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15th Annual Great Plains Infectious Disease
Meeting
November 4-5, 2016
University of Kansas
Lawrence, KS
Crystal Anniversary
The 15th Annual Great Plains Infectious Disease Meeting
We are pleased to host the 15th Great Plains Infectious Disease (GPID) Meeting which 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 160 participants from across the region with more than 30 posters being presented. We welcome participants from regional academic programs, industry, private firms, nonprofits, contract research organizations and government. This meeting has always been successful due to the generosity of our academic sponsors and selected vendors. Thank you all for attending! Wendy and Bill Picking – University of Kansas ACKNOWLEDGEMENTS Generous academic and industrial sponsors have contributed greatly to the success of this meetings. Many thanks to all of our 2016 GPID supporters!
15th Annual Great Plains Infectious Disease Meeting Program Schedule
November 4-5, 2016
University of Kansas-Lawrence
Friday, November 4, 2015 – Springhill Suites, 6th and New Hampshire, Downtown Lawrence, KS
4:00pm 6:00pm Welcome and Reception
6:00pm Dinner
6:30pm 9:30pm Science, Collaboration and Networking
Saturday, November 5, 2015 (Atrium, School of Pharmacy, West Campus )
7:15am 7:55am Check-In - Coffee, Juice and Doughnuts
8:00am 8:02am Opening Remarks – Wendy Picking
Session I: Epidemiology/Public Health/One Health (Room 2020 SOP)
8:02am 8:30am Phil Adam (KDHE) Bioterrorism and the laboratory response network
8:30am 9:00am Nancy Hanson (Creighton) Molecular surveillance of clinically important β-lactamase genes: A requirement for One Health
9:00am 9:30am Elizabeth Holzschuh (Johnson County DHE) Vaccine hesitancy and outbreaks
9:30am 10:00am Charley Cull (VBRS) Veterinary and comparative models for food safety research
10:00am 10:20am Refreshments and Networking Break (Atrium SOP)
Session II: Microbial Physiology (Room 2020 SOP)
10:30am 11:00am Christian Ray (KU) Regulation of bacterial growth in discrete steps and structured lineages
11:00am 11:30am Greg Somerville (UNL/Zoetis) Metabolism – the forbidden fruit of antibiotics
11:30am 12:00pm Torey Looft (USDA) Antibiotics modulate the microbiota and metabolomic profiles in turkey cecal microbiomes
12:00pm 12:30pm Donald Burke (MU) Overcoming aptamer-resistance in HIV-1 inhibition with broad-spectrum RNA apatemrs
12:30pm 1:30pm Lunch (First Floor Dining Area – Mortar & Pestle Cafe)
Session III: Biochemistry/Structural Biology (Room 2020 SOP)
1:30pm 2:00pm Christina Bourne (OU) Toxin-antitoxin modules: Functions in bacteria
2:00pm 2:30pm Brian Geisbrecht (KSU) Toward a structural understanding of the type-III secretion needle tip: Progress and challenges
2:30pm 3:00pm Michael Barta (KU) Using nanobodies to investigate the T3SS needle-tip complex of Shigella flexneri
3:00pm 3:45pm Bill Picking (KU) Structure-function relationships within the Shigella type-III secretion apparatus
Session IV: Poster Presentation
3:45pm 5:30pm Poster Session (Atrium) – Light Refreshments
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15th Annual Meeting
Oral Presentation
Abstracts
Session I
Epidemiology/Public
Health/One Health
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Philip Adam, Ph.D. Microbiologist
Kansas Health and Environmental Laboratories
Kansas Department of Health and Environment
Topeka, KS
Abstract:
Bioterrorism and the Laboratory Response Network
The Laboratory Response Network (LRN) is a nation-wide network of clinical labs
trained to detect and report events of biological and chemical terrorism. LRN lab roles
are stratified and consist of Sentinel facilities (hospitals, commercial labs), Reference
labs (State public health labs), and National labs (Centers for Disease Control and
Prevention (CDC)). Sentinel labs recognize and either rule-out or refer potential agents of
bioterrorism to Reference labs. Reference labs repeat Sentinel lab tests and perform
confirmatory testing according to LRN testing algorithms. Depending on the results of
the confirmatory testing, Reference labs will notify the CDC or Federal law enforcement
officials. National labs perform definitive characterization of isolates, especially when
Reference lab testing was inconclusive. The LRN is also the primary means of providing
rapid testing for emerging threats, such as Ebola virus and Zika virus.
Bio Summary:
Dr. Adam began his research career as an undergraduate research assistant in Dr. Wendy
Picking’s lab at the University of Kansas, investigating the binding of bile salts to
Invasion Plasmid Antigen D (IpaD) of Shigella flexneri. After graduating from KU with
a B.S. in Microbiology in 2009, he continued his research on Type Three Secretion in S.
flexneri at Oklahoma State University in Dr. William Picking’s lab, focusing on the pore-
forming effector protein, IpaB. Dr. Adam graduated in 2014 with a Ph.D. in
Microbiology and Molecular Genetics, and accepted a position with the Kansas
Department of Health and Environment Bureau of Laboratories in the Virology/Serology
Unit. After 7 months, he was promoted to a position within the Diagnostic Microbiology
Unit as the Principal Investigator to the select agent program, where he is now
responsible for the scientific and technical direction of the BSL-3 lab. Dr. Adam’s
academic achievements include 7 manuscripts, 2 book chapters, 14 awards for excellence
in presentation, and the Grula Distinguished Graduate Fellowship.
6
Nancy D. Hanson, Ph.D. Professor
Department of Medical Microbiology
Director: Center for Research in Anti-Infectives and Biotechnology
Creighton University School of Medicine
2500 California Plaza
Omaha, NE 68178
Abstract:
Molecular Surveillance of Clinically Important β-Lactamase Genes: A Requirement
for One Health
Antibiotic resistance is a global problem that affects not only human health, but animal
and environmental health as well. The One Health Approach is defined as ‘the
collaborative effort of multiple disciplines –working locally, nationally, and globally – to
attain optimal health for people, animals and our environment…’. Both Gram-negative
and Gram-positive organisms have developed a multitude of antibiotic resistance
mechanisms both plasmid and chromosomally mediated. Difficult resistance mechanisms
to detect in the clinical laboratory are mechanisms that confer resistance to β-lactam
antibiotics. There are over 2,000 different β-lactamases that can confer resistance to β-
lactam antibiotics. Many Gram-negative organisms produce multiple enzymes
simultaneously in addition to other mechanisms of resistance making these organisms
multi-drug resistant. This seminar will introduce 1) the global problem and One Health
Approach to antibiotic resistance, 2) how the production of β-lactamases by Gram-
negative pathogens can exclude the use of all β-lactam treatment for both human and
animal patients, and 3) introduce new PCR-based detection methods for β-lactamase
genes. Implementation of these PCR-based technologies can help control the spread of β-
lactamase producing pathogens, direct antibiotic stewardship programs, and guide
therapy.
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Nancy D. Hanson, Ph.D. Creighton University School of Medicine
Omaha, NE
Bio-summary:
Dr. Nancy D. Hanson is Professor and Director of the Center for Research in Anti-
Infectives and Biotechnology in the Department of Medical Microbiology and
Immunology at Creighton University. Dr. Hanson received her Ph.D. in Medical
Microbiology from the University of Nebraska Medical Center in 1991. She joined the
faculty of Creighton University in 1995. Her area of expertise is the study of molecular
mechanisms of antibiotic resistance in Gram-negative organisms such as E. coli, K.
pneumoniae, Salmonella spp. and Pseudomonas aeruginosa. Her research explores many
aspects of antibiotic resistance mechanisms including: 1) identification of the selective
pressures required for the emergence of resistance, 2) regulation of gene and protein
expression involved in resistant phenotypes, and 3) the development of PCR-based
diagnostic tests that can be used by laboratories to detect resistance genes in clinical
isolates. Dr. Hanson has published over 75 journal articles and holds 8 patents regarding
molecular diagnostics. In 2007, Dr. Hanson was awarded researcher of the year by the
Nebraska Chapter of the Cystic Fibrosis Foundation for her work on P. aeruginosa. In
2008, Dr. Hanson was part of an international colloquium for the American Academy of
Microbiology on Antibiotic Resistance in Annecy France. In 2013, Dr. Hanson received
the Distinguished Research Career Award from Creighton University School of
Medicine. Dr. Hanson was also selected as an American Society of Microbiology
Distinguished Lecturer for academic years 2014-2016. In June 2016, Dr Hanson was
invited to the Opening of the Australian Center for Antimicrobial Resistance Ecology as
a Keynote Speaker.
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Elizabeth Lawlor Holzschuh Epidemiologist II
Strategic Planning Division
Johnson County Department of Health and Environment
Olathe, KS
Abstract:
Vaccine Hesitancy Causes and Consequences
Vaccines are considered one of the top ten public health achievements and are credited with the
reduction in vaccine-preventable diseases (VPDs) in the 20th century. However, in the past
several decades there has been an increase in the number of individuals who are concerned about
the decision to vaccinate themselves or their children. This hesitancy is due to a variety reasons,
including the unfamiliarity with VPDs, fear of adverse health outcomes, and a distrust of
governmental agencies. The result of this has been an increase in the number of non-medical
exemptions to school immunization requirements along with an increase in outbreaks of VPDs
including measles, pertussis, and invasive Haemophilus influenza type B.
Bio-summary:
Elizabeth Holzschuh is the population health epidemiologist at the Johnson County Department
of Health and Environment, where she has worked since 2015. Her primary role is to provide
data for program and policy development and support department activities, including the
community health assessment. Prior to her current role, she served as an infectious disease
epidemiologist at the Kansas Department of Health and Environment. She obtained her Master’s
degree with a concentration in public health, microbiology, and emerging infectious disease from
the George Washington University.
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Charley A. Cull, D.V.M., Ph.D. Midwest Veterinary Services, Inc.
Veterinary and Biomedical Research Center, Inc.
9027 Green Valley Drive
Manhattan, KS 66502
Abstract:
Veterinary & Comparative Models for Food Safety Research
Societal and market driven dynamics, along with a continually progressing regulatory
environment have resulted in a novel food handling, preparation, storage, and production
practices. Congruently, businesses and consumers have increased their expectations of
producers at each segment of the food chain, from farm to fork. This new found intimacy
between consumers and their food has established the tangible need for a consistent approach to
food safety management that includes multifactorial and holistic methods, including production,
research, and intervention implementation strategies for a large and diverse industry. As such,
we have developed well characterized veterinary and comparative models for foodborne
pathogens including Salmonella sp., and Escherichia coli, to appropriately assess the efficacy
and safety of therapeutics and vaccines.
Bio Summary:
Dr. Charley Cull received his Doctor of Veterinary Medicine degree and his Ph.D. degree in
epidemiology/food safety, both from Kansas State University. He is recognized and has received
multiple honors for his food safety research in commercial feedlot cattle. Dr. Cull is a clinical
research associate of Midwest Veterinary Services, Inc. of Oakland, NE and Veterinary
Biomedical Research Center, Inc. of Manhattan, KS. In addition to providing data to support the
FDA and USDA approval of animal health products, a significant amount of his time and energy
is focused on biomedical research, production animal consulting, and internal food production
operations. Dr. Cull is a member of the Academy of Veterinary Consultants, the American
Association of Bovine Practitioners, American Association of Swine Veterinarians, and the
American Veterinary Medical Association.
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15th Annual Meeting
Oral Presentation
Abstracts
Session II
Microbial Physiology
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Christian Ray, Ph.D. Assistant Professor
Center for Computational Biology
Department of Molecular Biosciences
University of Kansas
Lawrence, KS
Abstract:
Regulation of bacterial growth in discrete steps and structured lineages
State-of-the-art clinical strategies targeting infectious disease pathways aim to kill or halt the
growth of the infectious agent. Paradoxically, the state of growth arrest in bacteria can render
cells tolerant to antibiotics (the persister state). Phenotypic heterogeneity allows a small fraction
of cells to enter growth arrest by randomly crossing a threshold, a form of bet-hedging that
allows the population of cells to survive even if future environments are inhospitable to actively
growing cells. Therapeutic targeting of growth arrested bacteria is a critical emerging strategy
during the current rising problem of antibiotic resistance and the continued challenge of treating
stubborn, chronic infections. We have created a new multifaceted approach that has opened new
avenues for under-standing persister formation with time-lapse microscopy and computational
models. Our experiments have shown a novel persister-forming condition. In this condition,
bacterial microcolonies discrete shifts in growth rate that correspond to fast molecular
reshuffling events. Analysis of cellular lineages in these conditions demonstrates that transitions
into growth arrest are not statistically independent: more closely related cells are more likely to
transition together. Our computational models are able to reproduce lineage correlations with a
remarkably simple set of assumptions. These results suggest an upper bound on the level of
phenotypic diversity that is attainable in conditions that produce persister cells. We discuss
implications of the novel persister phenotype for pathogens surviving in changing environments,
and new questions raised by our results.
Bio Summary:
I received my Ph.D. from the University of Michigan in Microbiology and Immunology and did
postdoctoral work in computational systems biology at Rice University and in experimental
synthetic biology at M.D. Anderson Cancer Center. I am now Assistant Professor in the Center
for Computational Biology and the Department of Molecular Biosciences at the University of
Kansas.
