September 26-27, 2014

University of Missouri - Columbia

13th Annual

Great Plains

Infectious Disease

Meeting

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The 13th Annual

Great Plains Infectious Disease Meeting

We are pleased to host the 13th Great Plains Infectious Disease (GPID) Meeting at the University of Missouri for the fifth 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 40 posters. This meeting has always been successful due to the generosity of our academic sponsors and selected vendors.

Thank you all for attending!

The GPID Programming Committee 2014

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13th Annual Great Plains Infectious Disease Meeting Program Schedule

September 26-27, 2014 University of Missouri – Columbia

Friday, September 26, 2014 – Monsanto Auditorium in Christopher S. Bond Life Sciences Center

4:00pm 5:00pm Keynote Presentation: Anthony James, Ph.D. University of California Irvine

6:00pm 7:00pm Reception (Stoney Creek Inn)

7:00pm 8:00pm Dinner (Stoney Creek Inn)

Saturday, September 27, 2014 (Adams Conference Center)

7:00am 8:00am Registration and Breakfast

8:00am 8:02am Opening Remarks

Session I: Host Pathogen (Adams Conference Center)

8:02am 8:30am Jerod Skyberg, University of Missouri

8:30am 9:00am Keer Sun, University of Nebraska Medical Center

9:00am 9:30am Francisco Martinez, University of Kansas

9:30am 9:50am Miqdad Dhariwala, University of Missouri

9:50am 10:20am Refreshments and Networking Break (Adams Conference Center)

Session II: Viruses and Vector Borne Pathogens (Adams Conference Center)

10:20am 10:50am Dana Vanlandingham, Kansas State University

10:50am 11:20am Wei Xu and Joshua Alinger, Washington University School of Medicine

11:20am 11:40am Asher Kantor, University of Missouri

11:40am Noon Minghua Li, University of Missouri

Noon 1:00pm Lunch (Adams Conference Center)

Session IV: Bacterial Pathogenesis (Adams Conference Center)

1:00pm 1:30pm Kevin Wilson, Oklahoma State University

1:30pm 2:00pm Huatao Guo, University of Missouri

2:00pm 2:30pm Jeff Bose, University of Kansas

2:30pm 2:50pm Sriram Varahan, University of Kansas

2:50pm 3:30pm Break (Adams Conference Center)

Session III: Poster Presentation

3:30pm 5:30pm Poster Session (Adams Conference Center)

5:30pm Pizza and Bonfire (Home of Dr. Anderson)

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SPEAKER ABSTRACTS

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Genetic Control of Malaria Parasite Transmission Anthony A. James Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA, 92697-4500; Department of Molecular Biology and Biochemistry, Francisco J. Ayala School of Biological Sciences, University of California, Irvine, CA, 92697-3900 USA Genetics-based approaches have been developed to control transmission of malaria parasites. Strategies for both population suppression and population modification of mosquitoes have benefitted from the development of transgenesis, site-specific recombination, insulator DNA sequences, specific gene ablation and targeted effector molecules. Proofs-of-principle with the Asian vector mosquito, Anopheles stephensi, have been demonstrated in the laboratory for flightless-female population suppression strains and modification strains engineered with pathogen-resistance genes for preventing transmission of Plasmodium falciparum. Population modification strategies have a potentially important role in the malaria eradication agenda. They can consolidate elimination gains by providing resistance to parasite and competent vector reintroduction, and allow resources to be focused on new sites while providing confidence that treated areas will remain malaria-free. Strategic planning in field-site selection, selection of mosquito strain and trial design and implementation is needed to achieve a successful first field trial. The trial should use local scientists and adhere to stringent community engagement and regulatory standards. The trial should have an epidemiological endpoint that results in local disease elimination.

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Immunopathology of Articular Brucellosis Jerod A. Skyberg Department of Veterinary Pathobiology and Laboratory for Infectious Disease Research, College of Veterinary Medicine, University of Missouri, Columbia, MO Brucellosis is one of the most common zoonotic diseases in the world. Osteoarticular complications are the most common focal manifestation of brucellosis and Brucella is a common cause of infectious arthritis in countries in which the disease is endemic. Little is known about the pathology of osteoarticular brucellosis due to the lack of relevant murine models. However, our laboratory has found that genetic- or antibody-mediated ablation of IFN-γ in mice results in colonization of the joint and articular inflammation during infection with B. abortus or B. melitensis. Histological analysis of affected joints reveals fibrinous and inflammatory infiltrates within the joint space co-localizing with Brucella antigen. Articular inflammation occurs in IFN-γ deficient mice infected with virulent Brucella regardless of the route of infection, indicating that Brucella has a tropism for articular tissue. In addition, adaptive immune cells are not essential for Brucella-induced articular inflammation as wild-type mice and mice lacking B and T cells develop articular inflammation at a similar rate and severity when infected with Brucella and depleted of IFN-γ. In contrast, we find increased numbers of neutrophils and macrophages in joints from mice with Brucella-induced arthritis. Also, the concentration of pro-inflammatory cytokines, such as IL-1, and chemokines, such as CXCR2 ligands, are strikingly enhanced in Brucella-infected joints. In addition, mice lacking the IL-1 receptor or CXCR2 display resistance to articular inflammation when infected with Brucella and depleted of IFN-γ. Collectively, these results indicate that adaptive immune cells are dispensable for Brucella induced- articular inflammation and that the targeting of chemokine or cytokine receptors may have potential as a complementary antibiotic therapy for the resolution of Brucella-induced arthritis.

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Expression of suppressor of cytokine signaling 1 (SOCS1) impairs viral clearance and exacerbates lung injury during influenza infection

Keer Sun1&2*, Sharon Salmon1, Vijaya Kumar Yajjala2, Christopher Bauer2 and Dennis W. Metzger1

1. Center for Immunology & Microbial Disease, Albany Medical College, Albany, NY, 12208

2. Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 68198-5900

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1-/- mice die within three weeks postnatally due to IFN- -induced hyperinflammation. Since it is well established that IFN-protection against influenza infection, we generated SOCS1-/-IFN- -/- mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1-/-IFN- -

/- mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN- -/- or WT animals. Enhanced viral clearance in SOCS1-/-IFN- -/- mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1-/-IFN- -/- mice had no significant effect on their enhanced resistance to influenza infection, while CD8+ splenocytes from SOCS1-/-IFN- -/- mice were sufficient to rescue RAG1-/- animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1-/- mice reconstituted with SOCS1-/-IFN- -/- adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN- protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.

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Subunit vaccine development: Type Three Secretion System of Shigella

Francisco J Martinez Becerra Dept. of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas Shigellosis is an important disease in the developing world, where about 90 million people are infected each year with Shigella spp. The Type Three Secretion Apparatus proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model when delivered together by intramuscular administration with MPL (monophosphoryl lipid A)/Alum or an intranasal administration with dmLT (double-mutant labile toxin) as the adjuvant. In order to simplify vaccine formulation and process development, we revised the vaccine design to incorporate both of the previously tested Shigella antigens in a single recombinant polypeptide chain. To determine if this novel fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice and compared the subsequent immunogenicity and efficacy. In the resulting in vivo studies, the DB fusion elicited comparable immune responses and retained the ability to protect mice in a lethal pulmonary challenge against S. flexneri 2a and S. sonnei, while providing moderate protection against S. dysenteriae. We revised the protective capacity of this protein using alternate administration routes and optimized the detergents present in the vaccine to enhance immunogenicity. In addition, we measured the protection of this vaccine in a novel intraperitoneal challenge model. This study exemplifies the process of vaccine development for recombinant proteins and its optimization.

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MyD88-independent TLR-7 signaling leads to type I interferon expression during plague Miqdad O. Dhariwala and Deborah M. Anderson Department of Veterinary Pathobiology, University of Missouri-Columbia Toll-like receptor 7 (TLR-7) is an intracellular pattern recognition receptor that becomes localized to endosomes/lysosomes in response to release of single stranded RNA by viruses and other microbes. Previous work has established a signaling pathway following TLR-7 activation involving the MyD88 adaptor protein and downstream activation of the transcription factors NF-κB and IRF-3 which in turn induce the expression of type I interferon and other interferon-stimulated genes. In this work, we describe a non-canonical pathway for TLR-7 signaling that is activated during bacterial infection and is independent of MyD88, but nevertheless leads to NF-κB- and IRF-3- dependent expression of Ifnβ. We further show that TLR-7 co-localizes with bacteria in a membrane-enclosed compartment suggesting bacteria may release small single stranded RNA as a pathogenesis mechanism during phagocytosis.

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Introduction and Distribution of Chikungunya Virus into the Americas Dana Vanlandingham Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, Kansas Arthropod borne viruses are an increasing threat to public health due to their emergence and re-emergence in both tropical and temperate areas. Human activities such as air travel and commerce have caused the geographic spread of both the vectors and viruses. The current introduction and distribution of chikungunya virus (CHIKV) into the Americas and the introduction and spread of West Nile virus (WNV) into the US since 1999 provide examples of how the geographic distribution of viruses can rapidly expand. The epidemiology, temporal and spatial distribution, and transmission cycles of two arboviruses will be compared. Following an overview of these two mosquito borne viruses, with particular reference to current situation in the US, the potential for the further spread of CHIKV in the US will be discussed.

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Mechanism of Cell-intrinsic Innate Immune Antagonism by Ebola Virus VP24

Wei Xu1, Megan R. Edwards2, Dominika M. Borek3, Alicia R. Feagins, Anuradha Mittal4, Joshua B. Alinger1, Kayla N. Berry1, Benjamin Yen2, Jennifer Hamilton2, Tom J. Brett5, Rohit V. Pappu4, Daisy W. Leung1, Christopher F. Basler2, and Gaya K. Amarasinghe1

1Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110. 2Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. 3Departments of Biophysics and Biochemistry, University of Texas Southwestern. Medical Center at Dallas, Texas. 4Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University, St Louis, MO 63110. 5Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110 Ebola virus are the causative agents of highly fatal hemorrhagic fever with case fatality rates of up to 90%. One of the key reasons behind the lethality Ebola viruses is their ability to rapidly and potently suppress the innate antiviral immune responses leading to uncontrolled viral replication and cytokine storm. Viral infections are detected by the innate immune system which signals the production of interferons (IFNs). These IFNs activate the JAK-STAT pathway to signal the phosphorylation of the signal transducer and activator of transcription (STAT) family of transcription factors. Phosphorylated STAT1 (PY-STAT1) is recognized by a subset of the karyopherin alpha (KPNA) family of nuclear transporters which shuttle PY-STAT1 to the nucleus and induce the expression of IFN stimulated genes (ISG) that confer an antiviral state. Previous studies show that Ebola virus VP24 (eVP24) protein can block the IFN signaling by binding to KPNA, but the molecular mechanism of this interaction is not clear. In this study, we solve the eVP24/KPNA5C complex structure, it revealed a unique binding site for eVP24 on KPNA5C which partially overlap with the PY-STAT1 binding site. This overlap provide a mechanism, where eVP24 binding to and completion with PY-STAT1 for KPNA results in the inhibition of STAT signaling. Together our results support a mechanism where nuclear translocation of PY-STAT1 and activation of ISG expression is inhibited by eVP24 binding to KPNA. Finally, our findings identify novel targets for therapeutic intervention against a biodefense agent with the potential to impact the American Warfighter.

