Download 14th Annual Great Plains Infectious Disease Meeting

14th Annual Great Plains
Infectious Disease
Meeting
A focus on: vaccines to prevent infectious
diseases, emerging infectious &
select agent threats, and the ugly
face of antibiotic resistance
November 6-7, 2015
University of Kansas
Lawrence, KS
The 14th Annual
Great Plains Infectious Disease Meeting
We are pleased to host the 14th Great Plains Infectious Disease (GPID) Meeting which is
returning to the University of Kansas after five strong years at the University of Missouri.
The GPID meeting was originally developed to promote collaborations in the Great Plains
region and to create a platform for networking among researchers. It continues to do so by
promoting student, postdoc and young faculty’s research by hosting a poster presentation,
and providing a platform for faculty, especially those new to the region, to give oral
presentations that provide an outline of their research programs and promote new
collaborative interactions. This year, we are proud to host more than 100 participants from
across the region with nearly 30 posters being presented. We also welcome participants
from the Kansas City Area Life Science Institute, the Animal Health Corridor and the USDA
who are embarking on new partnerships with our academic partners. This meeting has
always been successful due to the generosity of our academic sponsors and selected
vendors.
Thank you all for attending!
The GPID Programming Committee 2015
ACKNOWLEDGEMENTS
Generous academic and industrial sponsors have contributed greatly to the success of this
meetings. Many thanks to all of our 2015 GPID supporters!
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14th Annual Great Plains Infectious Disease Meeting Program Schedule
THE GOOD, THE BAD AND THE UGLY
November 6-7, 2015
University of Kansas-Lawrence
Friday, November 6, 2015 (Naismith Ballroom - Springhill Suites, 6th and New Hampshire, Downtown Lawrence, KS)
Welcome and Reception
Dinner
4:00pm 6:00pm
6:00pm 6:30pm
6:30pm 8:30pm
Plenary Session (Naismith Ballroom)
Wayne Carter (KCALSI) “Life Science Ecosystem in Kansas City Region”
Blake Hawley (IAH) “Leveraging the Animal Health Corridor and Human Research to Build a Company”
Dave Hustead (Boehringer Ingelheim) “It’s harder to decide which vaccines to give puppies and kittens
than which vaccine to give kids”
Saturday, November 7, 2015 (Atrium, School of Pharmacy, West Campus )
7:15am
8:00am
7:55am
8:02am
Registration and Breakfast
Opening Remarks
Session I - The Good: Immunology and Vaccine Development (2020 SOP)
8:02am 8:30am Jodi McGill (KSU) “The role of gamma delta T cells in immunity to respiratory infections in the
bovine”
8:30am 9:00am Brad Bearson (USDA) “DIVA Design: a cross-protective Salmonella vaccine”
9:00am 9:30am Tracy Nicholson (USDA) “Non-antibiotic Strategies to Prevent and Control Respiratory Bacterial”
9:30am 10:00am Mary Markiewicz (KUMC) “The influence of the innate immune receptor NKG2D on microbiota
composition and autoimmune diabetes"
10:00am 10:20am
Refreshments and Networking Break (Atrium SOP)
Session II - The Bad : Bad Bugs (2020 SOP)
10:30am 11:00am A. Paige Adams (KSU-Olathe) "Current Status of Vaccines for Venezuelan and Eastern Equine
Encephalitis."
11:00am 11:30am Jeff Adamovicz (MU, LIDR) “New RB51 Vaccine Schedule for Cattle, Impacts on Immunity and
Transmission Following B. abortus Infection.”
11:30am 12:00pm Eric Nicholson (USDA) “Thermodynamic insight into the basis of genetic prion disease in cattle”
12:00am 12:30pm Carl Gelhaus (MRIGlobal) “Animal Models for Tularemia Medical Countermeasures”
12:30
1:30pm
Lunch - Mortar and Pestle Cafe (First Floor Dining Area)
Session III - The Ugly: Antibiotic Resistance (2020 SOP)
1:30pm 2:00pm Josie Chandler (KU) “Quorum sensing-controlled antibiotics and antibiotic-resistance factors and their
role in interspecies competition”
2:00pm 2:30pm Brian Brunelle (USDA) “Antibiotic exposure can induce various bacterial virulence phenotypes in
multidrug-resistant Salmonella enterica serovar Typhimurium”
2:30pm 3:00pm Mario Rivera (KU) “Targeting iron homeostasis for developing novel antimicrobials”
3:00pm
3:45pm Jim Riviere (KSU) “Use of Mixed-Effect and Physiological Based Pharmacokinetic Models of
Antimicrobial Drugs as a Framework for Interspecies Extrapolations”
Session IV - Poster Presentations
3:45pm
4:00pm
5:30pm
6:00pm
Poster Session (Atrium SOP)
Light Dinner / Hors d'oeuvres
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14th Annual Meeting
Plenary Speaker Session
Abstracts
and
Bio-Summaries
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5
Wayne O. Carter, DVM, PhD, Dipl. ACVIM
President and Chief Executive Officer
Kansas City Area Life Sciences Institute
Abstract:
Life Science Ecosystem in Kansas City Region
The greater Kansas City region spanning from Kansas State University in Manhattan, KS
to the University of Missouri in Columbia, MO is home to a growing and vibrant life
science ecosystem comprising over 250 companies and employing over 24,000 people.
This region is also recognized as the Animal Health Corridor, home to the largest
concentration of animal health R&D assets in the world with companies representing
75% of the global sales and over $19 billion. The region contains over 90 contract
research organizations, is home of the $1.25 billion NBAF laboratory, and vibrant
entrepreneurial and collaboration networks. We are growing the life sciences looking for
nexus opportunities between human and animal diseases, health IT and convergent
technologies intersecting with the life sciences.
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Wayne O. Carter, DVM, PhD, Dipl. ACVIM
President and Chief Executive Officer
Kansas City Area Life Sciences Institute
Bio-Summary:
Dr. Wayne Carter is President and Chief Executive Officer of Kansas City Area Life
Sciences Institute that serves to advance human and animal health through translational
research, collaboration and commercialization.
Dr. Carter is an experienced life science executive with both human and veterinary
expertise spanning pharmaceutical and nutritional products. He is a technology
aficionado, passionate about applying technology to improve healthcare decisions. Dr.
Carter attended Purdue University, earning his BS degree in 1980, DVM in 1984 and
practiced veterinary medicine for 5 years. He completed advanced residency training in
internal medicine and board certification in 1992 and a PhD in cellular immunology at
Purdue University in 1994.
Dr. Carter's passion for technology applications developed from a group started at Pfizer
in 1997 focused on improving decisions in the drug development process. In that role, he
was responsible for translational research and led the company’s North American clinical
facilities in the development, validation and implementation of new clinical technologies
to accelerate development decisions for human pharmaceuticals. He drove decisions at
Pfizer by return on investment and grew the Clinical Technology group to span all phases
of drug development and all therapeutic areas. In 2007, Dr. Carter was hired as Vice
President of Research by Hill’s Pet Nutrition, a Colgate subsidiary, where he led
nutrigenomics research and applied gene expression profiling to drive nutrition decisions
for novel products in development.
Dr. Carter serves on several advisory boards. He serves on the board for the One Health
Commission. He is Chairman of BioKansas, a non-profit organization to promote
bioscience development in Kansas, a member of the Missouri Biotechnology Association
Board of Directors, a non-profit organization to promote bioscience development in
Missouri, a member of the KU Center for Research, Inc. Board of Trustees, a member of
the University of Missouri Research and Discovery Advisory Board, member of the
Kansas-State Olathe Advisory Board, a member of the MRIGlobal Board of Trustees,
and a member of the Biological Sciences Advisory Board for the University of Kansas.
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Blake Hawley, DVM, MBA
President and CEO
North and South America
Integrated Animal Health
Kansas City Animal Health Corridor
Abstract:
Integrated Animal Health (IAH) is commercialising disruptive, patented
technologies for the livestock sector and companion animal and horse space
History
Originally focused on a single technology platform, the IAH portfolio is expanding
rapidly. The current focus is on commercialisation of its natural feed inclusion
technologies which originated from licensing a human biotechnology firm’s asset.
Upon visiting the Animal Health Corridor Homecoming event in 2013, Founder Rob
Neely elected to set up an office in the US. Today, with headquarters in the BTBC
and stemming from a single technology, IAH is now approaching 30 technologies in
under two years, producing one of the most robust pipelines in privately held animal
health companies.
Products
IAH products range from natural feed inclusion technologies proven in feed trials
over 12,000 dairy cows, to equine nutrition products and supplements, calf scour
preventive and therapeutics, to poultry feed products, vaccines for livestock and pets,
fly and tick repellents, cancer therapies, peptide products for weight loss and joint
disease and much more. The vast majority originated from human health care and
research.
Overall Objectives
Leverage a lean, core team, their networks, and a strategic cohort of ‘early adopter’
commercial trial partners converted into active, paying customers. In parallel,
expand globally by leveraging a solid advisory board, university supported technical
white papers, evidence-based marketing materials, real-world testimonials and
established market channels and partners. Our biggest opportunities lie in the global
application and licensing of these technologies to enhance milk and meat production
worldwide, which will then fund development of our broader pipeline.
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Current Commercial Products: Problem Solved and Market Demand
Animal health management poses one of the greatest challenges to the financial viability of
producers and if not managed, is a threat to food safety, biosecurity and community health. By
the year 2050, the world will need to double food production to feed a global population
estimated to be 9.1 billion*. Meeting this need involves the cost-effective production of safe,
high-quality animal protein.
As livestock production continues to increase in response to rising demand and increased
standard of living, innovative animal health solutions are needed in greater volumes. There is
growing importance of livestock supplements, medicines and vaccines as a component of
global food supply. A key challenge for all players in the food ecosystem is how to meet the
so-called “productivity imperative” – how to produce more with less.
By providing ways for farmers and livestock producers to cost-effectively increase
productivity, Integrated Animal Health can help address this challenge. Vaccines and
medicines, feed additives, improved production practices and advanced techniques – all of
these assist producers with preventing diseases and optimizing the efficiency of the feed-tomeat conversion for protein and milk yield.
A major challenge for the industry, however, is the increasing scrutiny and regulation of
antibiotics given to animals. The use of antibiotics in the feed of dairy and their use as growth
promoters in livestock are a significant public health concern, with international pressure
building to abate or ban antibiotic use in animal production. The EU was the first region to
implement a ban in 2006. Developed countries such as the US, Canada and Australia are
under increasing pressure to follow suit and major corporations such as McDonald’s are
committing to antibiotic-free meats.
“…WHO has long recognized that antibiotic use in food animals contributes importantly to the
public health problem of antibiotic resistance”. – World Health Organization, March 12, 2012
Widespread scientific concern about the extensive use of antibiotics in the food chain is
creating opportunities for:
•
Non-antibiotic methods of enhancing animal productivity and profitability
• Increasing the innate immune function so disease doesn’t have so great an impact
• Helping prevent infections by eliminating exposure to insect-borne vectors such as flies
and mosquitoes
• Natural answers to improving food animal growth & feed conversion efficiency
IAH’s range of natural products solve multiple layers of these issues.
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Blake Hawley, DVM, MBA
President and CEO
North and South America
Integrated Animal Health
Kansas City Animal Health Corridor
Bio-summary:
Blake Hawley is the President and CEO for Integrated Animal Health, commercializing
disruptive, patented, natural technologies, biologics and biopharmaceuticals for the livestock
sector, companion animals and horses. Prior to joining IAH, he consulted for Jaguar Animal
Health, and served as Chief Commercial Officer for Kindred Biosciences, a pet
biopharmaceutical company he helped take public in 2014. Blake has also served as
Worldwide Director of Global Digital for Hill's Pet Nutrition, a subsidiary of ColgatePalmolive, working in various roles from 1998-2013 including Managing Director for Hill's
UK and Ireland business, Regional General Manager for Russia and Central-Eastern Europe
and General Manager-Australia and New Zealand. Before joining Hill's, he worked for Kaytee
Products Inc, servicing the pet bird, exotics and wild bird markets. He was Executive Director
of an environmental non-profit organization, and served on several boards. He was an adjunct
professor at the University of Wisconsin veterinary School. Today, he serves on the MBA
Advisory Board for KU, the Board of Directors for the Lawrence Humane Society and the
Board of Economic Development Corporation of Lawrence and Douglas County.
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David R. Hustead, DVM MPH DACVPM
Director
Veterinary Medical Affairs
Pet Division
Boehringer Ingelheim Vetmedica
Abstract:
It’s harder to decide which vaccines to give puppies and kittens than which vaccine
to give kids
Guidelines for the use of vaccines in pets direct veterinarians to give a small group of
vaccines to essentially every patient while at the same time directing them to give a much
larger set of vaccines only to those patients that have adequate risk of exposure. This is
in contrast to pediatricians who give most of the available vaccines to every child they
see while conducting risk assessment on only a very small number of vaccines. In the real
world of a busy veterinary practice, it is difficult to conduct a good risk assessment
interview during routine history taking. To assist veterinarians in this task, we provide a
web based interview tool which asks pet owners simple yes or no questions about pet
behaviors considered important to confer risk of exposure. The tool has a simple
decision system that can be configured by the veterinarian which informs the pet owner if
there is adequate risk of exposure to justify implementing a preventive program including
vaccination. The tool also provides the pet owner with disease information and advice
actions they can take to prevent these diseases in their pets and in the case of zoonotic
diseases to prevent these diseases in the human family, too.
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David R. Hustead, DVM MPH DACVPM
Director
Veterinary Medical Affairs
Pet Division
Boehringer Ingelheim Vetmedica.
