The 12th Annual Great Plains Infectious Disease Meeting We are pleased to host the 12th Great Plains Infectious Disease (GPID) Meeting at the University of Missouri for the fourth year in a row. In the past, the meeting has been hosted at the University of Kansas Medical Center and the University of Kansas. The GPID meeting was originally developed to promote collaborations in the Great Plains region and networking among researchers and it continues to do so. The meetings have promoted student and young faculty’s research by hosting a poster presentation, and providing a platform for faculty, postdoctoral researchers, and students to give oral presentations. This year, we are proud to host speakers from Nebraska, Missouri, Kansas, Illinois, and Oklahoma, almost 100 participants and approximately 30 posters. This meeting has always been successful due to the generosity of our academic sponsors and this year we are fortunate to add selected vendors to our growing list of sponsors. Thank you all for attending! The GPID Programming Committee 2013

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5:30pm 6:00pm6:00pm 7:00pm7:00pm 8:00pm

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Opening Remarks: Deborah Anderson, PhD. University of Missouri - Columbia

12th Annual Great Plains Infectious Disease Meeting Program ScheduleOctober 4-5, 2013

University of Missouri - Columbia

AgendaFriday, October 4, 2013 (Stoney Creek Inn)

Pre-meeting registrationDinner and Networking Session

Staphylococcus aureus Biofilm: A Complex Multicellular Organism

Saturday, October 5, 2013 (Adams Conference Center and Auditorium)Registration and Breakfast

Alexander Franz, PhD. University of Missouri - Columbia

Rollie Clem, PhD. Kansas State University

Jennifer Binning, Washington University School of Medicine

"The RNA Interference Pathway as a Modulator of Vector Competence for Arboviruses in Aedes aegypti "

"The Role of Apoptosis in Controlling Arbovirus Infection in Mosquitoes"

"Genetic Approach to H5N1 Influenza A virus Pathogenesis"

Session III: Poster Presentation

Lunch (Adams Conference Center)

Keynote Presentation: Kenneth Bayles, PhD. University of Nebraska Medical Center

"Effector Role of Invasion Plasmid Antigen D (IpaD) of the T3SA from Shigella flexneri "

Phil Hardwidge, PhD. Kansas State University"E. coli Virulence Factors and the Innate Immune System"

"Filoviral VP35 Protein as a Therapeutic Target"

Session I: Host Pathogen Interaction (Adams Auditorium)Vjollca Konjufca, PhD. Southern Illinois University

Mohammad Ayoub Mir, PhD. University of Kansas Medical Center

Jacco Boon, PhD. Washington University School of Medicine

Refreshments and Networking Break (Adams Conference Center)

"Internalization of Lumen Antigens by Epithelial Cells of the Small Intestine"

"Hantavirus Cap-snatching at the Cross Roads of Cellular P-bodies"

Session II: Viruses and Vector Borne Pathogens (Adams Auditorium)

Pizza and Bonfire (Home of Dr. Anderson)

Poster Session (Adams Conference Center)

Session IV: Bacterial Pathogenesis (Adams Auditorium)Erika Lutter, PhD. Oklahoma State University

Marianna Patrauchan, PhD. Oklahoma State University

Michael Barta, PhD. University of Kansas

Anna Cunningham, University of Missouri - Columbia

Olivia Arizmendi, Oklahoma State University

"Putting Chlamydia Under the Atomic Microscope"

"The Effect of Fish Oil Supplementation on Lyme arthritis and carditis"

"Role of the Myosin Phosphatase Pathway in Chlamydial Host-cell Egress"

"The Role of Calcium in Pseudomonas aeruginosa Virulence"

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Speaker Abstracts

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Kenneth Bayles, Ph.D. Professor Vice Chair for Research Director, Center for Staphylococcal Research Associate Vice Chancellor for Basic Science Research The University of Nebraska Medical Center Staphylococcus aureus biofilm: A complex multicellular organism Kenneth W. Bayles Center for Staphylococcal Research, The University of Nebraska Medical Center, Omaha NE

Studies of biofilm produced by Staphylococcus aureus have revealed that its formation is dependent on a variety of polymeric matrix molecules including extracellular genomic DNA (eDNA). The generation of eDNA is mediated by the autolysis-dependent release of the cellular contents from a subpopulation of cells via a Cid/Lrg-mediated process analogous to programmed cell death (PCD). Once released, this eDNA is subject to degradation by secreted staphylococcal nuclease (encoded by the nuc gene), which appears to limit biofilm accumulation. To gain a better understanding of the regulation of the cid, lrg, and nuc genes, we have taken advantage of BioFlux microfluidics technology that allows us to examine gene expression in time-course assays during biofilm development under shear-stress conditions. These studies revealed a previously unappreciated level of regulatory control that influences expression of these genes, including spatial, temporal, and stoichiometric mechanisms. For example, the cid operon is specifically expressed within tower structures in response to the hypoxic conditions produced as these structures grow in size and limit the exchange of oxygen from the surrounding growth medium. The lrg operon is also specifically expressed in tower structures but in a way that appears to be independent of oxygen levels. Strikingly, the nuc gene is induced well before tower formation, but only in an apparently random sub-population of cells. However, despite the fact that only approximately 1% of these cells are producing nuclease, the effects of this expression are felt throughout the population, as a result of the dispersal of a majority of the population. Overall, the patterns of gene expression observed in these studies hint at the existence of a complex pattern of cellular differentiation, common in more complex multicellular organisms, in which divisions of labor are organized for the benefit of the entire cellular population.

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Vjollca Konjufca, Ph.D. Assistant Professor Southern Illinois University Internalization of Lumen Antigens by Epithelial Cells of the Small Intestine S. E. Howe and V. H. Konjufca Department of Microbiology, Southern Illinois University, Carbondale IL Intestinal epithelial cells (IECs) overlying the villi establish a barrier that separates the internal milieu from the outside environment. IECs mediate the absorption of water and digested nutrients; however it is not known whether IECs play a role in the uptake of lumen antigens. Large particulate antigens are taken up mainly via M cells that are found in the Peyer’s patches, while soluble antigens enter the lamina propria (LP) of the small intestine via Goblet cell-associated passageways (GAPs). Among other factors, the route of antigen uptake and the nature of the antigen dictate the ensuing immune responses. Using in vivo imaging of the small intestine by two-photon microscopy we have found that IECs capture nanoparticles and further transport them to the LP. In contrast to this, small proteins and bacterial LPS enter the LP via GAPs, rather than via the IECs. The size of nanoparticles, rather than their chemistry appears to be important for their uptake by IECs in vivo. We have also identified at least one endocytic pathway by which IECs internalize nanoparticles in vivo. Blocking or conjugating particles with protein does not inhibit their uptake. Moreover, protein conjugated to nanoparticles is transported to the deeper lymphoid tissues in an immunologically relevant form and induces antigen-specific immune responses. Better understanding the mechanisms of antigen uptake in the small intestine has implications for understanding pathogenesis of small enteric viruses, and for designing more efficacious mucosal vaccines and therapies.

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Mohammad A. Mir, Ph.D. Assistant Professor University of Kansas Medical Center Hantavirus Cap-Snatching at the Cross Roads of Cellular P-bodies Cheng E and Mir MA Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS Abstract: Hataviruses are zoonotic tri-segmented negative strand RNA viruses. Their infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with mortalities as high as 50% in certain out breaks. Similar to other segmented negative strand RNA viruses, hantaviruses initiate the transcription of viral mRNA by a unique cap-snatching mechanism during which host cell mRNAs are cleaved by the viral RNA dependent RNA polymerase (RdRp) 10-14 nucleotides down-stream of the 5’ cap. The capped RNA segments are used as primers for transcription initiation. Unlike influenza virus, hantavirus replication occurs in cell cytoplasm where cellular decapping machinery removes the caps from host cell mRNAs after the completion of heir translation. Hantaviruses have to fight with the host decapping machinery to protect the host mRNA caps for the initiation of viral mRNA synthesis. We found that hantavirus nucleocapsid protein (N) bind to the host mRNA caps and protects them from the attack of cellular decapping machinery. N rescues the capped mRNA fragments up to 180 nucleotides in length in P-bodies, the discrete cytoplasmic foci where bulk mRNA degradation occurs. The capped RNA fragments stored in P-bodies by N are later efficiently used as primers by the viral RdRp. It is still unclear that how short capped primers of appropriate length and specificity are generated during cap snatching. We also found that hantaviruses efficiently snatch caps for host mRNAs containing a “G” residue located 14 nucleotides downstream of the 5’ cap. These studies have revealed new targets for therapeutic interventions.

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Philip R. Hardwidge, Ph.D. Associate Professor Kansas State University E. coli Virulence Factors and the Innate Immune System Philip R. Hardwidge Department of Diagnostic Medicine Pathobiology, Kansas State University, Manhattan KS Enteric bacterial pathogens cause diarrheal disease outbreaks, thus constituting enormous health burdens. The molecular mechanisms of how these pathogens inhibit innate immune responses to colonize their host are under intense investigation. The Shiga toxin-producing E. coli (STEC) use a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While T3SS effectors clearly play important roles in bacterial virulence, the mechanisms by which they subvert host functions to promote pathogen survival are incompletely characterized. Through studies of the mechanism of the NleB effector, an interaction between the mammalian glycolysis enzyme GAPDH, and an innate immunity scaffolding protein, TRAF2, was identified. TRAF2 regulates the pro-inflammatory NF-kB pathway. Maximal TRAF2 polyubiquitination and NF-kB activation requires the TRAF2-GAPDH interaction. NleB functions as a β-D-N-acetylglucosamine (GlcNAc) transferase that modifies GAPDH to inhibit its function in innate immunity. Protein O-GlcNAcylation regulates many cellular processes such as cell division and metabolism, but relatively little is known about the role of O-GlcNAc in intestinal immunity. Eliminating NleB O-GlcNAcylation activity attenuated Citrobacter rodentium colonization in a mouse infection model, confirming its significance to bacterial virulence.