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Effects of arbovirus infection on expression of matrix metallo-proteinases in Aedes aegypti Asher M. Kantor1, Nicole L. Held1, Shengzhang Dong1, Rollie J. Clem2, A. Lorena Passarelli2, Alexander W.E. Franz1

1Department of Veterinary Pathobiology, 303 Connaway Hall, College of Veterinary Medicine, University of Missouri, Columbia MO 65211 2Molecular, Cellular, and Developmental Biology Program, Division of Biology, 116 Ackert Hall, Kansas State University, Manhattan, KS 66506 It was previously shown that baculoviruses disseminate from midgut tissue of infected caterpillars by employing a novel biochemical pathway involving a baculovirus-encoded fibroblast growth factor homolog, matrix metalloproteinases (MMPs), and effector caspases. Activation of this pathway results in tracheal cell basal lamina remodeling, allowing baculoviruses to efficiently escape from the midgut during infection. In our current research effort we want to show whether arboviruses such as chikungunya virus (CHIKV, Togaviridae, Alphavirus) use a similar dissemination mechanism in Aedes aegypti. MMPs are zinc ion-dependent endopeptidases, which are excreted into the extracellular matrix. Besides playing a major role in developmental tissue remodelling and wound healing processes, MMPs are able to degrade collagen IV and laminin, components of the basal lamina. We annotated all and cloned full-length cDNAs for most of the MMPs from Ae. aegypti. The Ae. aegypti genome encodes nine putative MMPs and a single tissue-inhibitor of MMP, TIMP. All nine AeMMPs share no homologies with those of vertebrates. Five of the nine AeMMPs have homologs among Culex quinquefasciatus but not among any other arthropods. Based on domain mapping, we discovered five different principal MMP structures. Here we characterize the different MMPs and their expression patterns in different life stages of non-infected mosquitoes, and show how MMP expression patterns change in the presence of replicating CHIKV in Ae. aegypti. Alterations in MMP expression patterns in presence of replicating CHIKV may result in tissue barrier remodeling during the infection process.

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TIM-Family Proteins Inhibit HIV-1 Release

Minghua Li1, Sherimay Ablan2, Chunhui Miao1, Yi-Min Zheng1, Matthew S. Fuller1, Paul

D. Rennert3, Wendy Maury4, Marc Johnson1, Eric O. Freed2, and Shan-Lu Liu1

1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center,

University of Missouri, Columbia, MO, USA; 2Virus-Cell Interaction Section, HIV Drug

Resistance Program, National Cancer Institute-Frederick, Frederick, MD, USA; 3SugarCone Biotech LLC, Holliston, MA, USA; 4Department of Microbiology, University

of Iowa, Iowa City, IA, USA

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM)

proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins

strongly inhibit HIV-1 release, resulting in diminished viral production and replication.

Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the

plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1

abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding

deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be

incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-

positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1

entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as

that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in

differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The

inhibitory effects of TIM-family proteins on virus release are extended to other PS

receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-

family proteins to block the release of HIV-1 and other viruses by interaction with virion-

and cell-associated PS. Our work provides new insights into a virus-cell interaction that

is mediated by TIMs and PS receptors.

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Bacterial persisters survive antibiotics by remodeling their ribosomes Kevin Wilson Biochemistry and Molecular Biology, Oklahoma State University. Stillwater, OK 74078 When a population of bacterial cells is treated with an antibiotic, a small number of cells inevitably survive. After the antibiotic dissipates, the surviving cells can grow again. These survivors, called persisters, are phenotypically tolerant (but genetically sensitive) to the antibiotic because these cells are not actively growing and dividing. Despite their rarity, bacterial persisters are often sources of recalcitrant infection in humans. Because they survive, bacterial persisters may eventually become genetically resistant to antibiotics. We developed novel methodology to rapidly purify ribosome complexes from E. coli persisters. Our analysis revealed that ribosomes in persisters exist largely as inactive ribosomal subunits, whose ribosomal RNAs are progressively degraded by multiple ribonucleases. A small fraction of heterogeneous ribosomes remain mostly intact, except for reduced amounts of seven ribosomal proteins. These missing proteins are located on the exterior ribosome surface, where they play essential roles in translation initiation. Collectively, these findings reveal the mechanism for suppressing most protein synthesis and allow persister cells to survive extended exposure to antibiotics.

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Diversity-Generating Retroelements promote targeted evolution in bacterial and phage genes and may have broad applications for protein engineering Huatao Guo1,2, Longping V. Tse2, Angela W. Nieh2, Jorge Durand-Heredia1, Vincent Markovitz1, Elizabeth Czornyj2, Shufang Wang1, Brandon Kellogg1, Mari Gingery2, Diego Arambula2, Minghsun Liu2, Steven Williams3, Sabrina Oukil2, Vincent B. Liu2, and Jeff F. Miller2

1Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212; 2Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA; 3AvidBiotics Corporation, South San Francisco, CA Diversity-generating retroelements (DGRs) introduce vast amounts of sequence diversity into protein-encoding DNA genes. During DGR mutagenic homing, adenine residues are converted to random nucleotides in a unidirectional retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). The prototype DGR was discovered in a bacteriophage (BPP-1) infecting Bordetella species on the basis of its ability to promote phage tropism switching. DGRs have since been discovered in diverse phages and bacteria, which include human pathogens (Treponema, Legionella spp.), human commensals (Bacteroides, Bifidobacterium spp.), green sulfur bacteria (Chlorobium, Prosthecochloris spp.), cyanobacteria (Trichodesmium, Nostoc spp.), magnetotactic bacteria (Magnetospirillum spp.) and many other species. Using the Bordetella phage DGR as a model system, we studied DGR mutagenic homing and proposed that homing occurs through a target DNA-primed reverse transcription (TPRT) reaction. Deletion and mutation analyses determined the boundaries of the BPP-1 DGR target and demonstrated that a hairpin/cruciform structure downstream of VR is important for mutagenic homing. Using a kanamycin resistance gene as a reporter, we showed that the BPP-1 DGR can be engineered to target virtually any heterologous gene, suggesting they will have broad applications for protein engineering. Current work in the lab aims to deepen our understanding of the mechanism of DGR mutagenic homing and to develop them for practical applications.

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The Fatty Acid Kinase of Staphylococcus aureus controls virulence Jeffrey L. Bose Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, KS, USA During a screen of the Nebraska Transposon Mutant Library, we identified mutants in an operon encoding two hypothetical proteins that had a dramatic decrease in α-hemolysin production. Further analysis using non-polar mutations revealed that the second protein (termed VfrB) encoded in the operon was the primary contributor to conditionally-controlled α-hemolysin regulation as well as the expression of proteases and other secreted virulence factors. Bioinformatics suggested that VfrB may be a kinase, and it was found that this protein was essential for uptake of exogenous fatty acids. Furthermore, it was discovered that VfrB is a fatty acid kinase which works in cooperation with two additional previously hypothetical proteins (termed FakB1 and FakB2). Importantly, while biochemical evidence suggested the existence of such an enzyme, no protein performing this function had been described. While VfrB serves as a fatty acid kinase, FakB1 and FakB2 are fatty acid-binding proteins with differing specificity. In vivo studies using a murine model of dermonecrosis revealed that the vfrB mutant was hyper-virulent as observed by increased tissue damage, despite similar numbers of bacteria present. Together, these studies identify a previously undescribed fatty acid kinase complex found in gram-positive bacteria, and reveals the connection between fatty acid metabolism and virulence factor regulation in Staphylococcus aureus.

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Deletion of an RNPP family protein results in hyper biofilm formation in Enterococcus faecalis Sriram Varahan and Lynn E. Hancock Department of Molecular Biosciences, University of Kansas, Lawrence KS 66045 Enterococcus faecalis is an opportunistic pathogen that has emerged in recent years as one of the leading causes of nosocomial infections. Biofilm formation is considered to be one of the hallmarks of enterococcal biology and allows E. faecalis to cause persistent infections in the host such as catheter associated urinary tract infection (CAUTI) and surgical site infections. Several genes have been shown to be important for biofilm development. Here we describe a previously uncharacterized protein, EF0073, that is predicted to be a member of the RNPP family of transcriptional regulators in Gram-positive bacteria. Deletion of ef0073 resulted in hyper biofilm formation in comparison to the parental V583. When EF0073 was overexpressed using a high copy plasmid system, this resulted in a severe biofilm defect suggesting that this protein might be a repressor of biofilm formation in E. faecalis. Scanning electron microscopy analysis revealed the presence of an abundant exo-polymer matrix in the mutant biofilms in comparison to the parental V583 strain. The biofilms formed by the ef0073 deletion mutant was more resistant to proteinase K treatment compared to the wild-type which is indicative of an altered biofilm matrix composition. Elucidating the exact mechanism by which EF0073 regulates biofilm formation in E. faecalis is the focus of ongoing studies in the laboratory

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POSTER ABSTRACTS

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Last Name First Name Abstract Title

Arizmendi Olivia The Shigella flexneri T3SS tip protein IpaD elicits caspase-dependent cell death in macrophages

Barta Michael Hypothetical Protein CT398 (CdsZ) Interacts with σ54 (RpoN) and the Type III Secretion Export System in Chlamydia trachomatis

Behar Amanda Chlamydia trachomatis recruits Protein Kinase A and Protein Kinase A substrates during infection.

Brown Pamela Polar elongation during growth of A. tumefaciens cells

Coate Eric Poor Cardiac Function Underlies the Rapid Progression of Pneumonic Plague in Brown Norway Rats

Cho Junho Escherichia coli persister cells suppress translation by selectively disassembling their ribosomes

Elliott Ali Neutrophil depletion leads to increased disease severity following C. burnetii infection.

Fisher Mark Lessons from Nature: Strategies for Capturing, Transitioning and Imaging membrane inserted toxins from immobilized states.

