCLE und RACE für die Suszeptibilität und den Krankheitsverlauf bei Sepsis Friedrich-Schiller-Universität Jena Dettling, Anna Hyperspektrale Analyse von Single-Nucleotide-Polymorphisms auf Chipoberflächen am Beispiel von Chlamydia psittaci Fachhochschule Jena Dix, Andreas MicroRNA-Seq data analysis for age-related comparison of mouse and short-lived fish Nothobranchius furzeri Friedrich-Schiller-Universität Jena Fischer, Juliane Impact of selected histone H3 amino acid residues on Aspergillus nidulans secondary metabolism and TAP-tagging of SAGA/ADA complex Hochschule Lausitz (FH) Senftenberg Freitag, Janine Vorhersage von MicroRNAs in Candida albicans Friedrich-Schiller-Universität Jena Fricke, Markus UV-Laser ChIP-Seq in vivo: Bioinformatische Analyse von genomweiten Protein-DNA-Interaktionen Friedrich-Schiller-Universität Jena Hoffmann, Bianca Export dynamischer Modelle aus NetGenerator und TILAR in SBML Friedrich-Schiller-Universität Jena scriptomics of secondary metabolite gene clusters in filamentous fungi Friedrich-Schiller-Universität Jena Jacksch, Susanne Complement evasion of Candida albicans - Characterization of Pra1 Friedrich-Schiller-Universität Jena Kämpfer, Philipp Phylogeny of MADS-box transcription factors in fungi. Friedrich-Schiller-Universität Jena Kupis, Sabine Das embryonierte Hühnerei als Modellsystem für die molekulare Bildgebung bei Infektion mit Chlamydia psittaci Fachhochschule Jena Lauterbach, Tom Genom-basierte Suche nach Naturstoffen aus Rhizobium radiobacter Friedrich-Schiller-Universität Jena Manig, Steffi Molekularbiologische Untersuchungen zur Funktion von Cys3 und Cys4 in Candida albicans Fachhochschule Jena Meyer, Florian Synthese von Hydrazidomycinen und Strukturanaloga Friedrich-Schiller-Universität Jena Molina Rodríguez, Juan Camilo Investigation of iron acquisition genes predicted by a Candida albicans regulatory network model Friedrich-Schiller-Universität Jena Pflieger, Astrid Untersuchung und Optimierung der Netropsinbildung durch Streptomyces netropsis Fachhochschule Jena Raguse, Marina Identification of complement regulator binding sites and functional domains of Lipoamide dehydrogenase (Lpd) of Pseudomonas aeruginosa Friedrich-Schiller-Universität Jena Schaarschmidt, Barbara Charakterisierung monoklonaler Mausantikörper hinsichtlich ihrer Spezifität gegenüber verschiedener humanpathogener Pilze Friedrich-Schiller-Universität Jena Sehnert, Philipp Funktionelle Analyse der Genexpression in humanen B-Lymphozyten Friedrich-Schiller-Universität Jena Hornung, Bastian Comparative genomics and tran- Appendix HKI Research Report 2012 / 2013 225 Shabuer, Gülmire Strategie zur Aktivierung stiller Gencluster in Clostridium acetobutylicum und Clostridium cellulolyticum Friedrich-Schiller-Universität Jena Sheikh MD, Rajuiddin The role of methylthioadenosine phosphorylase in virulence and immune evasion of Candida albicans Friedrich-Schiller-Universität Jena Tennstedt, Sebastian Untersuchungen zur Einstellung von Temperaturregelkreisen an Bioreaktoren im pilottechnischen Maßstab Fachhochschule Jena Tovar Ballen, Miguel Angel Establishment of appropriate conditions for culturing Actinobacteria in picolitre droplets: a microfluidic approach for novel antibiotic screening Technische Universität Dresden Ulbricht, Andrea Biosynthese von Indol-3-Essigsäure in Basidiomyceten Friedrich-Schiller-Universität Jena Üzüm, Zerrim The role of chitinolysis and exopolysaccharide in Rhizopus microsporus and Burkholderia rhizoxinica endosymbiosis Friedrich-Schiller-Universität Jena Wagner, Lysett Light microscopical and molecular identification of terrestrial fungi from