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Alexander W.E. Franz, Ph.D. Assistant Professor University of Missouri – Columbia The RNA Interference Pathway as a Modulator of Vector Competence for Arboviruses in Aedes aegypti Alexander W.E. Franz Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO The arthropod-borne virus (arbovirus) transmission cycle involves arthropod vectors and vertebrate hosts. Following acquisition of a viremic bloodmeal from a vertebrate host, an arthropod vector needs to be persistently infected before it can transmit an arbovirus to a new vertebrate host. The mosquito, Aedes aegypti, is the major vector of dengue virus (DENV1-4; Flaviviridae; Flavivirus), but it can also transmit Sindbis virus (SINV; Togaviridae; Alphavirus) under laboratory conditions. RNA interference (RNAi) is the major antiviral pathway in mosquitoes that responds to replicating arboviruses such as DENV and SINV. We show that the mosquito’s RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. Transgenic Ae. aegypti of line Carb109 expressed an inverted-repeat RNA targeting DENV2 in midguts of bloodfed females in a homology-dependent manner. Carb109 mosquitoes have maintained their complete anti-DENV2 resistance phenotype for 33 generations under laboratory culture. We confirm the role of the mosquito’s RNAi machinery as a modulator of vector competence for arboviruses by impairing RNAi in Ae. aegypti in an inducible manner. Transgene-mediated silencing of dcr2, the sensor and initiator of RNAi, increased the replication dynamics of SINV above levels observed in wild-type mosquitoes. SINV titers were significantly increased in the RNAi-impaired mosquitoes and the virus overcame more rapidly dose-dependent midgut infection and –escape barriers. Our results show that the mosquito’s RNAi machinery has the ability to completely silence arboviruses, even though this does not seem to occur under natural conditions. A likely function of the mosquito’s RNAi pathway is to control arbovirus replication, thus preventing viruses from becoming detrimental to their vector.

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Rollie J. Clem, Ph.D. Professor Kansas State University The Role of Apoptosis in Controlling Arbovirus Infection in Mosquitoes Rollie J. Clem Division of Biology, Kansas State University, Manhattan, KS Arboviruses (Arthropod-BOrne viruses) such as dengue or West Nile virus must replicate in their mosquito vector in order to be transmitted between vertebrate hosts. Arboviruses are typically thought to cause persistent infections in mosquitoes, characterized by high levels of virus replication over the rest of the mosquito’s life, accompanied by little if any tissue damage. However, there have been reports of arboviruses inducing apoptosis (programmed cell death) in mosquito midgut or salivary gland tissues, and sometimes this pathology correlated with mosquito resistance to infection. Despite these observations, there was no direct evidence available to indicate whether or not apoptosis can play a role in mosquito defense against arbovirus infection. Over the past several years, we have directly tested the effects of inducing apoptosis during infection of Aedes aegypti mosquitoes with Sindbis virus, a model arbovirus (Family: Togaviridae). Using two different methods to induce apoptosis, we have found that apoptosis can have either a positive or a negative effect on Sindbis virus replication in A. aegypti, depending on whether apoptosis induction occurs in the mosquito in a widespread fashion, or is confined to only infected cells. We also observed strong negative selection against a Sindbis virus clone that was engineered to express a pro-apoptotic gene. Overall, the results the first direct evidence that apoptosis can have a strong negative effect on Sindbis virus replication in A. aegypti, if it is induced in infected cells. Whether apoptosis is naturally induced by arboviruses likely depends on the specific virus-vector combination.

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Jennifer M. Binning Graduate Student Washington University School of Medicine Filoviral VP35 Protein as a Therapeutic Target Jennifer M. Binning1,2,9,#, Tianjiao Wang2,#, Craig S. Brown1,2,9, Michael S. Lee3,4, Priya Luthra5, Reed S. Shabman5, Dominika M. Borek6,7, Gai Liu1, Wei Xu1, Daisy W. Leung1, Christopher F. Basler5, and Gaya K. Amarasinghe1

1Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, 2Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, 3Computational Sciences and Engineering Branch, US Army Research Laboratory, Aberdeen, MD, 4Integrated Toxicology Division, USAMRIID, 1425 Porter St., Ft. Detrick, MD, 5Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, 6Department of Biochemistry & 7Department of Biophysics, UT Southwestern Medical Center at Dallas, Dallas, TX, 8Biochemistry Graduate Program, Iowa State University, Ames, IA, 9Biochemistry Undergraduate Program, Iowa State University, Ames, IA Ebola viral protein 35 (eVP35) is a multifunctional double stranded RNA (dsRNA) binding protein that plays an important role in innate immune evasion, viral replication, and viral pathogenesis. The multifunctional nature of VP35 also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for non-enzymatic targets, are often challenging. We previously identified select conserved basic residues located within the C-terminal interferon (IFN) inhibitory domain (IID) of VP35 that are important for VP35 mediated IFN antagonism and for viral polymerase co-factor functions. Here, we describe our efforts to identify, validate, and characterize small molecule inhibitors and RNA aptamers that target functional sites within VP35 IID. Biochemical and structural studies reveal small molecules and RNA aptamers bind to a region of VP35 IID that is important for interaction with the viral nucleoprotein (NP) and replication complex formation. We also tested select compounds and aptamers for their ability to inhibit VP35-NP interactions in vitro and VP35 function in a minigenome assay. Taken together, our studies highlight the therapeutic potential of targeting the filoviral VP35 protein and provide an important framework for the development of antifiloviral compounds.

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Jacco Boon, Ph.D. Assistant Professor Washington University School of Medicine Genetic Approach to H5N1 Influenza A virus Pathogenesis Jacco Boon

Washington University School of Medicine, Departments of Medicine, Molecular Microbiology and Immunology and Pathology, St Louis, MO, USA The genotype of the host has been implicated in the severity of and susceptibility to influenza A virus infections. The exact mechanism and genetic polymorphisms responsible are currently unknown. Earlier work on recombinant inbred BXD mice, derived from resistant C57BL/6 and susceptible DBA/2 mouse strains, identified three genomic loci that were associated with resistance to severe highly pathogenic H5N1 disease and mortality. More recently we tested a chromosomal substitution strain set (CSS) derived from C57BL/6 and another susceptible mouse strain, A/J, in which a single chromosome of C57BL/6J is substituted with the homologous chromosome of A/J. Of the nineteen CSS strains tested, C57BL/6J-Chr. 4 A/J was significantly more susceptible to highly pathogenic H5N1 influenza A virus than the parental C57BL/6J, indicating that genetic polymorphisms on this chromosome are responsible for the difference in H5N1. The increased susceptibility of C57BL/6-Chr. 4 A/J mice was associated with higher virus titers at days 2, 7 and 9 post infection and increased expression of IFN-b1 and type I interferon induced proteins. To help identify the genetic polymorphism and host gene on chromosome 4, we have produced a series of congenic mouse strains. Preliminary analysis suggests that the polymorphism is located between 39 and 135Mb on chromosome 4. In conclusion, genetic polymorphisms in the genome of the host can predispose to severe influenza disease and systems biology and genetics approach allows us to identify essential host proteins involved in resistance to severe influenza disease.

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Erika Lutter, Ph.D. Assistant Professor Oklahoma State University Role of the Myosin Phosphatase Pathway in Chlamydial Host-cell Egress Erika Lutter Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK Chlamydia trachomatis, an obligate intracellular pathogen, is the leading cause of preventable blindness worldwide and the most common sexually transmitted bacterial infection in the United States. Inside the host, C. trachomatis resides within and undergoes multiple rounds of replication inside a parasitophorous vacuole, termed an inclusion, from which it must exit to initiate infection of neighboring cells and tissues. C. trachomatis host-cell exit can occur via cell lysis or extrusion, which is a specialized packaged release that results in extracellular membrane bound compartments filled with Chlamydia. During extrusion events C. trachomatis exploits the myosin phosphatase pathway, recruiting pathway components (myosin phosphatase and myosin light and heavy chains) to Src family kinase rich microdomains on the inclusion membrane. The phosphorylation states and recruitment of these proteins are enriched in late infection and depletion of the myosin phosphatase pathway components significantly decreases the number of C. trachomatis extrusion events. Utilization of myosin by C. trachomatis provides novel insights into host-cell egress mechanisms.

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Marianna A. Patrauchan, Ph.D. Assistant Professor Oklahoma State University The Role of Calcium in Pseudomonas aeruginosa Virulence. Marianna A. Patrauchan Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK Calcium (Ca2+) is a well-known signaling molecule that regulates a number of essential processes in eukaryotes. Abnormalities in cellular Ca2+ homeostasis have been implicated in many human diseases, including diseases associated with bacterial infections such as cystic fibrosis (CF) and endocarditis. Ca2+ plays a regulatory role in innate immune response, and its concentrations fluctuate in response to inflammation. We hypothesize that cellular Ca2+ balance in a host may provide an environmental cue for facultative pathogenic bacteria and trigger their virulence. Pseudomonas aeruginosa is a facultative human pathogen causing severe acute and chronic infections. We have shown that Ca2+ induces P. aeruginosa biofilm formation, motility, production of virulence factors, and the ability to cause disease. We identified a putative EF-hand Ca2+-binding protein, EfhP. The deletion of efhP caused multiple changes in the cytosolic proteome of the CF isolate FRD1 in response to Ca2+, including the reduced abundance of virulence factors and stress response proteins. The lack of efhP also reduced the degree of infection of FRD1 and its resistance to antibiotics and oxidative stress. These findings suggest that EfhP may be involved in Ca2+-induced signalling pathways triggering P. aeruginosa virulence and resistance in high Ca2+ environments. To enable further studies of the signalling role of Ca2+, we characterized Ca2+ homeostasis in P. aeruginosa and identified fifteen putative Ca2+ transporters, three of which are required for Ca2+ homeostasis. The lack of P-type ATPase PA3920 abolished Ca2+- induced swarming, suggesting the role of this protein in regulating P. aeruginosa response to Ca2+.

