South Central Branch Meeting

September 18-19, 2015

Trent Lott National Center The University of Southern Mississippi

Hattiesburg, Mississippi

WELCOME!

Dear Colleagues: It is my pleasure to welcome you to Hattiesburg, MS for the 2015 Annual Meeting of the South Central Branch of the American Society for Microbiology. The University of Southern Mississippi has graciously supported hosting this year’s meeting. I would like to thank the organizers of this year’s meeting, especially Dr. Mohamed Elasri and Ms. Jamie Lott. These two have been instrumental in pulling the meeting together. I am very grateful for the time and effort that all of the organizers and student volunteers have provided in hosting this conference. I am very excited about this year’s meeting! We have a fantastic keynote speaker, Dr. Michael Federle, from the University of Illinois. We also have wonderful speakers presenting from Mississippi, Arkansas, and Louisiana. In addition to these world-renowned speakers, it is also election year! Please consider nominating yourself or your colleagues for Secretary/ Treasurer and Vice President. Thank you also to the sponsors who have made this meeting possible. The acknowledgement page of the program has a list of all of those that have helped with this conference. Last, but not least, I would like to thank each of the attendees for continuing to support your ASM branch! THANK YOU ALL! Best wishes,

Janet R. Donaldson, SCB ASM President

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Table of Contents

South Central Branch Officers…………….….…………………………………………………………… 3 Meeting Agenda…………………………………………………………………………………………….. 4 Speaker Biographies Dr. Michael J. Federle..……..……………………………………………………………………. 5 Dr. Jeremy Kamil………………………………………………………………………………….. 6 Tick & Tick-Borne Disease Symposium….……………………………………………………………..... 7 Invited Speaker Sessions Applied & Environmental Microbiology…………………….....………………………………… 8 Pathogenic Microbiology………………..……..………………………….………………….……8

Immunology & Virology..……………..……….………….………………………………………. 9 Oral Presentation Sessions Immunology & Virology…………………………….……..………………………………………10 Immunology & Virology…..………….……………..…………………………………………….11 Applied & Environmental Microbiology……………………..…………………………………. 12

Pathogenic Micribiology……...…………………………………………………………………. 13 Applied & Environmental Microbiology…..……………………………………………………..14

Poster Session Applied & Environmental Microbiology.……………..……...………………………………… 15 Immunology & Virology………………...……………..……...………………………………… 16 Pathogenic Microbiology……………….……………..……...………………………………… 17

Conference Abstracts……………………...……………………………………………………………… 19 Past South Central Branch Meetings…………….……………………………..……………….……… 67 Charles C. Randall Lectureship, Past Recipients…........……………………..…………….………… 68 Sponsors………………………………………….………….…………………………………………….. 69 Acknowledgements…………………………...…………………………………………………………… 71

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2014-14 Officers, South Central Branch ASM

President

Janet Donaldson, Ph.D.

Department of Biological Sciences Mississippi State University

Box GY Mississippi State, MS 39406-0001

Phone: 601-266-6916 donaldson@biology.msstate.edu

Secretary-Treasurer

Andrew Yurochko, Ph.D.

Department of Microbiology & Immunology Louisiana State University

Health Sciences Center 1501 Kings Highway,

Shreveport, LA 71130-3932 Phone: 318-675-8332

ayuroc@lsuhsc.edu

Vice-President Karl Indest, Ph.D. U.S. Army Engineer Research and Development Center 3909 Halls Ferry Rd. Visksburg, MS 39180 karl.j.indest@usace.army.mil Councilor Mohamed Elasri Department of Biological Sciences University of Southern Mississippi 118 College Drive #5018 Hattiesburg, MS 39406 Phone: 601.266.6916 Mohamed.elasri@usm.edu

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Meeting Agenda

Friday, September 18, 2015

8:00-9:00 Registration, Coffee Vendor and Poster Setup

9:00-11:30 Tick & Tick Borne

Disease Symposium, Room 103C

Applied & Environmental Microbiology,

Invited Speakers Room 102

Immunology & Virology, Oral Presentations

Room 103A

11:30-1:00 Lunch on your own

1:00-3:30 Pathogenic Microbiology,

Invited Speakers Room 103C

Applied & Environmental Microbiology,

Oral Presentations Room 102

Immunology & Virology, Oral Presentations

Room 103A

3:30-5:00 Poster Session & Refreshments

5:00-6:30 Keynote Lecture, Room 103

6:30-7:30 Reception

Saturday, September 19, 2015

9:00-11:30

Pathogenic Microbiology,

Oral Presentations Room 103C

Applied & Environmental Microbiology,

Oral Presentations Room 102

Immunology & Virology, Invited Speakers

Room 103A

11:30-11:40 Break

11:40-12:40 Charles C. Randall Lecture, Room 103

12:40-1:40 Lunch (provided)