taxonomic groups in Mortierella (Zygomycetes) Friedrich-Schiller-Universität Jena Walther, Elisabeth Pathobiology of chlamydial epitheliocystis agents Fachhochschule Jena Wang, Ding Analyse und Visualisierung genomweiter DNA-Polymorphismusdaten in R Friedrich-Schiller-Universität Jena Weist, Corinna Immunevasion von Tumorzellen durch lösliche und membrangebundene Komplementregulatoren Friedrich-Schiller-Universität Jena Wolf, Thomas Analyse der transkriptionellen Regulation von SekundärmetabolitGen-Clustern Friedrich-Schiller-Universität Jena Ziereisen, Jana Charakterisierung der Komplementaktivierung und der Autoantikör- 226 per in den Seren von Patienten mit den Nierenerkrankungen EHECHUS und MPGN Friedrich-Schiller-Universität Jena Bachelor / Master / Diploma Theses 2013 Bachelor-/ Master-/ Diplomarbeiten 2013 Albán Proaño, María Cristina Role of Candida albicans Eed1 protein in morphogenesis and pathogenicity Friedrich-Schiller-Universität Jena Allert, Stefanie Der Einfluss spezifischer Gene der humanpathpogenen Hefe Candida glabrata auf das Überleben in Makrophagen Friedrich-Schiller-Universität Jena Baumeister, Tim Charakterisierung Anthranilatbasierter Naturstoffe aus Amycolatopsis nigrescens Friedrich-Schiller-Universität Jena Bick, Jochen Mapping non-coding RNAs in the transcriptome of a bovine Chlamydia psittaci strain DC15 Friedrich-Schiller-Universität Jena Böttger, Marco Biochemische Untersuchung der 6-Thioguanin-Biosynthese in Erwinia amylovora Fachhochschule Jena Bredy, Florian Funktionelle Charakterisierung von Complement Factor H related Protein 4 (CFHR4) A, B und C im alternativen Weg der Komplementaktivierung Friedrich-Schiller-Universität Jena Dunker, Christine Role of glutathione peroxidases in the oxidative stress resistance of the fungal pathogen Candida albicans Friedrich-Schiller-Universität Jena Fichtner, Maximilian Ermittlung spezies-spezifischer Virulenzfaktoren in pathogenen Candida-Spezies Friedrich-Schiller-Universität Jena Fischer, Daniel Mikroevolution von Candida glabrata: Untersuchungen zu genetischen Veränderungen eines hypervirulenten Stamms Friedrich-Schiller-Universität Jena Gröning, Stephanie RhiG, an unusual acyltransferase involved in rhizoxin biosynthesis Friedrich-Schiller-Universität Jena Hartmann, Anja Potential kryptischer BiosyntheseGencluster von Clostridium cellulolyticum Friedrich-Schiller-Universität Jena Helfrich, Eric MALDI imaging and genome mining for natural product discovery in Clostridia and Cyanobacteria Friedrich-Schiller-Universität Jena Hess, Carolin Charakterisierung der immunmodulatorischen Proteine Pra1 und Gpd2 von Candida albicans Friedrich-Schiller-Universität Jena Hofmann , Susann Herstellung und phänotypische Charakterisierung von Mutanten im Pentose-Phosphat-Weg des pathogenen Schimmelpilzes Aspergillus fumgiatus Fachhochschule Jena Jena Kaulfuß, Toni Untersuchungen zur Interaktion von humanen natürlichen Killerzellen mit dem pathologischen Hefepilz Candida albicans Fachhochschule Jena Koske, Iris Dihydrolipoamide dehydrogenase (Lpd) of Pseudomonas aeruginosa binds to extracellular matrix (ECM) proteins and mediates adherence to human cells Friedrich-Schiller-Universität Jena Kretzschmar, Tom Einfluss von löslichem Komplementrezeptor 2 auf die Aktivierung von Immunzellen Friedrich-Schiller-Universität Jena Manzke, Melanie Deregulation der Komplement C3 Konvertase durch ein CFHRHybridprotein Friedrich-Schiller-Universität Jena Meinel, Christian Characterization of the immune evasion proteins PspC and Tuf of two clinical Streptococcus pneumoniae isolates Friedrich-Schiller-Universität Jena Mohamed, Nizar Mustafa Yousif