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Michael L. Barta, Ph.D. Postdoctoral Fellow University of Kansas Putting Chlamydia under the Atomic Microscope Barta ML, Lovell S, Battaile KP and Hefty PS Department of Molecular Biosciences, University of Kansas, Lawrence KS Chlamydia trachomatis, an obligate intracellular human pathogen, is a prominent source of blinding trachoma and the leading cause of sexually transmitted disease worldwide. Even with this tremendous healthcare burden, factors controlling development and pathogenesis are poorly understood at the molecular and biochemical level. Several experimental constraints have limited progress in these areas, including the ability to directly target genes for disruption. Complicating the matter further is the evolutionary divergence of the C. trachomatis genome, leading to a large phylogenetic gap with better characterized bacterial systems. This gap limits the use of sequence homology between bacterial organisms to functionally annotate the Chlamydia proteome, leaving nearly 40% of the proteome with undefined roles (hypothetical protein). To address this challenge, we are applying the fundamental principle that two proteins sharing structural features frequently exhibit similar functions. We have initiated a limited structural proteomics project on the C. trachomatis hypothetical proteome. Over 80 proteins of unknown function have been screened for expression/solubility, with a ~60% success rate. Twenty-four targets have advanced to crystal screening, 11 have yielded diffraction quality crystals and 6 high resolution structures have been determined. Structural homology suggests these proteins could be involved in nucleic acid binding events, outer membrane stability, housekeeping and metabolism. Utilizing a newly developed chlamydial transformation system that allows tightly controlled expression of externally introduced DNA, we have begun identifying endogenous protein partners of these targets. Together these observations will reveal numerous mechanisms about the basic biology, development and pathogenesis of Chlamydia.

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Anna Cunningham Graduate Student University of Missouri - Columbia The Effect of Fish Oil Supplementation on Lyme Arthritis and Carditis Anna M. Cunningham, Darren S. Dumlao, Paul C. Norris, Jennifer Hughes Hanks, Kevin L. Fritsche, Edward A. Dennis, and Charles R. Brown. Department of Molecular Microbiology and Immunology, Department of Veterinary Pathobiology, University of Missouri, Columbia MO Eicosanoids regulate the development and resolution of Lyme arthritis resulting from infection with Borrelia burgdorferi (Bb). Eicosanoids are potent, bioactive lipids derived from the metabolism of fatty acids (FA) by a variety of enzymes, including the cyclooxygenases (COX) and lipoxygenases (LOX). A traditional Western diet contains high levels of ω-6 FA. ω-6 FA are metabolized into eicosanoids that are involved in the modulation of immunity. Growing evidence suggests that dietary fish oil (FO) is beneficial for the alleviation of a variety of inflammatory disorders. FO contains ω-3 fatty acids and is believed to exert anti-inflammatory effects by causing the preferential metabolism of ω-3 over ω-6 FA, resulting in the production of less bioactive eicosanoids. ω-3 FA can also directly inhibit immune signaling molecules such as NF-κB and the NALP3 inflammasome. Although the effect of dietary FO has been studied thoroughly in the context of dysregulated inflammation, its effect on inflammation resulting from infection is less known. Because Lyme arthritis is mediated by eicosanoids, we hypothesized that dietary fish oil would have profound effects on inflammation in this model. Therefore, we developed a diet in which the FA source was Menhaden FO, infected mice with Bb, and monitored disease. While dietary FO led to a dramatic shift in eicosanoid production, it did not effect the development of Lyme disease. Our data suggests that while FO is able to dampen dysregulated inflammation, it does not affect the ability of mice to mount an immune response and clear an infectious agent.

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Olivia Arizmendi Graduate Research Associate Oklahoma State University Effector Role of Invasion Plasmid Antigen D (IpaD) of the T3SA from Shigella flexneri Olivia Arizmendi, Nicholas E. Dickenson, Andrew J. Olive, William D. Picking, and Wendy L. Picking Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK Shigella flexneri is the most frequently isolated Shigella species and the causative agent of bacillary dysentery. Virulence of this organism depends on a type III secretion system (T3SS) which promotes pathogen invasion, evasion of the host immune system, and lateral spread through the intestinal epithelium. Functionality of this system relies on its type III secretion apparatus (T3SA), composed of a basal body and an extracellular needle. Invasion plasmid antigen D (IpaD) is a structural element at the tip of the needle that controls secretion of effectors to alter host cell functions. We propose IpaD has a novel effector role in addition to its structural function. To test this, we transfected a humanized ipaD gene into human cell lines and probed its expression and effect through confocal immunofluorescence microscopy (IF), co-immunoprecipitation (Co-IP) and phenotypic assays. IF revealed morphological changes in cells expressing IpaD, where this protein co-localizes with F-actin and induces formation of lamellipodia and filopodia. Co-IP and LC-MS/MS show several cytoskeletal binding partners, including actin and vimentin (an abundant intermediate filament protein). Sedimentation assays confirmed that a portion of IpaD is preferentially bound to F-actin. A pull-down assay with recombinant proteins confirmed binding of IpaD and vimentin. Invasiveness and plaque formation in a stable cell line expressing IpaD are highly impaired, suggesting a role in pathogenesis. These findings support an effector role of IpaD through binding to host cytoskeletal elements.

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Last NameFirst

NamePoster board #

Abstract page # Abstract Title

Adam Philip 1 18Characterizing the Protein-Lipid interaction of Invasion Plasmid Antigen B (IpaB) from Shigella flexneri

Akkaladevi Narahari 2 19Marching toward reality: The complete 18 Å structure of the extend Anthrax Protective Antigen Pore Translocon.

Burns Joshua 3 20The receptor binding domain of tetanus neurotoxin is dispensable for channel formation

Chen Xiaotong 4 21Biophysical characterization of a novel DB fusion complex from Shigella flexneri as a candidate subunit vaccine.

Choudhari Shyamal 5 22Biophysical characterization and stabilization of CagL, an antigenic protein from Helicobater pylori as a candidate subunit vaccine

Clem Rollie 6 23A potential role for effector caspases CASPS18 and CASPS19 in midgut escape of Sindbis virus in Aedes aegypti

Coate Eric 7 24 Natural history of pneumonic plague in Brown Norway rats

Davido David 8 25Identification of specific cellular kinases as potential regulators of HSV-1 ICP0 transactivation activity

Davido David 9 26HSV-1 ICP22 but not its Truncated Form US1.5 is Required for VICE Domain Formation and Efficient Acute Replication and Latent Infections in Mice

Dhariwala Miqdad 10 27TLR7- mediated induction of Type I Interferon by Intracellular Yersinia pestis Enhances Plague Pathogenesis

Duff Michael 11 28 Characterization of reassortant H1N2 variant influenza viruses in pigs

Elliott Ali 12 29 Guinea pig model of Coxiella burnetii infection

Harrison Kelly 13 30 Protective Efficacy of SPI-1 and SPI-2 Proteins in a Mouse Salmonella Model

Hefty Scott 14 31 Conditional Gene Expression in Chlamydia trachomatis using the Tet system

Johnson Cayla 15 32Site-Specific, Insertional Inactivation of incA in Chlamydia trachomatis Using a Group II Intron

Khanam Sharmily 16 33The role of RND transport systems in calcium-induced antibiotic resistance and virulence in Pseudomonas aeruginosa

Lambert Greg 17 34 Elucidation Of Binding By Clostridium difficile Toxin A

Lasky Carrie 18 35 Macrophage Polarization in Lyme Arthritis

Lotlikar Shalaka 19 36 Three Functional β-carbonic anhydrases in P. aeruginosa PAO1. Role in Calcification

Ma Wenjun 20 37 The H7N9 influenza virus emerged in China

Miao Chunhui 21 38 Triggering Ebola Virus for Membrane Fusion and Entry

Min Eun 22 39 Characterization of the Novel Exosporium Proteins of B. anthracis

Naik Subhash 23 40Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance

Pratt Carmela 24 41 Role of Cyclooxygenase (COX)-1 in Humoral Immunity

Rosche Kristin 25 42Infection with Salmonella Typhimurium alters the tissue architecture of the spleen and distribution of cell populations

Schoenlaub Laura 26 43 B Cell Infection by Coxiella burnetii Nine Mile Phase I and Nine Mile Phase II

Su Che-Min 27 44To determine if an antimicrobial compound from Eastern Red Cedar targets FtsZ in cell division of Staphylococcus aureus

Weidman Ruth 28 45Changes in the Type IVB Secretion System of C. burnetii in Response to Supplementation During Axenic Growth

Zheng Yi-min 29 46 Restriction of Ebola Virus Entry by IFITM2

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Characterizing the Protein-Lipid Interaction of Invasion Plasmid Antigen B (IpaB) from Shigella flexneri Philip R. Adam, Wendy L. Picking, and William D. Picking. Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK Shigella flexneri is a Gram-negative pathogen that uses its type III secretion system (T3SS) to invade host colonic epithelial cells. The T3SS is comprised of a basal body, a surface exposed needle, and a complex of proteins positioned on the distal end of the needle. The nascent tip complex is composed of invasion plasmid antigen D (IpaD), however, upon the appropriate environmental stimuli, the translocator proteins IpaB and IpaC are recruited to the needle tip and form a pore in the host cell membrane. We have found that in vitro, both oligomeric and monomeric IpaB are capable of interacting with liposomes but only oligomeric IpaB is capable of lysing them. We therefore asked the question: What regions of IpaB are involved in the protein:lipid interaction interface and which residues are exposed to solvent? To address this, we used a variety of biochemical techniques including fluorescence quenching, and limited proteolysis. Single cysteine substitutions were made at multiple positions throughout the IpaB sequence and subsequently labeled with fluorescein. The modified proteins were then used in fluorescence quenching experiments to determine the differential solvent accessibility of each residue for monomeric and oligomeric IpaB, both in the presence and absence of liposomes. Quenching experiments with the Cys mutants suggested the hydrophobic portion of IpaB was protected from solvent by liposomes, especially in the oligomeric form. Liposome flotation assays with proteolytic products of IpaB corroborated the quenching data. From these experiments, we have developed a model for IpaB regions involved in oligomerization and membrane association/insertion.