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Greg Somerville, Ph.D. (UNL/Zoetis)
Associate Professor
University of Nebraska-Lincoln
155 Veterinary Biomedical Sciences, East Campus
Lincoln, NE 68583-0905
Abstract:
Metabolism the forbidden fruit of antibiotics
As antibiotic resistance rises, the treatment of serious Staphylococcus aureus infections
becomes more challenging. To address this problem, pharmaceutical companies and
academic researchers tend to focus their efforts on two approaches: developing and/or
discovering new antibiotics, and re-purposing human use approved drugs. Metabolomic
studies of isogenic daptomycin susceptible and non-susceptible S. aureus strain pairs
(Antimicrob. Agents Chemother. 2015. 59:4226-4238) revealed that daptomycin non-
susceptible strains had decreased tricarboxylic acid cycle activity, increased synthesis of
pyrimidines and purines, and increased carbon flow to pathways associated with wall
teichoic acid and peptidoglycan biosynthesis relative to their daptomycin susceptible
counterparts. If these metabolic changes are necessary for the daptomycin non-
susceptible phenotype, then will altering the metabolome re-sensitize daptomycin non-
susceptible S. aureus to daptomycin?
Bio-summary:
Dr. Sommerville’s research focus is the elucidation of mechanisms by which bacteria
regulate virulence determinants in response to nutrient availability; specifically, my
interest is in the function of central metabolism in regulating pathogenesis. Currently, I
am an associate professor in the School of Veterinary Medicine and Biomedical Sciences
at the University of Nebraska-Lincoln. I earned a Ph.D. degree in Biology from the
University of Texas at Dallas under the supervision of Larry Reitzer. Prior to joining
UNL, I was a post-doctoral research fellow in the Laboratory of Human Bacterial
Pathogenesis at the Rocky Mountain Laboratories, in Hamilton, Montana.
15
Torey Looft, Ph.D. Research Microbiologist
USDA, ARS, NADC
Food Safety and Enteric Diseases Unit
Ames, IA
Adjunct Assistant Professor
Veterinary Microbiology Dept.
Iowa State University, Ames, IA
Abstract:
Antibiotics modulate the microbiota and metabolomic profiles in turkey cecal
microbiomes
Many antimicrobial compounds currently being used in U.S. for disease prevention or
treatment and feed efficiency in food producing animals will be withdrawn from the
market in 2017, highlighting the need to define their mode of action and aid the search
for alternatives. Here we describe the effects of bacitracin methylene disalicylate (BMD),
a commonly used antibiotic feed additive, on turkey microbial communities and
metabolomes over 14 weeks. Two-hundred-forty poults were divided into three treatment
groups (no antibiotic control, sub therapeutic BMD (50 g/ton), and therapeutic BMD (200
g/ton)). After euthanasia, cecal contents were collected to characterize microbial
population shifts using high-throughput 16S rRNA gene sequence analysis and evaluate
global metabolomic profiling. Both concentrations of BMD had immediate and lasting
impacts on the microbiota structure, reducing species richness in the BMD-treated
animals. Metabolomic analysis identified 712 named biochemical, including overlap
between the metabolic profiles of the therapeutic and subtherapeutic treatments, with 75
metabolites differentially present from the control animals (q < 0.1). These included
markers for increased protein and dietary triglyceride catabolism, as well as microbial
metabolism of complex plant carbohydrates such as hemicellulose and pectin. While
many effects were sustained, some metabolic changes in the therapeutic group were
transient. For example, birds fed both concentrations of BMD had early reductions in
metabolic pathways associated with growth (i.e. protein and aromatic amino acids
catabolism products), while only the therapeutic group had reduced metabolites
associated with glycolysis. These temporal metabolic effects may be due to an early
antibiotic disturbance followed by partial recovery of bacterial function even in the
presence of continued antibiotics. Connecting the microbiome structure and metabolomic
response during antibiotic disturbance may improve microbiota modulation
strategies. Metabolic shifts within the bacterial community resulting from antibiotic
consumption may be related to “beneficial” microbiome functions that can be targeted to
improve animal health and production.
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Torey Looft, Ph.D. USDA, ARS, NADC
Ames, IA
Bio-summary:
Dr. Looft obtained his undergraduate degree in biology in 2001 and his master’s degree
in conservation biology in 2005 from Bowling Green State University in Bowling Green,
Ohio. In 2012, he was awarded his Ph.D. degree in Microbiology from Iowa State
University in Ames, Iowa. Dr. Looft is currently a Research Microbiologist with the
USDA, ARS, NADC, Food Safety and Enteric Diseases Unit in Ames, Iowa. He also is
an Adjunct Assistant Professor in the Department of Veterinary Microbiology at Iowa
State University, Ames, IA. Dr. Looft’s research focuses on the characterization of
turkey microbiomes and he investigates the effects of antibiotic feed additives on the
expression and transmission of fitness and antimicrobial resistance genes in intestinal
microbial populations. He also evaluates the effects of environmental and host influences
on gut bacterial ecological niches and Campylobacter jejuni control strategies.
17
Donald H. Burke, Ph.D. Professor
Department of Molecular Microbiology & Immunology
Joint Appointments in Department Biochemistry, Department Biological Engineering
University of Missouri
Columbia, MO 65211-7310
Abstract:
Overcoming aptamer-resistance in HIV-1 inhibition with broad-spectrum RNA aptamer
RNA aptamers that bind HIV-1 Reverse Transcriptase (RT) inhibit HIV-1 replication in
human cells. Because RT must interact with diverse nucleic acids during HIV-1 replication,
the genetic threshold for eliciting resistance to some aptamers may be high. To evaluate the
impact of RT-aptamer binding specificity on virus replication, we engineered proviral
plasmids to encode phylogenetically diverse RT within a constant HIV-1NL4-3 (Subtype B)
background. Viruses that were inhibited by pseudoknot aptamers from the F1Pk family were
rendered resistant by the previously recognized R277K point mutation, providing the first
demonstration of aptamer-specific resistance. Naturally F1Pk-resistant viruses were rendered
sensitive by the inverse K277R mutation, thereby establishing RT as the genetic locus
responsible for aptamer-mediated HIV-1 inhibition. In contrast, aptamers with 6/5 asymmetric
loop (“6/5AL”) or “UCAA” structural motifs exhibited broad-spectrum inhibition of
replication across the entire recombinant panel. Inhibition was only observed when virus was
produced in aptamer-expressing cells, but not in target cells, indicating that prior encapsidation
is required. The magnitude of HIV-1 suppression correlated with the number of encapsidated
aptamer transcripts per virion, especially at low expression levels, with saturation occurring
around 1:1 stoichiometry with RT. Encapsidation specificity for inhibitory RNAs suggests that
it likely arises from cytosolic encounter with dimerized GagPol during viral assembly. High
expression levels promoted non-specific encapsidation of control RNAs without inducing
inhibition. Overall, this study provides important new insights into HIV-1’s capacity to resist
aptamer-mediated inhibition, the utility of broad-spectrum aptamers to overcome that
resistance and molecular interactions that occur during viral assembly.
18
Donald H. Burke, Ph.D. University of Missouri
Columbia, MO
Bio-summary:
Dr. Burke obtained his B.A. degree in Chemistry/Biology from the University of Kansas in
1986. His formal training included Ph.D. studies in biophysical chemistry and microbial
genetics at the University of California, Berkeley (with John Hearst) 1986-1992 and
postdoctoral training in RNA biochemistry and aptamer selection at the University of
Colorado, Boulder (with Larry Gold) 1992-1998.
From 1998 until 2005 Dr. Burke was an Assistant Professor of Chemistry & Biology at
Indiana University in Bloomington, IN. He currently serves as Professor, in the Department of
Molecular Microbiology & Immunology, University of Missouri School of Medicine and
holds joint appointments in the Departments of Biochemistry and Biological Engineering. He
has been a Present Bond Life Sciences Center Investigator at University of Missouri since
2005 and is Associate Chair of Department of Molecular Microbiology & Immunology at the
University of Missouri School of Medicine.
Dr. Burke has a broad background in the biology and biochemistry of RNA, with emphasis on
1) understanding the limits of RNA function and 2) developing novel RNA molecules for
biomedical and synthetic biology applications. A long-term objective of his lab's research is to
develop RNA aptamers for control and study of viral pathogens, cancer, and host
immunological responses to infection. As PI of several projects funded by NIH, NASA, NSF
and private foundations, Dr. Burke’s group has developed extensive expertise and
infrastructure for carrying out work in this area. One current project (NIH) evaluates antiviral
aptamers for potential gene therapy treatment of HIV. Another current project (NASA) is
developing new ribozymes that directly interface with the small molecule metabolome. The
current project is part of a major new initiative to develop new aptamers that bind surface
markers on viruses and on defined subsets of cells to aid efforts to study, detect and treat
human and animal diseases.
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15th Annual Meeting
Oral Presentation Abstracts
Session III
Biochemistry/Structural Biology
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Christina R. Bourne, Ph.D. Assistant Professor, Biochemistry and Structural Biology
Department of Chemistry and Biochemistry
University of Oklahoma
Norman OK 73019
Abstract:
Toxin-Antitoxin Modules: Functions in Bacteria
The process of bacterial cell survival during these treatments is termed tolerance, and it
results from adaptation of the bacterial cell metabolism to limit its susceptibility to
antibiotics. Toxin-Antitoxin (TA) modules are found in many prokaryotes, and are
interacting cytosolic protein pairs that can mediate metabolic adaptation shifts. They
function as regulatory switches to mediate rapid metabolic changes by interacting with
essential cellular machinery. The Bourne lab is focusing on two areas: TA module
function in the opportunistic pathogen P. aeruginosa, and the RelE/ParE superfamily of
toxins. P. aeruginosa is capable of growth in a wide variety of environments, a
testament to its flexible metabolic programming. TA modules may mediate parts of this
flexibility, which hinder treatment of infections by promoting antibiotic tolerance. The
RelE/ParE superfamily, of which P. aeruginosa contains two members, has conserved
protein folds but disparate cellular functions. Our preliminary data indicate selected
members of this superfamily can carry out overlapping functions. Our insights provide
an alternative understanding for the fundamental role of TA modules in metabolic
regulation, particularly that of P. aeruginosa, and the contribution to antibiotic tolerance
and chronic infections.
22
Christina R. Bourne, Ph.D. University of Oklahoma
Norman OK
Bio-summary:
Dr. Bourne, an Oklahoma native, completed her Ph.D. with Dr. Allen Edmundson at the
Oklahoma Medical Research Foundation, where she studied the structure and function of
human antibodies in diseases like Multiple Myeloma. She then worked with Dr. Adam
Zlotnick at the OU Health Sciences Center as a postdoc. In this position, Christina
mapped the binding site interactions of novel anti-viral inhibitors interacting with the
Hepatitis B virus capsid. This served as proof-of-principle of this approach, which is
now the focus of a spin-off company, Assembly Biosciences Inc. Christina then moved to
Stillwater, OK where she worked as a Research Scientist in the Barrow group at OSU.
She worked with local chemists to develop improved bacterial dihydrofolate reductase
inhibitors, and they succeeded in deriving a new compound that would work against B.
anthracis. During this time Dr. Bourne also worked on the screening pipeline and
developed assays to identify cellular targets of small molecules.
In January of 2014 she started her independent lab at the University of Oklahoma in the
Department of Chemistry and Biochemistry, and since joining she became a member of
the NIH CoBRE in Structural Biology. The focus of the lab is to understand how
bacterial cells can survive antibiotic treatments and persist to form chronic infections.
Dr. Bourne’s lab takes biochemical, microbiological, genetic and structural approaches to
find the right questions that may someday lead to those answers. For more information,
please see the lab website:
http://faculty-staff.ou.edu/B/Christina.Bourne-1/CRBourne/Welcome.html
23
Brian V. Geisbrecht, Ph.D. Professor
Departments of Biochemistry & Molecular Biophysics
Kansas State University
Manhattan, KS
Abstract:
Toward a Structural Understanding of the Type-III Secretion Needle Tip: Progress
and Challenges
Type Three Secretion Systems (T3SS) are essential virulence determinants in many
Gram-negative bacterial pathogens. Despite their significant sequence diversity, T3SS
share many anatomical, biochemical, and functional attributes with one another. While
the most prominent feature of the T3SS is an elongated proteinaceous tube that resembles
a molecular syringe, an equally important arrangement of proteins is found at the tip of
the maturing needle. This so-called ‘tip complex’ assembles in a step-wise process and is
required for completing the membrane-spanning conduit between the bacterial cytoplasm
and the target host cell. As part of a long-term collaboration, my laboratory has been
studying the components of the T3SS needle tip complex from Shigella flexneri in order
understand the structural features of its components and how these structures define - and
change - throughout the various steps of needle maturation. Work will be presented that
describes how the nascent needle tip protein, IpaD, senses small-molecule environmental
cues, and changes conformation allowing for subsequent exposure of the first translocator
protein, IpaB, to the tip of the maturing needle. The results of structural studies on a
major subdomain of IpaB will also be presented, as will the implications of these
structures on our understanding of how T3SS translocators may have evolved. Finally,
recent work toward the structure of progressively larger portions of IpaB will be shared.
Particular attention will be paid to the approaches needed to study a protein like IpaB,
which transitions from a chaperone-bound, soluble monomer to a membrane-spanning
tetramer during its life cycle.