Announcement of Awards, Room 103 1:40 Meeting Adjourned

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Keynote Speaker Dr. Michael J. Federle, University of Illinois

We are pleased to have Dr. Michael J. Federle as our American Society for Microbiology Distinguished Lecturer at this year’s branch meeting. Topic of lecture: Approaches to Identify Quorum Sensing Inhibitors Since pheromone signaling has a clear effect on bacterial gene expression and behavior, the potential to influence behavior may be possible through manipulation of bacterial communication. The Federle lab has initiated high-throughput screening for molecules that block Rgg-pheromone signaling with compounds available in both chemical and genetic libraries. Candidate screening utilizes luciferase and fluorescence reporters together with robotic liquid handling, phage display, and flow cytometry. Initial results have found compounds that specifically and directly bind to Rgg proteins and that compete with pheromone binding. BIOGRAPHICAL SKETCH Dr. Federle's ongoing research interests are in cell-cell communication among Gram-positive bacteria. His lab has helped identify a new class of quorum-sensing pathways that utilize proteins of the Rgg family and short, imported peptide pheromones. The lab’s work focuses on characterization of these pathways, identification of behaviors controlled by them, and development of

small molecule probes into novel therapeutics aimed at interfering with bacterial communication to treat disease. Dr. Federle has been awarded an R01 grant from the NIH and was recently named a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease. He has been an invited speaker for more than 30 seminars and conferences and has authored 26 articles and book chapters. Dr. Federle serves on the editorial board for Journal of Bacteriology, and provides ad hoc reviews for mBio, Infection and Immunity, and Applied and Environmental Microbiology. Visit Dr. Federle website for more info about his work: http://www.uic.edu/labs/federle/index.htm

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Charles C. Randall Lectureship

Dr. Jeremy Kamil LSU Health Sciences Center

Dr. Jeremy Kamil is a molecular virologist who is an Assistant Professor of Microbiology and Immunology at LSU Health Sciences Center (LSUHSC), Shreveport. Dr. Kamil first became interested in virology while studying for an undergraduate degree in biology at Cornell University. After graduation, he joined the laboratory of Dr. Hsing-Jien Kung at the University of California at Davis, and in his doctoral work identified and characterized a viral lipoprotein lipase homolog encoded by Marek’s disease virus. After obtaining his Ph.D. (2003), Dr. Kamil returned to Ithaca, joining the laboratory of Dr. Nikolaus Osterrieder at Cornell University, where he was awarded a USDA Postdoctoral Fellowship to study the role of the viral lipase homolog in the pathogenesis of Marek’s disease. Dr. Kamil then joined the laboratory of Dr. Donald Coen at Harvard Medical School, where he was supported by an NIH Ruth Kirschstein NRSA Fellowship to investigate the roles of a viral protein kinase (UL97) in the replication of human cytomegalovirus (HCMV). While in Dr. Coen’s laboratory, Dr. Kamil discovered a novel protein-protein interaction between UL97 and a major viral tegument protein, pp65, and demonstrated the physiological

relevance of UL97 as an inactivator of the retinoblastoma tumor suppressor protein. In 2010, Dr. Kamil joined the faculty at LSUHSC and established his laboratory that explores how HCMV coopts cellular tumor suppressor pathways to modulate its gene expression and how the virus can vary its cell tropism during infection. His ongoing work is funded by grants from the NIH supported Center for Molecular and Tumor Virology and the American Heart Association. His most recent findings have given new insight into how HCMV modulates the glycoprotein complexes on the virus surface that allow the virus to infect different cell types. His work will contribute to ongoing efforts to generate a vaccine to combat HCMV, which is a major cause of birth defects, plays a role in cardiovascular disease, and causes dangerous infections in persons with impaired immunity. The lectureship is named in honor of Dr. Charles C. Randall, Professor Emeritus, University of Mississippi Medical Center, Jackson, Mississippi. Dr. Randall conducted pioneering research in the area of viral structure, biochemistry, and molecular biology. As the former Chairman of the Department of Microbiology at the University of Mississippi Medical Center and former President of the South Central Branch, Dr. Randall made enormous contributions to the growth and development of the microbiological sciences within the South Central Branch. The South Central Branch, American Society for Microbiology, established the Charles C. Randall Lectureship, which is to be awarded annually to an “outstanding young faculty member” who will present a lecture on his or her research at the annual branch meeting. The awardee, selected by a panel of senior faculty members appointed by the Branch President, must hold the rank of Assistant Professor (or equivalent) in a scientific institution (public or private) within the geographic boundary of the South Central Branch of the ASM, and must be in the early stages of a research career with less than five years of experience since completing training, but be independent of a mentor.