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Marching Toward Reality: The Complete 18 Å Structure of the Extend Anthrax Protective Antigen Pore Translocon Narahari Akkaladevi, Srayanta Mukherjee, Rebecca Dillard, Yifei Qi, Wonpil Im, Edward P Gogol, Stephen Lutdke, Wah Chiu, R. John Collier, Mark T. Fisher Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City KS The full length 18 Å structure of the Anthrax toxin pore translocon containing an N terminal fragment of the lethal factor has been resolved by cryoEM microscopy. The previous structure nominally resolved at 25 Å, lacked the complete extended barrel structure, the location of a prominent domain (domain four) and most importantly, showed no internal lumen structure. The superior CryoEM structure provides a better platform to fit extended pore MD derived models into the EM defined electron density envelope. MDFF analysis indicates that the resolved internal connection within the barrel lumen defines the location of the critical protein translocation controlling element, the phe 427 clamp flexible loop. In addition to clearly resolving protein density that can only be defined as domain 4, the extended barrel region shows a prominent bulge, initially predicted by Fragment guided Molecular dynamics as a region where the positioning of extensive exterior electropositive residues contribute to a structural repulsion. These structural results indicate that the barrel of the anthrax pore is not as smooth as predicted by extended an extended hemolysin-like barrel models. This in turn may indicate that structure defined irregularities in the barrel lumen, results in a complex electrostatic interior, resulting in constrictions and vestibules which will control the kinetics and extended structural fluctuations of the translocating proteins.

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The Receptor Binding Domain of Tetanus Neurotoxin is Dispensable for Channel Formation Joshua R. Burns and Michael R. Baldwin Department of Microbiology and Molecular Immunology, University of Missouri, Columbia MO Tetanus neurotoxin (TeNT) – a prototypical member of the clostridial neurotoxin (CNT) family; causes neuroparalytic disorders (and death) by entering the neuronal soma to block the release of neurotransmitters. TeNT undergoes a membrane insertion/ translocation transition triggered by endosomal acidification as a key step in the cellular intoxication process. At present, the mechanistic basis of this pH-induced conformational switching is unclear. Current models of CNT translocation suggest that binding of the co-receptor GT1b enables toxin to sense low pH, undergo a significant change in secondary structure, and transform into a hydrophobic membrane protein. In order to establish a model for the formation of the membrane-competent state of TeNT, a series of biochemical and biophysical techniques were employed. Similar to other CNTs, TeNT undergoes significant secondary structural changes and transformation into a hydrophobic protein at low pH only in the presence of co-receptor GT1b. These data suggested that TeNT, similar to other CNT family members, acts as a coincidence detector for receptor and low pH, to undergo translocation. To further test this model, a variant of TeNT lacking the receptor binding domain (termed LHN) was created. Unexpectedly, LHN was found to mimic the behavior of TeNT in all assays. These observations suggest the interaction of GT1b with toxin is likely mediated by the ceramide moiety of the molecule. In conclusion, it is proposed that the TeNT T-domain associates with lipids in a pH-dependent manner, driving the transition to the pore-form of the toxin.

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Biophysical Characterization of a Novel DB Fusion Complex from Shigella flexneri as a Candidate Subunit Vaccine. Xiaotong Chen, Nicholas E Dickenson, Francisco J Martinez-Becerra, Shyamal P Choudhari, Jamie C Greenwood II, William D Picking and Wendy L Picking Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK Shigella spp. are the causative agent of bacillary dysentery, a human illness with high morbidity levels particularly among the elderly and infant populations. Shigella infects via the oral-fecal route, and its virulence is dependent on Type III Secretion System (T3SS). Two components of the exposed needle tip complex of T3SS, invasion plasmid antigen D (IpaD) and IpaB, have shown the potential to serve as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion (DB fusion) was created by joining the coding sequences of IpaD and IpaB with a minimal linker. The DB fusion protein co-expressed with IpaB’s cognate chaperone, IpgC for proper recombinant expression. The chaperon is later removed from the fusion complex with the mild detergent OPOE. The pure DB fusion protein was used for biophysical characterization using the following techniques: Circular Dichroism, Intrinsic Trp Fluorescence and Static Light Scattering . Using these data, we constructed an empirical phase diagram (EPD) that was used to determine the biophysical state of the protein as a function of both temperature and pHs. The EPD showed that the DB fusion is most stable at pH 7 and 35° C. Another mild detergent, LDAO, was used to remove the chaperone. We repeated the biophysical studies using the DB fusion in LDAO-containing buffer. We found that DB fusion protein with the presence of LDAO showed more plasticity and it allowed us to characterize a novel fusion protein, while providing information for appropriate vaccine formulation.

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Biophysical Characterization and Stabilization of CagL, an Antigenic Protein from Helicobater pylori as a Candidate Subunit Vaccine Shyamal P. Choudhari, Kirk P. Pendleton, Joshua D. Ramsey, Thomas G. Blanchard, Wendy L. Picking and William D. Picking Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK A major concern associated with protein subunit vaccines is loss of activity due to physical instability that may cause aggregation of the antigen proteins. This physical instability is mainly influenced by the effect of solution conditions like pH and temperature. Excipient screening aimed at improved protein stability is an essential step to finding an ideal vaccine formulation. CagL is a type IV secretion system protein from the gram negative bacterium Helicobacter pylori. This protein is involved in attachment of the bacterium to the host cell through contact with α5β1 integrin. This role makes it a potential candidate for a subunit vaccine against H. pylori. In the present study, CagL was subjected to various spectroscopic techniques like circular dichroism (CD), intrinsic fluorescence, static light scattering and extrinsic fluorescence under different solution conditions of pH and temperature. Protein stability at each pH condition was determined in terms of transition temperature (Tm) value. The data accumulated was incorporated into a color map called empirical phase diagram (EPD) that provided an overall view of physical stability of the protein. Additionally, aggregation assays were performed to screen excipients from a library of generally regarded as safe (GRAS) compounds. Excipients that inhibited protein aggregation were chosen to confirm their enhanced stabilizing effect by the spectroscopic techniques. The increased Tm value in the presence of certain excipients was considered to be a result of improved physical stability of the protein. These data allowed us to propose the use of select compounds as potential excipients for a CagL vaccine formulation.

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A Potential Role for Effector Caspases CASPS18 and CASPS19 in Midgut Escape of Sindbis Virus in Aedes aegypti Ning Huang, A. Lorena Passarelli, and Rollie J. Clem Division of Biology, Kansas State University, Manhattan KS The midgut epithelium is the first tissue infected by arboviruses when they invade the arthropod vector. To establish a disseminated infection, arboviruses must escape from the midgut by crossing the midgut basal lamina (BL), an extracellular layer that is secreted by epithelial cells and prevents passive diffusion by viruses. We are using Sindbis virus (SINV) and the mosquito vector Aedes aegypti to understand how arboviruses escape from the midgut. In lepidopteran larvae, baculovirus infection initiates a cascade of protease activation in which matrix metalloproteases (MMPs) activate effector caspases, leading to remodeling of the BL lining tracheal cells associated with the midgut, which allows baculovirus to escape the midgut. We hypothesize that the MMP-caspase-BL remodeling pathway is also used by arboviruses to escape the mosquito midgut. In this study we have focused on CASPS18 and CASPS19, effector caspase homologs related to Drosophila Decay. Although CASPS18 does not have enzymatic activity, it has been shown to act as a decoy caspase that is able to enhance the activity of CASPS19. The levels of CASPS18 and 19 transcripts and proteins in midgut were not altered by SINV infection, but silencing CASPS18/19 reduced the caspase activity observed in midgut, and also resulted in lower virus titers than control mosquitoes following an infectious blood meal containing SINV. Immunofluorescence studies revealed that CASPS18 and 19 were expressed in tracheal cells associated with midgut. SINV was also found in tracheal cells in SINV-infected midguts, suggesting that SINV may use the tracheal system to establish systemic infection.

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Natural history of pneumonic plague in Brown Norway rats Coate, EA*, Kocsis, AW*, Peters, KN*, Willix, JL, Dhariwala, MO, Bland, DM, Anderson, PE, Anderson, DM Department of Veterinary Pathobiology, Laboratory for Infectious Disease Research, University of Missouri, Columbia MO Yersinia pestis, the causative agent of plague, has been responsible for 3 worldwide pandemics, causing more deaths and socioeconomic impact throughout history than any other infectious agent. Though relatively well controlled in modern times, plague is endemic in 5 continents and is currently classified as a potential public health threat due to the well-documented ability of Yersinia to acquire and stably maintain foreign DNA. Due to the severity and lack of treatment options, well-characterized animal models that recapitulate disease in humans may be the only tool available for testing candidate vaccines or therapeutics for efficacy since large scale human clinical trials are not feasible given the limited number of patients who develop pneumonic plague each year. In this work, we describe the clinical course of pneumonic plague following inhalation exposure of rats with fully virulent Yersinia pestis. Telemetry implants were placed in the abdomen of each animal to monitor temperature, heart rate and activity of infected animals, and, in parallel, bacterial growth and tissue pathology were assessed every 24 hours. At 100xLD50, 95% of animals die within 3 days, between 48 and 72 hours post-infection. However, at 24 and 48 HPI, bacteria were frequently not recovered from the lungs or other tissues. In contrast, we monitored fever in 95% of the animals, and the appearance of fever was present in 100% of the animals that succumbed to the disease 16 hours before death. Together, the data indicate that fever reliably indicates disease severity and the Brown Norway rat develops pneumonic plague that follows a clinical disease course that is highly similar to humans.