Bio-summary:
Dr. Hustead is a graduate of Kansas State University. Following several years in
companion animal and equine practice, he joined Fort Dodge Laboratories as their first
technical service’s veterinarian specializing in companion animal and equine health
issues. During his tenure at Fort Dodge, he was responsible for both in-house and field
professional services, planned and supervised R&D product approvals, supervised
regulatory affairs activities in the US and internationally. He has served on several
committees including the American Association of Feline Practitioners Vaccination
Guidelines, American Veterinary Medical Association’s Council on Biologic and
Therapeutic Agents and represented US animal health manufacturers on the VICH Expert
Working Group on Pharmacovigilance. He is currently Director, Veterinary Medical
Affairs, Pet Division, at Boehringer Ingelheim Vetmedica. His team of veterinarians is
responsible for technical consultations with customers, technical training of sales
personnel, ensuring technical appropriateness of sales activities, post-approval clinical
studies, interactions with organized veterinary medicine and liaison with colleges of
veterinary medicine. Dr. Hustead recently obtained a Masters of Public Health from
University of Iowa/Iowa State University and is a diplomat in American College of
Veterinary Preventive Medicine.
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14th Annual Meeting
Oral Presentation
Abstracts
Session I
THE GOOD:
Immunology and Vaccine
Development
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Jodi L. McGill, Ph.D.
Assistant Professor
Immunology
Department of Diagnostic Medicine and Pathobiology
Kansas State University
Abstract:
The role of gamma delta T cells in immunity to respiratory infections in the bovine
γδ T cells are non-conventional T lymphocytes that form an important bridge between the
innate and adaptive immune systems. They are conserved in all vertebrate species
examined so far, but are particularly abundant in the immune system of the ruminant.
Thus, cattle are an ideal model for studying the role of nonconventional T cells in
immunity to infections and disease. We study the response of γδ T cells to infection with
Mycobacterium bovis, the causative agent of bovine and zoonotic tuberculosis; and to
bovine respiratory syncytial virus infection, a common respiratory viral infection of
young calves and human infants. We have shown that γδ T cells secrete a number of
inflammatory chemokines and cytokines in response to bacterial and viral infections both
in vitro and in vivo, and play an important role in immune cell recruitment and
inflammatory responses at the site of infection in the lungs.
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Jodi L. McGill, Ph.D.
Assistant Professor
Immunology
Department of Diagnostic Medicine and Pathobiology
Kansas State University
Bio-summary:
Jodi L. McGill received her B.S. in Microbiology from Iowa State University. She
received her M.S. in Pathology and Ph.D. in Immunology from the University of Iowa
studying dendritic cell biology and CD8 T cell immunity during influenza virus infection
in a murine model. She did her post-doctoral fellowship at the National Animal Disease
Center, USDA under the guidance of Dr. Randy E. Sacco studying the immune response
to bovine respiratory syncytial virus and bovine tuberculosis. Dr. McGill started in her
position as an Assistant Professor of Immunology in September 2014 in the Department
of Diagnostic Medicine and Pathobiology at Kansas State University. The focus of her
research is to elucidate the role of nonconventional T cells in shaping the innate and
adaptive immune response to respiratory infections in humans and animals. Dr. McGill
currently studies the response to several respiratory pathogens including bovine
respiratory syncytial virus, Mannheimia haemolytica and Mycobacterium bovis, the
causative agent of bovine and zoonotic tuberculosis.
17
Brad Bearson, Ph.D.
USDA, ARS Research Microbiologist
National Laboratory for Agriculture and the Environment (NLAE)
Ames, IA
Abstract:
DIVA Design: a cross-protective Salmonella vaccine
Salmonella is a leading cause of bacterial foodborne disease and food-related death in the
U.S. Greater than 50% of U.S. swine farms test positive for Salmonella, resulting in
issues for both animal production and food safety. Vaccination of swine against
Salmonella is a potential mitigation strategy to reduce or prevent pathogen colonization
of livestock. However, there are over 2,400 Salmonella serovars and current vaccines
may not provide adequate cross-protection against heterologous serovars. Furthermore,
vaccination against Salmonella may interfere with surveillance programs that monitor the
presence of Salmonella in swine herds. To overcome current vaccine limitations, we
rationally designed, constructed, and evaluated a Salmonella vaccine to provide broad
protection against a variety of Salmonella serovars while allowing the differentiation of
infected from vaccinated animals (DIVA). The attenuated Salmonella DIVA vaccine is
anticipated to protect animal health while enhancing food safety.
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Brad Bearson, Ph.D.
USDA, ARS Research Microbiologist
National Laboratory for Agriculture and the Environment (NLAE)
Ames, IA
Bio-summary:
Brad Bearson is a Research Microbiologist at the USDA, ARS, National Laboratory for
Agriculture and the Environment (NLAE) in Ames, IA. He received a B.S. in
Biomedical Sciences from the University of South Alabama (USA) in Mobile, AL and
earned his Ph.D. in Basic Medical Sciences with an emphasis in Microbiology from the
USA College of Medicine. He performed an American Society for Microbiology,
Clinical Microbiology Post-doctoral Fellowship at the University of California at Los
Angeles (UCLA). Brad was a Visiting Research Scientist at the Poultry Research Unit at
Mississippi State, MS prior to his move to Ames, IA where his initial position was as a
Research Molecular Biologist at the National Animal Disease Center. In 2004, Brad
assumed his current position at NLAE and investigates Salmonella colonization and
pathogenesis of the swine and turkey gastrointestinal tracts for the development of
interventions strategies to protect animal health, enhance food safety, reduce economic
losses for producers, and limit environmental risk. In conjunction with his wife, Shawn
Bearson, he has rationally designed, constructed and evaluated an attenuated Salmonella
enterica serovar Typhimurium vaccine that allows the differentiation of infected from
vaccinated animals (DIVA) while providing broad protection against multiple Salmonella
serovars. To date the attenuated S. Typhimurium vaccine has been evaluated in both
swine and turkeys for protection against S. Typhimurium and S. Choleraesuis or
multidrug-resistant S. Heidelberg, respectively. A PCT International Application has
been filed for the S. Typhimurium DIVA vaccine with a publication number of WO
2015/103104 A1.
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Tracy Nicholson, Ph.D.
Research Microbiologist
National Animal Disease Center
U.S. Department of Agriculture
Ames, IA
Abstract:
Non-antibiotic Strategies to Prevent and Control Respiratory Bacterial Infections in
Swine
The U.S. swine industry is the second largest producer of pork in the world, and
respiratory disease in pigs is the most important health concern for swine producers
today, making the development of efficacious vaccines and therapeutic interventions that
can protect against respiratory infections a top research priority. The primary bacterial
agents responsible for respiratory disease in swine include Mycoplasma hyopneumoniae,
Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, and Pasteurella multocida.
In addition to causing pneumonia, bacteria such as Haemophilus parasuis and
Streptococcus suis and can cause systemic diseases such as meningitis, polyserositis,
arthritis and septicemia. Antibiotics can be costly, of limited value against mixed
infections, and may eventually be limited in their use in domestic livestock. In addition,
several bacteria, such as Methicillin Resistant Staphylococcus aureus (MRSA), while
causing minimal disease in swine, are zoonotic agents with potential economic
consequences to the swine industry. The overall goals of my research are to: (1) identify
the transmission, genetic, and pathogenic mechanisms used by these bacterial pathogens
(2) develop and evaluate candidates for improved diagnostic tests, vaccines,
biotherapuetic, and novel non-antibiotic strategies to prevent, reduce, or eliminate
colonization and diseases caused by these pathogens.
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Tracy Nicholson, Ph.D.
Research Microbiologist
National Animal Disease Center
U.S. Department of Agriculture
Ames, IA
Bio-summary:
Tracy Nicholson is from San Antonio, Texas. Dr. Nicholson received a B.S. degree in
Biomedical Science from Texas A&M University in 1995. She stayed at Texas A&M for
graduate school where she worked in the laboratory of Dr. Andreas Bäumler studying
Salmonella Typhimurium pathogenesis and earned her Ph.D. in Medical Microbiology in
2000. After graduation, she spent 4 years working as a NIH Postdoctoral Research
Fellow with Dr. Richard Stephens at University of California, Berkeley, investigating
global gene expression in the obligate intracellular bacterium Chlamydia trachomatis
during active and persistent infections. In 2004, she began working for the USDA in
Ames, Iowa, as a Principal investigator and team member for the Strategies to Control
and Prevent Bacterial Infections in Swine research project. Her research encompasses
identifying and assessing virulence mechanisms of bacterial pathogens associated with
swine (Bordetella bronchiseptica, Haemophilus parasuis, Stretococcus suis, and
methicillin-resistant Staphylococcus aureus), countermeasures to prevent infection, and
host immune response to infection. She has authored/co-authored 34 peer-reviewed
publications, member of the Editorial Board for the journals Pathogens and Infection and
Immunity, and is currently serving as chair for Division Z Animal Health for the
American Society for Microbiology.
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Mary Markiewicz, PhD
Assistant Professor
Department of Microbiology, Molecular Genetics & Immunology
University of Kansas Medical Center
Abstract:
The influence of the Innate Immune Receptor NKG2D on Microbiota Composition
and Autoimmune Diabetes
Mary Markiewicz
Department of Microbiology, Molecular Genetics & Immunology,
University of Kansas Medical Center, Kansas City, KS USA
Type 1 diabetes is an autoimmune disease caused by T cell-mediated destruction of the
insulin-producing β cells in pancreatic islets. The current standard of care for type 1
diabetes patients is insulin replacement. While effective, this treatment is cumbersome
and the life expectancy of type 1 diabetics is still reduced compared with non-diabetics.
Therefore, novel prevention strategies and therapies are urgently needed. To reach this
goal, a better understanding of the molecular mechanisms of β cell destruction is needed.
Both human and animal studies implicate signaling through the NK group 2 member D
(NKG2D) immune receptor in autoimmune diabetes pathogenesis. However, the
mechanism by which NKG2D influences diabetes development is unclear. New data
from our lab utilizing the non-obese diabetic (NOD) mouse model suggest that NKG2D
interaction with its ligands affects β-islet cell destruction and diabetes via multiple and
opposing mechanisms. First, NKG2D-ligand interactions promote a diabetes-protective
intestinal microbiota composition. Second, NKG2D-ligand interaction via homotypic T
cell contact during CD8+ T cell differentiation into cytotoxic T lymphocytes (CTLs)
decreases diabetes-enhancing cytokine production. Third, NKG2D-ligand interaction via
homotypic T cell contact within pancreatic islets enhances lytic granule release and β islet cell killing. We are continuing studies with the NOD mouse model as well as
human pancreatic samples to clearly define these roles of NKG2D receptor-ligand
interaction in autoimmune diabetes pathogenesis.
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Mary Markiewicz, PhD
Assistant Professor
Department of Microbiology, Molecular Genetics & Immunology
University of Kansas Medical Center
Bio-summary:
Mary Markiewicz received her Ph.D. in Immunology from the University of Chicago
from her studies investigating the requirements for an effective tumor-specific CD8+ T
cell response. She continued her tumor immunology studies during a one-year
postdoctoral fellowship at the Cardinal Bernardin Cancer Center at Loyola University in
Chicago in Dr. Martin Kast’s laboratory. She next moved to St. Louis for a postdoctoral
fellowship in Dr. Andrey Shaw’s laboratory in the Department of Pathology and
Immunology at Washington University School of Medicine where she began her studies
concerning the role of the natural killer (NK) cell activating receptor NKG2D and its
ligands in various types of immunity. She then stayed on at Washington University as an
Instructor and Research Assistant Professor and focused her attention on determining the
role of NKG2D and its ligands in autoimmune diabetes and tumor immunity. In 2014 she
joined the faculty of the Department of Microbiology, Molecular Genetics &
Immunology at the University of Kansas Medical Center, continuing her studies to
understand the role of NKG2D signaling in autoimmunity and tumor immunity. She has
received funding for her work from the American Cancer Society, the American Diabetes
Association and the NIH.
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14th Annual Meeting
Oral Presentation
Abstracts
Session II
THE BAD:
Bad Bugs
24
25
A. Paige Adams, DVM, PhD
Research Assistant Professor
Kansas State University Olathe
Olathe, KS
Abstract:
Current Status of Vaccines for Venezuelan and Eastern Equine Encephalitis
A. Paige Adams, DVM, PhD
Kansas State University, Olathe, KS 66061
Venezuelan (VEEV) and eastern (EEEV) equine encephalitis viruses are closely related
single-stranded, positive-sense RNA viruses and are members of the Alphavirus genus of
the family Togaviridae. Both viruses are capable of causing encephalitis in animals and
humans when transmitted by mosquito vector or potentially via infectious aerosol.
Periodic re-emergence of Venezuelan equine encephalitis (VEE) in Central and South
America and the yearly threat of eastern equine encephalitis (EEE) in North America
emphasize the importance of these pathogens to public health and veterinary medicine.
Despite their importance and over 60 years of research on VEE and EEE, there are no
licensed human vaccines or effective antiviral treatments for human or equine disease,
and the concern that VEEV and EEEV may be used for biological terrorism has also
underscored the need for the development of improved, licensed vaccines and effective
antiviral treatments. Recent advances in genetic engineering and vaccine design have
been exploited to produce new vaccine candidates for VEE and EEE. This presentation
will review: 1) past and present approaches for the development of these vaccines, 2)
efficacy of vaccine candidates in various animal models, 3) limitations and advantages of
each approach, and 4) the potential use of these vaccine candidates in the future.