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Tick and Tick-Borne Disease Symposium September 18, 2015, 9:00-11:30 p.m., Room 103C Chair: Dr. Shahid Karim, Associate Professor, The University of Southern Mississippi 9:00 a.m. Welcome Dr. Shahid Karim, The University of Southern Mississippi 9:05 a.m.

Metagenomics as a route to understanding the tick and its microbiome Dr. Gregory Dasch, Center for Disease Control and Prevention

9:30 a.m. Rickettsial infection and dissemination in the arthropod host Emma Harris, Louisiana State University

9:55 a.m. Break

10:15 a.m. Rickettsial Interactions in the Amblyomma Maculatum-Rickettsia System Dr. Andrea Varela-Stokes, Mississippi State University

10:40 a.m. Vector Transmission Model of Monocytotropic Ehrlichiosis Dr. Tais Saito, University of Texas Medical Branch

11:05 a.m. Studying temperature-dependent gene regulation in the relapsing fever spirochete, Borrelia turicatae Dr. Jon Blevins, University of Arkansas Medical School

11:30 a.m. Concluding Remarks

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Invited Speaker Sessions

Applied and Environmental Microbiology September 18, 9:00-11:30 a.m., Room 102 Chair: Dr. Fiona Crocker, Engineer Research and Development Center

9:00 a.m. Field-Scale Demonstration of Aerobic Bioaugmentation of Explosive-Contaminated Groundwater Dr. Fiona Crocker, Research Microbiologist, Engineer Research and Development Center

9:30 a.m. A Microbial Ecosystem Beneath The Antarctic Ice Sheet Dr. Brent Christner, Associate Professor, Louisiana State University

10:00 a.m. Break 10:30 a.m.

The Use of Fungal Cultures for Biotransformation of Drugs Dr. John Sutherland, Research Microbiologist, National Center for Toxicological Research

11:00 a.m. Priming in the Microbial Landscape: Metabolic Stimulation of Litter-associated Microbial Heterotrophs by Periphytic Algae Dr. Kevin Kuehn, Associate Professor, The University of Southern Mississippi

Pathogenic Microbiology September 18, 1:00-3:30 p.m., Room 103C Chair: Dr. Gyan Sahukhal, The University of Southern Mississippi

1:00 p.m. Role of msaABCR Operon in Biofilm Development in Staphylococcus aureus Dr. Gyan Sahukhal, Bioinformatics Coordinator, The University of Southern Mississippi

1:30 p.m. Puma, a Critical Component of Innate Immunity Against Extracellular Pathogens Justin Thornton, Associate Professor, Mississippi State University

2:00 p.m. Break

2:30 p.m. Analyses of Gene Expression Profiles in Mycobacterium Marinum in an Activated Virulent State Don Ennis, Instructor, University of Louisiana at Lafayette

3:00 p.m. Ecology and Transmission Dynamics of Mycobacterium Ulcerans Heather Jordan, Assistant Professor, Mississippi State University

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Immunology and Virology September 19, 9:00-11:30 a.m., Room 103A Chair: Dr. Fengwei Bai, Assistant Professor, The University of Southern Mississippi 9:00 a.m.

IL-23-IL-17, A Bridge Links and Adaptive Immunity Against West Nile Virus Infection Dr. Fengwei Bai, Assistant Professor, The University of Southern Mississippi

9:30 a.m. The Battle Between Rotavirus and Its Host for Control of Innate Cellular Defenses Dr. Michelle Arnold, Assistant Professor, Louisiana State University Health Sciences Center

10:00 a.m. Break

10:30 a.m. A Clinical Perspective on the Immunobiology of Ovarian Cancer Dr. Martin J. Cannon, Professor, University of Arkansas for Medical Sciences

11:00 a.m. CD8 T Cells, Functional Diversity, and Pathogen Control Dr. Shannon Kahan, Postdoctoral Fellow, University of Alabama at Birmingham

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Oral Presentation Sessions

Friday, September 18 Immunology & Virology, Oral Presentations 9:00 -11:30 a.m., Room 103A 9:00 a.m.