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Identification of Specific Cellular Kinases as Potential Regulators of HSV-1 ICP0 Transactivation Activity Angela M. Fowler, Heather E. Shinogle, David S. Moore, and David J. Davido Department of Molecular Biosciences, University of Kansas, Lawrence KS The herpes simplex virus type 1 (HSV-1) immediate-early (IE) phosphoprotein, infected cell protein 0 (ICP0), is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. Published reports have shown that the phosphorylation state of ICP0 and the activities of a set of cellular kinases are important for ICP0’s transactivating activity. However, it is largely unknown what role specific cellular kinases play in the transactivator function of ICP0. To identify which host cell kinases regulate ICP0’s ability to stimulate viral gene expression, it was imperative to develop a high throughput screen to measure this activity. As a result, we designed a system using a set of HSV-1 GFP reporter viruses where the induction of the GFP gene is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24 h period by fluorescent microscopy. Subsequently, we adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescent imager. Using a small interfering RNA (siRNA) library against greater than 700 known and putative cellular kinases, we identified a small group of cellular kinases that appear to regulate HSV-1 ICP0 transactivation activity. Our results indicate that a high throughput assay is a valid and powerful method for monitoring the transactivating activity of ICP0. We are currently examining a subset of these identified kinases to determine how they affect the expression of ICP0 and its other biological activities.

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HSV-1 ICP22 but not its Truncated Form US1.5 is Required for VICE Domain Formation and Efficient Acute Replication and Latent Infections in Mice Heba H. Mostafa and David J. Davido Department of Molecular Biosciences, University of Kansas, Lawrence KS The herpes simplex virus type 1 (HSV-1) immediate early protein, infected cell protein 22 (ICP22), is required for efficient replication in restrictive cell lines, viral induced chaperone enriched (VICE) domain formation, and efficient expression of a subset of viral late proteins. Additionally, ICP22 is important for optimal acute viral replication in vivo and establishment of latency. Previous studies have shown that US1, the gene that encodes ICP22, also produces a second protein that is an in-frame N-terminally truncated form of ICP22 known as US1.5. Studies conducted to characterize the functions of ICP22 have not separated its functions from that of US1.5. To determine the individual roles that ICP22 and US1.5 play in viral infection, we made viral mutants that express either ICP22 (M90A) or US1.5 (3Xstop). Our studies showed that both mutants replicate to levels similar to that of wild type HSV-1 in the restrictive cell line, HEL, and both enhance the expression of the late viral proteins vhs and gC. However, in contrast to the M90A mutant, the 3Xstop mutant was unable to induce VICE domain formation in cell culture, replicate efficiently in the eyes and trigeminal ganglia of mice during acute infection, or efficiently establish a latent infection. Our data demonstrate that while both ICP22 and US1.5 can promote efficient replication and activate the expression of late genes in restrictive cells, only ICP22 induces VICE domain formation and enhances acute replication and establishment of latency in vivo.

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TLR7-mediated Induction of Type I Interferon by Intracellular Yersinia pestis Enhances Plague Pathogenesis Miqdad O. Dhariwala, Curtis J Pritzl, Bumsuk Hahm and Deborah M Anderson Department of Molecular Microbiology and Immunology, Department of Veterinary Pathobiology, University of Missouri, Columbia MO Yersinia pestis causes plague, a rapidly progressive and lethal disease. Extracellular Y. pestis relies on the type III secretion system to prevent activation of innate immune cells and induce programmed cell death in order to establish a replicative niche. Intracellular Y. pestis survives and replicates within a membrane-bound vacuole, and how this impacts the innate immune response is unknown. We recently demonstrated that respiratory infection of mice by Yersinia pestis induces type I interferon (IFN), a pro-inflammatory cytokine that is required for defense against viral infections, but causes increased susceptibility to plague. In fact, based on the pathogen, induction of type I IFN during bacterial infection leads to different outcomes, some of which result in clearance while others enhance pathogenesis. Data suggests that the cellular pathway exploited for the induction of type I IFN may influence its downstream effects. We therefore studied the mechanism whereby Y. pestis induces type I IFN in macrophages with the goal to understand how this cytokine enhances plague pathogenesis. We found that intracellular bacteria are recognized by toll-like receptor 7 (TLR7), leading to activation of type I IFN and increased host susceptibility in a murine model. TLR7 is known to localize to the endolysosome where it recognizes ssRNA and signals through the adaptor protein MyD88, which can activate NF-κB, IRF-3 and/or IRF-7, the latter two being transcription factors for type I IFN. We found that type I IFN production during Y. pestis infection was dependent on MyD88 as well as both NF-κB and IRF-3 suggesting both transcription factors are required for full induction of this response. Together the data suggest that, following phagocytosis, ssRNA of Y. pestis may be detected by TLR7 leading to a type I IFN response that enhances the pathogenesis of plague.

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Characterization of Reassortant H1N2 Variant Influenza Viruses in Pigs Michael Duff, Jingjiao Ma, Huigang Shen, Bhupinder Bawa, Qinfang Liu, Richard Hesse, Juergen Richt, Wenjun Ma Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan KS Many reassortant H1N2 swine influenza viruses (SIVs) have been detected worldwide since introduction of the 2009 pandemic H1N1 virus (pH1N1) into swine herds. Recently, reassortant H1N2 variant SIVs, that contain the M gene from pH1N1, have emerged and caused human infections in the United States. However, the pathogenicity and transmissibility of the H1N2 variant virus in pigs has not been investigated. Through passive surveillance, we have isolated two genotype reassortant H1N2 viruses with pH1N1 genes from diseased pigs in Kansas. One is a swine H1N2 variant (sH1N2v) with pH1N1 M gene; the other is a reassortant H1N2 virus (H1N2r) with six internal genes from pH1N1 and the remaining 2 genes are from endemic North American triple reassortant SIVs. In this study, we analyzed the pathogenicity and transmissibility of the sH1N2v, a human H1N2 variant (hH1N2v) and the H1N2r in pigs; an endemic triple reassortant H1N2 (H1N2e) SIV isolated in 2011 was used as a control. All viruses replicated efficiently in pigs’ lungs and successfully transmitted to sentinel animals. However, both human and swine H1N2 variants caused more severe lung lesions in infected pigs when compared to the H1N2r and H1N2e viruses. Although all four viruses were detected in the lungs of contact animals, the sH1N2v shed more efficiently than the other three viruses in contact animals. The hH1N2v displayed delayed and inefficient shedding kinetics in sentinel animals. Taken together, the H1N2 variant viruses are pathogenic and transmissible in pigs and could pose a threat to public and animal health.

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Guinea Pig Model of Coxiella burnetii Infection Ali Elliott, Ying Peng, Danielle Freches, Laura Schoenlaub, Guoquan Zhang Department of Veterinary Pathobiology, University of Missouri, Columbia MO Coxiella burnetii is an obligate intracellular bacterium that causes acute and chronic Q fever in humans. Currently there is no approved vaccine for Q fever in the United States. Most experimental vaccines against C. burnetii are tested using mouse models. The main phenotype observed in the mouse disease model is splenomegaly, however in humans, the most common symptom is fever. Guinea pigs infected with C. burnetii respond with fever and therefore may be a more suitable model to test vaccine protectiveness. The first step in testing vaccines is to establish a disease model, which we have done in this study. Hartley guinea pigs were infected IP with PBS, 2x104, or 2x106 C. burnetii (n=3/group). Daily body temperatures and weights were recorded, and necropsies were performed at 14d p.i. The high dose group had fevers from day 4 -7 and the low dose group had fevers from day 6-11. Body weights of both high and low dose groups decreased at the same rate. Splenomegaly was similar between the low and high dose groups, however bacterial burden was higher in the high dose group. Developing this model was a crucial step towards testing vaccines against C. burnetii in guinea pigs. Current studies are underway using guinea pigs to test the protectiveness of peptide vaccines which have been shown to be protective in mouse models.

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Protective Efficacy of SPI-1 and SPI-2 Proteins in a Mouse Salmonella Model Kelly Harrison Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK Salmonella enterica, with over 2500 serovars, is the causative agent of nearly 1.3 billion cases of disease annually in both humans and animals. Serovars Typhi and Paratyphi A and B cause enteric fever while non-typhoidal serovars such as Typhimurium cause gastroenteritis. In spite of these radical numbers of infection, a universal vaccine against many serovars is still absent. S. enterica possesses numerous pathogenicity islands, two of which encode type three secretion systems: SPI-1 and SPI-2. Their vital role in pathogenicity and their highly conserved sequence among different serotypes led us to investigate the protective efficacy of the type three secretion components SipB, SipD and SseB in S. enterica infections. Mice were immunized intranasally with the SPI-1 proteins SipB and SipD, the SPI-2 protein SseB, or all three proteins combined using monophosphoryl lipid A as the adjuvant. Immunogenicity was tested through antibody titers, enumerating antibody-secreting cells and levels of cytokine secretion. Mice were then challenged with a lethal dose of S. Typhimurium administered orogastrically. Up to 50% protection was observed for mice immunized with all three proteins. Our results indicate that these proteins provide protection against infection and provide information for further development of broad-range Salmonella vaccines.