Freches Danielle Mechanism of protection induced by a lipopolysaccharide-targeted peptide mimic vaccine against Coxiella burnetii

Harrison Kelly Analysis of Salmonella Type III Secretion System Proteins as Potential Vaccine Candidates in Murine, Bovine and Porcine Models

Hinshaw Kara Quorum sensing promotes defense and self-preservation during interspecies competition

Howe Savannah Protein-coated nanoparticles are internalized by the epithelial cells of the female reproductive tract and induce systemic and mucosal immune responses

Johnson Britney Biochemical characterization of Marburg virus VP24 interaction with Keap1 and its role in cytoprotective antioxidant responses

Kumar Amit Outer membrane proteins of Enterotoxigenic Escherichia coli with protective efficacy in intranasal mice challenge assay

Lacey Carolyn CXCR2 Signaling Facilitates Brucella-Induced Articular Inflammation

Lang Yuekun Characterization of Influenza B Viruses Resistant to Neuraminidase Inhibitors

Lasky Carrie Macrophage Polarization in Murine Lyme Borreliosis

Lazear Helen Interferon lambda restricts West Nile virus neuroinvasion by enhancing integrity of the blood-brain barrier

Lopez Lacey The Type VI Secretion System of Yersinia pestis Influences Intracellular Survival

Manoharan Shankar Role of Streptococcus mutans LiaF in the regulation of the liaFSR three component system

Miao Chunhui Ebola Virus Glycoprotein Induces Cell-cell tansmission at Neutral pH: Role of Cysteine Cathepsins and Niemann-Pick Type C1

Pascual Elizabeth Variability of Pseudomonas aeruginosa recovered from cystic fibrosis patients in different age groups

Pratt Carmela Characterization of Biphenotypic B/Macrophage cells in Vitro and in Pneumonic Francisella tularensis Infection

Richards Chris Transcription of T4ASS ORFs In Spotted Fever Group Rickettsia

Richner Justin Age-dependent defects in naïve CD4+ T cell trafficking and priming impair adaptive immunity and control of virus infection

Rimbey Jeanette Will a bacteriophage-based therapy be effective against the bacterial plant pathogen Agrobacterium tumefaciens?

Schoenlaub Laura The role of peritoneal B cells during primary infection with Coxiella burnetii

Silva Patricia Acyl-homoserine lactone-independent activation of an orphan LuxR in B. thailandensis and B. pseudomallei.

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Wang Gaochan Influence of NleH effector expression and host microbiota on Citrobacter rodentium colonization of mice

Weidman Ruth A Comparison of Coxiella burnetii Response to Supplementation During Growth in Axenic Media

Williams Graham Genomic Compatibility and Pathogenesis of Single Reassortant Viruses Derived from the 2009 Pandemic H1N1 and a North American Avian H7N3

Winford Edric The Role of 12/15 Lipoxygenase in a Murine Model of Lyme Disease

Yu Jingyou IFITM proteins inhibit HIV-1 cell-to-cell transmission

Willix Joshua Yersiniabactin evades IRF3-mediated protection during pneumonic plague

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The Shigella flexneri T3SS tip protein IpaD elicits caspase-dependent cell death in macrophages Olivia Arizmendi1, Francisco J. Martinez Becerra1, William D. Picking1 and Wendy L. Picking1‡

1Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047 Shigellosis, a type of bacillary dysentery, is a gastrointestinal infectious disease caused by Shigella spp. Approximately 165 million cases of shigellosis are reported every year around the world, most of them in developing countries. Shigella is able to colonize the human colonic epithelium and promote an inflammatory response only after inducing cell death of residing macrophages. All of these virulent actions are intimately tied to the Type III Secretion System (T3SS) of this organism. 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. IpaD has also been shown to induce cell death via apoptosis in B lymphocytes. We performed cytotoxicity, caspase activation and inhibition assays as well as a multiplex approach to study the cytokine response to IpaD. Furthermore, a possible binding partner was found. Together, this data allows us to conclude that IpaD is also able to elicit apoptosis in macrophages and more studies are needed to show the pathways that could enable IpaD to trigger these pro-apoptotic signals in macrophages.

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Hypothetical Protein CT398 (CdsZ) Interacts with σ54 (RpoN) and the Type III Secretion Export System in Chlamydia trachomatis Michael L. Barta1, Jason R. Wickstrum1, Frances S. Mandelbaum1, Kevin P. Battaile2, Scott Lovell3, P. Scott Hefty*1

1 Department of Molecular Biosciences, University of Kansas 2 IMCA-CAT, Hauptman-Woodward Medical Research Institute, Argonne, Illinois

3 Protein Structure Laboratory, Del Shankel Structural Biology Center, University of Kansas A significant challenge to bacteriology is the relatively large proportion of proteins that lack sufficient sequence similarity to support functional annotation (i.e. hypothetical proteins). The aim of this study was to apply protein structural homology to gain insights into a candidate protein of unknown function within the medically important, obligate intracellular human pathogen Chlamydia trachomatis. A crystal structure of CT398 was determined that displayed a high degree of structural similarity to FlgZ (Flagellar-associated zinc-ribbon domain protein) from Helicobacter pylori. This observation directed analyses of candidate protein partners of CT398, revealing interactions with the alternative sigma factor RpoN (σ54) and two paralogous Type III Secretion System (T3SS) ATPase regulators (CdsL and FliH). Employing a modified protocol that yielded highly efficient transformations in C. trachomatis, the functional role of CT398 was further examined by conditional gene expression and subsequent phenotypic evaluation. Overexpression of CT398 during mid/late developmental cycle resulted in significantly decreased inclusion volume, reduced levels of infectious progeny and grossly enlarged morphology. These studies provide the first phenotypic analyses of overexpressed genes in Chlamydia and suggest that CT398 functions in several key areas of chlamydial biology, including as a RpoN-chaperone and potentially in directing mRNAs for translation and secretion via the T3SS. We thus propose that CT398 be named CdsZ (Contact-dependent secretion-associated zinc-ribbon domain protein).

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Chlamydia trachomatis recruits Protein Kinase A and Protein Kinase A substrates during infection. Amanda Behar and Erika Lutter Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma The obligate intracellular pathogen, Chlamydia trachomatis, usurps many host cell-signaling pathways from within a membrane bound vacuole, deemed an inclusion. C. trachomatis has been previously shown to recruit and activate Src family kinases at discrete microdomains on the inclusion membrane. These microdomains are theorized to be regions for additional kinase activity. This study investigated the recruitment of Protein Kinase A (PKA) and PKA phosphorylated substrates to the inclusion membrane microdomains during C. trachomatis infection. PKA was found to be sequestered to the inclusion membrane microdomains and colocalizing with active Src family kinases at mid to late infection. Phosphospecific antibodies to PKA phosphorylated substrates demonstrated that PKA substrates also colocalized with Src family kinases. Pharmacological inhibition of PKA activity resulted in a loss of PKA phosphorylated substrate recruitment and localization, while PKA recruitment remained unaffected. These studies provide novel insights into the diverse role of PKA during C. trachomatis infection and suggest that the active Src family kinase rich microdomains function as highly active kinase regions on the inclusion membrane surface that may be involved in many essential chlamydial processes.

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Poor Cardiac Function Underlies the Rapid Progression of Pneumonic Plague in Brown Norway Rats Eric A Coate, Deborah M. Fine and Deborah M. Anderson Department of Veterinary Pathobiology1 and Veterinary Medicine and Surgery2, University of Missouri, Columbia, MO Background: Yersinia pestis is the etiologic agent of pneumonic plague, a devastating disease in humans characterized by acute bronchopneumonia, bacteremia, and sepsis. The bacteria is sensitive to a number of antibiotics in clinical use, yet the mortality rate for pneumonic plague remains over 50% even when the patient received antibiotics. In order to identify ways to extend the window where treatment can be effective, we have been studying the clinical course of pneumonic plague in Brown Norway rats infected by aerosol exposure to Y. pestis CO92. Methods: Telemetry transmitters were implanted in the abdomen of the rats and body temperature, heart rate and activity were recorded following infection. Animals uniformly developed fever before 48 hours post-infection and the fever period was short, followed by a pronounced hypothermia that occurred in all rats that later succumbed to disease. Upon death, a number of cardiac arrhythmias were observed. In this work, we used ECG Pattern Recognition Analysis (PRA) to localize the timing of heart arrhythmias. Results: With first order linear regression models, we examined the relationship between body temperature and heart rate in the fever and hypothermic stages of the infection. Strikingly, we found that decreased animal body temperature and heart rate were significantly correlated with heart arrhythmia development. The correlation range (r2) between body temperature and heart rate was 0.64 – 0.97 in the hypothermic stage compared to 0.12 in healthy animals. Conclusions: Overall, these data provide a mechanism underlying the rapid progression of disease following development of fever. We are therefore using this model to re-examine Y. pestis mutants and to test treatment regimens aimed at improving cardiac function. Together, these data may ultimately lead to methods that extend the life of plague victims such that effective antibiotic treatment can be administered.

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Polar elongation during growth of A. tumefaciens cells Jeremy Daniel, Matthew Howell and Pamela J.B. Brown1

1Division of Biological Sciences, University of Missouri, Columbia MO 65211 Elongation of many rod-shaped bacteria occurs by peptidoglycan synthesis at discrete foci along the sidewall of the cells. Yet, within the Rhizobiales there are many budding bacteria, in which new cell growth is constrained to a specific region. Indeed, the rod-shaped plant pathogen Agrobacterium tumefaciens grows unidirectionally from the new pole generated after cell division during elongation. The A. tumefaciens genome lacks the well-characterized genes mreBCD, pbpA, and rodA required for lateral cell elongation suggesting that A. tumefaciens utilizes a presently undescribed mechanism for constraining cell wall growth to the new pole. Efforts to reveal this mechanism by identifying proteins involved in this process are underway. We have identified a number of candidate proteins that co-localize with the active sites of peptidoglycan synthesis and are presently determining the function of these proteins in polar elongation. Notably, the Rhizobiales contains important pathogens of plants, animals, and humans. Thus, determining the mechanism of polar elongation in these bacteria will provide compelling insights that can be used to design novel anti-microbial strategies to control these pathogens.

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Escherichia coli persister cells suppress translation by selectively disassembling their ribosomes Junho Cho, Janet Rogers, Mark Kearns, Macall Leslie, Steven D. Hartson, and Kevin S. Wilson Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma

Persister bacteria survive exposure to multiple bactericidal antibiotics. Unlike antibiotic

resistant cells, persisters represent a subfraction of genetically identical antibiotic-

sensitive cells that transiently keep a physiologically dormant state. Previous studies

reported that protein synthesis is repressed in the dormant state, but the state of

ribosomes has not been studied in persister cells. Here, we investigated the ribosome

complexes in E. coli persister cells through mass spectrometry, ribosome profiling and

ribosomal RNA analysis. Our results revealed that ribosomes in persister cells are

heterogeneous. The majority of active ribosomes (i.e., polysomes) disappeared. The

ribosomal RNAs were progressively degraded by ribonucleases. Among the remaining

ribosomes, about 40% appeared to be intact, and the rest of them were heterogeneous

with seven ribosomal proteins depleted. Six of these proteins (S3, S5, S10, S11, S14,

and S21) are located on the surface of 30S ribosomal subunit. They are commonly late

binding proteins in the ribosome assembly, except S5 and play critical roles in translation

and transcription coupling. Protein L25 binds 5S ribosomal RNA to the 50S ribosomal

subunit. Collectively, a portion of the ribosome complexes in persister cells has immature

ribosome assembly or depleted ribosomal proteins. These results provide insights for

understanding the state of ribosome complexes and clues for how translation is

suppressed in persister cells.