26
A. Paige Adams, DVM, PhD
Research Assistant Professor
Kansas State University Olathe
Olathe, KS
Bio-summary:
Dr. Adams currently serves as Research Assistant Professor at K-State Olathe and is an
ancillary faculty member of the Department of Diagnostic Medicine and Pathobiology at
Kansas State University, College of Veterinary Medicine. Dr. Adams, a native Texan,
received her Bachelor of Science and Doctor of Veterinary Medicine degrees from Texas
A&M University. After graduating from veterinary school, she received large animal
internship training at the Atlantic Veterinary College, University of Prince Edward
Island, Canada, and large animal surgery residency training at the College of Veterinary
Medicine, University of Minnesota. Dr. Adams received a Ph.D. in immunology from
Cornell University with NIH support from institutional and individual National Research
Service Awards (NRSA). In 2004, she worked as a postdoctoral fellow in the laboratory
of Dr. Scott Weaver at the University of Texas Medical Branch, where she studied
pathogenesis of and vaccine development for Venezuelan (VEEV) and eastern (EEEV)
equine encephalitis viruses. In 2009, she joined the faculty at UTMB, where she
continued her work with VEEV and EEEV, including some work with arthralgic
alphavirus, chikungunya virus (CHIKV). In 2010, Dr. Adams received NIH K08 funding
to study the genetic factors that contribute to differences in neurovirulence of subtype IE
VEEVs. This award also concentrated on studies related to equine amplification of
VEEV as a major determinant of epidemic potential. Additionally, Dr. Adams served as
a co-investigator on a Western Regional Center of Excellence (WRCE) NIH grant to
study quantitative mapping of CD8+ T cell responses of nonhuman primates (NHP) to
vaccine strains of CHIKV. In 2013, Dr. Adams joined the faculty at K-State Olathe,
where she has been involved in teaching and supporting the Veterinary Biomedical
Science (VBS) Master of Science program. Dr. Adams is an active member of several
professional societies, including American Veterinary Medical Association (AVMA),
American Association of Veterinary Immunologists (AAVI), American Society for
Virology (ASV), and American Society of Tropical Medicine and Hygiene (ASTMH),
and she serves as a reviewer for several scientific journals.
27
Jeffery Adamovicz, Ph.D.
Associate Professor
Director, Laboratory of Infectious Disease Research
College of Veterinary Medicine Laboratory
University of Missouri
Abstract:
New RB51 Vaccine Schedule for Cattle, Impacts on Immunity and Transmission Following
B. abortus Infection
Jeff Adamovicz, Ph.D.
Department of Veterinary Pathobiology, Columbia, Missouri, United States
The current recommended vaccination for brucellosis in at-risk cattle is a single calf hood dose of
the live attenuated vaccine RB51. While this vaccine is successful in reducing infections in cattle
it is not fully infective in preventing vertical transmission, abortions or the development of
chronic infection. The hypothesis of this study is that RB51 booster vaccinations will provide
either increased cell mediated or humoral immunity as compared to a single calf-hood vaccination
alone. This research includes four specific aims; 1) that multiple doses of RB51 vaccine will
induce a measurable increase in cell-mediated and humoral immunity and that this will correlate
with a decreased incidence of infection and abortion at challenge; 2) that multiple doses of RB51
will not induce abortion when an adult booster vaccination is given during pregnancy; 3) that we
will be able to correlate cell-mediated immunity data with a predicted outcome at challenge to
establish an immune correlate for vaccination; 4) that RB51 titers to multiple doses of vaccine
will correlate with previous studies in terms of safety of the vaccine. For this study Black Angus
cattle were divided into four treatment groups; three treatment groups (n=7 each) and a control
group (n=4). Each treatment group was assigned to be vaccinated with zero, one, two, or three
doses of vaccine. All cattle were treated under normal ranching conditions and were vaccinated
for diseases following the usual protocols for the Wyoming region. Cell-mediated immunity was
measured through semi-quantitative PCR, cytokine suspension array, flow cytometry, and cell
proliferation and killing assays. Humoral immunity was measured through an RB51 ELISA. Cell
killing assays were used to determine the up-regulation in cytokine expression and CTL cell
killing. An immortalized bovine macrophage cell line was used to perform cell-mediated
immunity assays. Results will be presented for assays listed above. The results will reflect data
from all three RB51 vaccine doses. Vaccinated cattle and control cattle were challenged with wild
type B. abortus S2308 to determine the efficacy of multiple doses on bacterial burden, incidence
of vertical transmission and abortion and those results will be discussed along with an alternate
recommended vaccination schedule for at risk cattle.
28
Jeffery Adamovicz, Ph.D.
Associate Professor
Director, Laboratory of Infectious Disease Research
College of Veterinary Medicine Laboratory
University of Missouri
Bio-summary:
Jeff Adamovicz is from Des Moines, IA. He received his BA in Biotechnology from the
University of Northern Iowa and Ph.D. in the Department of Microbiology at the Uniformed
Services University of Health Sciences in Bethesda, MD. He did a post-doctoral fellowship in the
Bacteriology Division, USAMRIID. Subsequently he worked on development of a new
recombinant vaccine for plague which has just completed phase 3 clinical trials. After
completion of his 24 year military career including service as a U.N. weapons inspector, and
Chief of Bacteriology at USAMRIID Dr. Adamovicz became a senior medical product developer
at USAMRIID helping to develop the next generation anthrax vaccine which is in clinical trials.
He then worked for Midwest Research Institute for three years in central Asia as Chief Scientist,
to improve public health infrastructure and develop biocontainment facilities and procedures.
Following that he then worked for four years at the University of Wyoming as an Assistant
Professor with a focus on brucellosis vaccine development and diagnostic testing as the State
Veterinary serologist. He recently completed a study in cattle demonstrating improved vaccine
efficacy by altering the recommended schedule for the current RB51 vaccine. Upon moving to
the University of Missouri this past summer as an Associate Professor in the College of
Veterinary Medicine he took over as the new Director of the Laboratory of Infectious Disease
Research, a high containment biosafety laboratory facility. His research interests include
common pathogenic mechanisms of facultative intracellular bacteria and the development of
dendritic cell targeted vaccines.
29
Eric Nicholson, Ph.D.
Research Chemist
National Animal Disease Center
U.S. Department of Agriculture
Ames, IA
Abstract:
Thermodynamic insight into the basis of genetic prion disease in cattle
Prion diseases or transmissible spongiform encephalopathies (TSEs), are an unusual class
of infectious disease. The causative agent of TSEs is a misfolded form of a hostencoded protein. In humans and cattle, prion disease can originate through infectious,
spontaneous, and genetic processes. The most common genetic prion disease in humans
is caused by the substitution of a lysine for a glutamic acid at position 200 (E200K)
resulting in a heritable form of CJD. Rodent models of E200K CJD have proven
interesting but have significant limitations including the need to use human-mouse
chimeric protein constructs to reproduce a disease phenotype. The analogous amino acid
substitution in cattle (E211K) was first identified in a case of BSE in the U.S. in 2006 and
subsequently found in the only living offspring of that animal. This EK211 heterozygous
animal has been used to develop a herd of EE211, EK211, and KK211 cattle for research
on genetic TSEs. To date, our results do not faithfully replicate the observations from
transgenic rodent models. These results led us to pursue additional studies of the folding
and stability of E211K prion protein. Our studies implicate differences in the unfolded
state of the wild-type and E211K protein in genetic prion disease in cattle.
30
Eric Nicholson, Ph.D.
Research Chemist
National Animal Disease Center
U.S. Department of Agriculture
Ames, IA
Bio-summary:
Dr. Eric Nicholson grew up on the plains of western Kansas and received a B.S. degree
in Biochemistry from Kansas State University in 1993. He then pursued his Ph.D.
research in the lab of Dr. J. Martin Scholtz at Texas A&M University studying the
thermodynamics of protein folding, completing his degree in 1999. After graduation he
began post-doctoral research in the lab of Dr. Susan Marqusee at UC Berkeley as a
Damon Runyon Cancer Research Fellow investigating the folding and stability of the
prion protein. In 2004, he began working for the USDA in Ames, IA as a Principal
Investigator on the Transmission, Differentiation, and Pathobiology of Transmissible
Spongiform Encephalopathies research project. His research encompasses various
aspects of animal prion disease, including genetics of disease resistance/susceptibility,
biochemical methods of TSE strain differentiation, and folding/misfolding of the prion
protein.
31
Carl Gelhaus, PhD
Principal Scientist and Director of Bacteriology
MRIGlobal
Kansas City, MO
Abstract:
F. tularensis is a highly infectious bacteria of increased interest due to its potential use as
a biological/bioterror weapon. Tularemia, the disease caused by F. tularensis, can result
in severe morbidity and mortality. As such, therapeutics and vaccines are needed. Due to
the low natural incidence of tularemia, FDA licensure of tularemia therapeutics and
vaccines will require well-developed animal models of disease under the FDA Animal
Rule. We have developed well characterized animal models of tularemia which closely
resemble human tularemia. We are actively testing therapeutics and vaccines and present
a recent example.
32
Carl Gelhaus, PhD
Principal Scientist and Director of Bacteriology
MRIGlobal
Kansas City, MO
Bio-summary:
Dr. Gelhaus is Director of Biodefense Programs in the Medical Countermeasures of
MRIGlobal, where he directs cutting edge research to protect the world from biological
threats. Dr. Gelhaus has 17 years of experience in the field of Immunology, covering a
diverse range of topics. Dr. Gelhaus has 9 years of experience in infectious disease, with
emphasis on bioweapons and bioterror threats. In particular, Dr. Gelhaus is an
internationally recognized Burkholderia and tularemia research and has presented his
research around the globe. At the University of Colorado Health Sciences Center, Dr.
Gelhaus demonstrated that CD8 T cells were not necessary for islet allograft tolerance in
a mouse model of diabetes. At the United States Army Research Institute of Infectious
Diseases (USAMRIID), Dr. Gelhaus performed research on the role of toll-like receptors
(TLR) involved in the pathogenesis of diseases caused by gram-negative select agent
bacteria. Namely, the roles of TLR1, 2, 4, 6, and 9 were investigated in Burkholderia
mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. This work
was conducted both in vivo and in vitro, using mouse models, including bone marrow
derived dendritic cell cultures. Dr. Gelhaus participated in research efforts involving the
recombinant F1-V plague vaccine under development at USAMRIID. Dr. Gelhaus
investigated the role of quorum sensing in Y. pestis pathogensis. At Battelle, Dr. Gelhaus
was involved in the development of animal models as a Study Director for Good
Laboratory Practices regulated studies. This effort included development of associated
assay to support licensure of medical countermeasures through the US Food and Drug
Administration Animal Rule. Dr. Gelhaus has investigated the efficacy of vaccines and
drugs using these models, including doxycycline, and nitrogen-containing
bisphosphonates. Dr. Gelhaus has 9 years’ experience with bacterial select agents under
BSL-3 and ABSL-3 conditions, including Bacillus anthracis, B. mallei, B. pseudomallei,
F. tularensis, and Y. pestis. Dr. Gelhaus has also provided scientific support under
contract with a client company, including molecular biology database support and testing
antibacterial properties of household items under EPA regulations.
33
14th Annual Meeting
Oral Presentation
Abstracts
Session III
THE UGLY:
Antibiotic Resistance
34
35
Josephine R. Chandler, PhD
Assistant Professor
Department of Molecular Biosciences
University of Kansas
Lawrence, KS
Abstract:
Quorum sensing-controlled antibiotics and antibiotic-resistance factors and their
role in interspecies competition
Many Proteobacteria coordinate the production of virulence factors and other
extracellular goods using a type of density-dependent cell-cell signaling called quorum
sensing (QS). Extracellular products or ‘public goods’ are shared among the population
and vulnerable to cheating by non-producing individuals (e.g. QS-defective variants).
Cheaters do not incur any production costs and have a fitness advantage over cooperating
members of the population, which can ultimately cause the population to collapse. One
way to restrain the emergence of cheaters is by co-regulating “private goods” (goods that
only benefit the producing individuals) along with the public goods, which creates a
disadvantage to cheating. Some bacteria, such as the soil saprophyte Chromobacterium
violaceum, use QS to regulate secreted antibiotics that are important for interspecies
competition. Our group has been investigating the relationship between QS, cooperation,
and interspecies competition using a combination of laboratory evolution and dualspecies competition models. In addition to antibiotics, C. violaceum uses QS to regulate
antibiotic resistance determinants. In a C. violaceum laboratory evolution model QSdeficient cheaters readily emerge and increase in the population. However, when
antibiotics are added to the growth medium the cheaters are growth-restricted, because
they are more susceptible to the antibiotics. This improves the competitiveness of the
population, because it increases the frequency of antibiotic-producing individuals. Our
results demonstrate a mechanism where antibiotics may play an important role in cheater
restraint and the maintenance of QS. This may be important for survival in mixed
microbial communities or in antibiotic-treated infections.
36
Josephine R. Chandler, PhD
Assistant Professor
Department of Molecular Biosciences
University of Kansas
Lawrence, KS
Bio-summary:
Josephine Chandler grew up in Iowa City, IA and received her B.S. in Microbiology
from the University of Iowa and her Ph.D. in Microbiology at the University of
Minnesota. During her Ph.D., Dr. Chandler’s work focused on cell-cell signaling in the
opportunistic pathogen Enterococcus faecalis. After her Ph.D., Dr. Chandler moved to
Seattle, Washington and did a post-doctoral fellowship in the Department of
Microbiology at the University of Washington under the mentorship of Dr. E. Peter
Greenberg. As a postdoc Dr. Chandler was funded by an NIH NRSA to study cell-cell
communication (quorum sensing) in the soil bacterium Burkholderia thailandensis and its
close pathogenic relative, Burkholderia mallei. In 2012, she became a Research
Assistant Professor at the University of Washington, and in 2013 she moved to the
University of Kansas where she is currently an Assistant Professor in the Department of
Molecular Biosciences. Her current research program uses several bacterial species
including Burkholderia and Pseudomonas aeruginosa to understand how quorum
sensing-control of antibiotics and other competition factors may promote survival in
mixed microbial communities.