Immune signals activated in mammalian host cells during interaction with Chlamydiales Universitätsklinikum Freiburg und Friedrich-Schiller-Universität Jena Neuwirth, Toni Totalsynthese von Heterocyclen aus Aspergillus spp. Friedrich-Schiller-Universität Jena HKI Research Report 2012 / 2013 Appendix Opel, Viktoria Komplexe Polyketide aus Burkholderia gladioli Friedrich-Schiller-Universität Jena Pann, Patrick Interaction of the human-pathogenic fungus Aspergillus fumigatus with neutrophil granulocytes – the role of DNases Universität Rostock Piegsa, Judith Role of NF-kB in epithelial barrier maintenance during the interaction with C. albicans Friedrich-Schiller-Universität Jena Säuberlich, Marco Konstruktion und Charakterisierung von rekombinanten VHHAntikörpern auf der Basis des kameliden Antikörpers B10 zur Produktion in E. coli mittels Hochzelldichtefermentation Hochschule Zittau / Görlitz Schmidt, Ann-Katrin Participation of different innate immune reseptors in recognition of C. albicans in a whole blood model of infection Friedrich-Schiller-Universität Jena Schmidt, Franziska Untersuchung der intrazellulären Prozessierung von Aspergillus fumigatus Konidien durch Makrophagen Friedrich-Schiller-Universität Jena Schnell, Bastien Genome mining for new natural products in the bacterium Cupriavidus basilensis Université Paris-Sud XI Schreiner, Maria Genetische Ursachen für die Wiedererlangung der Filamentbildung einer evolvierten nicht filamentösen Candida albicans-Mutante Friedrich-Schiller-Universität Jena Schult, Kristin Expression und Lokalisation des altersabhängigen Makulardegeneration assoziierten Proteins ARMS2 in humanen Zellen Friedrich-Schiller-Universität Jena Schulze, Sylvie Prediction of a gene regulatory network of MAPK- and NFƙBsignaling pathway components under Candida albicans infection Friedrich-Schiller-Universität Jena Seibel, Elena Analyse und Manipulation der Indolosesquiterpen-Biosynthese aus einem Mangroven-Endophyten Friedrich-Schiller-Universität Jena Shopova, Iordana The effects of extracellular DNA on Aspergillus fumigatus biofilm formation Friedrich-Schiller-Universität Jena Thamm, Martin Versuche zur Messung des intrazellulären pH-Wertes in einzelnen Mono Mac 6-Zellen während der Infektion mit Aspergillus-Sporen Friedrich-Schiller-Universität Jena Timme, Sandra Automated estimation of motility parameters from agent-based simulations of host-pathogen interactions Friedrich-Schiller-Universität Jena Wallstabe, Lars Untersuchungen zur Regulation des Gliotoxin-Genclusters aus Aspergillus fumigatus Fachhochschule Jena Weiß, Elisabeth The effects of metal depletion on Candida albicans physiology: a fatty solution to high salt Friedrich-Schiller-Universität Jena Wichmann, Christina Bacterial Diversity in Indonesian Epinephelus fuscoguttatus faeces by 16S analysis Friedrich-Schiller-Universität Jena Zhang, Qi Respiration vs. Fermentation in dimorphen Pilzen Friedrich-Schiller-Universität Jena Zitzmann, Carolin Mathematical Modelling of Infections in XLA Patients under IgG Substitution Therapy Friedrich-Schiller-Universität Jena Appendix HKI Research Report 2012 / 2013 227 Inventions and Patents 2012/2013 Erfindungen und Schutzrechte 2012/2013 In addition to publications in peer reviewed journals, intellectual property rights are the main performance parameters reflecting the quality of research at the HKI. In 2012/2013 a remarkable number of inventions were filed both by departments focusing on natural product research and by technology-oriented groups, thus broadening the patent-portfolio of the institute. HKI patents resulted in a number of fruitful co-operations with