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Neutrophil depletion leads to increased disease severity following C. burnetii infection Ali Elliott, Danielle Freches, Laura Schoenlaub, Ying Peng, Guoquan Zhang Veterinary Pathobiology, University of Missouri, Columbia MO Coxiella burnetii, an obligate intracellular bacterium, is the causative agent in the zoonotic disease Q fever. The disease is spread through aerosol with the acute form causing flu-like symptoms, and the less common chronic form resulting in endocarditis. C. burnetii infects and replicates within host cells. A previous study from our lab revealed that C. burnetii infects neutrophils both in vitro, and in-vivo following aerosol infection. The current study is focused on discovering what role neutrophils play following infection. BALB/C mice were infected with C. burnetii NMI via intranasal route. Mice were injected with 0.25 mg of neutrophil depleting RB6 antibody or IgG control 1 day prior to infection and every other day following infection. Neutrophil depleted mice had significantly lower body weight on day 11 and 13 p.i. Mice were necropsied on days 3, 7, 14, 28, and 40 p.i. Neutrophil depleted mice had increased splenomegaly at all time points except 3 days p.i. and increased bacterial burden in the spleen on days 14, 28, and 40 p.i. Bacterial burden in the lungs was higher in neutrophil depleted mice on day 28 p.i. Histopathology revealed a higher infiltration of macrophages into the spleens of neutrophil depleted mice. MIP-1 was higher in the BALf collected from neutrophil depleted mice. These results suggest that neutrophils play an important role in the immune response to C. burnetii infection. Studying the interaction between C. burnetii and the innate immune system can provide a model system for understanding how this complex organism works.

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Lessons from Nature: Strategies for Capturing, Transitioning and Imaging membrane inserted toxins from immobilized states. Narahari Akkaladevi1, Deepa Patel2, R. John Collier2 and Mark T. Fisher1 1Department of Biochemistry and Molecular Biology. University of Kansas Medical Center. 2Department of Microbiology and Immunobiology, Harvard Medical School Bacterial toxin entry into the cell often requires an initial binding on the cell surface, followed by endocytosis where acid induced pH changes transition the benign toxin into a tranlocation competent state. At the molecular level, this acidification induces a limited unfolding /refolding and membrane insertion reaction. Our laboratories have devised methods to capture pretransitioned toxins on immobilized surfaces to orientate potential unfolding refolding transitions orthogonal to the immobilization surface. In some instances, a combination of vesicles or micelles and acidification induces this change, particularly in cases where the membrane insertion requires membrane curvature. In this work, cysteines were engineered onto the prepore cap alone and pH or urea 37oC induces pore formation of the PA alone. Initial negative stain EM analysis indicates that two cysteine PA heptamer variants could be immobilized using sulfhrydyl coupling, solubilized with lipid nanodics construction on the immobilized PA and released onto EM grids and visualized on electron micrographs. We propose that this general immobilization methodology will be useful to detect membrane inserted states of a whole host of membrane insertable toxins and viral proteins such as diphtheria toxin, Clostridia toxins (collaboration with Michael Baldwin U of Missouri) and influenzia hemagluttinin to visualize these complexes. More importantly, the ability immobilize toxins, induce transitions by acidification (~ pH 7- pH 5) and follow real time transitions of these toxins using Biolayer interferometry provides a unique detection platform that can be used to identify and validate ligand stabilizers to these transitions under endosomal pH conditions.

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Mechanism of protection induced by a lipopolysaccharide-targeted peptide mimic vaccine against Coxiella burnetii Danielle Freches, Ying Peng, Ali Elliott, Laura Schoenlaub, and Guoquan Zhang Veterinary Pathobiology, University of Missouri, Columbia MO Coxiella burnetii is an obligate intracellular bacterium that causes Q fever in humans. Although formalin-inactivated C. burnetii phase I (PI) vaccine provides near complete protection in animal models and humans, it can induce severe adverse reaction and is not authorized in the US. Our laboratory has developed an alternative candidate vaccine, m1E41920-KLH, which is composed of m1E41920, a synthetic peptide derived from PI-LPS, conjugated to the carrier protein keyhole limpet hemocyanin (KLH). Vaccination with m1E41920-KLH has been shown to confer a significant resistance against C. burnetii infection in mice. The current study investigated the mechanisms of protective immunity elicited by m1E41920-KLH and the long-term protection. These approaches included the passive transfer of mouse immune serum and adoptive transfer of a spleen cell subset isolated from immunized mice prior to bacterial challenge. Both immune serum and total splenocytes conferred protection against C. burnetii, whereas total T cells or CD4+ T cells did not. Moreover, the immunization did not induce a specific T cell response against C. burnetii suggesting that the antibody response plays a key role in m1E41920-KLH-mediated protection. However, immunization of CD4+-deficient mice showed that CD4+ T helper cells are required for synthesis of antibodies resulting in protection against C. burnetii infection. Furthermore, we show that m1E41920-KLH vaccination can induce a long-lasting protective immunity against C. burnetii.

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Analysis of Salmonella Type III Secretion System Proteins as Potential Vaccine

Candidates in Murine, Bovine and Porcine Models

Kelly Harrisona, Francisco J. Martinez-Becerraa, Todd Jacksonb, Scott Carterc, Shyamal P. Choudharia, Olivia Arizmendia, Daniel R. Pickingc, John D. Clementsd, Wendy L. Pickinga

aDepartment of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS; bCenter for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK; cDepartment of Animal Science, Oklahoma State University, Stillwater, OK; dDepartment of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA Over one billion cases of disease occur annually among humans and livestock due to infections with Salmonella enterica. Non-typhoidal serovars such as Typhimurium and Choleraesuis are capable of infecting humans along with their natural hosts: mice and pigs, respectively. With an ever increasing number of antibiotic resistances in Salmonella spp. and a push for their reduction in meat-production, the best approach for disease prevention is vaccination—yet there is currently no broadly protective vaccine available. S. enterica encode two Type Three Secretion Systems (T3SS) found on Pathogenicity Islands one and two (SPI-1 and SPI-2) with high sequence conservation among the different serovars. Because of this, we investigated the potential use of T3SS tip proteins SipB and SipD from SPI-1 and SseB from SPI-2, along with the adjuvants double mutant heat-labile toxin (dmLT) and monophosphoryl lipid A, as vaccine candidates in murine, bovine, and porcine animal models. Immunogenicity and protective efficacy were determined through antibody titers and a subsequent challenge: survival was monitored in mice while weight loss, shedding, and fever were monitored in the calf and pig challenges. High serum IgG levels were detected in all animal models as well as up to 50% protection in mice, and a marked reduction in fever and shedding in calves. No clear protection was observed in the pig model. Overall these models show that SPI-1 and SPI-2 proteins are immunogenic by a variety of immunization routes, challenge organisms, and hosts and set the groundwork for the development and enhancement of a broadly protective Salmonella vaccine.

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Quorum sensing promotes defense and self-preservation during interspecies

competition

Kara Hinshaw and Josephine R Chandler

Department of Molecular Biosciences, University of Kansas, Lawrence KS.

Many Proteobacteria use acyl-homoserine lactone mediated quorum sensing (QS) to

control the production of secreted public goods, such as antibiotics and virulence factors,

whose benefits are shared among all members of the population. However, QS is

susceptible to social cheaters, such as quorum-defective mutants, which benefit from the

public goods but do not incur the cost of production. In some species QS controls

transcription of efflux pumps and other cellular factors that may protect the cells from

antimicrobials. Such QS-controlled private goods may not have a shared benefit. To

investigate the importance of QS in defense during inter-species competition, we used a

competition model between two soil saprophytes, Chromobacterium violaceum and

Burkholderia thailandensis, that we previously described. We demonstrate that C.

violaceum QS mutants are more sensitive than wild type to a potent bactobolin antibiotic

produced by B. thailandensis. In laboratory growth media, C. violaceum QS mutants have

a growth advantage over their quorum-intact parent. However in ternary culture with

bactobolin-producing B. thailandensis, C. violaceum QS mutants are outcompeted by the

QS-intact C. violaceum cells, suggesting C. violaceum QS promotes defense against

bactobolin. The mechanism is as-yet unknown. Our results suggest that QS may have an

important role in defense during competition. This may be of significance during mixed-

species infections. Furthermore, QS-intact cells did not protect QS-deficient cells from

bactobolin suggesting that bactobolin-defense is a private benefit. Such regulation of

private goods may help to preserve QS in the population and protect against the

emergence of cheaters.

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Protein-coated nanoparticles are internalized by the epithelial cells of the female reproductive tract and induce systemic and mucosal immune responses Savannah E. Howe and Vjollca Konjufca Southern Illinois University, Carbondale, Illinois The female reproductive tract (FRT) includes the oviducts (fallopian tubes), uterus, cervix and vagina. A layer of columnar epithelium separates the endocervix and uterus from the outside environment, while the vagina is lined with stratified squamous epithelium. The mucosa of the FRT is exposed to antigens originating from microflora, and occasionally from infectious microorganisms. Whether epithelial cells (ECs) of the FRT take up (sample) the lumen antigens is not known. To address this question, we examined the uptake of 20-40 nm nanoparticles (NP) applied vaginally to mice which were not treated with hormones, epithelial disruptors, or adjuvants. We found that 20 and 40 nm NPs are quickly internalized by ECs of the upper FRT and within one hour could be observed in the lymphatic ducts that drain the FRT, as well as in the ileac lymph nodes (LNs) and the mesenteric lymph nodes (MLNs). Chicken ovalbumin (Ova) conjugated to 20 nm NPs (NP-Ova) when administered vaginally reaches the internal milieu in an immunologically relevant form, thus vaginal immunization of mice with NP-Ova induces systemic IgG to Ova antigen. Most importantly, vaginal immunization primes the intestinal mucosa for secretion of sIgA. Sub-cutaneous (s.c) boosting immunization with Ova in complete Freund’s adjuvant (Ova+CFA) further elevates the systemic (IgG1 and IgG2c) as well as mucosal (IgG1 and sIgA) antibody titers, which remain high for over 6 months after boosting immunization. These findings suggest that the modes of antigen uptake at mucosal surfaces and pathways of antigen transport are more complex than previously appreciated.