37
Brian Brunelle, Ph.D.
Research Microbiologist
ARS, USDA, Research Microbiologist
National Animal Disease Center
Ames, IA
Abstract:
Antibiotic exposure can induce various bacterial virulence phenotypes in multidrugresistant Salmonella enterica serovar Typhimurium
Salmonella is one of the most prevalent bacterial foodborne diseases in the United States
and causes an estimated 1 million human cases every year. Multidrug-resistant (MDR)
Salmonella has emerged as a public health issue as it has been associated with increased
morbidity in humans and mortality in livestock compared to antibiotic sensitive isolates.
It is known that antibiotics can have unintended consequences on bacteria, and the goal
of my research has been to characterize some of these collateral effects in MDR
Salmonella Typhimurium. We have found that various antibiotics can enhance a variety
of virulence phenotypes in MDR S. Typhimurium, such as inducing cellular invasion,
increasing antibiotic resistance, and stimulating horizontal gene transfer. These factors
may underlie some of the clinical observations associated with MDR Salmonella
virulence.
38
Brian Brunelle, Ph.D.
Research Microbiologist
ARS, USDA, Research Microbiologist
National Animal Disease Center
Ames, IA
Bio-summary:
Brian Brunelle was born and raised in western Massachusetts. He received his BS in
Biochemistry from Rensselaer Polytechnic Institute in upstate New York. After that, he
attended graduate school at the University of California in Berkeley and earned a PhD in
Infectious Diseases studying the phylogenetics and evolution of Chlamydia trachomatis.
He then did a post-doctoral research project at the National Animal Disease Center in
Iowa where he studied genetic factors in cattle associated with resistance and
susceptibility to bovine spongiform encephalopathy. Currently, he is a research
microbiologist in a Food Safety group at the National Animal Disease Center studying
how antibiotics impact virulence in multidrug-resistant Salmonella.
39
Mario Rivera, Ph.D.
Professor
Department of Chemistry
Courtesy Professor
Department of Molecular Biosciences
University of Kansas
Abstract:
Targeting iron homeostasis for developing novel antimicrobials
Mario Rivera
Department of Chemistry, University of Kansas
Most antibiotics target a limited number of crucial biological processes, including DNA
replication, protein translation, and cell wall biosynthesis. Thus, there is a need to
discover and validate novel biological targets in bacteria. To address this issue, we have
been exploring a new direction in the development of antimicrobials, which targets
bacterial iron homeostasis. Bacterial iron metabolism offers a major vulnerability
because: (i) iron is essential and must be obtained from the host, (ii) the concentration of
free iron in the host is vanishingly low (≈ 10-18 M), (iii) to survive, bacteria have evolved
mechanisms to “steal” iron from their host, which depend on well-regulated iron
homeostasis, and (iv) once in the bacterial cell, iron can be toxic and must be stringently
regulated. Hence, iron homeostasis, which is critical for infection, offers an excellent
target for anti-infectives. To disrupt bacterial iron homeostasis we are targeting the
protein bacterioferritin (BfrB), which is present in bacteria but not in humans. BfrB has a
spherical and hollow structure where ≈3,500 iron atoms can be stored in the form of an
Fe3+ mineral. BfrB functions by: (i) utilizing O2 or H2O2 to oxidize Fe2+ and store Fe3+ in
its internal cavity, and (ii) accepting electrons from Bfd to reduce Fe3+ in the internal
cavity and release Fe2+ to the cytosol. The talk will summarize our efforts at discovering
small molecules, which by targeting the BfrB/Bfd interaction disrupt iron metabolism in
Pseudomonas aeruginosa and render the opportunistic pathogen vulnerable.
40
Mario Rivera, Ph.D.
Professor
Department of Chemistry
Courtesy Professor
Department of Molecular Biosciences
University of Kansas
Bio-summary:
Dr. Mario Rivera is a Professor in the Department of Chemistry and a Courtesy
Professor in the Department of Molecular Biosciences at the University of Kansas. He
received his Ph.D. in Chemistry from the University of Arizona in 1991. The Rivera lab
has worked on the development of new NMR methodology for the study of heme
proteins in solution and has elucidated the structure, function and dynamics of a number
or proteins involved in bacterial heme-iron acquisition and in bacterial iron metabolism.
Their findings have contributed to shape current understanding of iron homeostasis in P.
aeruginosa. A current focus of the Rivera lab is to capitalize from their relatively recent
and unprecedented structural finding, which revealed the structure of P. aeruginosa
bacterioferritin (BfrB) in complex with its cognate partner (Bfd). The interaction between
BfrB and Bfd is necessary to regulate bacterial cytosolic iron concentrations by enabling
the crucial equilibrium between free Fe2+ and Fe3+ sequestered in BfrB. P. aeruginosa
mutants lacking BfrB or Bfd are growth impaired in iron-restricted media, significantly
less virulent than the parent strain in C. elgans host and hyper-susceptible to antibiotics.
Consequently, the Rivera lab, in collaboration with a multidisciplinary team with
expertise in synthetic organic chemistry (Richard Bunce, Oklahoma State University),
medicinal chemistry (Blake Peterson) and microbiology (Josephine Chandler and Bill
Picking, University of Kansas), is working toward validating the BfrB-Bfd binding
interaction as target for the development of novel antibiotics.
41
Jim E Riviere, DVM, PhD
The MacDonald Endowed Chair in Veterinary Medicine
University Distinguished Professor
Kansas Biosciences Eminent Scholar
Director, Institute of Computational Comparative Medicine
College of Veterinary Medicine
Kansas State University, Manhattan, KS
Abstract:
Use of Mixed-Effect and Physiological Based Pharmacokinetic Models of
Antimicrobial Drugs as a Framework for Interspecies Extrapolations
Out of necessity, veterinarians must work with multiple species of animals to assure that
administered drugs are both efficacious, safe and do not produce residues in edible tissues
of food producing animals. Making extrapolations across species is a daily task. Two
pharmacokinetic modeling techniques can be used to make such extrapolations;
physiological-based (PBPK) and mixed-effect population pharmacokinetics (PopPK).
Both allow incorporation of disease effects on distribution (protein binding), elimination
or biotransformation based on published literature. In fact, these approaches could be
considered meta-analysis tools that allow relevant data to be incorporated in appropriate
models. Efficiency of these approaches were assessed using published and new
experimental data from pharmacokinetic studies on penicillin-G, oxytetracycline and
flunixin in cattle and swine. The strengths and weaknesses of these approaches will be
discussed in the context of extrapolating antimicrobial drug disposition data across
laboratory animal and veterinary species to humans.
42
Jim E Riviere, DVM, PhD
The MacDonald Endowed Chair in Veterinary Medicine
University Distinguished Professor
Kansas Biosciences Eminent Scholar
Director, Institute of Computational Comparative Medicine
College of Veterinary Medicine
Kansas State University, Manhattan, KS
Bio-summary:
Dr. Jim E. Riviere is the MacDonald Endowed Chair, Kansas Bioscience Eminent
Scholar and a University Distinguished Professor at Kansas State University in
Manhattan where he is the founding director of the Institute of Computational
Comparative Medicine. He spent the first three decades of his career at North Carolina
State University in Raleigh as a Burroughs Wellcome Fund Distinguished Professor in
Pharmacology. He received his BS (summa cum laude) and MS degrees from Boston
College, his DVM and PhD in pharmacology as well as a DSc (hon) from Purdue
University. He is an elected member of the National Academy of Medicine, and serves
on the National Research Council (NRC) Board of Agriculture and Natural Resources.
He previously served on the Board of Scientific Counselors of the NIEHS National
Toxicology Program, the FDA Science Advisory Board as well as on numerous NIH
Study Sections, FDA Committees and journal editorial boards. He is the Editor of the
Journal of Veterinary Pharmacology and Therapeutics and confounder of the
USDA-supported Food Animal Residue Avoidance and Depletion (FARAD) program.
His honors include the 1999 O. Max Gardner Award from the Consolidated University
of North Carolina, the 1991 Ebert Prize from the American Pharmaceutical Association,
the Harvey W. Wiley Medal and FDA Commissioner’s Special Citation, and the
Lifetime Achievement Awards from both the European and American Association
of Veterinary Pharmacology. Dr. Riviere has been PI on over 20 million dollars of
extramural grants and published 550 full-length research papers and chapters, holds 6
U.S. Patents, and has authored/edited 11 books in pharmacokinetics, toxicology and food
safety. His current research interests relate to the development of animal models;
applying biomathematics to problems in toxicology, including the risk assessment of
chemical mixtures, pharmacokinetics, nanomaterials, absorption of drugs and chemicals
across skin; and the food safety and pharmacokinetics of tissue residues in food
producing animals. Jim has been married to fellow toxicologist Dr. Nancy MonteiroRiviere for 39 years and have three adult children.
43
14th Annual Meeting
Poster Abstracts
Session IV
Poster Presentations
44
45
14th Annual Great Plains Infectious Disease Meeting
University of Kansas - Lawrence Kansas
November 6 - 7, 2015
SESSION IV: POSTER PRESENTATIONS
Poster
#
Last
Name
First
Name
Abstract Title
1
Arizmendi Olivia
IpaD from the T3SS of Shigella Triggers Macrophage Apoptosis
2
Behar
Amanda
3
Cherla
Rama
4
Coate
Eric
Genetic Manipulation of Chlamydia trachomatis Inclusion Membrane
Protein CT228 using the Adapted TargeTron System
Coxiella burnetii infection inhibits Neutrophil Apoptosis through
activation of Erk1/2 and Anti-apoptotic protein Mcl 1
Identification and validation of antigens associated with lethal pneumonic
plague
5
Dhariwala
Miqdad
6
7
Dowdell
Eleshy
8
Fairlamb
9
Garcia
10
Hermanas
Alexander Localization of the B. burgdorferi Lipoproteome
Rawan
Antibiotic Resistance in Staphylococcus aureus Isolated From Cystic
Fibrosis Patients
Max
The Cra-FruK complex alters regulation of central metabolism of in
γ-Proteobacteria
Brandon
Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking
Activation of the C1 Complement Complex
Timothy
A c-di-GMP Signaling System in Bacillus anthracis Spores
11
Johnson
Lauren
Exploration of the Inhibitory Effects of Allicin on the Growth of
Staphylococcus aureus in an In Vivo Animal Model
12
Klaus
Jennifer
Regulation of an Antibiotic-Induced Virulence Gene Cluster in
Burkholderia pseudomallei
13
Krute
Christina
The Fatty Acid Kinase of Staphylococcus aureus Controls Virulence
14
Ledbetter
Lindsey
Mechanism of Vaccine-Induced Protection against Coxiella burnetii
15
Liang
Lingfei
Small-Angle X-ray Scattering Analysis of Bacteriophage Sf6 Structural
Protein gp8: a Molecular Hub that Mediates the Assembly of the Tail
Machine
16
Machen
Alexandra Novel and Rapid Development of Stabilizers Against Infectious Disease
Protein Toxins and Human Disease
Bacterial activation of TLR7 in macrophages stimulates a non-canonical
pathway for expression of type I interferon
46
17
Matocha
Kimberly
NKG2D Genotype Alters Gut Microbiome Composition and
Immunoglobulin A Production in The Large Intestine
18
McAllister Rachel
The Induction of Small Colony Variant Staphylococcus aureus in
Artificial Sputa Media
19
O’Neil
Pierce
Probing the Kinetic Stability of Tetanus Neurotoxin Using Biolayer
Interferometry along with a Novel Denaturant Pulse-Chaperonin Assay
with Parallel Confirmation Via Transmission Electron Microscopy
20
Olivarez
Nicholas
Bioinformatic Analysis of Antibiotic Resistance Mechanisms of Coxiella
burnetii
21
Olson
Rachel
Amino Acids T45 and R53 in Yersinia pestis YopK Putative Binding
Region Suppress Host Inflammasome Response
22
Park
So Lee
Thermostability of alphaviruses can be virus-specific
23
Pressnall
Melissa
Activation of Dendritic Cells by the subunit vaccine for Shigella
24
Sharma
Neekun
Constitutive Expression of the NKG2D Ligand RAE1ε Within Pancreatic
Islets Ameliorates Autoimmune Diabetes Development in Non-Obese
Diabetic Mice
25
Trembath
Andrew
Protective Roles for NKG2D in NOD Diabetes Development
26
Woehl
Jordan
Toward Understanding the Structural Basis of Inhibition of the Classical
and Lectin Pathways of Complement by S. aureus Extracellular
Adherence Protein
47
Abstract #1
IpaD from the T3SS of Shigella Triggers Macrophage Apoptosis
Olivia Arizmendi1, William D. Picking2 and Wendy L. Picking2
Dept. of Molecular Biosciences, University of Kansas, Lawrence, KS, USA; 2Dept. of
Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA
1
Abstract:
Shigellosis, a type of bacillary dysentery, is an infectious gastrointestinal disease caused
by Shigella spp. Approximately 165 million cases of shigellosis occur every year around
the world, the vast majority of them in developing countries. High levels of antibiotic
resistance, an increase in multidrug-resistant Shigella isolates and the lack of a licensed
vaccine are factors that situate shigellosis as a public health problem, especially among
young children. Shigella is able to cause death of resident macrophages in the gut to
avoid bacterial clearance early after infection. Shigella is then able to colonize the
intestinal epithelium and induce inflammation, which ultimately gives rise to the
symptoms of dysentery and bacterial shedding. The virulence of Shigella is intimately
tied to its Type III Secretion System (T3SS) for which invasion plasmid antigen D
(IpaD) is a structural element. IpaD was recently shown to induce apoptosis in B
lymphocytes in conjunction with an additional unknown factor. Furthermore, previous
studies have established that IpaD is secreted at levels beyond what is needed for its role as
the T3SS needle tip protein. Here, we present data substantiating our hypothesis that
IpaD secreted by Shigella triggers an apoptotic pathway in macrophages as an early
step in infection. To test this, we have applied a multidisciplinary approach that
includes cell biology, immunology and molecular pathogenesis. Macrophages exposed
to purified IpaD show clear morphological changes consistent with classical apoptosis
such as membrane blebbing and bundles of condensed chromatin. Furthermore, we
observed cytotoxicity as measured by LDH release and activation of specific caspases. A
multiplex assay also provided a clear understanding of the cytokine release profiles
caused by IpaD. We then tested our findings in Shigella infection of cultured
macrophages, which confirmed this role for IpaD in Shigella pathogenesis. A required
IpaD domain as well as possible binding partners were also identified. These findings
allow us to conclude IpaD is a contributing factor to macrophage cell death during
Shigella infection. Learning more about the ways T3SSs interact with host cells provides
potential new targets for antimicrobial drug design.