industry and affected the institute’s budget advantageously. Schutzrechte sind neben Originalpublikationen in referierten Fachjournalen ein wesentlicher Leistungsparameter für die Forschungsarbeit am HKI. Sowohl die naturstoffchemisch arbeitenden Abteilungen als auch Technologie-orientierte Gruppen trugen im Zeitraum 2012/2013 mit einer Reihe von Erfindungen zum Schutzrechts-Portfolio des Instituts bei. Vom HKI angemeldete Patente führten zu einer Reihe fruchtbarer Industriekooperationen und wirkten sich positiv auf das Budget des Instituts aus. Headed by Prof. Christian Hertweck, a group of scientists at the HKI has discovered a new antifungal substance. It is formed by a bacterium that triggers decay in cultivated mushrooms. The challenge was to search for an unknown substance that bacteria do not produce on standard cultivation conditions but only when they affect a mushroom. Hertweck and his colleagues sequenced the genome of the bacteria, scanned it for relevant biosynthesis genes and predicted anticipatory metabolite structures using bioinformatic methods. By means of this technique called genome mining, the researchers found a gene cluster called jag which could code for the biosynthesis of a new compound. In order to trigger its production, they injected slides of mushrooms with the bacterium and examined the emerging rotted spots using high-resolution mass spectrometry as an imaging technique. The researchers identified a mass peak that only appears at the affected spots. By adding pieces of mushroom and a special culture medium, they were able to extract and isolate the corresponding compound even in liquid culture in a larger quantity. Jagaricin – as the compound was called – is a novel lipopeptide consisting of a peculiar structure. Pure jagaricin induced symptoms of soft rot in mushrooms while bacteria with disabled jag genes are unable to induce soft rot. Eine Gruppe von Wissenschaftlern aus dem HKI entdeckte unter Leitung von Prof. Christian Hertweck einen neuen antifungalen Wirkstoff. Er wird von einem Bakterium gebildet, das beim Kulturchampignon Fäulnis auslöst. Die Herausforderung bestand darin, nach einer unbekannten Substanz zu suchen, die die Bakterien nicht unter Standard-Kulturbedingungen produzieren, sondern erst, wenn sie einen Pilz befallen. Hertweck und seine Kollegen sequenzierten das Genom der Bakterien, durchsuchten es nach einschlägigen Biosynthesegenen und machten mithilfe bioinformatischer Methoden Voraussagen über die zu erwartenden Metabolitstrukturen. Die Forscher fanden durch ein solches genome mining einen als jag bezeichneten Genabschnitt, der für den Biosyntheseapparat des gesuchten Stoffes codieren könnte. Um dessen Bildung anzuregen, impften sie Scheiben von Champignons mit dem Bakterium und untersuchten die entstehenden fauligen Stellen mithilfe bildgebender Massenspektrometrie. Die Forscher identifizierten einen Massenpeak, der nur auf befallenen Stellen auftritt. Durch Zugabe von Pilzstückchen und ein spezielles Nährmedium gelang es dann, die zugehörige Verbindung auch in flüssiger Kultur in größerer Menge zu gewinnen und zu isolieren. Bei Jagaricin – so wurde die Verbindung getauft – handelt es sich um ein neuartiges Lipopeptid mit ungewöhnlicher Struktur. Reines Jagaricin rief bei Pilzen die Symptome der Nassfäule hervor während Bakterien mit nicht funktionstüchtigen jagGenen keine Nassfäule auslösen können. Jagaricin is effective against Candida albicans, Aspergillus fumigatus and Aspergillus terreus and might be a starting point in developing a novel antifungal agent. The application for new patents is stringently evaluated within the