Efficiency of complement protein in saltwater hardhead catfish Ariopsis felis and freshwater yellow bullhead catfish Ameiurus melas Abby L. Adams, Nicholls State University

9:20 a.m. Monoclonal Antibodies CC34 and CC41 Bind Catfish Leukocyte Immune-Type Receptors Laura E. Blackmon, University of Mississippi Medical Center

9:40 a.m. Mechanism of HCMV-induced Monocyte-to-Macrophage Differentiation Stephen J. Cieply, Louisiana State University, Health Sciences Center-Shreveport

10:00 a.m. Break 10:30 a.m.

I Will Survive: HCMV Utilizes a Distinct Pro-Survival Mechanism in Infected Carrier Monocytes Donna K. Collins-McMillen, Louisiana State University, Health Sciences Center-Shreveport

10:50 a.m. Outer-membrane vesicles derived from Burkholderia pseudomallei drive innate immune responses in vivo in populations of antigen-presenting cells Dr. Christopher Davitt, Tulane University

11:10 a.m. HPV16 utilizes vesicular transport to deliver the viral DNA to the nucleus during mitosis Stephen A. DiGiuseppe, Louisiana State University, Health Sciences Center-Shreveport

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Immunology & Virology, Oral Presentations 1:00-3:30 p.m., Room 103A 1:00 p.m.

Viral binding induced signaling drives a unique and extended intracellular trafficking pattern during infection of primary blood monocytes Dr. Jung Heon Kim, Louisiana State University, Health Sciences Center-Shreveport

1:20 p.m. Determining the mechanism of rotavirus NSP1-induced degradation of host target proteins Dr. Lindy Lutz, Louisiana State University, Health Sciences Center-Shreveport

1:40 p.m. Characterization of an Oral Vaccine for M. marinum: Examination of Both Mucosal Immunity and Systemic Immunity in a Fish Model Lisa M. Shirreff, University of Louisiana at Lafayette

2:00 p.m. Break 2:30 p.m.

Growth Factor Signaling Pathway Regulation by HPV16 E5 Protein Matthew Scott, Louisiana State University, Health Sciences Center-Shreveport

2:50 p.m. The Regulation of Cellular and Viral Gene Expression by HPV16 E7 William Songock, Louisiana State University, Health Sciences Center-Shreveport

3:10 p.m. Human Cytomegalovirus Exploits Clathrin-Mediated Endosomal Trafficking for Maturation and Egress Dr. Ritesh Tandon, University of Mississippi Medical Center

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Applied & Environmental Microbiology, Oral Presentations 1:00-3:30 p.m., Room 102 1:00 p.m.

Presence of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes in Raw Source Water and Treated Drinking Water in Southeast Louisiana Scott Bergeron, Nicholls State University

1:20 p.m. Does pathogenic Rickettsia parkeri shape the native microbiota within the tick host? Khemraj Budachetri, The University of Southern Mississippi

1:40 p.m. Regulation of Glycine Rich Proteins in Rickettsia parkeri infected ticks Rebekah Bullard, The University of Southern Mississippi

2:00 p.m. Break 2:30 p.m.

Engineering Bacillus subtilis Shows Biofilm Promotes Cellulose Degradation Yijie Daniel Deng, The University of Southern Mississippi

2:50 p.m. Analyzing the Influence Oxygen Deprivation has on the Capability of Listeria monocytogenes to Induce Listeriosis in Gerbils Jillian L. Harris, Mississippi State University

3:10 p.m. Members of the Conserved DedA/Tvp38 Membrane Protein Family are required for growth of Escherichia coli at alkaline pH. Sujeet Kumar, Louisiana State University

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Saturday, September 19 Pathogenic Microbiology, Oral Presentations 9:00 -11:30 a.m., Room 103C 9:00 a.m.

The role of universal stress proteins in Edwardsiella ictaluri virulence Ali Akgul, Mississippi State University

9:20 a.m. The msaABCR Operon Activates Capsule Production in Staphylococcus aureus Justin L. Batte, The University of Southern Mississippi

9:40 a.m. AliC and AliD Enhance Virulence of Nonencapsulated Streptococcus pneumoniae Jessica L. Bradshaw, University of Mississippi Medical Center

10:00 a.m. Break 10:30 a.m.