48
Abstract #2
Genetic Manipulation of Chlamydia trachomatis Inclusion Membrane Protein
CT228 using the Adapted TargeTron System
Amanda Behar1, Cayla Johnson2, Derek Fisher2 and Erika Lutter1
1
Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA; 2 Department of Microbiology, Southern Illinois University
Carbondale, Carbondale, IL, USA
Abstract:
Chlamydia trachomatis is the most frequently reported bacterial sexually transmitted
infection. Even after a C. trachomatis infection is treated, there is an increased risk for
the development of pelvic inflammatory disease and cervical cancer, but the mechanisms
are poorly understood. As an obligate intracellular pathogen, C. trachomatis usurps
many host cell-signaling pathways from within a membrane bound vacuole, called an
inclusion. C. trachomatis is also known to synthesize and secrete via the type III
secretion system, inclusion membrane proteins (Incs) that insert into the inclusion
membrane and serve as the interface between Chlamydia and the host. C. trachomatis is
the first bacterial pathogen observed to recruit myosin phosphatase (MYPT1) for means
of host cell exit, and does so through the chlamydial Inc protein, CT228. In this study,
the chlamydial TargeTron system was used to genetically inactivate CT228 in the C.
trachomatis genome. TargeTron insertion was confirmed by PCR and expression of the
CT229-CT224 operon of the mutant was verified by RT-PCR to rule out polar effects.
The CT228 mutant was verified to be deficient in CT228 production and MYPT1
recruitment by immunofluorescence. This study demonstrates successful gene
inactivation of the chlamydial protein CT228 and confirms the role of CT228 in MYPT1
recruitment. Additionally, these studies provide a platform to further investigate the role
of CT228 in chlamydial pathogenesis.
49
Abstract #3
Coxiella burnetii infection inhibits Neutrophil Apoptosis through activation of
Erk1/2 and Anti-apoptotic protein Mcl 1
Rama Cherla*, Laura Schoenlaub, Yan Zhang, and Guoquan Zhang
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of
Missouri-Columbia, Columbia, Missouri, USA
Abstract:
Coxiella burnetii is an obligate intracellular pathogen that causes acute and chronic Q
fever in humans. It is mainly transmitted by inhalation of contaminated dust from
infected animals such as cattle and sheep as well as in petting zoos. Understanding the
host defense mechanisms will provide critical information for discovery of novel
therapeutic targets against C. burnetii infection. Neutrophils are the first responders to
migrate into the infection site during bacterial pathogen invasion. Previous studies from
our group showed that both virulent C. burnetii Nine Mile phase I (NMI) and avirulent
Nine Mile phase II (NMII) bacteria can infect mouse bone marrow-derived neutrophils
and NMI infection induced more severe disease in neutrophil-depleted mouse, suggesting
neutrophils play an important role in host defense against C. burnetii primary infection in
mice. However, the mechanisms of interaction between C. burnetii and neutrophils
remain unknown. In this study, we examined if there is a difference between NMI and
NMII in modulating host cell apoptotic signaling in mouse bone marrow-derived
neutrophils by MTS assay and TUNEL staining. Interestingly, both NMI and NMII
bacteria were able to inhibit neutrophil preprogramed apoptosis. Immunoblotting analysis
indicates that NMII infection induces a decreased caspase 3 cleavage and increased
phosphorylated p38 MAPK and Erk1/2 kinases. Inhibitors of p38 (SB203508) and
Erk1/2(PD98059) kinases were able to reduce NMII infection induced anti-apoptotic
activity. In addition, activation of Mcl1, a pro-survival Bcl2 family member was also
detected in NMII-infected neutrophils at different time points post-infection.
Collectively, these data suggest that C. burnetii infection induced anti-apoptotic activity
depends on activation of MAP kinase and Mcl1 signaling pathway.
50
Abstract #4
Identification and Validation of Antigens Associated with Lethal Pneumonic Plague
Eric A. Coate1 and Deborah M. Anderson1
1
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO
Abstract:
Inhalation of Yersinia pestis causes primary pneumonic plague, a rapidly progressing
disease that becomes untreatable shortly after symptom onset. We have recently shown
that lethal disease correlates with the development of cardiac arrhythmia in a Brown
Norway rat pneumonic plague model. This profound response is accompanied by
elevated IL-6, IL-1β, TNF-α and decreased platelets, consistent with a cytokine storm
severe sepsis enabled by bacterial growth in the lungs, and further suggesting that
arrhythmias could be caused by specific Y. pestis antigens. IL-6 and IL-1β are commonly
found during bacterial sepsis caused by toll-like receptor 2- or 4-dependent responses to
the bacterial cell wall or outer membrane lipopolysaccharide (LPS). However, host TLR2 and TLR-4 do not recognize Y. pestis and the antigens inducing this systemic
inflammatory response during infection are unknown. In this work, we sought to better
understand the mechanism underlying these arrhythmias and to explore the potential to
use this information to develop methods for early detection of primary pneumonic
plague. Towards these goals, we have compared electrocardiograms from rats infected
with WT or mutant Y. pestis strains in order to identify arrhythmias that were dependent
on specific Y. pestis virulence factors. Our data suggest that ST interval changes are
associated with the virulence factor YopJ and requires secretion, but not bacterial
transport, of the iron-binding siderophore Yersiniabactin. Furthermore, neither
bacteremia, fever nor serum cytokines were consistently associated with ST interval
changes. Together the data provide new insight into understanding the severity of
pneumonic plague, and support further investigation of the ECG as a diagnostic tool for
plague, and perhaps other infectious diseases.
51
Abstract #5
Bacterial Activation of TLR7 in Macrophages Stimulates a Non-Canonical Pathway
for Expression of Type I Interferon
Miqdad O Dhariwala1 and Deborah M Anderson1
1
Department of Veterinary Pathobiology, University of Missouri-Columbia, MO USA
Abstract:
Phagocytosis induces localization of Toll-like receptors (TLR) to endosomes where their
subsequent activation by nucleic acids triggers the expression of type I interferon (IFN).
TLR7 is known to localize to acidified- lysosome associated membrane protein 1
(LAMP1) positive vacuoles in the phagosomal pathway of dendritic cells where it
delivers an activation signal through the adaptor protein MyD88 followed by activation of
one or more interferon regulatory factors (IRFs) and NFκB. While this is viewed as a
general response to RNA released during phagocytosis, TLR7 is in many cases not the
main pattern recognition receptor (PRR) involved in initiating expression of type I IFN
and the issues underlying why TLR7 may only sometimes be used are not clear. In this
work, we show that TLR7 signaling in macrophages following phagocytosis of Yersinia
pestis leads to the MyD88-independent expression of IFNβ. Inhibition or absence of IRF3
or NFκB activation prevented IFNβ expression suggesting both transcription factors may
be activated through TLR7. Furthermore, we show that TLR7 localization to vacuoles
occurs following phagocytosis of Y. pestis, Escherichia coli or Staphylococcus aureus but
is evaded by the obligate intracellular pathogens Francisella tularensis and Brucella
abortus in a manner that is independent of phagosomal escape suggesting early events in
the phagocytic pathway may be involved in specifying the localization of TLR7. Together
these data identify a non-canonical TLR7 signaling pathway in macrophages activated by
the immune-evasive pathogen Y. pestis. Whether this pathway is affected by crosstalk due
to activation of other PRRs by the potent immune stimulators E. coli and S. aureus
remains unknown.
52
Abstract #6
Localization of the B. burgdorferi Lipoproteome
Alexander S. Dowdell1, Shiyong Chen1, Christina Azodi1, Max Murphy1, Wolfram R.
Zückert1
1
Department of Microbiology, Molecular Genetics & Immunology; University of Kansas
Medical Center; Kansas City, KS, USA
Abstract:
The spirochete bacterium Borrelia burgdorferi is the most common vector-borne
pathogen in the United States and a worldwide health risk. B. burgdorferi exhibits a
dualistic lifestyle, continually transitioning between arthropod vectors and vertebrate
hosts. In order to cope with such radical environmental changes, B. burgdorferi employs
many lipid-modified membrane proteins (lipoproteins) to serve in diverse roles ranging
from nutrient acquisition to immune evasion. The genes for these lipoproteins are
distributed across a highly fragmented genome, which is composed of a linear
chromosome and several circular and linear accessory plasmids. Until recently, research
has been primarily focused on a small subset of surface lipoproteins known to be
immunogenic that could serve as potential vaccine candidates. The majority of the
bacterium's lipoproteome has however been neglected, despite its crucial role to the
survival of the organism. Here, we used a tagged expression library and well-estabilished
techniques to localize each protein of the B. burgdorferi lipoproteome to a particular
cellular location. We demonstrate that the majority of lipoproteins are surface-exposed
and that the nonessential "accessory" plasmids are enriched in these surface lipoprotein
genes. Finally, we used in silico techniques to show conservation in multiple "families"
of lipoproteins and to explore the role of lipoproteins lacking experimental investigation.
Our work confirms previous paradigms of B. burgdorferi genome organization and lays
the groundwork for the future investigation of previously uncharacterized lipoproteins.
53
Abstract #7
Antibiotic Resistance in Staphylococcus aureus Isolated From Cystic Fibrosis
Patients
Rawan G. Eleshy1 and Erika Lutter1
1
Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA
Abstract:
Cystic fibrosis (CF) is one of the most common autosomal recessive genetic diseases
caused by a mutation in the CFTR gene. Mutations within this gene disrupt the function
of the ion channels and inhibit the transport of chloride ions across the epithelial
membranes. This leads to dehydrated thick mucus and impaired mucocillary clearance.
As a result, the environment within the CF lung airways becomes ideal for bacterial
colonization by several bacterial species. Staphylococcus aureus is one of the first
pathogens isolated from the CF lung and is prevalent throughout the life of CF patients.
It is very adept at developing resistance to antibiotics and as such is of great concern to
the medical community. This study investigates the prevalence of antibiotic resistance
and presence of antibiotic resistance genes within a subset of S. aureus isolates recovered
from CF patients of various ages: adults (over 18), adolescents (13-18), and children
(under 13). Prior studies have identified nine genes that are correlated with antibiotic
resistance in CF patients. Isolates were tested for the presence of any of these resistance
genes by PCR. Some isolates expressed up to four resistance genes, while others did not
express any of them. Therefore, susceptibility tests were performed to determine if these
isolates show a resistance phenotype. Surprisingly, some of the isolates that contained the
resistance genes did not show resistance in the susceptibility tests. In addition, some
isolates showed resistance on Kirby-bauer plates without expressing the genes.
Understanding the antibiotic resistance mechanisms of Staph aureus isolates from CF
patients will provide significant insights into the complexity of CF infections and may
help in future patient treatments.
54
Abstract #8
The Cra-FruK Complex Alters Regulation of Central Metabolism in
γ-Proteobacteria
Max Fairlamb1, Dipika Singh1, Chamitha Weeramange1, Benjamin Rau1, Sarah
Meinhardt1, Sudheer Tungtur1, Dr. Aron Fenton1, Dr. Liskin Swint-Kruse1
1
Department of Biochemistry and Molecular Biology, University of Kansas Medical
Center, Kansas City, KS, USA
Abstract:
The Gram negative γ–proteobacteria include E.coli, Shigella, plague-causing Yersinia,
and Vibrio cholarae. In these organisms, one protein that is required for pathogenicity is
the catabolite repressor activator (“Cra”). Cra deletion is not lethal, but the bacteria are no
longer robust enough to become pathogenic. This is probably related to its role as a
central regulator of carbon metabolism: Cra directly regulates more than 100 genes,
including those of glycolysis, gluconeogenesis, and the Kreb’s cycle. In regulating these
pathways, Cra enables the bacteria to switch between aerobic and anaerobic metabolism.
Cra is a LacI/GalR homolog and has a function analogous to LacI: Cra binds to operator
DNA sequences to regulate transcription of downstream genes; and DNA binding is
diminished when Cra binds fructose metabolites. Of these, fructose-1-phosphate (“F1P”)
is a strong inducer at micromolar concentrations. F1P is created when fructose is
transported into the cell via a phosphotransferase system. In addition, fructose-1,6bisphosphate (“F16BP”, a central metabolite in glycolysis) has been reported to be a
weak (millimolar) inducer in E. coli, although not in Yersinia. Further, if F16BP acts as a
simple inducer like F1P, it is difficult to rationalize the switching mechanism between
aerobic and anaerobic metabolism.