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Biochemical characterization of Marburg virus VP24 interaction with Keap1 and its role in cytoprotective antioxidant responses Britney Johnson1, Megan R. Edwards2, Chad E. Mire3, Wei Xu1, Reed S. Shabman2,4, Hao Zhang5, Michael Gross5, Daisy W. Leung1, Thomas W. Geisbert3, Christopher F. Basler2 and Gaya K. Amarasinghe1

1Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110; USA. 2Dept. Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029; USA. 3Galveston National Laboratory, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555; USA. 4Current Address: J. Craig Venter Institute, Rockville, Maryland, USA 20850. 5Department of Chemistry, Washington University, St. Louis, Missouri 63130; USA Recently two groups, Basler and colleagues (Edwards, M. R. et al. Cell Reports 6, 1017–1025 (2014)) and Volchkov and colleagues (Page, A. et al. Cell Reports 6, 1026–1036 (2014)), have shown that Marburg virus (MARV) VP24 (mVP24) protein interacts with Kelch-like ECH-associated protein 1 (Keap1). Keap1 is an ubiquitin E3 ligase specificity factor that regulates antioxidant responses by targeting transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) for ubiquitination and proteasomal degradation. Although oxidative stress has long been considered as an important component in the host response to cellular stress, its role in filoviral infections were unclear. These new studies provide insight into a novel mechanism, where direct interaction between mVP24 and the Kelch domain of Keap1 results in the release of Nrf2 from Keap1, resulting in Nrf2 nuclear accumulation and activation of antioxidant response elements (AREs). ARE genes provide cytoprotection, and expression of mVP24, but not Ebola (EBOV) VP24 (eVP24), suggesting a specificity for mVP24 in this process. Modeling based on the Zaire eVP24 structure identified in mVP24 an acidic loop (K-loop) critical for Keap1 interaction. Transfer of the K-loop to eVP24, which otherwise does not bind Keap1, confers Keap1 binding and Nrf2 activation. In order to better understand the molecular mechanism that leads to a more cytoprotective state during Marburg virus infections, we are using a biochemical approach to characterize mVP24 binding to Keap1. Here we will describe a molecular mechanism by which mVP24 activate Nrf2, including an allosteric model for mVP24 binding to Keap1.

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Outer membrane proteins of Enterotoxigenic Escherichia coli with protective efficacy in intranasal mice challenge assay

Amit Kumar, Mike Hays, Philip Hardwidge

Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, Kansas Enterotoxigenic Escherichia coli (ETEC) causes traveler’s diarrhea and is a major endemic health threat in underdeveloped nations. Despite the significant effort that has been employed in vaccine trials using ETEC colonization factors, none of these vaccines have been especially effective in mediating cross-protective immunity. These studies have also uncovered significant confounding differences among volunteers that will further complicate human challenge studies, unless new protective antigens are discovered. We used quantitative proteomics to discover secreted and surface-exposed ETEC proteins that are conserved among ETEC strains that express different colonization factors. We initially characterized ETEC H10407, a strain isolated from a patient with a severe cholera-like diarrhea. We used quantitative proteomics to identify 24 proteins that differed in abundance in secreted and outer membrane protein preparation of wild-type vs. a type II secretion system mutant of ETEC. We expressed and purified a subset of these proteins and identified 9 antigens that generated significant immune responses in mice. Out of the 9 antigens, sera obtained from mice against the MltA-interacting protein MipA, a homolog of the ATP-binding protein CcmA, or the long-chain fatty acid outer membrane transporter ETEC_2479, all reduced the adherence of multiple ETEC strains that differ in colonization factor expression to intestinal epithelial cells. In intranasal ETEC challenge assays, immunization with ETEC_2479 protected mice from an otherwise lethal challenge with ETEC H10407. Immunization with MipA or CcmA provided an intermediate degree of protection. This study has identified several proteins that are conserved among heterologous ETEC strains and may thus improve cross-protective efficacy if incorporated into future vaccine designs.

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CXCR2 Signaling Facilitates Brucella-Induced Articular Inflammation Carolyn A. Lacey1,2, Lauren L. Keleher1,2, Charles R. Brown1,2 and Jerod A. Skyberg1,2

1Department of Veterinary Pathobiology and 2Laboratory for Infectious Disease Research, University of Missouri, Columbia, MO, 65211 Brucella spp. cause brucellosis, one of eight neglected zoonotic diseases according to the World Health Organization. These facultative intracellular bacteria may also be employed as biological weapons infecting not only humans but a wide range of agricultural species. Brucellosis is chronic in some individuals and displays manifestations such as endocarditis and osteoaticular inflammation. It has been previously found that IFN-γ-deficient mice infected with Brucella melitensis or abortus display arthritis and swelling in joints similar to human chronic brucellosis. The purpose of this study is to understand the primary mediators of joint inflammation and determine the signaling of leukocyte recruitment. Here, we report Brucella-induced arthritis is not initiated through an adaptive response. Rag1-/- mice, deficient in B and T cells, along with µMT-/- mice, deficient in B cells, developed inflammation at the same rate and severity as wild type animals. Mice infected with B. melitensis and depleted of IFN-γ displayed elevated levels of neutrophils and macrophages in their joints. Also, joints from B. melitensis infected IFN-γ-/- mice expressed increased levels of the CXCR2 ligands, MIP-2 and KC and the CCR2 ligand MCP-1. CXCR2-/- mice had delayed onset of inflammation and did not develop severe focal swelling as compared to IFN-γ depleted wild type mice. In contrast, CCR2-/- mice did not show a reduction in their inflammation score and incidence. Collectively, these results demonstrate CXCR2 is necessary for an innate inflammatory response in the joint and targets of CXCR2 could be used as potential therapies when treating chronic articular brucellosis.

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Characterization of Influenza B Viruses Resistant to Neuraminidase Inhibitors Yuekun Lang1, Zhiguang Ran2, Jingjiao Ma1, Qinfang Liu1, Bhupinder Bawa1, Richa Sood2, Laihua Zhu2, Broddy Edman2, Victor Huber3, Juergen Richt1, Feng Li2, and Wenjun Ma1

1Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA. 2Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD, USA. 3Department of Basic Medical Sciences, University of South Dakota, Vermillion, SD, USA Influenza B virus (IBV) causes influenza epidemic in humans annually. Like influenza A virus (IAV), effective treatment of IBV infections in humans relies on administration of neuraminidase (NA) inhibitors (NAIs). In contrast to IAV, antiviral drug resistance in IBV is not fully studied. To gain insights into this important topic, we generated nine IBV viruses containing NAIs-resistant mutations (E117A, R150K, D197E, I221T, I221V, S249G, H273Y, R292K, and R374K) in the genetic background of B/Yamanashi/166/1998 using reverse genetics based on previous findings. These viruses were then characterized for drug susceptibility, virus replication in vitro, and in vivo using a mouse model. Results of our studies identified one complete cross-resistance mutation (E117A) and two moderate mutations (D197E and I221T) to NAI Zanamivir and Oseltamivir. Interestingly, mutations S249G and R292K conferred high-level resistance to Zanamivir but not to Oseltamivir. Conversely, R150K and H273Y mutations induced moderate resistance only to Oseltamivir. Negligible levels of NAIs resistance were found in IBVs harboring I221V or R374K mutation (< two-fold resistance). Furthermore, in vitro experiments revealed that all mutated viruses replicated to a level similar to that of wild-type IBV, despite the R150K mutant exhibiting slightly lower virus titers at some time points tested. Significantly, the mouse experiment showed that three NAI cross-resistant IBVs (E117A, D197E, and I221T) were replication-competent in mice. Although disease severity was less profound overall, infections of mice with drug resistant viruses indeed resulted in significant weight loss, lung lesions, and various levels of mortality. Finally, our structural modeling showed that E117A and I221T mutations likely affect NAIs binding to NA protein, while D197E mutation confers drug resistance probably through influencing the structural rearrangement of NA or NAIs entry to their binding pocket.

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Macrophage Polarization in Murine Lyme Borreliosis Carrie E. Lasky, Rachel M. Olson, Charles R. Brown Department of Veterinary Pathobiology, University of Missouri, Columbia MO Infection of susceptible mouse strains with the causative agent of Lyme disease, Borrelia burgdorferi, reliably produces an infectious arthritis and carditis that peaks and spontaneously resolves within 60 days. The exact mechanisms that drive the development and resolution of inflammation during infection with B. burgdorferi are not well understood. Macrophage polarization has been suggested to drive inflammation, the clearance of bacteria, and tissue repair and resolution in a variety of infectious models. During Lyme disease it is well understood that macrophages are capable of clearing Borrelia spirochetes as well as exhausted neutrophils. However, to our knowledge macrophage phenotype has not been analyzed in a murine model of Lyme disease. Using flow cytometry, we show there are significantly more macrophages in both the joints and hearts of infected C3H/HeJ mice, as compared with uninfected controls. Using classical (M1) and alternative (M2) specific markers, we set out to describe the phenotype of macrophages throughout the infection time course. We found that throughout the infection M2 macrophages dominate, but M1 macrophage numbers become elevated during the peak of inflammation. Interestingly, we see a significant increase of resolution phase macrophages (rM) that possess characteristics of both M1 and M2 macrophages once both inflammation and spirochetes have been cleared.

37

Interferon lambda restricts West Nile virus neuroinvasion by enhancing integrity of the blood-brain barrier

Helen M. Lazear1†, Brian P. Daniels4†, Amelia K. Pinto1, Albert C. Huang5, Sarah C.

Vick1‡, Sean E. Doyle6, Michael Gale Jr.5, Robyn S. Klein1,2,4†, and Michael S. Diamond1,

2, 3†*

1Department of Medicine, Washington University School of Medicine, St Louis, MO

63110. 2Department of Pathology & Immunology, Washington University School of

Medicine, St Louis, MO 63110. 3Department of Molecular Microbiology, Washington

University School of Medicine, St Louis, MO 63110. 4Department of Anatomy &

Neurobiology, Washington University School of Medicine, St Louis, MO 63110. 5Department of Immunology, University of Washington School of Medicine, Seattle, WA

98195. 6ZymoGenetics, a Bristol-Myers Squibb company, Seattle, WA 98102

†These authors contributed equally to this work.

‡Current address: Department of Microbiology and Immunology, University of North

Carolina, Chapel Hill, NC 27599

Although type III interferons (IFN), also known as IFN-λ or IL28/IL-29, restrict infection by several viruses, their mechanism of inhibitory action has remained uncertain. We used recombinant IFN-λ and mice lacking the IFN-λ receptor (IFNLR1) to evaluate the effect of IFN-λ on infection with West Nile virus (WNV), an encephalitic flavivirus. Cell culture studies in keratinocytes and dendritic cells showed no direct antiviral effect of exogenous IFN-λ even though ISGs were induced. Correspondingly, we observed no differences in WNV burden between wild type and Ifnlr1-/- mice in the draining lymph node, spleen, and blood. However, we detected earlier dissemination and increased WNV infection in the brain and spinal cord of Ifnlr1-/- mice, yet this was not associated with a direct antiviral effect on infection of neurons. Instead, an increase in blood-brain barrier (BBB) permeability was observed in Ifnlr1-/- mice. Accordingly, treatment of mice with pegylated IFN-λ2 resulted in decreased BBB permeability and reduced WNV infection in the brain without impacting peripheral viral burden. An in vitro model of the BBB showed that IFN-λ signaling in brain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movement across the barrier, and modulated tight junction protein localization in a protein synthesis-independent manner. Our data establish a novel indirect antiviral function of IFN-λ in which signaling through IFNLR1 tightens the BBB and restricts viral neuroinvasion and pathogenesis. This finding suggests new clinical applications for IFN-λ in treating viral or autoimmune diseases.