24
Brian V. Geisbrecht, Ph.D. Kansas State University
Manhattan, KS
Bio-summary:
Dr. Geisbrecht is a Professor in the Department of Biochemistry & Molecular
Biophysics at Kansas State University in Manhattan, Kansas. He received his B.S. in
Chemistry/Biochemistry in 1996 from Saint Vincent College, Latrobe, PA and his Ph.D.
in Biological Chemistry in 2004 from The Johns Hopkins University School of Medicine,
Baltimore, MD.
Dr. Geisbrecht spends the majority of his time as a principal investigator in applying a
structure-intensive, but broadly interdisciplinary approach to analysis of innate immune
evasion by Staphylococcus aureus. Working both independently and through synergistic
collaborations, his group has made significant contributions to understanding
mechanisms within this process at a molecular level. They have published nearly twenty
crystal structures of S. aureus innate immune evasion proteins, both free and bound to
their targets, and have extensively used solution structural approaches such as NMR,
small angle X-ray scattering, and Hydrogen/Deuterium exchange mass spectrometry
(HDX-MS) in their work. Dr. Geisbrecht’s laboratory has actively investigated different
means for transforming the wealth of structural, functional, and mechanistic information
on the complement system into targeted anti-inflammatory inhibitor development since
2013.
Aside from these areas described above, Dr. Geisbrecht has maintained an ~8 year
collaboration with the groups of Bill and Wendy Picking (University of Kansas) that has
centered upon understanding the structural/function and mechanism of maturation for the
Shigella flexneri Type-III Secretion Needle. Their collaborative work in this area has
helped define the conformational changes that allow for step-wise assembly of the
maturing needle tip complex in response to environmental cues. Dr. Geisbrecht’s
laboratory also provided the initial high-resolution structural information on a large
proteolytic fragment of the membrane-spanning translocator protein, IpaB, and its
homolog, SipB, from the closely related bacterium Salmonella typhimurium. Beginning
in 2013, his group’s work in this field has focused on developing approaches and reagent
systems that will ultimately provide structure/function information on increasingly larger
portions of IpaB, including the full-length protein.
Understanding the mechanisms through which microbes subvert and inhibit the innate
immune response is a significant and compelling problem that simultaneously impacts
several fields in the life and health sciences. Meeting at the crossroads of bacterial
pathogenesis and immunology, these studies have the potential to change how we think
about the co-evolution of pathogens and their hosts. They also provide new information
on how to prevent and treat infectious disease, and offer a unique perspective into the
regulation/inhibition of inflammatory networks. These latter goals are particularly
significant from the perspective of translational medical research, given the widespread
nature of antibiotic resistance among common pathogens and the limited panel of
therapeutic options for targeted manipulation of the human innate immune/inflammatory
system.
25
Michael L. Barta, Ph.D. Research Assistant Professor
Higuchi Biosciences Center
University of Kansas
Abstract:
Using nanobodies to investigate the T3SS needle-tip complex of Shigella flexneri
Numerous Gram-negative pathogens are capable of causing infections in eukaryotes through the use of a
unidirectional protein delivery apparatus termed a type III secretion system (T3SS). The type III
secretion apparatus (T3SA) is comprised of a basal body spanning both bacterial membranes, an
extracellular filamentous needle and a tip complex located at the distal end of the needle responsible for
assembly of the pore-forming translocon. Shigella spp., the causative agents of bacillary dysentery or
bloody diarrhea, utilize a T3SS to invade the colon, spread from cell to cell and cause a productive
infection. The tip complex of S. flexneri is comprised of invasion plasmid antigen (IpaD) D, which
initially regulates the T3SS secretion state and during active invasion, provides a physical platform for
IpaB and IpaC to form a pore in the host membrane. The tip complex currently represents a promising
point for therapeutic intervention across multiple important pathogens. Here we have generated a panel
of single-domain antibodies (VHHs or nanobodies) that recognize distinct epitopes within IpaD. These
VHHs display a diverse ability to recognize the in situ tip complex and modulate the infection of
cultured mammalian cells by Shigella. In tandem with this data, structural elucidation of several IpaD-
VHH complexes has enabled novel insights into tip complex formation and assembly, with potential
applications across other T3SS-possessing pathogens.
Bio-summary:
Dr. Barta is currently a Research Assistant Professor of the Higuchi Biosciences Center working at the
Kansas Vaccine Institute (KVI) at the University of Kansas. Dr. Barta has extensive research
experience studying mechanisms of virulence in pathogenic bacteria using structural biology
approaches. He obtained his Ph.D. in Structural Biology from the University of Missouri Kansas City in
2011 under the mentorship of Brian Geisbrecht, studying the structure of type III secretion system
components. He then spent 3 years as a Postdoctoral Researcher in Dr. Scott Hefty’s lab at the
University of Kansas, studying proteins of unknown function from Chlamydia trachomatis.
26
William D. Picking, Ph.D. Foundation Distinguished Professor
Department of Pharmaceutical Chemistry
Director, Higuchi Biosciences Center
Director, Kansas Vaccine Institute
University of Kansas
Lawrence, KS
Abstract:
Structure-Function Relationships within the Shigella Type III Secretion Apparatus
Type III secretion systems (T3SS) are nanomachines used by some Gram-negative
bacteria to communicate directly with targeted eukaryotic cells. A typical T3SS is
comprised of a basal structure (a syringe) that spans the entire bacterial envelope and
serves as the anchor for an external needle that possesses a tip complex (TC). The TC
recognizes host cell surfaces and, upon sensing contact, inserts the proteins into the host
membrane to create the translocon pore. It is through the translocon that effector proteins
are injected to alter normal cellular functions. The Shigella flexneri T3SS senses contact
with human intestinal cells to inject effector proteins that promote pathogen entry as the
first step in causing life threatening bacillary dysentery (shigellosis). Our group was the
first to identify the components of the Shigella TC and to work out the events that occur
within the TC that lead to induced secretion. That work has evolved into an ongoing
investigation of the biochemistry, structure and function of the translocon pore
component invasion plasmid antigen B (IpaB) which is anchored to the T3SS needle tip
by IpaD. In parallel, we are exploring the structure and function of a large cytoplasmic
complex that we propose forms the sorting platform at the base of the envelope-spanning
basal body. The sorting platform is essential for effector protein selection and needle
assembly, but it remains largely uncharacterized. We are working with other groups to
use high throughput cryo-electron tomography to visualize the T3SS of Shigella flexneri
in situ so that we can develop mechanistic models of Shigella type III secretion function
and activation.
27
William D. Picking, Ph.D. University of Kansas
Lawrence, KS
Bio-summary:
William D. Picking received his B.S. in Microbiology (Biology) from Kansas State
University in 1984 and then joined the laboratory of Dr. David Paretsky at The
University of Kansas to complete his Ph.D. in Microbiology in 1989 working on host
responses to infection by Coxiella burnetii. He then became a postdoctoral fellow with
Boyd Hardesty in the Department of Chemistry at the University of Texas at Austin to
develop skills in protein chemistry and the use of biophysical methods for studying
protein structure and function. Dr. Picking’s first faculty position was in the Department
of Biology at Saint Louis University in 1992 where he started his current line of
investigation on the molecular basis for the pathogenesis of Shigella flexneri. He
returned to The University of Kansas in 1999 to become faculty in the Department of
Molecular Biosciences where his research program expanded significantly. In 2009, he
became head of the Department of Microbiology and Molecular Genetics at Oklahoma
State University where he oversaw a tripling of undergraduate enrollment, a 50%
increase in graduate student enrollment and a 50% increase in research funding. In 2014,
Dr. Picking once again headed back to Kansas when he was hired at The University of
Kansas as the third of 12 Foundation Professors and joined the faculty of the Department
of Pharmaceutical Chemistry. He is currently developing the new Kansas Vaccine
Institute and is an advisor for its affiliated Immunology Core Laboratory. He now also
serves as Director of the Higuchi Biosciences Center which oversees the biomedical
research effort at the KU-Lawrence campus. Dr. Picking continues research on Shigella
for which he has been funded by the NIH since 1997 and he works closely with Dr.
Wendy Picking who is developing novel vaccine strategies for bacterial pathogens that
are serious causes of diarrheal disease in the developing world.
28
29
15th Annual Meeting
Poster Abstracts
Session IV
Poster Presentations
30
31
15th Annual Great Plains Infectious Disease Meeting
University of Kansas - Lawrence Kansas
November 4 - 5, 2016
SESSION IV: POSTER PRESENTATIONS
Poster #
Last Name First Name Abstract Title
1 Allen Noah Identification of Chlamydial Extrusions Shed from the Murine Cervicovaginal Tract
2 Arizmendi Olivia Correlates of Protection Induced by a Serotype‐Independent Vaccine Against Shigellosis: The Role of Dendritic Cells in a Mouse Model
3 Ayers Victoria North American Culex Species Mosquitoes were Refractory to Zika Virus
4 Eleshy Rawan Detection and Characterization of Antibiotic Resistant S. aureus from Cystic Fibrosis Patient Isolates
5 Eslick Carley Characterization of the Role of the BAS0384‐390 Operon Genes in Affecting Assembly of Exosporium Proteins of Bacillus anthracis
6 Gallaway Erin Silver(I) Cyanoximates as Novel Antimicrobials
7 Gelhaus Carl Reduced Mass of Pups Born to Zika Virus Infected Mice
8 Hermanas Timothy Cutaneous anthrax modeling in Sprague Dawley rats and Hartley Guinea pigs
34 Hinshaw Kara Quorum Sensing Control of Antibiotic Resistance Protects Cooperating Bacterial Cells during Interspecies Competition
9 Hossain Mohammad Evaluation of Antibody Response against Bovine Viral Diarrhea Virus (BVDV) Recombinant Antigens Generated from Envelope Glycoproteins
10 Johnson David Computational Chemical Biology Core, a Chemical Biology of Infectious Disease COBRE Core Laboratory
11 Jordan Lorne RNPP Family Transcription Factors in Enterococcus faecalis
12 Kayastha Biraj Calcium Binding Protein; EfhP; Plays Role in Virulence of Pseudomonas aeruginosa
13 Klaus Jennifer An Antibiotic‐Activated LuxR Family Quorum Sensing Regulator in Burkholderia pseudomallei
14 Kumar Prashant Characterization and Protective Efficacy Assessment of Potential Subunit Vaccine‐S1S2 Against Salmonella enterica
15 Li Jiaqin High Resolution Structures of the Regulatory Domain of the AraC Family Transcriptional Activator RhaR
16 Liang Lingfei High‐Resolution Structure of Bacteriophage Sf6 Tail Adaptor Protein gp7 Reveals the Conformational Switch that Mediates the Sequential Assembly of the Phage Tail
17 Ma Minh Degrading Chlorinated Dioxins Using a Bacillus thuringiensis Spore Expression System
18 Machen Alexandra Optimization of Sample Preparation and Initial CryoEM Structure of Anthrax Toxin with Singly Bound Lethal Factor Inserted into a Nanodisc Bilayer
32
Poster #
Last Name First Name Abstract Title
19 Massa Nicole Development of a High Throughput Assay to Identify Inhibitors of Oligopeptide Permeases in Enterococcus faecalis
20 ONeil Pierce Investigating the Structure of Tetanus Neurotoxin using Electron Microscopy
21 Park So Lee Experimental Infection of North American Sus scrofa domesticus with Japanese Encephalitis Virus
22 Parthasarathy Srivatsan Development of a Drip‐Flow Biofilm Competition Assay for Screening Enterococcus faecalis Mutants
23 Paudyal Anuja Polymorphisms in the PrP prion protein gene in domestic pigs from the FHSU farm
24 Perera Chamani The Synthetic Chemical Biology Core (SCB): A Resource for Research in Chemical Biology
25 Rogers Rendi Examining Calcium Binding in the EF‐Hand Protein; EfhP; Regulating Calcium‐Dependent Virulence in Pseudomonas aeruginosa
26 Roy Anuradha Infectious Disease Assay Development Core: High Throughput Screening Laboratory at the University of Kansas
27 Russ Breeanna The Effect of Environmental Factors on Swarming Motility in a Human Pathogen Pseudomonas aeruginosa
28 Sah Prakash Chlamydia trachomatis Manipulation of Protein Kinase C
29 Starr William Antibiotic Resistance of Pseudomonas aeruginosa Recovered From Cystic Fibrosis Patients
30 Villanueva Cecilia Bactericidal Activity of Immunized Animal Sera Against Shigella and Salmonella
31 Wang Gaochan Identification of Enterotoxigenic Escherichia coli Secreted Protein That Prevents TNF‐induced NF‐B Activation in HCT‐8 Cells
32 Wu Miaomiao Do T3SS effectors other than NleH1 inhibit RPS3 nuclear translocation?
33 Yang Yang Vaccinating with Conserved Escherichia coli Antigens Does Not Alter the Mouse Intestinal Microbiome
33
Abstract #1
Identification of Chlamydial Extrusions Shed from the Murine Cervicovaginal Tract
Noah A. Allen1, Amanda R. Behar2, Erika I. Lutter2, and Jennifer H. Shaw1
Department of Integrative Biology, Oklahoma State University, Stillwater, OK, USA1;
Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA2
Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and
preventable blindness. Infection is often unrecognized, re-infection is common and,
whether treated or not, frequently leads to permanent complications, such as pelvic
inflammatory disease, ectopic pregnancy, tubal infertility and an increased risk for cervical
cancer. The murine cervicovaginal infection model is established for the study of
Chlamydial infection, but with variations in infectious dose, chlamydial strains, mouse
strains and timing of assessment it can be difficult to compare results of different studies.