Investigating the role of transition metals in Streptococcus pneumoniae colonization Lindsey R. Brown, Mississippi State University

10:50 a.m. Determining the Impact of Bile Induced Damage on Listeria monocytogenes Oindrila Paul, Mississippi State University 11:10 a.m.

Deletion of the Polyamine Transporter potABCD Attenuates the Virulence of Encapsulated but Not Nonencapsulated Streptococcus pneumoniae Haley R. Pipkins, University of Mississippi Medical Center

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Applied & Environmental Microbiology, Oral Presentations 9:00 -11:30 a.m., Room 102 9:00 a.m.

An insight into the antioxidant responses to exogenous oxidative stress agents and a glimpse into the role of catalase in the reproductive fitness of the Gulf-Coast tick (Amblyomma maculatum) Deepak Kumar, The University of Southern Mississippi

9:20 a.m. E. coli PBP5 active site mutations alter cell shape William John MacCain, University of Arkansas Medical School

9:40 a.m. Elucidating the Functional Role of the H2O2-Generating Dual Oxidase (Duox) in the Gulf Coast Tick, Amblyomma maculatum Virginia C. Meyers, The University of Southern Mississippi

10:00 a.m. Break 10:30 a.m.

Physiological & Genetic Characterization of Lipid Accumulation in Gordonia sp. KTR9” Dr. Karl Indest, U.S. Army Engineer Research & Development Center

10:50 a.m. Metagenomic study of bacteriophage diversity from the gut of the Formosan subterranean termite, Coptotermes formosanus Shiraki Chinmay V. Tikhe, Louisiana State University

11:10 a.m. Ehrlichia chaffeensis –Vector-Host-Pathogen Interactions Jaclyn B. Williams, The University of Southern Mississippi

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Poster Sessions

September 18, 3:00-5:00 p.m.

Applied & Environmental Microbiology, Poster Session 1.1 Dissemination of Ehrlichia chaffeensis using an Artificial Membrane Feeding System Paige E. Allen, The University of Southern Mississippi 1.2 Presence of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes in Different Salinity

Gradients in Water from Bayou Petit Caillou in Southern Louisiana Ryan Brown, Nicholls State University 1.3 The Microbial Clock: Tracking Bacterial Translocation through Successional Host Decomposition Zachary M. Burcham, Mississippi State University 1.4 Effect of Diet and Geography on the Horse Gut Microbiota and the Potential for Rapid

Growth of ‘Bloom’ Taxa in Fecal Samples Kalie F. Beckers, Southeastern Louisiana University 1.5 Superoxide dismutases modulate the native microbiota of Amblyomma maculatum, a

vector of Rickettsia parkeri Gary Crispell, The University of Southern Mississippi 1.6 From Microbes to Mosquitoes: An Interdisciplinary, Multi-Institutional Approach

to Student Engagement in the Ecological and Microbiological Sciences Dr. William Dees, McNeese State University 1.7 Presence of Vibrio vulnificus and V. parahaemolyticus in Louisiana Seafood. Richard Grabert, Nicholls State University 1.8 An Assessment of the Microbiological Impacts of Aerobic Septic Systems in Southwest Louisiana Brittany N. Joseph, McNeese State University 1.9 Comparison of antibiotic resistance in bacteria isolated from Atlantic Bottlenose Dolphins inhabiting Barataria

Bay, LA, and Sarasota Bay, FL Shuo Shen, The University of Southern Mississippi, Gulf Coast Research Laboratory 1.10 Antimicrobial Evaluation of Essential Oils Encapsulated in Latex Nanoparticles D.N. Amato, D.V. Amato, The University of Southern Mississippi 1.11 Analysis of Rhizosphere Microbial Communities from the Verkhnekamsk Salt Mining Region of Russia Dmitri Mavrodi, The University of Southern Mississippi 1.12 Biosynthesis of Phenazine Metabolites in Burkholderia Spp. Samuel Hendry, The University of Southern Mississippi 1.13 Pseudomonas/Brachypodium as a Model System for Understanding Rhizosphere Plant-Microbe Interactions