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The Type VI Secretion System of Yersinia pestis Influences Intracellular Survival Lacey R. Lopez, Miqdad O. Dhariwala, Deborah M. Anderson Department of Veterinary Pathobiology, University of Missouri – Columbia, MO 65211 Yersinia pestis, causative agent of the plague, is a bacterium that has been responsible for the death of over 200 million people. The ability of this pathogen to counteract host immune response is due to several virulence factors including, but not limited to, the utilization of secretion systems. The lesser-studied type VI secretion system (T6SS) is thought to influence the interactions between Y.pestis and the host cell, particularly during phagocytosis. This particular virulence factor functions by secreting proteins known as “effectors” in response to environmental stimuli. Ultimately, we aim to understand the mechanism(s) by which this secretion system contributes to the pathogenesis of plague. In order to discern how this T6SS interacts with eukaryotic host cells, phagocytosis assays were performed to study bacterial uptake and intracellular survival. A transposon mutant (EO-84), isolated from a screen to identify bacterial genes important to killing macrophages, was used for these assays. Using this mutant and its parental strain, CO92 pCD1-, RAW 264.7 macrophages were infected with a multiplicity of infection (MOI) of 20. The ability of the bacteria to survive intracellular stresses was then studied at 2, 8, and 24 hours post-infection. Here, we demonstrate that both strains of bacteria declined in intracellular survival over the 24 hour time period. However, the mutant strain had a higher phagocytosis and survival rate than CO92 pCD1-. These data suggest that the T6SS limits host phagocytosis activity, implicating a role for the T6SS in immune evasion by Yersinia pestis. Furthermore, Lactate Dehydrogenase (LDH) assays were performed to determine the ability of these strains to kill host cells at 8 and 24 hours post-infection. These data suggest that the T6SS evades cell death mechanisms in the eukaryotic cell. Together these data enhance our understanding of the role of the T6SS in immune evasion during the pathogenesis of plague.

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Role of Streptococcus mutans LiaF in the regulation of the liaFSR three component

system

Jarosław E. Król, Shankar Manoharan and Indranil Biswas

Department of Microbiology, Molecular Genetics and Immunology, University of Kansas

Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160.

Streptococcus mutans is an aggressive colonizer of the human oral cavity, where it

overcomes host-imposed stresses, outcompetes other microbiota and initializes the

formation of polymicrobial biofilms thereby leading to caries development. The LiaFSR

three component system (TCS) is one of the major TCS in S. mutans known to be critical

for host colonization and disease establishment. Our study identifies LiaF, a membrane

bound protein as the negative regulator of the liaSFR operon. In addition, we employed

a bacterial two hybrid system to demonstrate physical interaction between LiaF and the

sensor kinase, LiaS. We also show that the topology of LiaF in the membrane is critical

for LiaS binding. While it still remains unclear what role the interaction of LiaS and LiaF

could play in the regulation of this TCS, we speculate that LiaF could reversibly modify

LiaS thereby negatively modulating downstream gene expression.

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Ebola Virus Glycoprotein Induces Cell-cell tansmission at Neutral pH: Role of Cysteine Cathepsins and Niemann-Pick Type C1 Chunhui Miao1, Minghua Li1, Yi-Min Zheng1, Fredric S. Cohen2, and Shan-Lu Liu1*

Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. It is currently poorly understood how EBOV enters host cells. We use a highly sensitive cell-cell transmission assay to interrogate the viral and cellular factors governing EBOV glycoprotein (GP)-mediated transmission. We showed that EBOV GP induces cell-cell transmission at neutral pH, and that acidic pH does not enhance its transmission activity. Overexpression of human Niemann-Pick type C1 (NPC1) potentiates cell-cell transmission of EBOV GP. In contrast, treatment of cells with cathepsin B/L inhibitors or the lysosomotropic agent NH4Cl blocks cell-cell transmission of EBOV GP. In further support for a role of NPC1, we demonstrated that knockdown of NPC1 expression significantly reduces cell-cell transmission mediated by GP, and that EBOV GP mutants deficient for receptor binding were unable to induce efficient cell-cell transmission. We further showed that EBOV entry inhibitor 3.47, known to block the binding of EBOV GP to NPC1, potently inhibits cell-cell transmission of EBOV GP. Altogether, our study provides the evidence that EBOV GP induces cell-cell transmission at neutral pH, and that NPC1 and cellular proteases, especially cathepsin B and L, are critical for the fusion to occur.

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Variability of Pseudomonas aeruginosa recovered from cystic fibrosis patients in different age groups Elizabeth Pascual*1, James Royall2, and Erika Lutter1 Oklahoma State University1, Stillwater, Oklahoma; Oklahoma Cystic Fibrosis Center2, Oklahoma City, Oklahoma Cystic fibrosis (CF) the most common autosomal recessive genetic disorder resulting in faulty chloride ion channels in the lungs. As a result, the lungs are severely compromised with viscous secretions leading to chronic and repeated infections and the colonization of a diverse microbial community. Pseudomonas aeruginosa is one of the primary pathogens in the CF lung and prior research has demonstrated a high degree phenotypic heterogeneity among adult isolates in comparison to control strains. This study uses subsets of P. aeruginosa isolates obtained from two patients in each age category to investigate the variability of isolates within a patient and between patients. P. aeruginosa CF isolates were recovered from CF sputa originating from three age categories: children (under 13), adolescents (13-18), and adults (over 18). Isolates were assayed for a number of virulence factors including surface motility (swim, swarm, and twitch) and production of hydrogen cyanide, biosurfactant, casein protease, and various fluorescent pigments. Isolates from most patients showed a high degree of consistency in producing hydrogen cyanide, fluorescent pigments, protease and biosurfactant. There was, however, variability was seen in the different surface motilities of isolates between the different age groups. These results provide insights to the variability of P. aeruginosa between age groups and confirm differences between P. aeruginosa isolated from newly infected CF patients and those chronically infected for many years.

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Characterization of Biphenotypic B/Macrophage cells in Vitro and in Pneumonic

Francisella tularensis Infection

Carmela L. Pratt, Jerod A. Skyberg, Carrie Lasky, and Charles R. Brown

Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211

Biphenotypic B/Macrophage cells are a unique cell type derived from B-cells that co-express both B and macrophage cell surface markers (B220, IgM, F4/80, and CD11b). Following treatment with M-CSF and GM-CSF, splenic B-2 B cells can be induced to transition into biphenotypic cells. As the cells transition, the expression of CD19 becomes down-regulated, CD11b and F4/80 become up-regulated, and B220 and IgM expression remain static. mRNA microarray analysis of in vitro biphenotypic cells demonstrates the expression of several monocyte and lymphocyte chemotactic receptors. Biphenotypic cells are capable of phagocytosing opsonized murine RBCs and Borrelia burgdorferi. To evaluate the presence of these cells in an infectious model, B6 WT mice were intratracheally inoculated with the LVS strain of Francisella tularensis, a pulmonary pathogen. Biphenotypic cells within the pulmonary tissue were significantly elevated as compared to uninfected mice on days 10 and 14 (p<0.0001, p<0.001, respectively) post-infection, and corresponded to the resolution of disease. Preliminary data suggests intratracheal M-CSF administered post infection may alter recovery times from pneumonic Tularemia. Further functional studies are underway to elucidate the role of biphenotypic cells in disease, which may provide the premise for devising therapies to enhance or attenuate their development.

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Transcription of T4ASS ORFs In Spotted Fever Group Rickettsia Chris Richards and Edward Shaw Microbiology & Molecular Genetics, Oklahoma State University. Stillwater, OK 74078 Rickettsiae are Gram-negative obligate intracellular bacteria spread by arthropod vectors. The genus Rickettsia is divided into three distinct groups: Spotted Fever, Typhus, and Belli. The Spotted Fever Group Rickettsia (SFGR) is the largest and most pathogenically diverse group. SFGR virulence ranges from R. rickettsii, the highly virulent causative agent of Rocky Mountain Spotted Fever (RMSF) to several avirulent species. Rickettsia species possess a Type 4A Secretion System (T4ASS) homologous to T4ASS found in several pathogens as well as Agrobacterium tumefaciens, which has the best characterized T4ASS. The T4ASS is a critical virulence determinant for many pathogens, secreting effector proteins into host cells during infection. Eighteen open-reading-frames (ORFs) have been identified in the Rickettsia T4ASS with several genes having paralogs. Fifteen of the ORFs are in close proximity to each other in two separate loci. Many of these ORFs are oriented in the same direction, suggesting co-expression may exist. To investigate the expression of SFGR T4ASS ORFs, total RNA was harvested from R. parkeri (virulent) and R. amblyommii (avirulent) infected Vero cultures growing at either 34°C or 37°C. Using reverse transcriptase-PCR, the presence of transcripts for T4ASS ORFs was determined using primers internal to the ORFs. Primer pairs spanning between ORFs were used to detect potential polycistronic mRNA. Many ORFs were confirmed as being transcribed as well as the identification of co-transcribed ORFs, suggesting that expression of this critical virulence determinant may be regulated during infection of host cells.

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Age-dependent defects in naïve CD4+ T cell trafficking and priming impair adaptive immunity and control of virus infection Justin M. Richner1, Grzegorz B. Gmyrek1, Gerritje J. W. van der Windt2, Jennifer Govero1, Yizheng Tu1, Johannes Textor4, Erika L. Pearce2, Mark J. Miller1, and Michael S. Diamond1,2,3 Departments of Medicine1, Pathology & Immunology2, Molecular Microbiology3, Washington University School of Medicine, St. Louis, MO. Department of Theoretical Biology & Bioinformatics4, Utrecht University, The Netherlands. Although adaptive B and T cell responses decline with age, the mechanistic basis for impaired immunity of the elderly against viruses remains uncertain. Herein, in the context of infection with West Nile virus (WNV), we identify a novel mechanism for compromised adaptive immunity that occurs with aging. Old mice had blunted humoral immune responses to WNV, increased viral burden, and enhanced mortality. This decline in immunity and enhanced vulnerability to infection was associated with decreased trafficking of naïve CD4+ T cells but not B cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within draining lymph nodes (DLN). These trafficking defects correlated with diminished respiratory capacity of old naïve CD4+ T cells, as well as reduced cytokine and chemokine production in the DLN of old mice. Our results support a model in which age-dependent cell-intrinsic and -extrinsic defects result in delayed immune cell recruitment and antigen recognition in the DLN, which compromises induction of adaptive immune responses and contributes to the susceptibility of old animals to viral infections.