As such, a side-by-side comparative study of different Chlamydial strains in the same
infection model utilizing identical conditions is essential for an appropriate comparison.
Within this study three Chlamydial strains were compared for infectivity and sequelae in
the cervicovaginal infection model using C3H/HeJ mice. We characterized the (i) time
course of infection and morphology of Chlamydia shed, (ii) mucosal and systemic immune
response to infection, and (iii) gross and histopathology following clearance of active
infection. A key finding herein is the first identification of chlamydial extrusions shed from
host cells in an in vivo model. Extrusions, a recently appreciated mode of host-cell exit
and potential means of dissemination, had been previously observed solely in vitro. The
results of this study demonstrate that chlamydial extrusions exist in vivo thus warrant
further investigation to determine their role in chlamydial pathogenesis.
34
Abstract #2
Correlates of Protection Induced by a Serotype-Independent Vaccine Against
Shigellosis: The Role of Dendritic Cells in a Mouse Model
Olivia Arizmendi1, Melissa Pressnall2, Wendy L. Picking2, Francisco J. Martinez-
Becerra1
1Higuchi Biosciences Center and 2Department of Pharmaceutical Chemistry, University
of Kansas, Lawrence, KS
Diarrheal diseases are a major cause of morbidity and mortality worldwide and are
common in settings where there is inadequate sanitation, poor hygiene and contaminated
water. In industrialized countries, diarrheal diseases are more commonly seen as a result
of the contamination of food sources. In a multicenter study conducted in Africa and Asia,
it was observed that infections with rotavirus, Shigella spp., Cryptosporidium spp. and ST-
ETEC (E. coli) accounted for the majority of cases of moderate-to-severe diarrhea in
children.
No commercially available vaccine exists against shigellosis, and immunity to the
pathogen is commonly serotype-restricted. The variety of serotypes (over 50 serotypes
across 4 different species of Shigella), along with the geographical overlap between them
highlight the need for a broadly protective vaccine. This need drives our efforts on the
development of a subunit vaccine against this enteric disease.
Our research group has previously shown the Type Three Secretion System (T3SS)
proteins IpaB and IpaD are protective antigens in mouse models of infection. These
proteins are highly conserved among all Shigella serotypes and are essential to virulence
and pathogenesis.
In order to optimize vaccine formulation we generated a fusion protein (DBF) that includes
IpaD and IpaB in the same polypeptide chain and used the adjuvant dmLT. Testing of
different administration routes, as well as other related experiments, revealed the protection
elicited by our vaccine formulation did not correlate with antibody titers and it did with T
cell responses. Therefore, we have proposed a mechanism of protection based on
recognition of the vaccine by APCs of the immune system and presentation to T cells,
which would mimic some of the responses found upon natural infection that allow bacterial
clearance in the gut.
35
Abstract #3
North American Culex Species Mosquitoes were Refractory to Zika Virus
Victoria B. Ayers,1,2 Yan-Jang S. Huang,1,2 Amy C. Lyons,1,2 Isik Unlu,3,4 Barry W.
Alto,5 Lee W. Cohnstaedt,6 Stephen Higgs,1,2 and Dana L. Vanlandingham1,2
1 Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, Kansas. 2 Biosecurity Research Institute, Kansas
State University, Manhattan, Kansas. 3 Center for Vector Biology, Rutgers University,
New Brunswick, New Jersey. 4 Mercer County Mosquito Control, Trenton, New Jersey. 5
Florida Medical Entomology Laboratory, University of Florida, Vero Beach, Florida. 6
Arthropod-Borne Animal Disease Research Unit, Agriculture Research Service, United
States Department of Agriculture, Manhattan, Kansas.
Zika Virus (ZIKV) is an emerging flavivirus that has caused significant disease burden in
the Americas since 2015. Although it is well-accepted that ZIKV is vectored by
mosquitoes, specifically Aedes aegypti for its urban cycles, viral isolates of ZIKV from
Culex species have raised concern on the potential expansion in the number of competent
vector species. Therefore, determining the vector competence of medically important
Culex species mosquitoes has become a top priority in studying the vector biology of ZIKV
as it is expected to provide the critical information needed for the formulation of vector
control strategies. In this study, Culex quinquefasciatus from Vero Beach, FL, Culex
pipiens from Anderson, CA, and Culex pipiens from Ewing, NJ were evaluated for their
competence for ZIKV after per os infection. The results demonstrated the Culex species
mosquitos tested in our study were highly refractory to ZIKV. Our results suggest that
ZIKV will not be transmitted by these species in North America. Vector control strategies
for ZIKV should remain concentrated on Aedes species mosquitoes, especially Aedes
aegypti and Aedes albopictus.
36
Abstract #4
Detection and Characterization of Antibiotic Resistant S. aureus from Cystic
Fibrosis Patient Isolates
Rawan G. Eleshy1, Nighat F. Mehdi2, and Erika I. Lutter1
1Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA; 2Oklahoma Cystic Fibrosis Center, University of Oklahoma Health
Sciences Center, Oklahoma City, OK, USA
Cystic fibrosis (CF) is a common genetic disease caused by a mutation in the cystic fibrosis
transmembrane conductance regulator gene (CFTR). Mutations within this gene inhibit the
function of the chloride ion channels across epithelial membranes. This leads to the
formation of thick mucus within the lung airways of CF patients. Therefore, the CF lung
becomes an excellent environment for bacterial colonization. S. aureus is the first pathogen
to colonize the lungs and tends to persist throughout the lives of CF patients. S. aureus is
known for its ability to develop resistance against antibiotics. Antibiotic resistance is one
of the biggest problems faced in medicine today. This study aims to detect and characterize
the resistance of S. aureus obtained from CF patients of various age groups to a panel of
clinically relevant antibiotics. Based on findings from previous studies, there are nine
antibiotic resistance genes in S. aureus that have been correlated with CF patients. Using
PCR amplification, we checked if any of these resistance genes are present in the CF
isolates. In addition, we performed antibiotic susceptibility tests to determine if these
isolates exhibit a resistant phenotype. Minimum inhibitory concentrations (MIC’s) of each
antibiotic to each isolate were determined to further confirm resistance. In conclusion, the
presence of resistance genes and susceptibility to antibiotics differ among CF patients. CF
isolates showed both susceptibility and resistance to the tested antibiotics, but the
percentage of resistant isolates was higher. The interesting finding was that resistance to
antibiotics, in some isolates, did not correlate with the presence of resistance genes. The
lack of resistance genes in isolates that showed a resistant phenotype to antibiotics suggests
that S. aureus is using other mechanisms to acquire resistance. This study shines the light
on understanding S. aureus as a CF pathogen and its resistance within the CF lung. This
will aid in enhancing treatment options for CF patients to help them live longer and more
productive lives.
37
Abstract #5
Characterization of the Role of the BAS0384-390 Operon Genes in Affecting
Assembly of Exosporium Proteins of Bacillus anthracis
Carley Eslick1,2, Hsin-Yeh Hsieh2,3, George C. Stewart2,3
Department of Biology1, Bond Life Sciences Center2, Department of Veterinary
Pathobiology3, University of Missouri, Columbia, MO, USA
Anthrax is a highly fatal disease primarily of sheep, cattle, goats, and wild ruminants. It is
caused by the Gram-positive, rod-shaped bacterium, Bacillus anthracis. Endospores are
the infectious form of Bacillus anthracis. The outer layer of this infectious spore is the
exosporium. It is composed of a basal layer and an external hair-like nap that are made up
largely of Bacillus collagen-like protein, BclA.
In a previous study from our laboratory, a mutant B. anthracis strain with a deletion of the
seven gene BAS0384-0390 operon exhibited defects in the exosporium incorporation of
BclA. To determine if any of the seven proteins encoded in the operon are structural
components of the exosporium, or are needed for proper exosporium assembly, we
analyzed the expression of each gene from BAS0384-0390 fused with mCherry reporter
gene during the sporulation process. BAS0386, BAS0387, and BAS0389 fluorescent
fusion proteins were spore-associated in both the wild-type Sterne strain and the
BAS0384-0390 deletion strain as measured by epi-fluorescence microscopy. The results
suggest that these genes encode proteins that are spore structural components. To verify
that these are spore proteins, isolated spores were subjected to detergent extraction and
western blot analysis using antiserum raised against the mCherry component of the fusion
proteins and anti BclA antibody. The results demonstrated the presence of these three
proteins in the outer layer of the spore, but not on the exosporium layer.
38
Abstract #6
Silver(I) Cyanoximates as Novel Antimicrobials
Erin Gallaway1, Treyon Grant1, Snow Popis2, Nikolay Gerasimchuk2
and Marianna A. Patrauchan1
1Oklahoma State University, Department of Microbiology 2Missouri State University
Biofilms are microbial communities that grow on surfaces and are embedded into
extracellular polymeric matrices, which consist of polysaccharides, proteins, and
extracellular DNA. Biofilms induce multiple virulence factors and horizontal gene transfer
making them more resistant to antimicrobials and host factors. The ability to form a biofilm
plays a major role in the development of infections. The increase in microbial resistance
introduces an important clinical challenge, particularly in cases associated with implants,
which have a high predisposition for developing infections. This requires the development
of alternative antimicrobial practices to prevent infections. We have synthesized a series
of novel silver(I) cyanoximates that have remarkable resistance to high intensity visible
light, UV, and heat with a broad range of water solubility. The goal is to be able to use
these compounds as potential additives to implant materials, for example as a part of UV–
radiation-curable polymeric glues used during joint replacement. We have incorporated
these compounds into polymeric composites, and tested their antimicrobial activities
against planktonic and biofilm growth of several diverse human pathogens, such
as Pseudomonas aeruginosa, the most frequent gram negative agent infecting implants,
and Staphylococcus aureus, which is highly resistant to multiple antibiotics. Biofilm
quantification by crystal violet biofilm assay followed by scanning electron microscopy
confirmed the high antimicrobial potential of the compounds, particularly AgPiCO yellow
and AgPiCO red. Currently we are testing possible synergistic effect of the compounds in
combination with tobramycin and trimethoprim, the antibiotics commonly used to treat P.
aeruginosa and S. aureus infections.
39
Abstract #7
Reduced Mass of Pups Born to Zika Virus Infected Mice
Cheryl Nevins1, Macy Potts2, H. Carl Gelhaus1
1Medical Countermeasures Division, MRIGlobal, Kansas City, MO, USA 2 College of
Bioscience, Kansas City University of Medicine and BioScience, Kansas City, MO, USA
Zika virus (ZIKV) is an emerging arbovirus, first isolated in Uganda in the 1950’s. ZIKV
was considered of minor importance, as 80% of infected patients are asymptomatic and the
infection being self-limiting in symptomatic patients. In June 2015, ZIKV autochthonous
infections were reported for the first time in Brazil. Co-incident with the Brazil ZIKV
outbreak, a 20 fold increase in microcephaly prevalence was reported and in 2016, ZIKV
has spread throughout Central America. As of October 5, 2016, 105 locally-acquired and
3,712 travel-associated ZIKV infections have been reported in the United States, with all
locally-acquired cases occurring in Florida; all 50 states having at least one travel-
associated infection. It is now apparent that ZIKV is associated with birth defects and given
the rapid spread of ZIKV through the Americas, medical countermeasures are desperately
needed. Animal models of ZIKV infection are critical to the development of medical
countermeasures to eliminate or reduce the morbidity associated with infection. There has
been a rapid response to develop animal models but BALB/c, C57Bl/6, and SJL mice
infected through different routes have been examined with different results. We describe
initial systematic investigations of ZIKV infection in pregnant mice and the impact on
pups. Previous work showed non-pregnant Swiss Webster mice have a transient viremia
following intraperitoneal (IP) infection with ZIKV. We infected pregnant Swiss Webster
dams with diluted ZIKV culture supernatants IP on embryonic development day 7. On
post-natal day 0 (P0) and P7 and P14, each pup's body weight was measured. Brains were
harvested and weighed from selected pups. Pups from ZIKV infected mice had lower body
weight on P7 and P14 compared to pups from uninfected dams, but no difference on P0.
Also, there was no evidence of decreased brain weight between pups from infected and
uninfected dams without a corresponding decreased body weight. These data suggest that
this model of restricted mouse pup growth within the first two weeks’ post-partum
following vertical transmission from dam to pup may be suitable for MCM testing and
continues to be refined.
40
Abstract #8
Cutaneous anthrax modeling in Sprague Dawley rats and Hartley Guinea pigs.