We serendipitously discovered a new, tight protein-protein interaction between Cra and
fructose-1-kinase (“FruK”). This kinase interconverts F1P and F16BP and is itself
regulated by Cra. Thus, we hypothesized that the interaction must have a critical role in
regulating central carbon metabolism. Experiments with purified proteins show that the
Cra-FruK complex enhances DNA binding relative to Cra alone. Further, whereas F16BP
acts as an inducer for the Cra-DNA complex, it acts as an anti-inducer for Cra-FruKDNA complex. In contrast, F1P induces both Cra-DNA and Cra-FruK-DNA. Thus, the
Cra-FruK interaction (+/- F16BP) provides the missing event for switching E. coli
between aerobic and anaerobic metabolism.
55
Abstract #9
Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation
of the C1 Complement Complex
Brandon L. Garcia1,3, Hui Zhi2, Beau Wager2, Magnus Höök2,3, and Jon T. Skare2
1
Department of Biochemistry & Molecular Biophysics, Kansas State University,
Manhattan, KS, USA; 2Department of Microbial Pathogenesis and Immunology, College
of Medicine, Texas A&M Health Science Center, Bryan, TX, USA; 3Center for Infectious
and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health
Science Center, Houston, TX, USA
Abstract:
Microbial pathogens have evolved a sophisticated arsenal of secreted and membrane
associated proteins capable of recognizing, exploiting and/or inactivating host immune
responses. Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt
strategies to evade human innate immune defenses, notably the human complement
system. Through recruitment of host regulators of complement to their own surface many
human pathogens are able to escape complement-mediated attack. The etiologic agent of
Lyme disease, Borrelia burgdorferi, expresses a number of surface exposed proteins that
bind directly to factor H related molecules, which function as the dominant negative
regulator of the alternative pathway of complement. Relatively less is known about how
B. burgdorferi evades initiation of the classical pathway of complement despite the
observation that some sensu lato B. garinii isolates are sensitive to classical complement.
Furthermore, the classical pathway is involved in the development of the adaptive
immune response that is quelled following borrelial infection. Here we report that the
borrelial lipoprotein BBK32 is capable of potent and specific inhibition of the classical
pathway by binding with high affinity to the initiating complex, the first component of
complement (C1). Using a biochemical and biophysical approach we localized the anticomplement activity of BBK32 to its globular C-terminal domain. Mechanistic studies
reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting
the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical
pathway. To our knowledge this is the first report of a spirochetal protein acting as a
direct inhibitor of the classical pathway and is the only example of a biomolecule capable
of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of
complement evasion this study greatly enhances our understanding of how pathogens
subvert and potentially manipulate host innate immune systems.
56
Abstract #10
A c-di-GMP Signaling System in Bacillus anthracis Spores
Timothy M. Hermanas1; Chung-Ho Lin2, and George C. Stewart1
1
2
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO
Center for Agroforestry, University of Missouri, Columbia, MO
Abstract:
Bacillus anthracis is a gram-positive spore-forming organism that causes fatal disease in
humans, cattle, sheep and goats. B. anthracis spore stability, combined with ability to
inflict lethal disease, has resulted in its use as a biowarfare agent. The 2001 Amerithrax
attacks, utilizing spores concealed in letters, resulted in five deaths and over $320 million
dollars spent decontaminating areas of release. The B. anthracis spore structure and
physiology, especially involving the outermost exosporium layer, are incompletely
understood. The exosporium plays a role in spore uptake and intracellular survival in the
host. Utilizing transposon mutagenesis, we identified a gene in the Sterne strain, bas3594,
as important for the proper assembly of the exosporium. The bas3594 mutant spores
exhibited decreased BclA deposition and microarray data revealed an expression profile
similar to that of bclA, the gene encoding the major surface glycoprotein of the spore.
BAS3594 is a GGDEF/EAL-domain-containing protein. GGDEF/EAL domains are
typically found in proteins that regulate intracellular levels of the messenger c-di-GMP,
which modulates gene function and host immune responses. The B. anthracis genome
also encodes nine other proteins that contain GGDEF/EAL domains. Expression of
mCherry-GGDEF/EAL-protein fusions revealed seven of these putative cyclic di-GMP
regulatory proteins are expressed during specific phases of B. anthracis growth and
sporulation. A subset of these proteins is contained within mature spores and incubation
of B. anthracis spores with GTP resulted in the generation of c-di-GMP showing
evidence diguanylate cyclase activity in mature spores.
57
Abstract #11
Exploration of the Inhibitory Effects of Allicin on the Growth of Staphylococcus aureus in
an In Vivo Animal Model
Lauren J. Johnson1, Fawn Beckman1, J. David McDonald Ph.D1
1
Department of Biological Sciences, Wichita State University, Wichita, KS USA
Abstract:
The antimicrobial properties of Allicin, while long known, require further investigation in order
to evaluate this garlic-derived chemical as an anti-infective agent against Staphylococcus aureus
wound infection. S. aureus is well adapted to live on skin as either normal flora or as a pathogen.
Indeed, it is carried as normal flora by approximately one-third of all people. Thus, there is an
important ongoing clinical problem with wound infection by this pathogen along with the fact
that antibiotics continue to lose their effectiveness against strains. This has motivated us to
explore alternative methods to deal with this common clinical problem and Allicin quickly
emerged as an agent worthy of testing in a standardized wound infection model. Using this
mouse model, we plan to follow wound progression in the presence of different levels of Allicin
applied at the wound site, and compare that to uninfected and untreated controls. We will follow
the progression of this infection in a number of ways: visually (by periodic photography of the
wound site), through the quantitative determination of inflammatory cytokine gene expression by
the mouse host, through the quantitative determination of virulence factor gene expression by the
pathogen, and histologic staining and microscopic analysis of wound tissue. These forms of
analysis will serve as the basis for determining whether Allicin is effective at controlling wound
infection and, if so, to perhaps yield important clues about the mechanism by which it operates.
These clues may be useful in designing more effective control on wound infection in the future.
58
Abstract #12
Regulation of an Antibiotic-Induced Virulence Gene Cluster in Burkholderia
pseudomallei
Jennifer R. Klaus1, Patricia Silva1, and Josephine R. Chandler1
1
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
Abstract:
The bacterium Burkholderia pseudomallei is a Category B select agent and opportunistic
pathogen that causes the deep-lung disease melioidosis, an often-fatal and difficult-totreat condition. The disease mechanisms of B. pseudomallei are poorly understood.
Because B. pseudomallei is a BSL-3-restricted pathogen, studies on B. pseudomallei
disease mechanisms are often carried out using a closely related non-pathogen,
Burkholderia thailandensis. We are interested in the mal gene cluster, which has been
shown to be important for virulence in several animal models. The mal genes are
comprised of the polyketide biosynthesis genes malA-M, and malR. The DNA element
containing the mal genes and malR is highly conserved in B. thailandensis and B.
pseudomallei, and most of the Mal proteins have >80% sequence identity. MalR is a
predicted member of the LuxR family of transcriptional regulators that are typically
activated by acyl-homoserine lactone (AHL) signals. Although MalR has all of the
conserved amino acids of AHL-binding LuxR-family proteins, we have shown that the
MalR from B. thailandensis is an AHL-independent transcriptional activator of the mal
genes. Expression of the mal genes is induced by certain antibiotics (e.g. trimethoprim)
that activate MalR by driving malR expression. In standard laboratory conditions malR
and the mal genes are not expressed. We have begun experiments to elucidate mal gene
regulation in B. pseudomallei using an attenuated, BSL-2-approved strain. Our results
show that in B. pseudomallei the mal genes and malR are activated by trimethoprim,
which is clinically used to treat melioidosis infections. Our results suggest that the
regulation of the mal cluster may be similar in B. thailandensis and B. pseudomallei. Our
results also suggest the possibility that this virulence cluster may be activated by
antibiotics during infections, which we aim to test in future experiments.
59
Abstract #13
The Fatty Acid Kinase of Staphylococcus aureus Controls Virulence
Christina N. Krute1 and Jeffrey L. Bose1
1
Department of Microbiology, Molecular Genetics and Immunology, University of
Kansas Medical Center, Kansas City, KS, USA
Abstract:
Virulence factor regulation in Staphylococcus aureus is under complex transcriptional
and post-transcriptional control. During a screen of the Nebraska Transposon Mutant
Library, we identified mutants in an operon encoding two hypothetical proteins, named
VfrA and VfrB, which had a dramatic decrease in α-hemolysin production. Further
analysis using non-polar mutations revealed that the second protein, VfrB, was the
primary contributor to conditionally-controlled α-hemolysin, protease production, and
expression of other secreted virulence factors. This regulation may be mediated by VfrB
activation of SaeRS, as α-hemolysin production in a vfrB mutant is restored in S. aureus
strain Newman, which encodes a constitutively active SaeRS system. Additionally,
expression of saeRS-controlled genes is down-regulated in vfrB mutants. In conjunction
with a role in virulence, VfrB was identified as a fatty acid kinase essential for uptake of
exogenous fatty acids. When proposed active site residues of VfrB are mutated, the
hemolytic activity of the enzyme is abolished, indicating a role for these residues in
kinase activity. Furthermore, it was discovered that VfrB works in cooperation with two
additional previously hypothetical proteins (termed FakB1 and FakB2). While VfrB
serves as a fatty acid kinase, FakB1 and FakB2 are fatty acid-binding proteins with
differing specificity. In vivo studies using a murine model of dermonecrosis revealed that
the vfrB mutant was hyper-virulent as observed by increased tissue damage, despite
similar numbers of bacteria present. Together, these studies identify a previously
undescribed fatty acid kinase complex found in gram-positive bacteria, and reveal the
connection between fatty acid metabolism and virulence factor regulation in S. aureus.
60
Abstract #14
Mechanism of Vaccine-Induced Protection against Coxiella burnetii
Lindsey E. Ledbetter1 and Guoquan Zhang1
1
Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA
Abstract:
Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes the
zoonosis Q fever in humans. This disease manifests as an acute flu-like illness, although
it can escalate to a chronic and often fatal disease. It is also considered an occupational
hazard, therefore prophylactics should be studied to protect individuals who are at risk of
exposure to infected materials. There is currently a licensed whole-cell vaccine available,
however it is not approved for use in the United States due to adverse reactions in
previously sensitized individuals. Safe use of this vaccine requires multiple screening
procedures, which precludes a mass vaccination program. This whole-cell vaccine has
been shown to be protective against C. burnetii challenge, although the mechanism of
protection remains unknown. Previous work from our laboratory has demonstrated that
this protection is reliant on B cells. In addition, we have shown that these B cells produce
protective IgM independent of T cell help. Here we look further into the mechanism of
protection and show that it is specifically marginal zone B cells that proliferate upon
vaccination. This class of B cells is likely responsible for the majority of T cellindependent IgM produced. Future studies will identify the innate cells responsible for
priming this adaptive immune response. By understanding the mechanism behind this
vaccine-induced protection, we will be able to develop safer, more effective vaccines.
61
Abstract #15
Small-Angle X-ray Scattering Analysis of Bacteriophage Sf6 Structural Protein
gp8: a Molecular Hub that Mediates the Assembly of the Tail Machine
Lingfei Liang*, Tsutomu Matsui§, Thomas M. Weiss§, Haiyan Zhao* and Liang
Tang*
*Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA;
§
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator
Laboratory, Stanford University, Menlo Park, CA, USA
Abstract:
Most double-stranded DNA (dsDNA) bacteriophages contain a highly specialized and
highly efficient tail structure responsible for attachment to host cells and injection of
phage DNA into host cytoplasm. The tail of dsDNA bacteriophage Sf6 is a multiprotein, multi-functional molecular machine with a mass of 2.8 million Dalton and
consisting of 5 gene products present in as many as 51 subunits arranged with different
symmetries. Here we show that a tail component, gp8, of phage Sf6, exists in solution
in equilibrium of a monomer, a dimer and a hexamer. Small-angle X-ray scattering
(SAXS) analysis of the purified gp8 monomer shows a brick-shaped, globular protein
with a small protrusion. Fitting of the gp8 SAXS model into the electron cryomicroscopy map of the entire tail machine defines molecular boundaries among gp8
monomers and with adjacent subunits of other tail components. Each gp8 monomer
interacts with two adaptor protein monomers, two head-binding domains and two
receptor-binding domains from two adjacent tailspike trimmers. The N-terminal
portions of the adaptor protein and the tailspike head-binding domain extend towards
gp8, which may generate additional intermolecular interactions. These interactions
unveil various symmetry mismatches and hold up the ultra-stable tail machine. These
results suggest that gp8 serve as a molecular hub that mediates the assembly of the tail
machine and integrates multiple functions.
62
Abstract #16
Novel and Rapid Development of Stabilizers Against Infectious Disease Protein
Toxins and Human Disease
Alexandra Machen1, Pierce O’Neil1, and Mark T. Fisher1
1
University of Kansas Medical Center / CHAPRx LLC, Kansas City, KS, USA
Abstract:
CHAPRx has developed a patent pending proprietary detection platform to rapidly and
rationally identify and validate protein stabilizers to prevent deleterious protein folding
reactions. We demonstrate that our accelerated platform can 1) validate small molecule
protein stabilizers to block protein toxin transitions during bacterial and viral infections,
2) reverse and prevent folding diseases, or 3) provide industry solutions to stabilize
protein therapeutics. The ability to directly and rapidly inhibit toxin transitions that
change folding shapes during infection has wide applications in human and animal
health. Our technology can rapidly identify and validate inhibitors of infection dependent
shape changes of numerous bacterial/viral protein toxins (e.g. Anthrax, Diphtheria,
Botulinum, Ricin, Tetanus, Shiga toxin, influenza, MRSA). Our technology platform can
rapidly develop stabilizers to prevent protein folding diseases. We have successfully
demonstrated stabilization against proteins of Parkinson’s disease, cystic fibrous, amyloid
diseases (transthyretin), breast cancer, bleeding disorders as well as in rare orphan
diseases such as Friedrich’s ataxia. The common thread between bacterial/viral protein
toxins and protein folding diseases is that they both undergo folding changes that then
result in disease.