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Will a bacteriophage-based therapy be effective against the bacterial plant pathogen Agrobacterium tumefaciens? Jeanette Rimbey, George Smith, and Pamela Brown Division of Biological Sciences, University of Missouri, Columbia, MO 65211 Plant disease is a growing problem in the agriculture industry both at a national and world wide level. The bacterium Agrobacterium tumefaciens causes crown gall disease in many flowering plants which cause tumors to form on roots and stems. Crown gall is frequently diagnosed on the roots of apple, peach, and walnut trees, as well as blackberry bushes, potentially reducing the yield of these fruits. Presently, there are limited options for the prevention and treatment of crown gall disease. Furthermore, bacterial resistance to antibiotics is a growing threat in our world. Thus, we propose that bacteriophages might serve as an alternative therapeutic tool. Bacteriophages are viruses that can infect bacterial cells, but have no effect on animals or humans. These viruses are specific to one or a limited number of bacteria. Due to this specificity, bacteriophages which infect Agrobacterium tumefaciens should have minimal negative effects on other bacteria in the environment. The long-term goal of our project is to determine if pre-treatment with a combination of bacteriophages will act as a biocontrol agent to limit plant infection by A. tumefaciens. Because A. tumefaciens are common soil dwelling bacteria, we expect to find bacteriophages in similar environments, including soils and water sources. Using standard techniques we have amplified bacteriophage capable of infecting A. tumefaciens. After amplification, bacteriophages were isolated from plaques which formed on a petri dish containing a lawn of A. tumefaciens. Finally, we sub-cloned the phage from one selected plaque in order to ensure its purification and to concentrate it. As a result we have found a bacteriophage, called AP2 that originated from waste-water of the local Columbia, Missouri treatment plant. Future work will focus on understanding how AP2 interacts with A. tumefaciens, testing this bacteriophage to determine if they can be used to prevent or treat crown gall disease, and finally continuing to isolate additional bacteriophages.

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The role of peritoneal B cells during primary infection with Coxiella burnetii Laura Schoenlaub, Alexandra Elliott, Danielle Freches, William J. Mitchell, and Guoquan Zhang 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. It is known that T cells and interferon gamma production are crucial for disease clearance during primary infection. It has been suggested that B cells may play a role in regulating Th1 responses during acute infection, but this remains unclear. We sought to determine the effect of B cells during primary infection with C. burnetii. For these experiments, we used peritoneal B cells, as these cells can secrete large amounts of regulatory cytokines such as interleukin-10 (IL-10) and are also capable of phagocytosing and killing bacterial cells. Indirect immunofluorescence indicated that peritoneal B cells can take up NMI, into LAMP-1 positive vacuoles. However, these cells do not appear to kill C. burnetii after uptake. A subset of peritoneal cells, known as B1a cells, secreted IL-10 during stimulation with NMI. To determine the role of these cells in vivo, B cell deficient µMT mice and SCID mice were adoptively transferred with peritoneal B cells prior to challenge with NMI. While SCID mice were not protected with the B cell transfer, µMT mice had significantly higher body weights during early infection compared to PBS controls. These data indicate that B cells alone are not sufficient for protection during primary infection, but likely play a role in regulating T cell responses in vivo via production of IL-10.

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Acyl-homoserine lactone-independent activation of an orphan LuxR in B. thailandensis and B. pseudomallei Patricia Silva1, Thao Thoung2, Mohammed Seyedsayamdost3, Peter Greenberg2 and Josephine Chandler1. 1.Molecular biology, University of Kansas (Lawrence, KS); 2. Department of Microbiology, University of Washington (Seattle, WA); 3. Department of Chemistry, Princenton University (Princenton, NJ). LuxR family members with no genetically linked acyl-homoserine lactone (acyl-HSL) signal synthase are called solos or orphans. A subset of orphans do not respond to acyl-HSLs, and instead respond to as-yet unknown signals derived from plantsWe have found an orphan LuxR, BtaR4 in Burkholderia thailandensis, with all nine of the conserved LuxR-family residues that does not require acyl-HSLs to activate its target genes. Previous work demonstrated BtaR4 is required for virulence in C. elegans and we show that BtaR4 directly regulates gene for the virulence factor malleilactone. Although B. thailandensis encodes three acyl-HSL synthases, these are not required for BtaR4 to activate transcription of the malleilactone biosynthetic genes. Instead, BtaR4 activity correlates with its cellular level, which is influenced by the addition of particular growth-inhibitive antibiotics. BtaR4 activation of target genes requires a lux-box motif in the promoter region of the malliolactone operon (mal) promoter, similar to other LuxR-family proteins. BtaR4 is unique to B. thailandensis and two of its close relatives, Burkholderia pseudomallei and Burkholderia mallei. The B. pseudomallei BtaR4 homolog BpsR4 is also important for virulence in C. elegans and regulates a malleilactone-like gene cluster in response to growth-inhibitive antibiotics, similar to BtaR4. It is unclear if BtaR4/BpsR4 activates its target genes in a ligand-independent manner or if it is activated by a ligand that is not an acyl-HSL. Our finding that a conserved orphan LuxR does not require acyl-HSLs to activate its target broadens our current view of the LuxR family.

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Influence of NleH effector expression and host microbiota on Citrobacter rodentium colonization of mice Gaochan Wang, Philip R. Hardwidge Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State Univesity, Manhattan, KS 66506, USA Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are human pathogens that cause diarrheal disease. They employ Type III Secretion System (T3SS) effector proteins to subvert host innate immune responses and to colonize the host intestine. Intestinal microbiota regulate host resistance to enteric bacterial infections. In this study, Citrobacter rodentium, a mouse pathogen that is similar to EHEC and EPEC, was used to investigate the contribution of NleH (a T3SS-secreted effector) and the intestinal microbiota to bacterial colonization in mice with different genetic backgrounds. Mice of 3-4 weeks age were treated with antibiotics to remove the existing microbiota, were transplanted with microbiota from homologous or heterologous mouse strains, and then infected with C. rodentium. Mouse colons were collected and the pathogen load in the intestine was quantified. Our preliminary data suggest both that NleH expression affects C. rodentium colonization and that transplantation of microbiota between C57BL/6J and C57BL/10ScNJ mice affects host resistance to infection.

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A Comparison of Coxiella burnetii Response to Supplementation During Growth in Axenic Media Ruth Weidman, Brandon Luedtke, and Edward Shaw. Microbiology and Molecular Genetics, Oklahoma State University. Stillwater, OK 74078 Coxiella burnetii is a gram-negative, pleomorphic, obligately intracellular pathogen that requires a functional Type IVB secretion system (T4BSS) for productive infection. C. burnetii causes Q fever, a zoonotic illness that is endemic worldwide. In humans, Q fever manifests as either an acute flu-like illness or chronic illnesses such as endocarditis, pneumonia, or hepatitis. The C. burnetii T4BSS’s closest homolog is the T4BSS of Legionella pneumophila, upon whose model the C. burnetii T4BSS was originally developed. While the T4BSS model continues to be informed, it is far from complete and lacks regulatory information. Previously we have shown that components of the T4BSS are released into axenic media in response to supplementation with Bovine Serum Albumin (BSA) and/or an emulsified, chemically defined lipid concentrate (CDLC). Here we add further comparisons of the extracellular proteome between C. burnetii cells grown in differently supplemented media to show that the presence of biological stimulants trigger release of T4BSS components and changes in the total extracellular proteome of C. burnetii. Using LC-MS/MS, we analyzed the contents of growth media (ACCM-2) for released proteins following either supplementation with CDLC and/or BSA compared with no supplementation. We observed a release of DotA in response to the presence of CDLC as well as an increase in the release of IcmX in the presence of CDLC further suggesting lipid structures stimulate the release of C. burnetii T4BSS components.

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Genomic Compatibility and Pathogenesis of Single Reassortant Viruses Derived from the 2009 Pandemic H1N1 and a North American Avian H7N3 Graham D. Williams and Adrianus C. Boon Microbiology, Washington University in Saint Louis, St. Louis, MO Reassortment of co-circulating strains of Influenza A (IAV) is an important strategy for the evolution of this segmented RNA virus and is responsible for generating emergent viruses including pandemic and pre-pandemic IAV. In recent decades, multiple zoonotic events introduced novel avian viral subtypes (H7N3, H7N9, H5N1) into an immunologically naive human population.. We sought to characterize the replication and pathogenesis of a North American Avian Influenza Virus, A/Shorebird/Delaware/22/2006 (H7N3), and the genomic compatibility of this virus with the current dominant human strain, derived from the 2009 H1N1 Pandemic, using the model isolate A/California/04/2009 (H1N1). Utilizing a recently developed in vitro model of reassortment we found multiple 2009 H1N1 gene segments, previously implicated in mammalian pathogenesis and transmission, were preferentially selected by an otherwise fully H7N3 background. Additionally, introduction of these segments, NA, M, or PB2 to H7N3 by reverse genetics did not attenuate viral replication in tissue culture nor in vivo in a mouse model of infection. Conversely addition of 2009 H1N1’s PA, PB1, NP, or NS resulted in decreased morbidity. Next, we sought to determine if host response to H7N3 and 2009 H1N1 were equivalent and quantified this by cytokine array. The avian isolate and reassortant derivatives induced hypercytokinemia relative to 2009 H1N1. Taken together our study demonstrates that a North American Avian virus, H7N3, is capable of inducing morbidity in a mammal without prior adaptation. Moreover, introduction of 2009 H1N1 segments previously implicated in mammalian transmission does not attenuate the virus.

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Yersiniabactin evades IRF3-mediated protection during pneumonic plague Joshua Willix, Ami A. Patel, and Deborah Anderson Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211 Yersinia pestis causes plague, a lethal disease characterized by immune evasion and rapid bacterial growth. Virulence is largely enabled by two major virulence factors, the type III secretion system and a pigmentation locus that is largely responsible for iron scavenging. Yersiniabactin (Ybt) is an iron-binding siderophore that is produced by Yersinia pestis and other Gram-negative human pathogens such as Klebsiella pneumoniae and Escherichia coli. The biosynthesis of Ybt and transport Fe-bound Ybt into the bacterial cytosol are encoded by a pathogenicity island located on a large mobile genetic element. Although Ybt is one of many iron acquisition mechanisms encoded in the genome of Y. pestis, it is uniquely required for bacterial pathogenesis. Here we describe the role for Ybt in mediating evasion from the mammalian immune regulator Interferon Regulatory Factor 3 (IRF-3). In a murine pneumonic plague model, we show that Y. pestis lacking the ability to synthesize or transport Ybt are sensitive to immune defense mediated by IRF-3, with uncontrolled bacterial growth and massive production of pro- and anti-inflammatory cytokines. When Ybt was expressed, however, IRF-3 did not contribute to host defense, even when effector proteins of the type III secretion system were absent. Following in vitro infection of dendritic cells or macrophages, no Ybt-dependent regulation of cytokine production was observed. In contrast, the iron-regulated gene heme oxygenase (Hmox1) was activated by Ybt- Y. pestis but only in the presence of IRF-3. These data suggest that IRF-3 may control infection of Ybt- Y. pestis through the activation of Hmox1 and perhaps other genes that mediate iron homeostasis in the host.