Timothy M. Hermanas1,2, Michael K. Fink1, Sarah A. Hansen1 and George C. Stewart1,2
1Department of Veterinary Pathobiology and 2Bond Life Sciences Center,
University of Missouri, Columbia, MO
Anthrax is an infection caused by the bacterium Bacillus anthracis. The type of disease can
vary depending upon the route of exposure. The three major forms of anthrax occur due
to inhalation, ingestion, and cutaneous exposure to the spores. The most lethal forms of
the disease are inhalational and gastrointestinal anthrax, which lead to death of the host
within 48-72 hours due to toxemia. With humans, 95% of naturally occurring cases are
cutaneous. To date, the modeling of cutaneous anthrax has been limited due the lack of an
animal model that accurately reflects human cutaneous anthrax. A murine model of
cutaneous anthrax was developed using the toxigenic, yet non-encapsulated Bacillus
anthracis Sterne strain. However, this model differs from clinical cutaneous anthrax in
that it has a high mortality rate and fails to produce the characteristic skin lesion (malignant
pustule) seen with human patients. We conducted a pilot study to investigate new
cutaneous anthrax models using Sprague Dawley rats (relatively resistant to anthrax
infections) and Hartley Guinea pigs (used extensively in vaccine protection studies) and
the fully virulent B. anthracis Ames strain. We shaved and applied spores to both species,
using the same method, and had differing results. While the Sprague Dawley rats were
resistant to the cutaneous infections, the Hartley Guinea pigs succumbed to systemic
infection of B. anthracis.
41
Abstract #9
Evaluation of Antibody Response against Bovine Viral Diarrhea Virus (BVDV)
Recombinant Antigens Generated from Envelope Glycoproteins
Mohammad M. Hossain1, and Raymond R. Rowland1
1Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas
State University, Manhattan, KS, USA
Introduction: Bovine viral diarrhea virus infections are enzootic in the cattle population and one
of the world’s most costly diseases. It continues to cause significant economic losses to the beef
and dairy industries worldwide. For effective disease surveillance, rapid and sensitive assays are
required. A novel assay was developed for the rapid detection of antibody against pestiviruses, i.
e., bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) by using fluorescent
microsphere based immunoassay (FMIA). We describe here the construction of fourteen
recombinant antigens from BVDV glycoproteins E2 and Erns and antibody responses to these
antigens were evaluated. The aim of the present study was to determine the serological responses
against BVDV E2 and Erns antigens using multiplex FMIA.
Methods: E2 and Erns play an important role in immune response. Full length E2 (1-375 aa) and
Erns (1-233 aa) have been fragmented into 7 and 5 small pieces respectively. The recombinant
protein fragments were expressed in BL-21 (DE3) Escherichia coli and purified proteins were
covalently coupled to Luminex MagPlex® polystyrene, carboxylated microsphere beads. The
target antigens were assembled into a single multiplex and tested for antibodies in cattle infected
with BVDV. The results were reported as mean fluorescent intensity (MFI) and then converted to
positive per sample (S/P) ratio.
Results: The results demonstrate that all the protein fragments of E2 (8 including full length) and
Erns (6 including full length) were highly responsive to IgA, IgG and IgM in BVDV infected cattle
sera. Antibody response to BVDV antigens were IgG> IgM> IgA. The use of non-species-specific
conjugates such as Protein A (PA), G (PG), and (PA/G) derived from bacterial cell wall in place
of species specific IgG showed E2 and Erns specific antibody response with very low negative
background. However, the full length E2 and Erns showed full potency of antibody response
relative to the fragmented proteins. The results clearly indicate that the assay provides a novel,
robust and highly sensitive and specific method for improved detection of antibody specific to
BVDV antigens.
Conclusions: In this study, multiplex detection of BVDV IgA, IgG, and IgM in cattle is a novel
confirmatory diagnostic approach. Further, in the absence of species specific reagents the
incorporation of proteins PA, PG, and PA/G provide a suitable substitute and are useful in a
veterinary diagnostic laboratory.
42
Abstract #10
Computational Chemical Biology Core, a Chemical Biology of Infectious Disease COBRE
Core Laboratory
David K. Johnson
Computational Chemical Biology Core Laboratory
University of Kansas, Lawrence, KS, USA
The University of Kansas Computational Chemical Biology Core (CCB) provides the
computational resources and expertise to enhance the productivity of researchers studying
infectious diseases. The CCB is able to provide or assist with virtual screening, protein-small
molecule docking, binding site prediction, protein modeling and design, prediction of protein
stability changes upon mutation, fragment based probe design, as well as preparation of
presentation graphics. The core utilizes the KU Community Cluster at the Advanced Computing
Facility for its high-performance computing needs. The KU Community Cluster offers 458
compute nodes with a total of 8,568 compute cores, including 17 nodes that offer GPU-accelerated
computing. The CCB specializes in initial hit identification of non-traditional drug targets such as
protein-protein or protein-RNA interfaces by offering high-throughput virtual screening via pocket
optimization with exemplar screening at protein-protein interfaces and hotspot pharmacophore
mimicry of protein-RNA interactions.
The CCB works in collaboration with the Molecular Graphics and Modeling Laboratory.
43
Abstract #11
RNPP Family Transcription Factors in Enterococcus faecalis
Lorne D. Jordan, Nancy Schwarting, Lynn E. Hancock
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, United States
Infections caused by enterococci are a serious threat to human health, as they represent one of the
three most common hospital-acquired pathogens in the United States and around the world. A
dichotomy exists between the many benefits of enterococci as a commensal organism and the
harmful effects caused to its host in an infective state. Due to its opportunistic nature, enterococci
readily transition from being commensals to pathogens. We recently discovered a peptide
transporter, PptAB, involved in the export of small peptide pheromones that induce a mating
response in plasmid harboring donor cells. This same peptide transporter was also shown to
contribute to biofilm formation and recent evidence suggests that it contributes to virulence in a
catheter-associated UTI (CAUTI) model. In Gram-positive bacteria, the RNPP family of
transcription factors are known to contribute to a variety of cellular processes, including biofilm
formation and pathogenesis and their activity is regulated by binding to small peptides. Here we
identify five uncharacterized and predicted RNPP homologs in E. faecalis V583 by querying the
genome for characteristic elements of the RNPP family. We generated gene deletion mutants for
each predicted RNPP homolog and examined these mutants for affects on biofilm development
and compared their role in pathogenesis using a mouse model of a CAUTI by using a mixed
infection of fluorescently labeled parental and mutant strains. We found that one such homolog
displayed an increase in biofilm biomass and resulted in a significant increase in catheter and
bladder colonization compared to the parental strain.
44
Abstract #12
Calcium Binding Protein, EfhP, Plays Role in Virulence of Pseudomonas aeruginosa
Biraj B. Kayastha1, Rendi Rogers1, Mariette Barbier2 and Marianna Patrauchan1
1Oklahoma State University Stillwater, OK 2West Virginia University School of Medicine, Morgantown, WV
Pseudomonas aeruginosa, an opportunistic pathogen, is the main cause of chronic lung
infection and mortality in the individuals with cystic fibrosis. Earlier, we have shown that
its virulence and antibiotic resistance is induced by calcium (Ca2+). Also, we identified a
putative Ca2+-binding calmodulin-like protein EfhP containing two EF-hand motifs. We
characterized and established role of the protein in Ca2+-induced plant infectivity,
production of pyocyanin, formation of biofilm, oxidative stress resistance, and intracellular
Ca2+ homeostasis. Based on bioinformatic analysis, we predicted that EfhP is anchored into
the inner membrane facing its EF-hands into the periplasm and that it preferentially binds
Ca2+. We hypothesize that EfhP senses environmental Ca2+ and upon binding Ca2+
undergoes conformational changes and regulates cellular responses. By using wax worm
and murine macrophage infection model, we showed that EfhP contributes to the
intracellular survival and virulence of the pathogen. Currently, to understand the
relationship between Ca2+ and EfhP, we are aiming to study the changes in the
transcriptional profile of efhP in response to Ca2+and other host factors. For this, the 300
bp region upstream of efhP with a predicted promoter sequence has been cloned upstream
of the promoterless lux operon in the reported plasmid pMS402. The temporal effect of
Ca2+on the promoter activity will be measured at the various levels of the ion. This
observation will be further confirmed by RT-qPCR. To determine the effect of the
intracellular Ca2+ signaling on the transcriptional level of efhP, we will also measure the
efhP promoter activity in the mutants with disrupted Ca2+ transporters early shown to have
either increased or abolished intracellular Ca2+ responses. We expect this study to establish
the role of EfhP in Ca2+ signaling in P. aeruginosa.
45
Abstract #13
An Antibiotic-Activated LuxR Family Quorum Sensing Regulator in Burkholderia
pseudomallei
Jennifer R. Klaus1, Patricia Silva1, Jakki Stevens1 and Josephine R. Chandler1
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
LuxI-R-type quorum sensing systems enable cell density-dependent gene regulation. In
many soil bacteria, LuxR-type quorum sensing signal receptors regulate antibiotic
production and may be important for competing with other bacteria in mixed soil
communities. In some opportunistic pathogens, LuxR proteins also play an important role
in infections. We are interested in understanding how LuxR proteins are used in free-living
and host-associated lifestyles and in shifting between them. Here, we focus on an unusual
LuxR-type protein, MalR, in the soil-dwelling opportunistic pathogen Burkholderia
pseudomallei. We show that MalR activates a cluster of genes (the mal genes) in response
to certain antibiotics, but does not respond to typical quorum sensing signals. Thus MalR
is an atypical LuxR-type protein that does not appear to be cell density-dependent. The
MalR-regulated mal genes are homologous to genes encoded in the close relative
Burkholderia thailandensis where they are important for biosynthesis of a polyketide that
is toxic to eukaryotic and prokaryotic cells. MalR and the mal cluster are also important
for virulence in C. elegans. Altogether the available results support that MalR is an
important virulence factor in B. pseudomallei and a uniquely regulated LuxR family
member. Our results also suggest the possibility that MalR might be important for
responding to antibiotics produced by competing bacteria in multispecies soil
communities. Future studies on MalR might provide a window into the mechanisms of B.
pseudomallei adaptation to different environments and significantly broaden our view of
how LuxR proteins benefit bacteria that switch between lifestyles.
46
Abstract #14
Characterization and Protective Efficacy Assessment of Potential Subunit Vaccine-
S1S2 Against Salmonella enterica
Prashant Kumar1, Francisco J. Martinez Becerra1, Olivia Arizmendi1,
Russell Middaugh2, William D. Picking1, Wendy D. Picking1
1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA; 2Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, KS,
USA
Diarrhea caused by Salmonella enterica is an important public health problem. It is difficult
to develop a broadly protective vaccine for S. enterica due to the presence of multiple
serotypes and immunodominance of LPS. We have designed novel subunit vaccines, S1
(SipD-SipB) and S2 (SseB-SseC) fusion proteins, based on the highly conserved type-III
secretion systems (T3SS) of Salmonella pathogenicity islands SPI-1 and SPI-2,
respectively. The proteins were characterized using spectroscopic techniques to understand
their structural and biophysical properties. Far-UV circular dichroism (CD) indicated both
possess predominant alpha-helical secondary structure. Tertiary and quaternary structures
were monitored using fluorescence and static light scattering (SLS) techniques,
respectively. The resulting data sets from the spectroscopic techniques were collectively
viewed using multi-index empirical phase diagrams and radar charts for assessment of
protein structural integrity as a function of pH and temperature. S1 was found to be stable
at lower temperature (below 25 ⁰C-30 ⁰C) while S2 was more thermostable (50 ⁰C - 55 ⁰C)
over a wide pH range. We immunized mice with these proteins intramuscularly with Alum
and MPL as adjuvants. Serum antibodies against the individual Tip proteins were
measured. In addition, immunization with both fusion proteins yielded significant
protection against Salmonella enterica serovar Typhimurium and Enteritidis. Since both
the fusion proteins were tested for their broad range protection in different studies, their
pre-formulation biophysical characterization is vital for further developing this protective
Salmonella vaccine.
47
Abstract #15
High Resolution Structures of the Regulatory Domain of the AraC Family
Transcriptional Activator RhaR
Jiaqin Lia, Haiyan Zhaoa, Graham Wehmeyera, Deena Shaatha, Scott Lovell b, Kevin P.
Battaile c and Susan M. Egana*
a Department of Molecular Bioscience, 1200 Sunnyside Avenue, University of Kansas,
Lawrence, Kansas 66045, USA; b Shankel Structure Biology Center, 2034 Becker Drive,
University of Kansas, Lawrence, Kansas 66047, USA; and c IMCA-CAT, Hauptman-
Woodward Medical Research Institute, 9700 S. Cass Ave, Bldg 435A, Argonne, IL,
60439, USA
The crystal structure of the rhamnose-binding and dimerization (regulatory, NTD) domain
of the Escherichia coli AraC-family transcriptional activator protein RhaR was determined
in the presence and absence of L-rhamnose. The structure in the presence of rhamnose was
solved at 2.05 Å resolution. The structure shows that the RhaR-NTD has a fold that is
similar to the corresponding domain of AraC, binding its respective sugar within a cupin-
superfamily β-barrel. In addition, a Ni2+ ion, which has not been seen in other AraC family
protein structures, is present in the sugar-binding pocket of RhaR. The dimerization of
RhaR-NTD is mediated by an antiparallel coiled-coil motif and a loop region. A rhamnose-
free structure was solved at a resolution of 1.73 Å. In this structure, a loop region that is
involved in rhamnose binding, and is located in the β-barrel, is completely disordered, and
a second loop region showed minor structural changes. Each of the two regions with
rhamnose-dependent structural changes is predicted to be at the interface between the RhaR
NTD and DNA-binding domain, suggesting their potential involvement in rhamnose
allosteric signalling. The structures in the presence and absence of rhamnose showed no
differences in the RhaR N-terminal arm region. We propose a model for RhaR rhamnose-
dependent allosteric signalling that shares some features with the ‘light switch’ model of
AraC, but differs in other features.