under Abiotic Stress Janiece Rawalt, The University of Southern Mississippi

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Immunology & Virology, Poster Session 2.1 The Effect of Route and Anatomical Location On Antigen-Specific Immune Responses in the Respiratory Tract Following Parenteral Immunization Sarah Margaret Baker, Tulane University 2.2 Improving the Annotation of Bovine herpesvirus-1 Genome Using Experimental Data Kaley A. Barber, Mississippi State University 2.3 HCMV pUL93 Interacts with pUL77 to Determine Late Events in Virus Maturation Bernadette DeRussy, University of Mississippi Medical Center 2.4 Expression of platelet-derived growth factor receptor alpha in the embryonic bursa of Fabricius Dr. Claire L. Fellman, Mississippi State University 2.5 Host Cell Splicing Factors hnRNP A/B and hnRNP H3 are induced and re-localized

after infection with Bovine Herpesvirus type 1 Kathryn Elizabeth Foster, Mississippi State University 2.6 Investigating the role of the minor capsid protein L2 in delivery of HPV16 genome to

PML nuclear bodies Lucile G.M. Guion, Louisiana State University, Health Sciences Center-Shreveport 2.7 Novel bioconjugated hybrid gold-graphene oxide nanoparticles for the treatment of

cytomegalovirus infection Mohammad H. Hasan, University of Mississippi Medical Center 2.8 Partial characterization of a chicken B-cell antigen recognized by monoclonal antibody EIV-E12 Nikhil Nuthalapati, Mississippi State University 2.9 Evaluating vector, route, and dose effects on the adjuvant capacity and safety of flagellin

when encoded in DNA or adenovirus vaccines Dr. Hamada F. Rady 2.10 Identification and Characterization of Interleukin-6 in Channel Catfish, Ictalurus punctatus David Spencer, University of Mississippi Medical Center 2.11 Regulation of Paracrine Signaling Factors by HPV16 Brittany Woodby, Louisiana State University, Health Sciences Center-Shreveport 2.12 The Delta Peptide region of Ebola Virus Soluble Glycoprotein (sGP) is a Potent Viroporin. Jing He, Tulane University

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Pathogenic Microbiology, Poster Session 3.1 Evaluation of Baron Based Molecules in Listeria Monocytogenes LapB Protein Nawar Al-Janabi, Mississippi State University 3.2 The use of bacteriophage to control biofilm formation of Vibrio parahaemolyticus in aquaculture Ashleigh Aubin, Nicholls State University 3.3 Observing the Mold-Specific Gene, M46 Expression During the Morphological Shift from Mold to Yeast and Yeast

to Mold in Histoplasma capsulatum Ashly Claiborne, Alcorn State University 3.4 Determining the Role of Alterations to the Cell Membrane Lipid Components in Improving Survival Against Bile

Induced Damage in Listeria monocytogenes Dominique Clark, Mississippi State University 3.5 The Contribution of Pyruvate Oxidase to Pneumolysin Release in Streptococcus pneumoniae Ridge C. Dabbs, Mississippi State University 3.6 Emerging Infectious Diseases and Entomological Intelligence: A Biosurveillance Initiative Dr. William Dees, McNeese State University 3.7 Effect of Pneumococcal H2O2 on Epithelial Cell Gene Expression Karien N. Dixon, Tougaloo College 3.8 The Emerging Pathogen Nonencapsulated Streptococcus pneuominae: the Case of a 2-year-old with Chronic

Adenoiditis Cheshil Dixit, University of Mississippi Medical School 3.9 Role of msaABCR in Antibiotic Susceptibility during Biofilm Development in Staphylococcus aureus Latoyia P. Downs, The University of Southern Mississippi 3.10 Virulence of the novel species Streptococcus tigurinus in a Galleria mellonella model Amal Fadil, Mississippi College 3.11 Susceptibility of Neisseria gonorrhoeae to the Plant Compound Neem In Vitro and In Vivo Cathryn Frey, McNeese State University 3.13 The Overexpression of the Mold Specific Gene, MS88, in the Pathogenic Dimorphic Fungi Histoplasma

capsulatum Mariah L. Lloyd, Alcorn State University 3.14 Rapid detection using tdh, trh-induced hemolysis on human erythrocytes and urease production as biomarkers

for screening pathogenic Vibrio parahemolyticus strains Maryam K. Mohammadi, Mississippi State University 3.15 Early innate immune response to a polyamine transport deficient pneumococcus in a mouse model of

pneumococcal pneumonia Bindu Nanduri, Mississippi State University 3.16 Enterotoxin Production in Clostridium perfringens is affected by antimicrobial agents and bile acids Dr. Fatemeh Rafii, United States Department of Health & Human Services