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Conditional Gene Expression in Chlamydia trachomatis Using the Tet System Jason Wickstrum, Lindsey Sammons, Keasha Restivo, and P. Scott Hefty Department of Molecular Biosciences, University of Kansas, Lawrence KS Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. Using this system, a key sigma factor (Sigma 28) was expressed at selected times of the developmental cycle and the phenotype and regulon was determined. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia.

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Site-Specific, Insertional Inactivation of incA in Chlamydia trachomatis Using a Group II Intron Cayla M. Johnson and Derek J. Fisher Department of Microbiology, Southern Illinois University, Carbondale IL Chlamydia trachomatis is a Gram negative, obligate intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. Methods now exist to allow stable introduction of foreign DNA using the chlamydial cryptic plasmid as an expression platform and to screen for mutants using Tilling-based approaches. However, the field remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron’s substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA- mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at an MOI >1. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron and ability to select for mutants makes this platform a powerful tool to add to the growing chlamydial molecular toolbox.

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The role of RND Transport Systems in Calcium-induced Antibiotic Resistance and Virulence in Pseudomonas aeruginosa Sharmily S Khanam*, Dirk L. Lenaburg, Ryan C. Kubat, and Marianna A. Patrauchan Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK Pseudomonas aeruginosa is a facultative pathogen infecting lungs of cystic fibrosis patients and causing severe nosocomial and device-related infections. It is resistant to all antimicrobials available for clinical treatments, and therefore represents a great challenge in medicine. Calcium (Ca2+) is a well-established signaling molecule that regulates essential processes in eukaryotes, including innate immune responses. Earlier we showed that Ca2+ triggers biofilm formation and production of virulence factors in P. aeruginosa. By using both 96-well plate assays and E-test strips, we determined that Ca2+ increases minimal inhibitory concentrations of tobramycin and polymixin B at least tenfold in P. aeruginosa PAO1. Analyses of PAO1 membrane and extracellular proteins by using 2D PAGE-MS/MS and LC-MS/MS-based spectrum counting identified five RND multidrug efflux pumps MexAB-OprM, MexGHI-OpmD, TriABC-OpmD and MuxABC-OpmB, MexVw-OprM, whose abundance was affected by Ca2+. To study the role of RND systems in PAO1 Ca2+-induced antibiotic resistance and virulence, we used transposon mutants obtained from the University of Washington. We characterized the effect of Ca2+ on the mutants’ antibiotic resistance and infectivity in a plant model. The lack of all RND genes, except mexB, impaired Ca2+- induced virulence. The lack of mexXY, mexB, mexV, mexC, mexE, or czcB reduced Ca2+-induced tobramycin resistance, and mexC, mexQ and mexJ decreased the Ca2+-induced polymyxin B resistance. Furthermore, RT-qPCR transcriptional analysis showed that mexB, mexV, mexI and mexJ were up-regulated in response to Ca2+. Overall, the findings suggest that multiple RND systems respond to Ca2+, and play role in Ca2+-induced resistance and virulence in P. aeruginosa.

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Elucidation Of Binding By Clostridium difficile Toxin A Greg Lambert and Michael Baldwin Department of Molecular Microbiology and Immunology, University of Missouri, Columbia MO Nosocomial infection by Clostridium difficile has become a common problem in healthcare environments. TcdA (a 308kDa glucosyltransferase protein toxin) is one of two toxins produced by Clostridium difficile during infection, and contributes to damage of the intestinal epithelium. It was previously believed that the C-terminal CROPS domain was the only domain responsible for toxin binding, but recent evidence indicates that a putative secondary binding domain may exist. Truncation mutants of TcdA will be characterized with respect to binding capability, in order to locate the putative secondary binding domain.

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Macrophage Polarization in Lyme Arthritis Carrie E. Lasky, Anna M. Cunningham, Charles R. Brown Department of Molecular Microbiology and Immunology, Department of Veterinary Pathobiology, University of Missouri, Columbia MO Macrophages play an important role in the clearance of spirochetes in mice infected with the causative agent of Lyme Disease, Borrelia burgdorferi. There are a significant number of macrophages in the joints of infected C3H/HeJ mice. Due to the recent advances in the study of proinflammatory and anti-inflammatory macrophages, we set out to describe the phenotype of macrophages throughout the infection timecourse. To our knowledge, an in vivo study of infectious arthritis analyzing numbers of classically activated M1 macrophages and alternatively activated M2 macrophages has not yet been accomplished. Using flow cytometry and specific proinflammatory (M1) and anti-inflammatory (M2) macrophage markers, we found that M1 macrophages outnumber M2 macrophages at the peak of inflammation. Interestingly, even at resolution, M1 macrophage numbers remain elevated.

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Three Functional β-carbonic Anhydrases in P. aeruginosa PAO1. Role in Calcification S. R Lotlikar1, S. B. Hnatusko1, M. Pinard2, R. McKenna2, and M.A. Patrauchan1 1Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, 2Department of Biochemistry and Molecular biology, University of Florida, Gainesville, FL Calcium (Ca2+) is a key signaling molecule in eukaryotes. Abnormalities in Ca2+ homeostasis may lead to soft tissue calcification commonly associated with human diseases. However, the origin, molecular mechanisms of calcium precipitation and the role of this process in bacterial physiology remains unknown. Our hypothesis is that Pseudomonas aeruginosa carbonic anhydrases (CAs) are involved in CaCO3 deposition and thus contribute to the virulence of this human pathogen. Quantitative analysis of the extracellularly deposited Ca2+ showed that PAO1 precipitates Ca2+ in a concentration dependent manner. Transmission electron microscopy with X-Ray elemental analysis demonstrated that PAO1 grown at 10mM Ca2+ forms 0.1 µm deposits containing Ca2+. Bioinformatic analysis identified three genes PA0102, PA2053, PA4676 encoding β-CAs, designated psCA1, psCA2 and psCA3, respectively. The CAs were purified and confirmed to have specific CA activity. To study the structure and function of the β-CAs as potential targets for developing novel antimicrobials against P. aeruginosa infections, X-ray crystallographic structural studies have been initiated. Two crystal forms (A and B) have been obtained for psCA3, which diffracted to a resolution of 2.9 A˚ and 3.0 A˚, respectively. Structure analysis confirmed the open confirmation of the protein active site. Initial electron-density maps suggested that psCA3 is folded as a dimer of dimers in both crystal forms. The current work is focused on refining the protein structures for understanding the catalytic mechanisms, and determining the role of P. aeruginosa β-CAs in the formation of Ca2+ deposits and Ca2+-induced virulence.

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The H7N9 Influenza Virus Emerged in China Wenjun Ma Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan KS Influenza A virus is a zoonotic pathogen which is able to infect and cause diseases in humans and a wide range of avian and mammalian species. To date, 17 hemagglutinin (H) subtypes of influenza A viruses have been identified from birds and mammalian hosts. Recently, the novel H7N9 virus emerged in China responsible for more than one hundred thirty cases of human infection, has raised concerns that this strain has pandemic potential. Furthermore, many of the avian H7N9 viruses isolated from humans contain amino acid substitutions in HA and PB2 genes that are believed to be important in the adaptation of avian viruses to humans and other mammals. To date, the animal reservoir, routes of transmission and the scope of the spread of this virus among people and animals remain unclear. In this talk, I will report current situation on this novel H7N9 virus, and our and published research in different animal models on this novel H7N9 virus. Taken together, the novel H7N9 virus emerged in China has the potential for further adaptation to human/mammalian hosts and sustained human-to-human transmission.

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Triggering Ebola Virus for Membrane Fusion and Entry Chunhui Miao1, Minghua Li1, Lorraine M. Albritton2, Shan-Lu Liu1 1 Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, Columbia MO, 2 Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN The entry of Zaire Ebola virus (EBOV), a deadly Class A agent that causes hemorrhagic fever, is mediated by the envelope glycoprotein (GP). In order to initiate viral and host membrane fusion, EBOV GP must undergo protease cleavage that is mediated by cellular cysteine proteases, Cathepsin B/L, as well as requires binding to the specific cellular receptor, Niemann-Pick C1 (NPC1) present in endosome. Currently, the exact triggers for membrane fusion of EBOV as well as the modes of its regulation are not known. Here, we provide evidence that the protease cleavage, low pH, as well as the NPC1 binding are all required for the inactivation of EBOV infectivity. Aided by structure-based design, we characterized some EBOV GP mutants, and found that thermolysin cleavage can inactivate some of stabilizing GP mutants but not that of the WT. Interestingly, despite high levels of expression in transfected cells, most stabilizing and destabilizing mutants are less efficiently incorporated into retrovirus-based pseudoviral particles. KZ52, a neutralizing antibody, which is known to bind to the prefusion structure of EBOV GP, were able to effectively neutralize and inactivate the viral infectivity of GP mutants. Altogether, these data suggest conformational differences between these GP mutants and WT, and raise the possibility that these mutants may be useful for generating novel neutralizing antibody and subunit vaccines to combat EBOV infection.

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Characterization of the Novel Exosporium Proteins of B. anthracis Eun Min1,3, Hsin-Yeh Hsieh2,3, George Stewart2,3 1. Department of Bioengineering, University of Missouri, 2.Department of Veterinary Pathobiology, University of Missouri 3. Bond Life Sciences Center, University of Missouri, Columbia MO Bacillus anthracis is a spore-forming bacterium that is an infectious agent of the disease anthrax. The outermost layer of the spore, the exosporium, is responsible for the initial contact with the host, and it is covered with a hairlike nap layer consisting of BclA glycoprotein. In previous study from our laboratory, a mutant B. anthracis strain with a deletion of an operon carrying BAS0384-0390 genes was created and was found to have structural defects on the exosporium layer in the production of BclA. In order to observe how the genes of this operon would affect the assembly of the exosporium layer, each gene in the operon was fused with the mCherry reporter protein gene and introduced to the wild type Sterne strain of B. anthracis by electroporation, and the strains carrying the fusion protein were observed under fluorescent microscopy and photographed. Out of seven proteins encoded in the operon, three fluorescent fusion proteins localized to the exosporium. The results suggest that these genes encode proteins that are likely to be associated to the exosporium. For further investigation, the fusion construct of each gene will be introduced to the operon knockout strain to begin to study inter-dependence among the proteins encoded in this operon.