63
Abstract #17
NKG2D Genotype Alters Gut Microbiome Composition and Immunoglobulin A
Production in The Large Intestine
Kimberley N. Matocha1, Devin Koestler2 and Mary Markiewicz1
1
The Department of Microbiology, Molecular Genetics & Immunology, University of
Kansas Medical Center, Kansas City, KS; 2The Department of Biostatistics, University of
Kansas Medical Center, Kansas City, KS
Abstract:
The NKG2D receptor and its ligands have been implicated in mediating the progression
of multiple autoimmune diseases including type I diabetes, colitis, rheumatoid arthritis,
chronic obstructive pulmonary disease, and a variety of cancers. Our lab uses the nonobese diabetic (NOD) mouse model to ascertain the effects of NKG2D genotype on type
I diabetes and, more recently, its influence on the gut microbiome composition. In mice,
the NKG2D receptor is expressed on all natural killer cells, activated CD8+ T cells and T
cell subsets, CD4+ T cells, natural killer T cells, and gamma-delta T cells. Mouseexpressed NKG2D ligands include retinoic acid inducible genes-1 (RAE1α-ε), minor
histocompatibility antigen (H60a-c), and mouse UL16-binding protein-like transcript
(MULT1). With the exception of intestinal epithelial cells, most healthy tissues do not
express NKG2D ligands. Given the unique property of NKG2D ligands being
constitutively expressed by intestinal epithelial cells, we investigated whether the
NKG2D genotype affects the gut microbiome composition. 16S Sequencing was
performed on a small portion of fecal pellet aseptically extracted from the colon.
Preliminary data suggests the gut microbiome composition is altered due to NKG2D
genotype. To further understand the mechanism with which NKG2D modifies the gut
microbiome composition, we assessed the role of mucosal-associated immunoglobulin A
(IgA). Via ELISA analysis, colonic fecal homogenate supernatants were analyzed for
IgA concentration and normalized to per gram feces. Our findings support NKG2D
genotype significantly alters IgA concentrations, specifically, in the large intestine thus
may prove to be quintessential for maintaining the gut microbiome composition.
64
Abstract #18
The Induction of Small Colony Variant Staphylococcus aureus in Artificial Sputa
Media
Rachel McAllister1, Elizabeth Pascual1 and Erika Lutter1
1
Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA
Abstract:
Cystic fibrosis (CF) is a genetic disease that results in increased mucus formation in lungs
and pancreas making individuals with CF prone to chronic infections by a number of
pathogens including Staphylococcus aureus. S. aureus is one of the first pathogens to
infect the respiratory tract of patients being the most prevalent pathogen in children and
early teens. Infection with S. aureus can persist into late adulthood. During infection
phenotypic variants, small-colony variants (SCVs), of S. aureus can emerge. SCVs are
believed to be caused by the selective pressure of antibiotics and are of great concern as
they have been increasingly isolated from CF patients. The aim of this study is to
determine if S. aureus isolated from CF patients grown in artificial sputum medium or
minimal media exhibit an altered SCV phenotype. It was found that growth of S. aureus
and prevalence of SCVs was enhanced when S. aureus was grown in artificial sputa
media compared to minimal media. This suggests conditions mimicking the environment
of the CF lung are more favorable for growth and SCV formation of S. aureus.
65
Abstract #19
Probing the Kinetic Stability of Tetanus Neurotoxin Using Biolayer Interferometry
along with a Novel Denaturant Pulse-Chaperonin Assay with Parallel Confirmation
Via Transmission Electron Microscopy
Pierce T. O’Neil1, Alexandra J. Machen1, Josh Burns2, Michael R. Baldwin2, and Mark
T. Fisher1
1
Department of Biochemistry and Molecular Biology, University of Kansas Medical
Center, Kansas City, KS, USA; 2Department of Molecular Microbiology and
Immunology, University of Missouri School of Medicine, Columbia, MO, USA
Abstract:
Tetanus neurotoxin (TeNT) is a virulence factor produced by Clostridium tetani. TeNT,
synthesized as a single polypeptide, has three distinct domains: heavy chain C-terminus
(HC), heavy chain N-terminus (HN) and the light chain (L). HC acts to target the toxin to
neurons via acidic gangliosides and protein receptors. Correct receptor binding facilitates
endocytosis, retrograde transport, and discharge into the spinal intersynaptic space. After
HC-independent endocytosis into pre-synaptic inhibitory neuron endosomes, the pH
decreases. The low pH triggers conformational change in the HN domain allowing it to
insert into the endosomal membrane acting as a cation channel and translocon for the L
chain. Once in the cytosol, the disulfide link between L and HN is reduced, and the L
chain is now neurotoxic.
To better understand the conformational change of HN associated with pH drop, we
investigated the kinetic denaturation isotherm of the complete toxin. Using bio-layer
interferometry (BLI), the toxin is attached onto a biosensor tip. This tip serves two
purposes: 1) the toxin is oriented in the same manner to produce maximal signal, and 2)
the toxin cannot interact with each other, thus preventing aggregation, a common hurdle
when studying membrane toxins with exposed hydrophobic regions. Once the His-tagged
TeNT is immobilized on the Ni-NTA biosensors, it is briefly exposed to urea. Following
the urea pulse, the tip is submerged into a GroEL solution. GroEL, a bacterial chaperonin,
ubiquitously binds to any exposed hydrophobic patches. Kinetic stability isotherms are
generated by plotting urea concentration against GroEL binding signal. In parallel, we
varied the pH (6.5-7.5) of the urea to see a shift in the isotherm curve. Using negative
stain transmission electron microscopy, the TeNT-GroEL complexes were confirmed.
This was achieved by releasing TeNT from the biosensor directly onto glow discharged
grids into microliter volumes. Using the images captured, potential regions of instability
were noted. With greater knowledge of conformational change, we hope to provide
insight into directed design for inhibitors against TeNT. Any identified compounds can
be tested using this methodology, where any leftward shift in the resulting isotherm
and/or diminished GroEL binding indicates small molecule stabilizers of the toxin.
Ultimately, we hope the inclusion of small molecules leads to novel anti-toxin drug
therapies.
66
Abstract #20
Bioinformatic Analysis of Antibiotic Resistance Mechanisms of Coxiella burnetii
Nicholas P. Olivarez1, Guoquan Zhang1,2
1
Department of Molecular Microbiology & Immunology, University of Missouri,
Columbia, MO, USA; 2Department of Veterinary Pathobiology, University of Missouri,
Columbia, MO, USA
Abstract:
The obligate intracellular bacterial pathogen Coxiella burnetii is the causative agent of
the zoonotic disease, Q Fever. This pathogen is extremely resistant to environmental
damage and is easily transmitted by aerosolized particles from fluids of infected hosts.
Current therapeutic procedures for acute infections requires two weeks of treatment with
tetracycline class antibiotics; for chronic infections antibiotic use for a minimum of
eighteen months is required to resolve infections. Despite being the focus of continuous
research for over eighty years, little is known of the mechanisms behind this antibiotic
resistance, specifically, whether resistance is attributed to how the antibiotics interact
with the bacterial targets or if the acidic environment of the intracellular vacuole that the
bacteria replicate within plays a role in reducing the efficacy of antibiotics. To address
this, a thorough bioinformatic analysis of the putative antibiotic resistance proteins of C.
burnetii are compared to antibiotic resistance mechanisms of other well characterized
pathogens. This approach has established novel relationships at the protein level between
C. burnetii and well known pathogens. This work facilitates the modeling of predicted
protein structures of this important pathogen of humans and livestock to identify unique
domains on antibiotic resistance proteins that can better inform the development of
therapeutics by minimizing the duration of antibiotic treatment while maximizing
treatment efficacy.
67
Abstract #21
Amino Acids T45 and R53 in Yersinia pestis YopK Putative Binding Region
Suppress Host Inflammasome Response
Rachel M Olson1,2, Kristen N Peters1,2, Deborah M Anderson1,2
Veterinary Pathobiology, University of Missouri, Columbia, MO, USA 2Molecular
Microbiology and Immunology, University of Missouri, Columbia, MO, USA
Abstract:
Yersinia pestis infection has caused an estimated 200 million deaths in multiple
pandemics worldwide. Plague has been recognized as a re-emerging infectious disease
with naturally occurring antibiotic resistance. Coupled with its potential for aerosol
transmission and high mortality rate, this makes Y. pestis a dangerous potential
bioterrorism agent. Yersinia species have a virulence plasmid encoding a type III
secretion system (T3SS), which translocates effectors to subvert host defenses. The T3SS
itself is sufficient to activate the inflammasome. YopJ, YopM and YopK are known to
manipulate the inflammasome, but their relationship to each other is unclear. While
deletion of yopJ or yopM has a very small effect on virulence, the deletion of yopK
produces a severe virulence defect. YopK is a 21kDa protein with no known sequence
homologs. We recently showed that YopK was involved in promoting YopJ-dependent
macrophage apoptosis, suggesting YopK either has direct effector function or modulates
the activity of YopJ and perhaps other Yop effectors. In this work, we studied this
hypothesis by constructing a yopK-yopJmutant. We show that this strain causes a
significant increase in IL-1β secretion by macrophages and dendritic cells compared to
deletion of either yopJ or yopK alone, as well as WT and T3SS null bacteria. This result
suggests that YopK prevents inflammasome activation caused by one or more elements
of the T3SS. To further understand the potential role of YopK on inhibiting this host
response, native or point mutant YopK-expressing plasmids were added back to the yopJyopK mutant. The suppression of IL-1β by YopK was dependent on amino acids T45 and
R53 in the putative binding region of YopK. The critical amino acid for other known
YopK functions is D46, suggesting multiple binding surfaces of YopK or a novel host
target for the suppression of the inflammasome response. Together the results support a
model whereby YopK is required to modulate host cell death responses during Y.
pestis infection that is necessary to block protective inflammatory responses.
68
Abstract #22
Thermostability of Alphaviruses Can Be Virus-Specific
So Lee Park1,2, Yan-Jang S. Huang1,2, Susan M. Hettenbach2, Stephen Higgs1,2, and
Dana L. Vanlandingham1,2
1
Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, KS, USA; 2Biosecurity Research Institute, Kansas
State University, Manhattan, KS, USA
Abstract:
Alphaviruses are important veterinary and human pathogens with significant epidemic
potential and disease burden. Whilst infections can cause incapacitating clinical diseases
and symptoms, detecting asymptomatic infections by laboratory diagnosis is critical for
disease surveillance prior to the epidemics. Serological assays are important methods for
laboratory diagnosis of alphavirus infections. Currently, plaque reduction neutralization
test (PRNT) is considered as the gold standard for serological assays because of the
demonstration of neutralizing capacity in serum samples. Heat inactivation of serum
samples at 56oC for 30 minutes is a required procedure before the neutralization tests to
eliminate complement activity and adventitious viruses. Although such method is
effective for the complete inactivation of flaviviruses, such parameters have recently been
demonstrated to be insufficient for Western equine encephalitis and chikungunya viruses.
Residual viruses can yield false negative PRNT results and represent a risk to laboratory
personnel. Despite its significance in diagnostic results and laboratory safety, it is not
known whether or not this high thermotolerance is a phenotype shared by all
alphaviruses. In this study, the thermostability profiles of Ross River, Barham Forest,
chikungunya, and o’nyong-nyong viruses were examined to determine the optimum
parameters for complete heat inactivation of these alphaviruses.
69
Abstract #23
Activation of Dendritic Cells by the Subunit Vaccine for Shigella
Melissa Pressnall1, Francisco Martinez-Becerra1, Olivia Arizmendi-Perez2, Wendy
Picking1, William Picking1.
1
2
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA;
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
Abstract:
Shigellosis is a disease with severe global impact yet it has no approved vaccine. This
gastrointestinal disease effects ~90 million people per year with 100,000 deaths primarily
in young children in developing countries. Shigella infects humans by utilizing a type
three secretion system (T3SS) which is highly conserved over the ~50 Shigella serotypes.
The T3SS works like a needle and syringe that injects bacterial protein effectors into the
host epithelial cells in the colon to promote bacterial entry. The internalized bacteria then
lyse the resulting phagosome which allows bacterial replication in the host cell
cytoplasm. Two proteins, IpaB and IpaD, control the activity of the T3SS from the tip of
the needle. Using a lethal pulmonary mouse challenge model, we have shown that
vaccination with IpaB + IpaD or a genetic fusion of these proteins (the DB Fusion) along
with the adjuvant dmLT is protective against a Shigella flexneri challenge as well as a
heterologous challenge by Shigella sonnei. In addition, we found that using both antigens
(either combined or in the DB Fusion) gives rise to higher cytokine activation and
protection profiles. We are working to determine the protective mechanism of the DB
Fusion vaccine and to identify the pathway for antigen presentation. The first step in this
effort is to analyze the effect of these proteins in antigen presenting cells such as
dendritic cells. In this study, we are stimulating dendritic cells with IpaB, IpaD,
IpaB+IpaD, or the DB Fusion, with or without dmLT to measure subsequent cytokine
release as well as the up-regulation of activation markers. Identification of the activation
markers and cytokines involved will help to describe a plausible mechanism for the
response of dendritic cells incubated with our candidate vaccine proteins.