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The Role of 12/15 Lipoxygenase in a Murine Model of Lyme Disease Edric D. Winford, Carrie E. Lasky, Carmela L. Pratt and Charles R. Brown Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211 Borrelia burgdorferi (Bb) is the causative agent of Lyme disease and is spread in nature by the bite of the Ixodes scapularis tick. When susceptible mouse strains are infected with Bb, they develop arthritis of the large joints that primarily localizes to the ankles. About one week after the onset of arthritis, carditis will develop. Both manifestations of disease will resolve about 60 days after infection. The immunological mechanisms that drive both the onset and resolution of inflammatory diseases are unknown. Eicosanoids are bioactive lipids that are known to regulate inflammatory processes and have been shown to play critical roles in certain infectious disease models. In addition to their primary pro-inflammatory role, eicosanoids have recently been shown to also have anti-inflammatory properties. The enzyme 12/15 lipoxygenase (LO) is a component of the eicosanoid pathway that is thought to produce primarily anti-inflammatory mediators. Others have reported more severe inflammation in 12/15 LO deficient mice using the K/BxN serum transfer model of arthritis compared to control mice, suggesting an anti-inflammatory role for 12/15 LO in arthritis. Our lab uses a mouse model of Lyme arthritis and a role for 12/15 LO in the development of Lyme arthritis or carditis has not been established. We hypothesize that infection of 12/15 LO-deficient mice with B. burgdorferi will lead to exacerbated disease. We will monitor arthritis development by measuring ankle swelling and disease severity will be assessed on H&E stained histological sections of joints and hearts. Clearance of bacteria from tissues will be assessed using quantitative real-time PCR. Preliminary data shows no differences in swelling curves of either arthritis-resistant B6 or –susceptible C3H 12/15 LO-deficient mice compared to their wild type counterparts. However, ankle swelling is not always indicative of underlying tissue damage, so histological analysis will be critical. In the future, a pathologist will assess our H&E stained slides for severity. In addition, protein and lipids will be extracted to assess concentrations of pro-inflammatory and anti-inflammatory mediators. A better understanding of this pathway may lead to the development of treatments for Lyme and other inflammatory diseases.

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IFITM proteins inhibit HIV-1 cell-cell transmission Jingyou Yu, Minghua Li, and Shan-Lu Liu Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA It has been well established that HIV-1 cell-cell transmission is more efficient than the cell-free virus to spread in vitro and in vivo. We recently showed that interferon-inducible transmembrane (IFITM) proteins, especially IFITM1, strongly inhibit HIV-1 replication; however, the detailed molecular mechanism is not understood. In this study, we used an intron Gaussia luciferase- and a green fluorescence protein (GFP)-based reporter assays and demonstrated that IFITMs inhibits HIV-1 cell-cell transmission. We found that expression of IFITMs in effector cell is more potent than its expression in target cells to inhibit HIV-1 cell-to-cell transmission.IFITM3 is most potent (more than 90% inhibition), compared with IFITM1(50% inhibition) and IFITM2 (80% inhibition). Mutagenesis analysis revealed that the C-terminal end of IFITM1 is critical for this inhibition. Interestingly, we observed that the potency of IFITM inhibition of HIV-1 cell-to-cell transmission can be modulated by expression or knockdown of some tetraspanins such as CD81 and CD63, suggesting that tetraspanins or its associated microdomain may be important for IFITMs inhibition of HIV-1 cell-to-cell transmission. Altogether, our data demonstrate that IFITMs effectively prevent HIV-1 cell-cell transmission, which likely contributes to its restriction of HIV-1 replication.

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Last Name First Name Email Institution

Alam Khalid kkafy3@mizzou.edu University of Missouri

Anbazhagan Rajesh rajesh.anbazhagan@bacterioscan.com BacterioScan

Arizmendi Olivia olivia.arizmendi@ku.edu University of Kansas

Barta Michael barta.michael@ku.edu University of Kansas

Beerntsen Brenda BeerntsenB@Missouri.edu University of Missouri

Behar Amanda amanda.rain.behar@okstate.edu Oklahoma State University

Boon Jacco jboon@dom.wustl.edu Washington University

Bose Jeffrey jbose@kumc.edu The University of Kansas Medical Center

Brown Charles brownchar@missouri.edu University of Missouri

Brown Pamela brownpb@missouri.edu University of Missouri

Burke Donald burkedh@missouri.edu University of Missouri

Cho Junho junho.cho@okstate.edu Oklahoma State University

Choudhari Shyamal shyamalchoudhari@ku.edu University of Kansas

Coate Eric eackr3@mail.missouri.edu University of Missouri

Dhariwala Miqdad modcp6@mail.missouri.edu University Of Missouri-Columbia

Elliott Ali elliottal@missouri.edu University of Missouri

Fales Susan J Falesw@Missouri.edu University of Missouri CVM

Fales William H Falesw@missouri.edu University of Missouri CVM

Fisher Mark mfisher1@kumc.edu University of Kansas Medical Center

Freches Danielle frechesd@missouri.edu University of Missouri

Ganjam Irene ganjami@missouri.edu VMDL - University of Missouri

Gregory Michelle GregoryML@missouri.edu University of Missouri

Guo Huatao guohua@missouri.edu University of Missouri Columbia

Hancock Lynn lynnh@ku.edu University of Kansas

Hardwidge Philip hardwidg@gmail.com Kansas State University

Harrison Kelly kelly.harrison@ku.edu University of Kansas

Hefty Scott pshefty@ku.edu University of Kansas

Hinshaw Kara k699h433@ku.edu University of Kansas

Howe Savannah savannah.howe@siu.edu Southern Illinois University, Carbondale

James Anthony aajames@uci.edu University of California

55

Johnson Britney bjohnson@path.wustl.edu Washington University School of Medicine

Kantor Asher amkt33@missouri.edu University of Missouri

Kocher Elena kocherelena@gmail.com University of Missouri-Columbia

Konjufca Vjollca vjollca@micro.siu.edu Southern Illinois University, Carbondale

Kumar Amit docamit2004@gmail.com Kansas State University

Labrie Scott scottlabrie@ku.edu University of Kansas

Lacey Carolyn calm55@mail.missouri.edu University of Missouri

Lambert Greg gsl7cf@mail.missouri.edu University of Missouri-Columbia

Lameira Elsa elsa.lameira@okstate.edu Oklahoma State University

Lang Yuekun yuekun@ksu.edu Kansas State University

Lasky Carrie cel9h3@mail.missouri.edu University of MIssouri-Columbia

Lazear Helen hlazear@dom.wustl.edu Washington University

Lee Jinhwa jinhwa@vet.k-state.edu Kansas State University

Li Minghua mlmmc@mail.missouri.edu University of Missouri-Columbia

Li Nengzhang lich2001020@163.com Kansas State University

Liu Shan-Lu liushan@missouri.edu University of Missouri

Lopez Lacey lrl437@mail.missouri.edu University of Missouri-Columbia

Lutter Erika erika.lutter@okstate.edu Oklahoma State University

Manoharan Shankar smanoharan@kumc.edu University of Kansas Medical Center

Markiewicz Mary mmarkiewicz@kumc.edu University of Kansas Medical Center

Marshall Dana dana.marshall@bacterioscan.com BacterioScan

Martinez Becerra Francisco francisco.martinez@ku.edu University of Kansas

Matocha Nicole kmatocha@kumc.edu University of Kansas Medical Center

Miao Chunhui cmp55@mail.missouri.edu University of Missouri-Columbia

Noden Bruce Bruce.noden@okstate.edu Oklahoma State University

Pascual Elizabeth elizabeth.pascual@okstate.edu Oklahoma State University

Patel Ami Patelam@missouri.edu University of Missouri

Picking Wendy wendy.picking@ku.edu University of Kansas

Picking William picking@ku.edu University of Kansas

Pratt Carmela clp446@mail.missouri.edu University of Missouri

Reinholtz Linda lindar@shenandoah-bt.com Shenandoah Biotechnology

Richards Chris ricch@okstate.edu Oklahoma State University

56

Richner Justin justin.m.richner@gmail.com Washington U. in St. Louis

Rimbey Jeanette jnrd54@mail.missouri.edu University of Missouri

Schneider Olivia olivias@shenandoah-bt.com Shenandoah Biotechnology

Schoenlaub Laura ls33d@mail.missouri.edu University of Missouri

Sharma Neekun nsharma4@kumc.edu The University of Kansas Medical Center

Shaw Ed ed.shaw@okstate.edu Oklahoma State University

Silva Patricia pmdsmartins@gmail.com Kansas University

Skyberg Jerod skybergj@missouri.edu University of Missouri

Soest Rhonda rhonda.soest@bacterioscan.com BacterioScan

Stewart George stewartgc@missouri.edu University of Missouri

Sun Keer keer.sun@unmc.edu Univerisity of Nebraska Medical Center

Trembath Andrew atrembath@kumc.edu University of Kansas Medical Center

Vanlandingham Dana dlvanlan@vet.k-state.edu Kansas State University

Varahan Sriram sriram89@ku.edu University of Kansas

Wang Gaochan wang2034@k-state.edu Kansas State University

Wang Songjie wangso@missouri.edu University of Missouri

Weidman Ruth ruth.weidman@okstate.edu Oklahoma State University

Williams Graham williamsgraham@wustl.edu Washington University in Saint Louis

Wilson Kevin kevin.s.wilson@okstate.edu Oklahoma State University

Winford Edric edwrh7@mail.missouri.edu University of Missouri

Wolfe Annie Aewmb7@mail.missouri.edu University of Missouri

Wright Kathleen kathleenlwright@gmail.com University of Missouri

Xu Wei weixu@path.wustl.edu Washington Universion School of Medicine

Yu Jingyou jypn4@mail.missouri.edu University of Missouri-Columbia

Zhang Guoquan zhangguo@missouri.edu University of Missouri

Zhang Yan zhanyan@missouri.edu University of Missouri

Zhukovsky Mikhail mikhail.zhukovsky@physik.uni-saarland.de University of Missouri

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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

MU Laboratory for Infectious Disease Research 1817 E Campus Loop Columbia, MO 65211 Email: lidr@missouri.edu Phone: 573-884-3808 Fax: 573-884-0791 Website: www.lidr.missouri.edu

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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

Equipment include: • Cell- and tissue-culture specific incubators and dedicated bio-safety cabinets • Imaging capabilities for bright field and fluorescence microscopy • Micro centrifuges, hanging bucket and fixed angle centrifuges and Ultra centrifuges • Thermo cyclers, electroporation unit and spectrophotometers

Aerobiology Core

Immunology Core

Infectious Disease Expertise