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Monitoring the Kinetics of the pH Driven Transition of the Anthrax Toxin Prepore to the Pore by Biolayer Interferometry and Surface Plasmon Resonance Subhashchandra Naik, Susan Brock, Narahari Akkaladevi, Jon Tally, Wesley Mcginn-Straub, Na Zhang, Phillip Gao, Edward P. Gogol, Brad L. Pentelute, R. John Collier, Mark T.Fisher Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City KS Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

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Role of Cyclooxygenase (COX)-1 in Humoral Immunity Carmela L. Pratt and Charles R. Brown Department of Veterinary Pathobiology, University of Missouri, Columbia MO Non-steroidal anti-inflammatory drugs (NSAIDs) have a multitude of uses, causing them to be more widely prescribed. Cyclooxygenase (COX) enzymes are most commonly known for their roles in inflammation; however, recently a focus has been directed on their role in antibody production and development of adaptive immunity. Our lab has previously demonstrated that following Borrelia burgdorferi infection, mice deficient in COX-1 activity developed normal levels of Borrelia-specific immunoglobulin (Ig)M but decreased levels of Borrelia-specific IgG production due to defective germinal center formation. Further investigation has revealed normal B-cell maturation, follicular helper T-cell numbers, B-cell receptor expression, and similar deposition of plasma cells in the bone marrow in COX-1-/- mice as compared to wild-type mice. Further evaluation of plasma cells revealed the number of short-lived plasma cells in the spleen of COX-1-/- mice was statistically greater (p=0.004) than in wild-type mice at 14 days post-infection. They remain elevated 45 days post-infection in COX-1-/- mice as compared to wild-type, although not statistically significant. Future experiments will include exploration of alterations in transcription factors involved in plasma cell development to further delineate the cause for increases in splenic plasma cells in COX-1-/- mice. Additionally, further evaluation of signaling defects in dendritic cells and T-cell-dependent pathways involved in B-cell activation may explain attenuation of antibody class switching. A better mechanistic understanding by which COX-1 influences the immune system in an infectious model will identify more selective utilization of NSAIDs and the potential for development of alternative drug targets applicable to all fields of medicine.

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Infection with Salmonella Typhimurium Alters the Tissue Architecture of the Spleen and Distribution of Cell Populations Kristin Rosche and Vjollca Konjufca Department of Microbiology, Southern Illinois University, Carbondale IL Salmonella Typhimurium (Salmonella) is a food-borne pathogen that causes self-limiting gastroenteritis, and often bacteremia and enlargement of the spleen (splenomegaly). Splenomegaly is attributed to increased recruitment of phagocytes, expansion of CD4 and CD8 T cells and massive extramedullary erythropoiesis, characterized by expansion of immature CD71+Ter119+ reticulocytes. It is not known however, whether Salmonella alters the architecture of the spleen, which could dramatically affect the immune responses to and the clearance of Salmonella. We used four color immunofluorescence microscopy (IMF) to characterize the in situ changes in splenic architecture and changes in cell population profiles during Salmonella infection in C57BL/6 mice. We show that splenomegaly is evident within the first week of infection and is accompanied by dramatic alterations of spleen architecture. In addition, splenomegaly leads to drastic changes in the proportion of cells in the tissue. Most notably, there is a significant increase in the populations of macrophages, and CD71+ and Ter119+ reticulocytes. In contrast to this, there is a significant decrease in proportions of B and T lymphocytes. Within the first week of infection, over 90% of Salmonella is cleared from the spleen by the innate immune system. It is not known how the destruction of splenic architecture caused by splenomegaly affects the generation of adaptive immunity to Salmonella. Understanding the mechanisms by which Salmonella causes splenomegaly will have important implications in the development of effective Salmonella-based vaccines and a better understanding of Salmonella pathogenesis.

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B Cell Infection by Coxiella burnetii Nine Mile Phase I and Nine Mile Phase II Laura Schoenlaub, Ali Elliott, and Guoquan Zhang Department of Molecular Microbiology and Immunology, Department of Veterinary Pathobiology, University of Missouri, Columbia MO Coxiella burnetii is an obligate intracellular bacterium that causes acute Q fever and occasional chronic infections in humans. Although it is known to infect a wide variety of cells, including macrophages and epithelial cells, its ability to infect B cells has not been investigated. A subset of B cells found in the peritoneal cavity known as B1b cells have demonstrated the ability to phagocytose particles, including C. burnetii Nine Mile phase II. We sought to determine whether these cells can phagocytose virulent Nine Mile phase I and whether C. burnetii NMI and NMII are capable of replicating in peritoneal B cells. Confocal and fluorescence microscopy showed NMI and NMII are capable of entering peritoneal B cells. NMII also showed the ability to replicate within purified peritoneal B cells using real-time PCR analysis. This data indicates that C. burnetii NMII is capable of infecting and replicating in peritoneal B cells. Further studies will be performed to determine whether NMI is capable of replicating in B cells and whether B cells have the ability to kill C. burnetii or act as antigen presenting cells.

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To Determine if an Antimicrobial Compound from Eastern Red Cedar Targets FtsZ in Cell Division of Staphylococcus aureus Che-Min Su1,2, Hsin-Yeh Hsieh2,3, Chung-Ho Lin1,4, and George C. Stewart2,3

1Center for Agroforestry, 2Bond Life Sciences center, 3Department of Veterinary Pathobiology, 4Department of Forestry, University of Missouri Staphylococcus aureus is a bacterium commonly found on human skin and is a frequent cause of skin and respiratory infections. Some S. aureus strains have developed resistance to multiple antibiotics, including beta-lactam antibiotics, and are known as methicillin resistant Staphylococcus aureus or MRSA. The CDC has reported over 80,000 invasive MRSA infections and a 14% mortality rate in 2011. Although the peptide antibiotics like cephalosporins can treat MRSA effectively, some newly discovered MRSA strains exhibit resistance to these drugs. Therefore, the need of the new antibiotics against MRSA is urgent. Our laboratory identified a natural antimicrobial compound synthesized by Eastern Red Cedar (Juniperus virginiana), named diterpenoid A that was reported to be inhibitory to the cell division process of Gram-positive bacteria, such as Bacillus subtilis and S. aureus, by inhibiting the action of the essential tubulin-like cell division protein FtsZ. We used ethylmethanesulfonate to create diterpenoid A-resistant mutants of Staphylococcus aureus, and the genomic DNA of the mutant strains along with sensitive parent strain were sent for Illumina high throughput genomic sequence analysis and data processed. Mutations unique to the resistant strains were identified, however, interestingly, no point mutation mapped to ftsZ. In order to observe how FtsZ protein may be affected by diterpenoid A, the S. aureus ftsZ gene was fused with the eGFP reporter protein gene and introduced to the S. aureus 8325-4 strain by transduction. To determine the efficiency of diterpenoid A against S. aureus in terms of FtsZ inhibition, a minimum inhibitory concentration test was conducted with the strain carrying the fusion protein and also the wild-type strain for comparison. To determine if diterpenoid A inhibited FtsZ activity during cell division, the strain carrying the fusion protein and the wild-type strain were grown to OD at 540 nm = 0.3, then treated with diterpenoid A of 50 μg/ml and incubated at 37°C. Samples were collected at 0, 1, 2, and 4 hours, fixed, washed, and observed under fluorescent microscopy and photographed. The MIC tests showed no difference in sensitivity between the wild-type strain and the strain carrying the fusion protein. The fluorescent microscopy showed, after 4 hours of diterpenoid A treatment, the S. aureus cells in the culture could still form the FtsZ ring while the treated culture optical density had decreased to 0.1 but the control culture’s optical density was increased to 2.0.

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Changes in the Type IVB Secretion System of C. burnetii in Response to Supplementation During Axenic Growth Ruth Weidman, Brandon Luedtke, and Edward Shaw Department of Microbiology & Molecular Genetics, Oklahoma State University, Stillwater OK In nature, Coxiella burnetii is an intracellular pathogen that causes the zoonotic disease Q-fever. C. burnetii resides in a nascent, host-derived parasitophorous vacuole (PV) that is reminiscent of an autophagolysosome. From the PV, C. burnetii modulates the host cell using effector proteins secreted by an essential type IVB secretion system (T4BSS). It is not clear what signals induce the C. burnetii T4BSS to deliver effectors across the PV membrane, but it has been shown that virulence secretion systems in other pathogens undergo physical changes based on host signals prior to secretion of effectors. We have previously shown that DotA and IcmX are released from C. burnetii during growth in the first generation acidified citrate cystine medium (ACCM-1) and that DotA localizes to the PV membrane and cytoplasmic vessicles of the host during infection. Using immunoblot assays, we show that DotA and IcmX are not released during C. burnetii growth in the second generation ACCM (ACCM-2). To identify components signaling DotA and IcmX release in ACCM-1, supplements were added to ACCM-2 grown C. burnetii. These assays revealed that lipid (fatty acid micelles) recovered the release of DotA and some IcmX. The addition of BSA induced release of larger amounts of IcmX and no DotA suggesting a series of signals may induce changes to the C. burnetii T4BSS. Work to further define these interactions and potential gene regulation in response to induction may reveal the nature of the signals present on host cell membranes/PV that induce C. burnetii’s T4BSS during infection.