70
Abstract #24
Constitutive Expression of the NKG2D Ligand RAE1ε Within Pancreatic Islets
Ameliorates Autoimmune Diabetes Development in Non-Obese Diabetic Mice
Neekun Sharma1, Mary Markiewicz1
1
Department of Microbiology, Molecular Genetics, & Immunology, University of Kansas
Medical Center, KS, USA
Abstract:
Type 1, or autoimmune, diabetes is driven by T cell-mediated destruction of the insulinproducing β cells in pancreatic islets. The natural killer cell activating receptor NKG2D
and its ligands are implicated in this process. However, the mechanism by which
NKG2D-NKG2D ligand interaction affects diabetes development is poorly understood.
Transgenic expression of the NKG2D ligand retinoic acid early transcript 1ε (RAE1ε) in
β-islet cells of the pancreas induces the recruitment of cytotoxic T lymphocytes (CTLs)
to the islets in mice. Surprisingly, however, we report here that the severity of
spontaneous autoimmune diabetes is decreased in non-obese diabetic (NOD) mice
expressing RAE1ε in islets (RIP-RAE1ε NOD). Despite enhanced infiltration of
lymphocytes to the pancreas, NKG2D expression is reduced on the surface of T cells
within the pancreas of RIP-RAE1ε NOD mice compared with control NOD mice. This
suggests that transgenic expression of RAE1ε in pancreatic islets leads to the downregulation of NKG2D surface expression on T cells, decreasing the ability of the cells to
respond to NKG2D engagement by ligands naturally expressed in the pancreas. We
further demonstrate that the NKG2D ligand H60a is expressed on T cells within the
pancreas of wild-type NOD mice. Together, these data suggest that interaction between
NKG2D and H60a expressed on T cells within the pancreas plays a critical role in the
development of NOD diabetes and that blocking this interaction may be therapeutically
useful in type 1 diabetes.
71
Abstract #25
Protective Roles for NKG2D in NOD Diabetes Development
Andrew P. Trembath1, Neekun Sharma1, Kimberly N. Matocha1, Mary A. Markiewicz1
1
Department of Microbiology, Molecular Genetics & Immunology, University of Kansas
Medical Center, Kansas City, KS, USA
Abstract:
Type I diabetes (TID) is a debilitating autoimmune disorder which requires ongoing insulin
injections, and is associated with numerous long-term health consequences. This disease is
mediated by autoreactive T cells that destroy the insulin-secreting β-islet cells within the
pancreas. While it is clear that many genes contribute to the development of type 1 diabetes, its
cause remains unknown. Both human and murine studies implicate the activating immune
receptor natural killer group 2 member D (NKG2D) in enhancing TID development. However,
the mechanism by which NKG2D signaling influences diabetes development has been unclear.
Here we report that NKG2D signaling influences spontaneous autoimmune diabetes development
in non-obese diabetic (NOD) mice via multiple, opposing mechanisms. First, we found that
NKG2D influenced diabetes development via alteration in the microbiota composition. Second,
when we eliminated this microbiota effect, we observed an unexpected protective role for
NKG2D in NOD diabetes development. To determine how this effect was mediated, we
examined NOD pancreatic islets for expression of NKG2D ligands and found that T cells within
the pancreas of pre-diabetic NOD mice expressed the NKG2D ligand H60a. In vitro we observed
both H60a and NKG2D on activated NOD CD8+ T cells. This suggested a possible role for
NKG2D-ligand engagement during homotypic T cell-T cell interaction during both CD8+ T cell
differentiation into cytotoxic T cells (CTLs) and during CTL effector function. We tested this
hypothesis in vitro using CD8+ T cells from mice with various NKG2D expression levels and
blocking antibodies against H60a. During differentiation, NKG2D-H60a interaction influenced
the CTL effector cytokine response, but not cytotoxic granule release. In contrast, NKG2D-H60
interaction during the CTL effector phase did not affect cytokine production, but increased
cytotoxic granule release. These results are the first to describe a role for NKG2D-ligand
engagement via homotypic T cell-T cell interactions. Our findings suggest there are two roles for
these interactions in NOD CTL responses. First, there is a dominant protective role for these
interactions during NOD CD8+ T cell differentiation into CTL within pancreatic lymph nodes,
second, there is a detrimental role for these interactions within the pancreatic islets where they
increase β-cell killing.
72
Abstract #26
Toward Understanding the Structural Basis of Inhibition of the Classical and Lectin
Pathways of Complement by S. aureus Extracellular Adherence Protein
Jordan L. Woehl1, Brandon L. Garcia1, Kasra X. Ramyar1, John K. Walker2, Michal
Hammel3, Brian V. Geisbrecht1
1
Department of Biochemistry and Molecular Biophysics, Kansas State University,
Manhattan, KS, USA; 2St. Louis University School of Medicine, St. Louis, MO, USA;
3
Lawrence Berkeley National Lab, SIBYLS High Throughput SAXS, Berkeley, CA, USA
Abstract:
The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human
innate immunity by expressing a series of secreted inhibitory proteins. A number of these
proteins have been shown to inhibit the complement system. We recently reported that S.
aureus Eap inhibits the classical and lectin pathways of the complement cascade by a
previously undescribed mechanism (Woehl et al (2014) J. Immunol.). Specifically, Eap
binds with nanomolar-affinity to complement C4b, and thereby blocks binding of the
Classical and Lectin pathway pro-protease, C2. This effectively eliminates formation of
the CP/LP C3 proconvertase, which is required for downstream complement activity.
Although Eap’s mechanism of action has been demonstrated, little is known about how
Eap interacts with complement C4b at the structural level. The full-length, mature Eap
protein consists of four ~97 residue domains, which adopt a similar beta-grasp fold, and
are connected through a short linker region. Through multiple assays, we have identified
the 3rd and 4th domains as being critical for interacting with C4b and inhibiting the
complement cascade. Co-crystallization efforts of Eap34 bound to a large fragment of
C4b yielded ordered crystals, but they diffracted X-rays poorly; this has hampered our
ability to obtain a satisfactory structural model. We therefore explored an alternative
approach that takes advantage of the abundance of surface exposed lysines in the Eap
protein. Through this lysine-acetylation footprinting technique, we have tentatively
mapped and identified two key areas in Eap domains 3 and 4 that show protection upon
being bound to C4b. Further experiments are underway to test this provisional
identification of the C4b-binding site within Eap domains 3 and 4.
73
14th Annual Great Plains Infectious Disease Meeting
University of Kansas - Lawrence Kansas
November 6 - 7, 2015
MEETING REGISTRANTS
Last Name
Adam
Adamovicz
Adams
Agarwal
Al-Murrani
Amachawadi
Anderson
Arizmendi
Ashitey
Ault
First Name
Phil
Jeffrey
Paige
Sanjeev
Sam
Raghavendra
Deborah
Olivia
Pearl
Kevin
Email Address
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
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[email protected]
[email protected]
[email protected]
Barta
Bearson
Michael
Brad
[email protected]
[email protected]
Behar
Amanda
[email protected]
Berkland
Bhattacharya
Cory
Somanon
[email protected]
[email protected]
Bodugam
Bose
Mahipal
Jeffrey
[email protected]
[email protected]
Bourne
Brunelle
Christina
Brian
[email protected]
[email protected]
Carter
Wayne
[email protected]
Carvalho
Claudia
[email protected]
Chandler
Chen
Cherla
Josephine
Kangming
Rama
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74
Institution Affiliation
Kansas Dept. of Health
University of Missouri
Kansas State University
KU
Prommune, Inc.
Kansas State University
University of Missouri
University of Kansas
University of Kansas
University of Kansas
Medical Center
University of Kansas
USDA; ARS; National
Laboratory for
Agriculture and the
Environment
Oklahoma State
University
University of Kansas
University of Missouri
Kansas City
University of Kansas
University of Kansas
Medical Center
University of Oklahoma
National Animal Disease
Center
Kansas City Area Life
Sciences Institute
Fort Hays State
University
University of Kansas
Kansas State University
College of Veterinary
Medicine; University of
Missouri-Columbia;
Choi
Coate
Seong-O
Eric
[email protected]
[email protected]
Coyle
Davis
Dean
DeDonder
Kathryn
Heather
Bartholomew
Keith
[email protected]
[email protected]
[email protected]
[email protected]
DeDonder
Sarah
[email protected]
DeKosky
Dhariwala
Brandon
Miqdad
[email protected]
[email protected]
Dowdell
Alexander
[email protected]
DurandHeredia
Eleshy
Jorge
[email protected]
Rawan
[email protected]
Eshelman
Esquivel
Kate
Brooke
[email protected]
[email protected]
Fairlamb
Max
[email protected]
Fisher
Mark
[email protected]
Ganguly
Gao
Garcia
Geanes
Arghya
Philip
Brandon
Eric
[email protected]
[email protected]
[email protected]
[email protected]
Gelhaus
Hawley
Hefty
Hermanas
Carl
Blake
Scott
Timothy
[email protected]
[email protected]
[email protected]
[email protected]
Hettenbach
Hilliard
Horvat
Susan
Kinsey
Rebecca
[email protected]
[email protected]
[email protected]
Hsieh
Hulangamuwa
Hustead
Hsinyeh
Wasundara
David
[email protected]
[email protected]
[email protected]
75
Kansas State University
University of MissouriColumbia
University of Kentucky
Kansas State University
university of Kansas
Veterinary and
Biomedical Research
Center, Inc.
Kansas Department of
Agriculture
NIAID
University Of MissouriColumbia
University of Kansas
Medical Center
University of Missouri at
Columbia
Oklahoma State
University
University of Kansas
University of MissouriKansas City
University of Kansas
Medical Center
University of Kansas
Medical Center
University of Kansas
KanPro Research Inc.
Kansas State University
University of MissouriKansas City
MRIGlobal
Integrated Animal Health
University of Kansas
University of Missouri at
Columbia
Kansas State University
University of Missouri
University of Kansas
Med Center
University of Missouri
Kansas State University
Boehringer Ingelheim
Vetmedica
Jafarain
Javed
Jha
Jimenez
Sohaila
Salim
Jay
Wanda
[email protected]
[email protected]
[email protected]
[email protected]
Johnson
Jordan
Kamath
Lauren
Lorne
Divya
Keleher
Klaus
Krute
Lauren
Jennifer
Christina
Kumar
Lane
Ledbetter
Prashant
Abigail
Lindsey
Liang
Lin
Lutter
Lingfei
Chungho
Erika
Ma
Machen
Manley
Markiewicz
Wenjun
Alexandra
Michael
Mary
Marks
Kayla
Martinez
Becerra
Massa
Matocha
Francisco
Kansas State University
University of Kansas
University of Kansas
Fort Hays State
University
[email protected]
Wichita State University
[email protected]
University of Kansas
[email protected]
University of MissouriKansas City
[email protected]
University of Missouri
[email protected]
University of Kansas
[email protected]
University of Kansas
Medical Center
[email protected]
University of Kansas
[email protected]
Kansas State University
[email protected] University of MissouriColumbia
[email protected]
University of Kansas
[email protected]
University of Missouri
[email protected]
Oklahoma State
University
[email protected]
Kansas State University
[email protected]
KUMC
[email protected]
KU Medical Center
[email protected]
University of Kansas
Medical Center
[email protected]
University of MissouriColumbia
[email protected]
University of Kansas
Nicole
Nicole
[email protected]
[email protected]
McAllister
Rachel
[email protected]
McDonald
McGill
McKinney
Miriyala
MonteiroRiviere
Moore
Dave
Jodi
Megan
Nagaraju
Nancy
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Kaitlin
[email protected]
Moral
Mario
[email protected]
76
The University of Kansas
The University of Kansas
Medical Center
Oklahoma State
University
Wichita State University
Kansas State University
University of Kansas
University of Kansas
Kansas State University
Fort Hays State
University
University of Kansas
Nicholson
Eric
Nicholson
Nowak
Olivarez
Olson
Tracy
Martha
Nicholas
Rachel
ONeil
Pierce
Park
Parsel
So Lee
Suzanne
Patel
Ami
Paudyal
Anuja
Picking
Picking
Pratt
Pressnall
Qiu
Rezac
Wendy
William
Miranda
Melissa
Iris
DJ
Rivera
Riviere
Rosa-Molinar
Sease
Sharma
Mario
Jim
Eduardo
Rebecca
Neekun
Stein
Stewart
Taylor
Trembath
Sydney
George
Anna Marie
Andrew
Vanlandingham
Vishwakarma
Wakefield
Ward
Whitaker
Woehl
Yao
Zhang
Zhang
Dana
Vikalp
Susan
Claire
Neal
Jordan
Huili
Xuehan
Guoquan
[email protected]
National Animal Disease
Center; USDA
[email protected]
USDA
[email protected]
K-State Olathe
[email protected] University of Missouri
[email protected] University of MissouriColumbia
[email protected]
University of Kansas
Medical Center
[email protected]
Kansas State University
[email protected]
Kansas State University
Olathe
[email protected]
Laboratory for Infectious
Disease Research
[email protected]
Fort Hays State
University
[email protected]
University of Kansas
[email protected]
University of Kansas
[email protected]
The University of Kansas
[email protected]
University of Kansas
[email protected]
The University of Kansas
[email protected]
Veterinary and
Biomedical Research
Center, Inc.
[email protected]
University of Kansas
[email protected]
Kansas State University
[email protected]
The University of Kansas
[email protected]
Kansas State University
[email protected]
University of Kansas
Medical Center
[email protected]
University of Missouri
[email protected]
University of Missouri
[email protected]
Kansas State University
[email protected]
University of Kansas
Medical Center
[email protected]
Kansas State University
[email protected]
University of Kansas
[email protected]
University of Kansas
[email protected]
Kansas State University
[email protected]
University of Kansas
[email protected]
Kansas State University
[email protected]
University of Kansas
[email protected]
Kansas State University
[email protected]
University of Missouri
77