48
Abstract #16
High-Resolution Structure of Bacteriophage Sf6 Tail Adaptor Protein gp7 Reveals
the Conformational Switch that Mediates the Sequential Assembly of the Phage Tail
Lingfei Liang, Haiyan Zhao and Liang Tang*
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
Most DNA bacteriophages possess a multi-component tail structure that encodes multiple
functions essential for attachment to host cells and injection of phage DNA into host
cytoplasm. The tail of bacteriophage Sf6 is a macromolecular assembly of ~2.8
megadalton, consisting of 5 types of polypeptides with 51 protein subunits, among which
is a tail adaptor protein that interacts with the portal, the tailspike and a tail distal-end
protein. Assembly of such a molecular machine has been thought to occur in a sequential
manner to ensure proper molecular interaction and avoid aberrant products. Here we report
the high-resolution crystal structure of the tail adaptor protein gp7 from Shigella phage Sf6.
The structure shows a conserved fold for adaptor proteins from podoviruses, siphoviruses,
and probably myoviruses. The structure exhibits a negatively charged surface on one side
and a positively charged patch on the other, suggesting that assembly of the dodecameric
ring from monomers occurs through arrangement of a bipolar molecule in a head-to-tail
manner. A model of the dodecameric ring of gp7 shows an entirely negatively charged
surface, suggesting that binding of the tailspike and the tail distal-end protein to gp7 is
mediated by charge:charge interaction. Comparison with the homologous protein from
phage P22 reveals two distinct conformations for a conserved N-terminal portion
encompassing helix-loop (residues 7-33), which may represent the states prior to and after
assembly into the tail respectively and is made possible by alternating positioning of a
sequence motif in this region. The N-terminal portion of gp7 may serve as a molecular
switch that enables attachment of the tail distal-end protein only after gp7 has assembled
with the portal. Additionally, the C-terminal portion of gp7 shows two conformations in
the crystal, indicating an induced fit upon binding to the portal protein. These results
provide insight into the mechanism for the gp7-mediated, sequential assembly of the tail at
atomic detail.
49
Abstract #17
Degrading Chlorinated Dioxins Using a Bacillus thuringiensis Spore Expression
System
Minh Ma1,2, Hsin-Yeh Hsieh1,3, Shu-Yu Hsu4,5, Chung-Ho Lin4, George Stewart1,3
1Bond Life Sciences Center; 2Department of Biochemistry; 3Department of Veterinary
Pathobiology; 4Center for Agroforestry, University of Missouri, Columbia, MO, U.S.A. 5Department of Marine Environment and Engineering, National Sun Yat-sen University,
Kaohsiung, Taiwan, R.O.C.
Dioxins are persistent organic pollutants due to their long half-life in nature and
contamination by these pollutants is a substantial public health problem worldwide. In US
and Taiwan, dioxins are mainly by-products of a wide range of manufacturing, such as
herbicides, pesticides or chlorine bleaching of paper pulp. Although formation of dioxins
is local, its distribution can be found throughout the world in the environment. Thermal
desorption is the most frequently used technique to remediate many Superfund sites but it
is energy consuming with high cost. So, a more cost effective and efficient approach is
needed so that contaminated areas can be safely repopulated.
A dioxin-degrader bacterium, Pseudomonas mendocina strain NSYSU, was isolated from
a heavily contaminated site in Taiwan and was sent to our lab for further studying. A
Bacillus thuringiensis spore expression system was previously developed in out lab to act
as a support platform for various fused proteins for enhanced activity and long term
stability for soil bioremediation purposes. The Pseudomonas mendocina NSYSU’s
genomic sequence was determined and candidate genes encoding enzymes involved in
dioxin degradation were identified, and will be expressed in our spore-based system to
produce spores. The putative dioxin degrading enzyme decorated spores can then be
applied to contaminated soil to degrade dioxins.
Genes encoding ferredoxin and 4,4a-dioxygenase to initiate the dioxin degrading pathway
were identified, PCR amplified and cloned to a protein expression vector. The ferredoxin
protein transfers electrons from NADH oxidation to dioxin dioxygenase. Both of the
recombinant proteins were produced and purified for testing. A NADH dehydrogenase
activity assay was performed to determine the function of the ferredoxin recombinant
protein. Next we will set up the dioxin degradation reaction with the ferredoxin and
dioxygenase recombinant proteins for HPLC analysis. Once the results confirm the
function of both proteins, the recombinant spores will be added to dioxin-contaminated soil
samples to assess activity in soil.
50
Abstract #18
Optimization of Sample Preparation and Initial CryoEM Structure of Anthrax Toxin
with Singly Bound Lethal Factor Inserted into a Nanodisc Bilayer
Alexandra J Machen1, Pierce O’Neil1, Narahari Akkaladevi2, Tommi A White2,3,
and Mark T Fisher1
1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center,
Kansas City, KS, USA. 2Department of Biochemistry, University of Missouri, Columbia, MO,
USA. 3Electron Microscopy Core Facility, University of Missouri, Columbia, MO. USA
The lethality of anthrax, a zoonotic disease and bioterrorism agent, is due to the anthrax toxin.
One form of the toxin consists of protective antigen (PA) and lethal factor (LF). PA83 binds to
a host cell receptor and is cleaved by proteases leaving PA63 to self-association and form the
heptameric PA prepore. Up to three molecules of LF bind to the PA prepore. This complex is
endocytosed. As the intracellular endosome is acidified to pH 5.0 in the late maturation stage,
the prepore PA-LF complex transitions to a PA pore capable of translocating LF across the
endosomal membrane into the cytosol. The translocation event is initiated when the N-terminal
polylysine tail of LF feeds into the negatively charged pore lumen. A hypothesized directed
Brownian ratchet mechanism of translocation relies on the electrostatic nature of the pore
lumen and appears to be gated by the Phe clamp. Our working hypothesis states differences in
the PA pore lumen with and without singly bound LF in different pH environments will result
in functional changes in 1) the electrostatic potential of the pore lumen and 2) the conformation
of the Phe clamp region. To test our hypothesis we succeeded in constructing PA pore
complexes with single bound LFN, the N-terminal domain of LF, for cryo electron microscopy
analysis. With recent advancements of cryo EM, the main bottleneck in obtaining atomic
structure resolutions structure is now sample preparations, especially for aggregation prone
and membrane proteins. In order to solve the atomic structure using cryo EM, large quantities
of soluble PA pore with single bound LF are needed to avoid imposing sevenfold symmetry
while obtaining diverse conformational coverage. To this end, LFN-PA pore complexes were
produced using an immobilized bead based system and solubilized using lipid bilayer
nanodiscs. A new approach where LFN and PA prepore were incubated prior to immobilization
was developed to reduce the number of complexes with multiple bound LFN. The assembly of
singly bound LFN PA nanodisc complexes were first confirmed and optimized using negative
stain EM. LFN PA nanodisc complexes were then vitrified to for single particle cryo EM under
optimal ice conditions, imaging was accomplished using a FEI Tecnai F30 G2 Twin
Transmission Electron Microscope. Classification and single particle analysis was performed
using EMAN2. The structures that are emerging from this controlled complex formation
should lead to a better understanding of the directional translocation mechanism.
51
Abstract #19
Development of a High Throughput Assay to Identify Inhibitors of the Oligopeptide
Permease in Enterococcus faecalis
Nicole L. Massa1 and Lynn E. Hancock1
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS, United
States of America
Enterococcus faecalis uses peptide based quorum signals for cell-to-cell communication
and these signals are involved in conjugation and biofilm development. The generation
of peptide signals require several processes related to peptide processing, secretion, and
importation. Through a genetic screen our laboratory identified two complementary
peptide importation pathways and disruption of both pathways blocks peptide signaling.
In order to identify chemical compounds that inhibit peptide-based signaling we
developed a whole cell luciferase-based assay and have begun to screen small chemical
compound libraries for inhibitors of oligopeptide permease.
52
Abstract #20
Investigating the Structure of Tetanus Neurotoxin using Electron Microscopy
Pierce O’Neil1, Joshua Burns2, Tommi White3,4, Michael Baldwin2, Mark Fisher1
1Department of Biochemistry and Molecular Biology, University of Kansas Medical
Center, Kansas City, KS, USA .2Molecular Microbiology and Immunology, School of
Medicine, University of Missouri, Columbia, MO, USA. 3Department of Biochemistry,
University of Missouri, Columbia, MO, USA. 4Electron Microscopy Core Facility,
University of Missouri, Columbia, MO, USA
Tetanus neurotoxin (TeNT) is a virulence factor produced by Clostridium tetani, which
causes paralysis by inhibiting neuronal vesicle release. TeNT consists of a single
polypeptide which is activated by backbone cleavage resulting in a light (50kDa) and heavy
(100kDa) chain connected by a single disulfide bond. The heavy chain (H) has two
functional domains. The heavy chain C-terminus (HC) has both a jelly roll and a β-trefoil
topology to mediate ganglioside (a neuronal-specific lipid) binding, which is the first step
for neurotoxicity. After binding to the neuronal membrane, the entire TeNT complex is
endocytosed and the vesicle acidifies. A portion of the heavy chain called the translocon
domain undergoes a temperature (37°C) and pH dependent (5.0) unfolding/refolding
reaction resulting in a part of the translocon inserting into the membrane to form an as yet
undefined pore. The inserted structure transports the light chain (LC) across the endosomal
membrane by an unknown mechanism. Once the nicked LC is transported into the cytosol,
the linking disulfide is reduced by thioredoxin releasing the LC zinc endopeptidase, leading
to neurotoxicity. Inhibition of a number of pH-induced refolding reactions would prevent
the toxic activity of TeNT and therefore represents a therapeutic target. To better
understand the structural rearrangements of the TeNT neurotoxin associated with pH drop,
the 3D structure of TeNT both membrane bound and membrane inserted needs to be solved.
Although the LC and HC have been crystalized, the structure of membrane associated and
membrane inserted forms are not readily crystalized. To gain insight into the structure of
TeNT, we used I-TASSER modeling, hydropathy analysis, optimized negative stain
transmission electron microscopy (TEM), and individual particle electron tomography
(IPET). Our previous work on TeNT structure/stability revealed the chaperonin GroEL will
bind to the toxin under ambient temperature. This lead to the hypothesis that GroEL may
be capturing a transient pre-transition conformation of TeNT which exposes a hydrophobic
face. For this reason, we first focused on the structure of TeNT alone and after temperature
activation to capture a potential hydrophobic transition TeNT structure bound to GroEL.
The elevated temperature is both physiologically relevant and increases protein dynamic
breathing. Structures of the TeNT-GroEL complex may reveal regions in TeNT that must
partially unfold in order to interact with GroEL, eventually leading to regions that may
serve as target sites for developing novel anti-toxin stabilizers.
53
Abstract #21
Experimental Infection of North American Sus scrofa domesticus with Japanese
Encephalitis Virus
So Lee Park1, 2, Victoria Ayers1, 2, Yan-Jang S. Huang1, 2, Susan M. Hettenbach2, Amy
Lyons1, 2, D. Scott McVey1,3, Kenneth R. Burton4, Stephen Higgs1, 2,
Dana L. Vanlandingham1, 2
1Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, KS, USA 2Biosecurity Research Institute, Kansas State University, Manhattan, KS, USA
3Arthropod-Borne Animal Diseases Research Unit, Center for Grain and Animal Health
Research, Agricultural Research Service, United States Department of Agriculture,
Manhattan, KS, USA 4National Agricultural Biosecurity Center, Kansas State University, Manhattan, KS, USA
Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted primarily from the
bites of infected mosquitoes. With about 50,000 annual cases and a 40% case mortality
rate among unvaccinated individuals, JEV is one of the leading causes of pediatric viral
encephalitis in Asia. Whilst humans are considered incidental hosts that may succumb to
severe encephalitic disease, transmission and maintenance of JEV in nature primarily
involves viremic swine and avian species. In addition to developing viremia, pigs can
experience species-specific JEV clinical manifestations, such as infertility, stillbirths, and
abnormal fetal development. Due to the agricultural importance of swine species in many
countries, reproductive losses in pigs can have a highly devastating impact on local and
global economics. Despite its significance as a swine pathogen, there is limited knowledge
on JEV pathogenesis in pigs. Additionally, susceptibility to JEV by swine species in the
New World remains undetermined; therefore, the knowledge on the potential for its
introduction and the establishment of a zoonotic transmission cycle is limited. In this
study, commercially available domestic pigs from North America were inoculated with
JEV to investigate their susceptibility to the disease. Blood and tissue samples were titrated
for virus isolation. Results indicate that despite the low viremic profile, North American
pigs are susceptible to systemic infection after IV inoculation.
54
Abstract #22
Development of a Drip-Flow Biofilm Competition Assay for Screening
Enterococcus faecalis Mutants
Srivatsan Parthasarathy, Christine E. Schultz and Lynn E. Hancock
Department of Molecular Biosciences, University of Kansas
Lawrence, KS 66045
Enterococcus faecalis is a common Gram-positive commensal of the intestinal tract, but is
also well known as a leading cause of nosocomial infections. E. faecalis utilizes biofilm
formation as one of the successful infection strategies, and this strategy has been implicated
in several infectious diseases including endocarditis and urinary tract infections (UTI).