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Restriction of Ebola Virus Entry by IFITM2 Yi-Min Zheng1, Chunhui Miao1, Kartik Chandran2 and Shan-Lu Liu1 1 Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, Columbia MO, 2 Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York The interferon-inducible transmembrane (IFITM) proteins have been recently identified to broadly inhibit entry of a wide spectrum of enveloped viruses, including influenza A virus, West Nile virus, Dengue fever virus, and HIV. Currently, the underlying mechanism of IFITM-mediated viral inhibition remains elusive. Here we showed that IFITM2 strongly inhibits entry of Ebola virus (EBOV), a highly pathogenic infectious agent with a fatality rate up to 90%. By using forward genetic strategy and recombinant VSV (rVSV) encoding GP, we recovered several EBOV glycoprotein (GP) mutants that are virtually resistant to IFITM2 restriction. We found that some of these escape mutants are less sensitive to the inhibitors of cathepsin B, an essential factor that is required for cleavage of EBOV GP and priming the fusion activation. The effects of these GP mutants on membrane fusion of EBOV GP have also been examined. Results from this work will facilitate better understanding of IFITM-mediated host restriction of EBOV infection, and have important implications for antiviral therapy.

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The University of Missouri Laboratory for Infectious Disease Research

MU LIDR is a NIH/NIAID Regional Bio-containment Laboratory (RBLs) that serve as a critical resource for researchers involved in the discovery and development of vaccines and therapeutics to prevent and treat infectious diseases. LIDR faculty focus on the interdisciplinary study of immunity, vector-borne disease, and microbial pathogenesis. LIDR is a part of our nation’s effort to protect public health and houses laboratory facilities that support BSL-2, BSL-3 and ABSL-3 research. LIDR provides professional research services in Aerobiology, Immunology, Microbiology and Pathology using experimental models of disease.

The Core offers custom designed aerosol services for BSL-2 and BSL-3 agents. All challenges are performed within a custom-designed and –built GERMFREETM Class III biological safety cabinet for optimal containment and user protection. Aerosols are generated via Collison nebulizer or Sparging Liquid Aerosol Generator.

The staff working at Immunology Core have expertise and several years of experience in study of immune response performing analysis and sorting of variety of bacterial pathogens and immune cells. The Core utilizes a powerful, 3-laser 8-color Beckman Coulter MoFlo XDP high speed cell sorter to perform flow cytometry and cell sorting in the study of infection response in fixed or live infected samples.

Laboratory space is available for scientists and collaborators to perform infectious disease research and vector-borne transmission study in BSL-2 and -3 laboratories. Research is emphasized on animal model & vaccine development, early immune response to study innate and adaptive immunity, vector-borne disease development, and host-pathogen interaction. Pathogens include Bacillus anthracis, Brucella, Burkholderia, Coxiella burnetii, Francisella tularensis, Yersinia pestis, and vector-borne viral pathogens such as dengue, chikungunia, sindbis.

Services include: CH TechnologiesTM inhalation exposure system for research on lower respiratory tract infections Pathogen and small animal aerosol model development Characterization of particle size distribution and real-time monitoring via aerosol spectrometer Dose determination, viability of infectious agents and animal evaluation services

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Services include: • Consultation and experimental design of multi-parameter panels • Four-way bulk cell sorting • Single-cell sorting into multi-well plates or onto microscope slides • Analysis of infected samples with a BSL-2 and BSL-3 select agents

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Last Name First Name Email Address InstitutionAdam Philip philip.adam@okstate.edu Oklahoma State UniversityAljasham Alanoud gollanar@siu.edu Southern Illinois University Amin Divya damin@kumc.edu University of Kansas Medical CenterAnderson Christina christina_anderson@bio-rad.com Bio-RadAnderson Deborah andersondeb@missouri.edu University of MissouriArizmendi Olivia oarizme@okstate.edu Oklahoma State UniversityBaldwin Mike baldwinmr@missouri.edu University of MissouriBarta Michael barta.michael@ku.edu University of KansasBayles Ken kbayles@unmc.edu University of Nebraska Medical CenterBeerntsen Brenda BeerntsenB@missouri.edu University of MissouriBermudez Carla cjpmbc@mail.missouri.edu University of MissouriBinning Jennifer jbinning@path.wustl.edu Washington University School of MedicineBland David dmb896@mail.missouri.edu University of MissouriBoon Jacco jboon@dom.wustl.edu Washington UniversityBouyoukos NIck nick.bouyoukos@midsci.com MIDSCIBrown Charles brownchar@missouri.edu University of MissouriBurns Joshua jrbf2b@mail.missouri.edu University of MissouriCamargo Carolina ccxd8@mail.missouri.edu University of MissouriCasey Mary Mcc6x2@mail.missouri.edu University of MissouriChen Xiaotong xiaotong.chen@okstate.edu Oklahoma State UniversityCheng Erdong mmir@kumc.edu University of Kansas Medical CenterChoudhari Shyamal shyamal.choudhari@okstate.edu Oklahoma State UniversityClem Rollie rclem@ksu.edu Kansas State UniversityCoate Eric eackr3@mail.missouri.edu University of MissouriCunningham Anna amrvd7@mail.missouri.edu University of MissouriDavido David ddavido@ku.edu University of KansasDhariwala Miqdad modcp6@mail.missouri.edu University of MissouriDuff Michael duff@vet.k-state.edu Kansas State UniversityElliott Ali elliottal@missouri.edu University of MissouriFales William H Falesw@missouri.edu University of MissouriFales Susan Falesw@missouri.edu University of MissouriFisher Derek fisher@micro.siu.edu Southern Illinois UniversityFisher Mark mfisher1@kumc.edu University of Kansas Medical CenterFranz Alexander franza@missouri.edu University of MissouriGater Susan gaters@missouri.edu University of MissouriGregory Michelle mlgt24@mail.missouri.edu University of MissouriHardwidge Philip hardwidg@gmail.com Kansas State UniversityHarrison Kelly hkellys@okstate.edu Oklahoma State UniversityHefty Scott pshefty@ku.edu University of KansasHermanas Timothy tmh3m8@mail.missouri.edi University of MissouriHowe Savannah savannah.howe@siu.edu Southern Illinois UniversityHsieh Hsinyeh hsiehh@missouri.edu University of MissouriHuang Xiaohong xhhuang@vip.sina.com Kansas State UniversityJohnson Cayla cjohnson@siu.edu Southern Illinois UniversityKantor Asher amkt33@missouri.edu University of MissouriKeleher Lauren keleherl@missouri.edu University of MissouriKhanam Sharmily khanam@okstate.edu Oklahoma State UniversityKim Kee Jun kkim@kumc.edu University of Kansas Medical CenterKocher Elena ejke99@mail.missouri.edu University of MissouriKonjufca Vjollca vjollca@micro.siu.edu Southern Illinois UniversityLambert Greg gsl7cf@mail.missouri.edu University of MissouriLang Yuekun yuekun@ksu.edu Kansas State University

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Last Name First Name Email Address InstitutionLasky Carrie cel9h3@mail.missouri.edu University of MissouriLea Wendy wlea@kumc.edu University of Kansas Medical CenterLee Jinhwa jinhwa@vet.k-state.edu Kansas state universityLi Minghua mlmmc@mail.missouri.edu University of MissouriLin Chungho linchu@missouri.edu University of MissouriLiu Shan-Lu liushan@missouri.edu University of MissouriLotlikar Shalaka shalaka.lotlikar@okstate.edu Oklahoma State UniversityLutter Erika erika.lutter@okstate.edu Oklahoma State UniversityMa Wenjun wma@vet.k-state.edu Kansas State UniversityMa Jingjiao jjma@vet.k-state.edu Kansas State UniversityMiao Chunhui cmp55@mail.missouri.edu University of MissouriMin Eun Ehm887@mail.missouri.edu University of MissouriMir Mohammad mmir@kumc.edu University of Kansas Medical CenterNaik Subhashchandra subhashnaik@gmail.com University of Kansas Medical CenterNguyen D.M Phuong pdnwf9@mail.missouri.edu University of MissouriOlson Sarah solson@datasci.com Data Sciences InternationalPatel Ami patelam@missouri.edu University of MissouriPatrauchan Marianna m.patrauchan@okstate.edu Oklahoma State UniversityPeng Ying pengy@missouri.edu University of MissouriPennella Min pennellami@missouri.edu Universityof MissouriPicking Bill william.picking@okstate.edu Oklahoma State UniversityPratt Carmela clp446@mail.missouri.edu University of MissouriPritzl Curtis pritzlc@missouri.edu University of MissouriRohrer Jennie jrohrer@datasci.com Data Sciences International (DSI)Rosche Kristin roschekl@siu.edu Southern Illinois UniversitySchoenlaub Laura ls33d@mail.missouri.edu University of MissouriSkyberg Jerod skybergj@missouri.edu University of MissouriStewart George stewartgc@missouri.edu University of MissouriSu Che-Min cs9n9@mail.missouri.edu University of MissouriWang Zekun zwang2@kumc.edu University of Kansas Medical CenterWang Juan wangj@missouri.edu University of MissouriWasala Lakmini lwxz4@mizzou.edu University of MissouriWeidman Ruth ruth.weidman@okstate.edu Oklahoma State UniversityYu Jingyou jypn4@mail.missouri.edu University of MissouriYu Hai haiy@vet.k-state.edu Kansas state univeristyYuan Liguo liguo@vet.k-state.edu Kansas State UniversityZhang Guoquan zhangguo@missouri.edu University of MissouriZhang Yan zhanyan@missouri.edu University of MissouriZheng Yi-min zhengyim@missouri.edu University of Missouri

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