HKI and focuses on novel biologically active natural products and their (bio-)synthetic derivatives. Since 2006, the HKI co-operates with Ascenion GmbH, Munich, to further optimize technology transfer and commercialization activities. This project is funded by the Federal Ministry of Education and Research. Jagaricin ist gegen Candida albicans, Aspergillus fumigatus und Aspergillus terreus wirksam und könnte ein Ansatzpunkt für ein neues Antimykotikum sein. Die Anmeldung neuer Schutzrechte unterliegt einer strengen hausinternen Evaluation und konzentriert sich auf neue, biologisch aktive Naturstoffe und deren (bio-)synthetische Derivate. Das HKI arbeitet zur effektiven Verwertung der Schutzrechte seit 2006 erfolgreich mit der Ascenion GmbH, München zusammen. Bretschneider T, Graupner K, Hertweck C, Lackner G, Scherlach K (2012) Jagaricin, derivatives and uses thereof EP 12 006 113.0 Dahse HM, Hertweck C, Kirchner H, Kusebauch B, Scherlach K, Fiebig HH, Kelter G, Maier A (2012) Neue Rhizoxinderivate DE 10 2012 000 956.9 Ding L, Hertweck C (2012) Bixiamycins US 61/684,941 Hertweck C, Ueberschaar N, Scherlach K, Dahse HM, Bretschneider T, Metsä-Ketelä M (2012) Novel chartreusin analogues EP 12 005 528.0 Höfs S, Hube B, Wilson D, Naglik J, Moyes D, Richardson J, Tang S (2013) Peptides and binding partners therefor GB 1306588.3 228 HKI Research Report 2012 / 2013 Appendix The antifungal lipopeptide jagaricin is formed by Janthinobacterium agaricidamnosum, the pathogen of soft rot in cultivated mushrooms. External Funding 2012/2013 Drittmittel 2012/2013 2012 T€ 1,400 Total receipts: 4,849 T € 1,200 1,000 800 600 400 200 0 DFG BMBF/BMWi EU Industry Leibniz Competition ZIK Septomics Free State of Thuringia Others 2013 T€ 1,400 Total receipts: 4,426 T € 1,200 1,000 800 600 400 200 0 DFG BMBF/BMWi EU Industry Leibniz Competition ZIK Septomics Free State of Thuringia Third-party funds during the period reported: composition of the sources of income Drittmittelausgaben im Berichtszeitraum: Verteilung über die Drittmittelgeber Appendix HKI Research Report 2012 / 2013 229 Others Maps | Lagepläne 230 HKI Research Report 2012 / 2013 Maps 1 HKI Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie – Hans-Knöll-Institut 2 Leibniz-Institut für Altersforschung – Fritz-Lipmann-Institut 3 Abbe-Zentrum Beutenberg 4 Fraunhofer-Institut für Angewandte Optik und Feinmechanik 5 Leibniz-Institut für Photonische Technologien 6 Institut für Angewandte Physik (Friedrich-Schiller-Universität Jena) 7 Max-Planck-Institut für Biogeochemie 8 Max-Planck-Institut für chemische Ökologie 9 Technologie- und Innovationspark 10 BioInstrumentezentrum 11 Zentrum für Molekulare Biomedizin (Friedrich-Schiller-Universität Jena) Maps HKI Research Report 2012 / 2013 231 232 Imprint | Impressum HKI Leibniz Institute for Natural Product Research and Infection Biology e. V. – Hans Knöll Institute – Beutenbergstraße 11a 07745 Jena, Germany fon +49 (0)36 41-532 1011 fax +49 (0)36 41-532 0801 [email protected] www.hki-jena.de Editorial Board Prof. Dr. Axel A. Brakhage Dr. Michael Ramm Dr. Christine Vogler Design and Layout Bernd Adam Copyright © 2014 HKI Jena The copyright for any material created by the HKI is reserved. Any duplication or use of objects such as texts, diagrams, photos or other artwork in electronic or printed publications is not permitted without the HKI’s prior written agreement. 233 HKI Leibniz Institute for Natural Product Research and Infection Biology e. V. – Hans Knöll Institute – Beutenbergstraße 11 a | 07745 Jena, Germany | Fon +49 (0)36 41 532-1011 | Fax +49 (0)36 41 532-0801 [email protected] | www.hki-jena.de 234