Recent evidence suggests that static biofilms do not closely mimic relevant in vivo
conditions seen in endocarditis and UTI. The drip-flow bioreactor, which grows biofilm in
a continuous supply of growth media, provides a physiologically more relevant
experimental model as there is continuous flow of nutrients over the biofilm surface, and
this also provides a moderate level of sheer stress. This also provides an environment to
grow multiple strains in the same run for directed competition assays. We have engineered
a system to express fluorescent markers with non-overlapping spectra in order to conduct
directed competition assays in the drip-flow reactor. We have focused our efforts on
quorum sensing systems, including Fsr and a series of mutants predicted to encode
members of an emerging family of transcription factors, namely the RNPP family. We
provide evidence that this system allows a sensitive measure of differences in how biofilms
form under semi-flow conditions.
55
Abstract #23
Polymorphisms in the PrP prion protein gene in domestic pigs from the FHSU farm
Paudyal Anuja1 and Gillock, E. T2.
Department of Biological Sciences, Fort Hays State University, KS
A group of conditions that leads to neurological disorders in a variety of mammals
including human is known as prion disease. Study of prion proteins in pig (Sus scrofa) is
important because they are widely used in human daily life. More than a billion pigs are
consumed by human every year. Although natural TSE have not yet been identified in pigs
but there is a possibility that these prion disease resistant pigs can be clinically carrier of
replicating prions. Pigs are also considered to be excellent organ donors for human because
humans and pigs share similar physiology. There is another possibility that prion disease
can be transmitted to humans during xenotransplantation.
PrP gene polymorphisms in 50 pigs belonging to 3 breeds namely Hampshire, Yorkshire,
Blue Butt Cross (blue spotted cross from a Hampshire X Yorkshire) were studied. They
are used nationwide for commercial purpose. Prp genes of pig that code for prion proteins
were sequenced and thus obtained sequences were compared with the prp genes of other
mammals. Based on the obtained PrP variants, NJ phylogenetic tree was constructed using
MEGA 7 softwareTotal six polymorphic sites were observed. Deletion of octapeptides
(WGQPHGGG) were observed in samples 9-1, 9-2, 9-4, 9-5, 9-6, 9-7, 9-8, 9-9,,9-11, 23-
6, 23-7, and 23-8 that belongs to the crossbreed Dark Cross sow 71 by Blue Butt Boar
(Gummy bear). The presence of octapeptide is correlated with increased susceptibility of
prion disease as shown in the previous study done in transgenic mice expressing bovine
PrPc with four otapeptide repeats as well as humans with nine octapeptide repeat insertion
in the PNRP gene.
56
Abstract #24
The Synthetic Chemical Biology Core (SCB): A Resource for Research in Chemical
Biology
Chamani T. Perera1, Benjamin Neuenswander1, Digamber Rane1, Blake R. Peterson1,2
and Thomas E. Prisinzano1,2
1Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA;
2School of Pharmacy, University of Kansas, Lawrence, KS, USA
The Synthetic Chemical Biology Core strives to provide comprehensive synthetic
chemistry capabilities to investigators under one roof. The synthetic expertise of the core
includes, but is not limited to, novel and commercially unavailable small molecules,
fluorescent molecules and peptides. The core assists in identifying hits for medicinal
chemistry optimization in infectious disease targets and provides synthesis capabilities for
structure activity studies of said hits. The core staff will work with investigators to design
and synthesis novel molecular probes to facilitate their research. SCB core encompasses
the Purification and Analysis Laboratory (PAL) that provides purification, analysis and
quality control of compounds via HPLC-MS. The core utilizes automated mass directed
fractionation for purification in both reversed and normal phases (including chiral
separations), and also provides relative purity analysis by UPLC coupled to a high-
resolution mass spectrometer for structure confirmation.
57
Abstract #25
Examining Calcium Binding in the EF-Hand Protein, EfhP, Regulating Calcium-
Dependent Virulence in Pseudomonas aeruginosa
Rendi Rogers, Biraj Kayastha, Marianna A. Patrauchan
Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA
Pseudomonas aeruginosa is a human pathogen that, along with causing other various
types of infections, is the leading cause of death in patients with cystic fibrosis. Research
in our lab has shown that calcium (Ca2+) induces virulence in P. aeruginosa. Aiming to
identify the main components of Ca2+ signaling and regulatory networks in P.
aeruginosa, our lab predicted several putative Ca2+-binding proteins and characterized
their role in P. aeruginosa virulence. One of them is EfhP protein, whose sequence
contains two canonical EF-hand domains. The EF-hand motif has been studied in
eukaryotes and is shown to bind Ca2+. We hypothesize that EfhP binds Ca2+ and plays
role in P. aeruginosa Ca2+-induced virulence. The gene encoding for EfhP (PA4107) was
successfully cloned, expressed in E. coli, and the protein was purified. The identity and
the predicted size of the protein was confirmed by mass spectrometry. The quantitative
analysis of Ca2+ by Inductively Coupled Plasma-Optical Emission Spectroscopy detected
3 calcium ions to be bound to the molecule of the protein. Current studies aim to assess
the Ca2+ binding capabilities and specificity of EfhP. Using isothermal titration
calorimetry, a Ca2+-binding constant will be calculated and the Ca2+ binding affinity will
be compared to that of Mg2+. Further, the amino acid residues involved in the Ca2+
binding process will be identified by studying mutants with point mutations based on
predictions from previous studies. Future studies will aim to detect whether EfhP
undergoes conformational changes when binding Ca2+, and identify any protein partners
EfhP may have to transduce Ca2+ signals.
58
Abstract #26
Infectious Disease Assay Development Core: High Throughput Screening
Laboratory at the University of Kansas
Anuradha Roy
High Throughput Screening Laboratory, University of Kansas, Lawrence, KS, USA
The University of Kansas High Throughput Screening Laboratory (KU-HTSL) is a fee-
for-service, state-of-the-art facility dedicated to providing academia, not-for-profit
institutions, biotech, and pharmaceutical industries with exceptional assay development
and high throughput screening services at economical rates. The staff has experience in
executing cell-based, biochemical, siRNA as well as high content screening campaigns
against a plethora of target classes. Clients have the option of using our collection of
300,000 compounds and/or a client's own chemical library. Our chemical library is
augmented with the KU Medicinal Chemistry department's legacy compound collection.
KU-HTSL is innovative and flexible in providing superior service to the drug discovery
research community, including assay development, screening, compound profiling and
data mining. The integrated automation of the HTS system allows us to screen about
100,000 compounds in two days for endpoint assays, or one week for kinetic assays, at an
affordable cost. KU-HTSL further leverages the strengths of the KU Core facilities and the
KU Medical Center's Institute for Advancing Medical Innovations to support your new
lead discovery research.
59
Abstract #27
The Effect of Environmental Factors on Swarming Motility in a Human Pathogen
Pseudomonas aeruginosa
Breeanna C. Russ, Amber E. Price, Michelle M. King, Marianna A. Patrauchan
Department of Microbiology, Oklahoma State University, Stillwater, OK, USA
Pseudomonas aeruginosa is a ubiquitous bacterium that can be found in a variety of
environments such as soil, water, plants, and animals. It is also a human pathogen causing
severe infections, and a leading cause of death in Cystic Fibrosis (CF) patients. P.
aeruginosa possesses many virulence factors, one of which is motility. There are three
types of motility in this organism: swimming, twitching, and swarming. Swarming motility
is required for biofilm formation, which is another important determinant of P. aeruginosa
virulence. Here we characterized the effect of several conditions, commonly associated
with human lung environment, on P. aeruginosa swarming. We have determined that
elevated Ca2+ increased swarming distance and induced pyocyanin production in the
swarming cells. Changing Mg2+ concentrations and humidity did not impact swarming
distance, but the swarming pattern showed unique concentric circles at low Mg2+ and
elevated Ca2+. Lowering phosphate levels had no effect in the presence of MgSO4, but
significantly reduced swarming in the presence of MgCl2. Low Fe2+ showed no effect at
ambient levels of CO2, but significantly increased swarming at 5 % CO2. These results
demonstrate that host environment regulates swarming motility of P. aeruginosa and thus
defines its ability to spread and cause a disease.
60
Abstract #28
Chlamydia trachomatis Manipulation of Protein Kinase C
Prakash Sah1, Ted Hackstadt 2, Erika Lutter1
1Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA; 2Laboratory of Intracellular Parasites, NIAID, NIH Rocky
Mountain Laboratories, Hamilton, USA
Chlamydia trachomatis is responsible for causing a range of diseases such as blinding
trachoma and urogenital infections leading to serious complications. Inside a host cell, C.
trachomatis lives in a parasitophorous vacuole called an inclusion from where it is able to
secrete various effectors to manipulate host-cellular functions to its benefit. Currently, not
much is known about Chlamydial manipulation of host kinases such as Protein Kinase C
(PKC). PKCs are members of AGC family of kinases and involved in regulating various
cellular functions such as, growth and proliferation, migration, survival and apoptosis. We
hypothesize that C. trachomatis manipulates PKC pathways to regulate intracellular
development inside the host, as PKCs are important in regulating various cellular functions.
Indirect immunofluorescence of infected cells verified recruitment of multiple PKC
isoenzymes to microdomains (Src-family kinases rich regions) on the inclusion.
Recruitment of PKC substrates, including Marcks, was also confirmed. Inhibition of PKC
activity with Staurosporine at various time points resulted in decreased recoverable
infectious progeny. These results confirm PKCs are important for intracellular growth and
development of C. trachomatis.
61
Abstract #29
Antibiotic Resistance of Pseudomonas aeruginosa Recovered From Cystic Fibrosis
Patients
William Starr¹, Rawan Eleshy¹, Nighat Mehdi², and Erika Lutter¹
¹Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK; ²Oklahoma Cystic Fibrosis Center, University of Oklahoma Health
Sciences Center, Oklahoma City, OK
Cystic Fibrous (CF) patients produce dehydrated thick mucus in their lungs and lack the
ability to clear this mucus due to mutations in the cystic fibrosis transmembrane
conductance regulator gene (CFTR gene). The mucus provides an optimal environment for
bacterial infections. It is reported that P. aeruginosa infects up to 50% of children and
80% of adults with CF. Once the infection has been acquired, eradication of P. aeruginosa
from the CF lung is rare. P. aeruginosa is naturally resistant to many antibiotics and
acquires antibiotic resistance during the infection process. This study aims to determine
resistance profiles of P. aeruginosa clinical isolates from patients of various ages. Kirby-
Bauer tests were performed on 52 isolates using nine different antibiotics which represent
multiple antibiotic classes. To determine if resistance was due to genetic factors genomic
DNA was extracted from the CF isolates and PCR was performed to verify the presence of
eight antibiotic resistance genes. The results showed that all of the isolates had resistance
to at least one of the nine antibiotics; however, not all of isolates showed the presence
antibiotic resistance genes by PCR. The results also showed at higher dosing of antibiotics
is needed for CF patients due to infections being able to survive the immune system,
smaller antibiotic treatments, and swapping of genetic material between bacterial species.
By understanding antibiotic resistance of P. aeruginosa from CF patients regards to the
mechanisms in which this resistance is acquired , treatment options for CF patients can be
more specialized and targeted based on age, infection type, and susceptibility or resistance
to certain antibiotics.
62
Abstract #31
Identification of Enterotoxigenic Escherichia coli Secreted Protein That Prevents
TNF-induced NF-B Activation in HCT-8 Cells
Gaochan Wang1, Brian Geisbrecht2 and Philip R. Hardwidge1
1Department of Diagnostic Medicine/Pathobiology, 2Department of Biochemistry and
Molecular Biophysics, Kansas State University, KS, USA
Enterotoxigenic Escherichia coli (ETEC) is defined by production of a heat-labile (LT)
and/or heat-stable (ST) enterotoxin causing childhood diarrhea in developing countries and
diarrhea in people traveling to developing world, which is characterized by watery diarrhea
ranging from mild, self-limiting to severe complications. The nuclear factor-B (NF-B)
has long been considered a prototypical pro-inflammatory signaling pathway, which is
activated by pro-inflammatory cytokines such as interleukin 1 and tumor necrosis factor
(TNF). Our laboratory previously reported that ETEC secretes a heat-stable, proteinaceous
factor (ESF) that blocks NF-κB signaling normally induced by TNF in human colon
carcinoma cell line (HCT-8). In this study, we employed fast protein liquid
chromatography and mass spectrometry technology to identify the ESF, which directed our
focus on FliC. ETEC H10407 fliC mutant lost the ability to prevent HCT-8 cells from
TNF-induced NF-B activation. ETEC H10407 fliC mutant complemented with a
plasmid expressing fliC restored the ability to prevent HCT-8 cells against TNF-induced
NF-B activation. Recombinant FliC was able to inhibit TNF-induced NF-B activation.
These results suggested that flagellin (FliC) was responsible for prevention of HCT-8 cells
from TNF-induced NF-κB activation.
64
Abstract #32
Do T3SS effectors other than NleH1 inhibit RPS3 nuclear translocation?
Miaomiao Wu1, Philip R. Hardwidge1
1Department of Diagnostic Medicine/Pathobiology
Kansas State University, Manhattan, KS
Enterohemorrhagic Escherichia coli (EHEC) and other attaching/effacing bacterial
pathogens cause severe diarrhea in humans. These pathogens use a type secretion
system (T3SS) to inject virulence proteins (effectors) into host cells. EHEC NleH1 from
EHEC disrupts host immune responses by binding to the ribosomal protein RPS3 to block
its nuclear translocation, resulting in repression of RPS3-dependent gene transcription.
RPS3 is a co-activator of many NF-B dependent genes, many of which are involved in
innate immunity. We hypothesize that effectors other than NleH1 also bind to RPS3 to alter
RPS3 nuclear translocation in host cells.
We cloned 12 effectors from Salmonella, Shigella, and E. coli with proved rules in
suppressing innate immune response. We transfected HEK293T cells with these effectors
and quantified RPS3 nuclear translocation, and we found that SseL, SopE, SptP, OspZ,
OspG, IpaH9.8, and OspF inhibit RPS3 translocation. To determine whether these effectors
bind directly to RPS3, we also performed GST pulldown assays and found that SseL binds
directly to RPS3. We confirmed this interaction between SseL and RPS3 by performing
co-immunoprecipitation assay. Taken together, we propose that inhibition of RPS3 to block
NF-B activity might be a common strategy for pathogens utilize T3SS.
65
Abstract #33
Vaccinating with Conserved Escherichia coli Antigens Does Not Alter the Mouse
Intestinal Microbiome
Michael P. Hays1, Aaron C. Ericsson2, Yang Yang1, and Philip R. Hardwidge1
1College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA 2University of Missouri (MU) Mutant Mouse Resource and Research Center, College
of Veterinary Medicine, University of Missouri, Columbia, MO, USA
Enterotoxigenic Escherichia coli (ETEC) causes hundreds of millions of cases of
diarrhea annually. Its antigenic and structural heterogeneity complicates vaccine
development efforts. We have utilized type II secretion system (T2SS) mutants to screen
ETEC surface proteins and identified three vaccine candidates (MipA, Skp, and
ETEC_2479) that protected mice in intranasal ETEC challenge assays. However, these
surface proteins are conserved not only in multiple ETEC isolates, but also in
commensal bacteria. In this study, we characterized the changes to mouse intestinal
microbiomes as a function of vaccination. We failed to observe any significance in
changes of mouse weight gain, diversity or richness of mouse intestinal microbiomes,
as well as overall community structure before and after vaccination. Thus, we
concluded that despite the conservation of MipA, Skp, and ETEC_2479 among Gram-
negative bacteria, vaccination with these antigens fails to alter significantly the host
intestinal microbiome.
66
Abstract # 34
Quorum Sensing Control of Antibiotic Resistance Protects Cooperating Bacterial
Cells during Interspecies Competition
Kara C. Hinshaw, Ellen B. Nasseri and Josephine R. Chandler.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA.
Many bacteria have quorum-sensing (QS) systems that coordinate expression of genes in
a population-wide manner. Most of the gene products are public goods, such as proteases
and antimicrobials, which can be used by any community member. Public goods are
vulnerable to cheating by nonproducers, such as QS-defective mutants. This creates a
burden on the rest of the cooperating population that negatively impacts the fitness of the
population during interspecies competition. Various mechanisms have evolved to restrain
cheaters, such as QS-controlled private goods which only benefit the producer and are
often cellular products. Because many antimicrobial resistance factors are cellular, we
hypothesized that QS-control of antimicrobial resistance might protect against QS-
defective cheaters. Growth on casein media requires the production of a secreted QS-
controlled protease. In cooperating populations passaged on casein as a sole carbon
source, QS-defective mutants naturally arise. We show that the addition of select
antibiotics to casein-passaged Chromobacterium violaceum cultures prevents the
emergence of evolved QS-defective cheaters due to a QS-controlled antibiotic resistance
mechanism. To determine if Chromobacterium QS-defective mutants are also suppressed
during interspecies competition, we used a laboratory competition model between
Burkholderia thailandensis and C. violaceum. We competed Burkholderia with a mixture
of wild-type and mutant Chromobacterium. Wild-type Chromobacterium increased in
frequency relative to the mutant during growth with the bactobolin-producing
Burkholderia. However, the mutant increased during growth with a Burkholderia
bactobolin-deficient strain. Because the wild-type cells are also antibiotic producers, this
resulted in a net increase in the competitiveness of the Chromobacterium population only
in the presence of antibiotic-producing Burkholderia. Thus interspecies competition may
be important for selection and maintenance of QS. This may have important implications
for the evolution of intraspecies cooperation in mixed microbial communities.
67
15th Annual Great Plains Infectious Disease Meeting
University of Kansas - Lawrence Kansas
November 4 - 5, 2016
MEETING REGISTRANTS
Last Name First Name Email Address Institution Affiliation
Acharya Rashmi [email protected] Fort Hays State University
Adam Philip [email protected] Kansas Dept. of Health and Environment
Adamovicz Jeff [email protected] University of Missouri
Adams Paige [email protected] Kansas State University ‐ Olathe
Aillon Kristin [email protected] MRIGlobal
Allen Noah [email protected] Oklahoma State University
Amachawadi Raghavendra [email protected] Kansas State University
Anderson Paul [email protected] University of Missouri
Anderson Deborah [email protected] University of Missouri
Arizmendi Olivia [email protected] University of Kansas
Atobatele Mori [email protected] Kansas State University
Ayers Victoria [email protected] Kansas State University
Baldwin Mike [email protected] University of Missouri
Barta Michael [email protected] University of Kansas
Berg Amy [email protected] Viracor‐IBT Laboratories
Boan Daniel [email protected] Kansas City University of Medicine and Biosciences
Bose Jeffrey [email protected] University of Kansas Medical Center
Bourne Christina [email protected] University of Oklahoma
Burke Donald [email protected] University of Missouri
Calhoun Katherine [email protected] University of Kansas
Carter Wayne [email protected] Kansas City Area Life Science Institute (KCALSI)
Carvalho Claudia [email protected] Fort Hays State University
Casillas Robert [email protected] MRIGlobal
Cull Charley [email protected] Veterinary and Biomedical Research Center, Inc. (VBRC)
Davido David [email protected] University of Kansas
DeDonder Keith [email protected] Veterinary and Biomedical Research Center, Inc. (VBRC)
DeMars Zachary [email protected] University of Kansas Medical School
Dimond Zoe [email protected] University of Kansas
Duan Qiangde [email protected]‐state.edu Kansas State University
Egan Susan [email protected] University of Kansas
68
Last Name First Name Email Address Institution Affiliation
Eleshy Rawan [email protected] Oklahoma State University
Eslick Carley [email protected] University of Missouri‐Columbia
Fang Ying [email protected]‐state.edu Kansas State University
Faulk Sara [email protected] Viracor‐IBT Laboratories
Fisher Mark [email protected] University of Kansas Medical Center
Fitzpatrick Erica [email protected] University of Kansas
Flynn Tony [email protected] The University of Kansas Medical Center
Forge Kurt [email protected] GE Healthcare
Gallaway Erin [email protected] Oklahoma State University
Ganjam Irene [email protected] University of Missouri VMDL
Gao Philip [email protected] University of Kansas
Geisbrecht Brian [email protected] Kansas State University
Gelhaus Carl [email protected] MRIGlobal
Gillock Eric [email protected] Fort Hays State University
Guerra‐Maupome Mariana [email protected]‐state.edu Kansas State University
Hancock Lynn [email protected] University of Kansas
Hanson Nancy [email protected] Creighton University
Hardwidge Philip [email protected] Kansas State University
Harrison Kelly [email protected] University of Kansas
Hermanas Timothy [email protected] University of Missouri
Hinshaw Kara [email protected] University of Kansas
Hoffman Nikki [email protected] Olathe School District
Holzschuh Elizabeth [email protected] Johnson County
Hossain Mohammad [email protected]‐state.edu Kansas State University
Hsieh Hsinyeh [email protected] University of Missouri
Hsu Shu [email protected] University of Missouri
Huang Jiachen [email protected]‐state.edu Kansas State University
Hulangamuwa Wasundara [email protected] Kansas State University
Hunter Rob [email protected] One Medicine Consulting
Huynh Hien [email protected] University of Missouri
Johnson David [email protected] University of Kansas
Jordan Lorne [email protected] University of Kansas
Kayastha Biraj B [email protected] Oklahoma State University
Klaus Jennifer [email protected] University of Kansas
Klozenbucher Kolin [email protected] Fort Hays State University
Krausz Kelsey [email protected] University of Kansas Medical Center
Krute Christina [email protected] University of Kansas Medical Center
Kumar Prashant [email protected] University of Kansas
LaBrie Scott [email protected] University of Kansas
Layton Sierra [email protected] University of Kansas
Li Jiaqin [email protected] University of Kansas
69
Last Name First Name Email Address Institution Affiliation
Liang Lingfei [email protected] University of Kansas
Lin Chungho [email protected] University of Missouri
Looft Torey [email protected] USDA, ARS, MWA, NADC
Lutkenhaus Joe [email protected] University of Kansas Medical Center
Lutter Erika [email protected] Oklahoma State University
Ma Minh [email protected] University of Missouri
Machen Alexandra [email protected] University of Kansas Medical Center
Markiewicz Mary [email protected] University of Kansas Medical Center
Martinez Becerra Francisco [email protected] University of Kansas
Massa Nicole [email protected] University of Kansas
McKinney Megan [email protected] University of Kansas
Mehojah Justin [email protected] University of Kansas
Mendez Karina [email protected] Kansas State University
Miller Julie [email protected] Olathe Public Schools
Moral Mario [email protected] University of Kansas
Nowak Martha [email protected] Kansas State University ‐ Olathe
Olivarez Nicholas [email protected] University of Missouri ‐ Columbia
Olson Rachel [email protected] University of Missouri ‐ Columbia
ONeil Pierce [email protected] University of Kansas Medical Center
Park So Lee [email protected]‐state.edu Kansas State University, College of Veterinary Medicine
Parthasarathy Srivatsan [email protected] University of Kansas
Patel Ami [email protected] Laboratory for Infectious Disease Research
Paudyal Anuja [email protected] Fort Hays State University
Perera Chamani [email protected] University of Kansas
Peterson Blake [email protected] University of Kansas
Peterson Rebecca [email protected] University of Kansas
Picking William [email protected] University of Kansas
Picking Wendy [email protected] University of Kansas
Potts Macy [email protected] Kansas City University
Pressnall Melissa [email protected] University of Kansas
Qiu Iris [email protected] The University of Kansas
Ray Christian [email protected] University of Kansas
Rezac DJ [email protected] Veterinary and Biomedical Research Center, Inc. (VBRC)
Richardson Ralph [email protected] Kansas State University ‐ Olathe
Richt Juergen [email protected]‐state.edu Kansas State University
Rivera Mario [email protected] University of Kansas
Rogers Rendi [email protected] Oklahoma State University
Rossi Davide [email protected] FitBark
Roy Anuradha [email protected] University of Kansas
70
Last Name First Name Email Address Institution Affiliation
Russ Breeanna [email protected] Oklahoma State University
Sah Prakash [email protected] Oklahoma State University
Seawell Nichole [email protected] University of Kansas Medical Center
Self Adrian [email protected] Kansas State University
Sharma Neekun [email protected] University of Kansas Medical Center
Shaw Jennifer [email protected] Oklahoma State University
Skaar Ryan [email protected] Simpson College
Smith Sydni [email protected] Oklahoma State University
Somerville Greg [email protected] University of Nebraska ‐ Lincoln
Soules Katelyn [email protected] University of Kansas
Spangler Julie [email protected] University of Kansas
Spears Jacqueline [email protected] Kansas State University
Starr William [email protected] Oklahoma State University
Stewart Jason [email protected] University of Kansas
Stewart George [email protected] University of Missouri
Strawderman Oather [email protected] Lawrence Public Schools
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Student OSHS [email protected] Olathe South High School
Tachiyama Shoichi [email protected] University of Kansas
Tiemann Gail [email protected] University of Kansas
Trembath Andrew [email protected] University of Kansas Medical Center
Treml Jack [email protected] University of Kansas ‐ Edwards Campus
Turocy Ken [email protected] CTL Analyzers
Ulapane Kavisha [email protected] University of Kansas
Vande Garde Blake [email protected] Erickson Kernell IP
Villanueva Cecilia [email protected] University of Kansas
Wang Gaochan wang2034@k‐state.edu Kansas State University
Wang Zili [email protected] Kansas State University
Wang Michael Zhuo [email protected] University of Kansas
Ward Claire [email protected] Kansas State University
Witters Jeff [email protected] Olathe Public Schools
Wu Miaomiao [email protected] Kansas State University
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Last Name First Name Email Address Institution Affiliation
Xing Minli [email protected] University of Kansas
Yang Yang [email protected] Kansas State University
Yao Huili [email protected] University of Kansas
Yount Shaylee [email protected] Kansas City Area Life Science Institute (KCALSI)
Zekarias Bereket [email protected] Ceva
Zhang Weiping [email protected]‐state.edu Kansas State University
Zheng Qi [email protected] University of Kansas
Zimmerman Drew [email protected] Fort Hays State University
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