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3.17 Characterizing the Extensive Heterogeneity of Mycobacteriosis Disease Progression in Controlled Animal Model Caroline Y. Sciarrillo, University of Louisiana at Layfayette 3.18 Understanding the Regulation of MsaB production from msaABCR operon in Staphylococcus aureus Sarah Simmons, The University of Southern Mississippi 3.19 The Regulatory Relationship Between Transcriptional Regulators CodY and MsaB in

Staphylococcus aureus Brittany L. Trunell, The University of Southern Mississippi 3.20 The role of GenF in the asymmetric distribution of the virulence protein, IcsA in Shigella flexneri Birendra Sharma, Mississippi University for Women

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Conference Abstracts Tick and Tick-Borne Disease Symposium September 18, 2015, 9:00-11:30 p.m., Room 103C Chair: Dr. Shahid Karim, Associate Professor, The University of Southern Mississippi [9:05 a.m.] Metagenomics as a Route to Understanding the Tick and its Microbiome

Gregory A Dasch, Michael J. Pung, Robert Z. Holmes, Maria L. Zambrano, and Amanda Jo Williams-Newkirk Rickettsial Zoonoses Branch, Centers for Disease Control, Atlanta, Georgia

Background: After mosquitoes, ticks are the most important arthropod vectors because of the diversity of viral, bacterial and parasitic agents which they transmit and their efficiency as reservoirs for these agents. Many surveys of tick-borne agent prevalence are based on group specific or specific PCR assays but this methodology and the great abundance of some agents can limit our ability to detect novel agents. New methods for the direct and complete genetic characterization of novel and uncultivable agents are needed. Methods: We have used indexed 16S rRNA amplicon sequencing on the 454 and Ion Torrent for tick microbiome analysis and deep sequencing with the Ion Torrent and Illumina HiSeq platforms to characterize different bacterial targets and tick mitochondrial genomes directly from field-caught ticks. The sequences were assembled with CLC Genomics Workbench and Geneious. Amplicon sequences were analyzed in Mothur, and bacterial and mitochondrial contigs were annotated with Prokka and MITOS, respectively. Results: Tick microbiomes were characterized from questing Amblyomma americanum, A. maculatum, Ixodes scapularis, Dermacentor variabilis, and flat and engorged Rhipicephalus sanguineus. Deep 16S rRNA coverage can be limited by the presence of dominant, highly abundant bacteria so sequence specific blocking primers are being evaluated. Three complete tick mitochondrial genomes, and the genome sequences of R. bellii, and Coxiella-like and Francisella-like endosymbionts have been obtained. Sequencing costs have dropped to the point that obtaining the complete genome sequences of many important ticks is possible. Conclusion: While small bacterial and mitochondrial genomes can be readily assembled from individual tick samples, the targets must be abundant or suitable for enrichment. Consequently, low abundance members of microbiome communities of ticks are still a challenge to sequence. Assembly of large, repeat-rich tick genome sequences will require the creation of several types of libraries and the ability to organize and map scaffolds to the individual chromosome level. Novel advanced methods of eukaryotic genome assembly which may help to simplify this task have been developed recently. [9:30 a.m.] Rickettsial infection and dissemination in the arthropod host

Emma Harris1, J.A. Macaluso1, V.I. Verhoeve1, K.H. Banajee1, A.F. Azad2, and K.R. Macaluso1

Louisiana State University, Baton Rouge, Louisiana1

University of Maryland Baltimore, Baltimore, Maryland2

Background: Rickettsia, the Gram negative, obligate intracellular bacteria is divided into four groupings based on biological, genetic, and antigenetic properties. The spotted fever group of Rickettsia (SFGR), which contains R. rickettsii, the causative agent of Rocky Mountain spotted fever, is well known for its close association with ticks. Experimental evidence and field reports show only competent ticks are able to act as vectors for select rickettsial species. However, the geographical overlap of multiple Rickettsia and tick species coinciding with the molecular detection of an assortment of rickettsial agents in what may be novel tick hosts is becoming a common occurrence. Little is currently known concerning vector competence of these tick hosts for various rickettsial species. Knowledge of rickettsial proteins contributing to dissemination of Rickettsia within the tick host is also limited. Methods: Adult, female D. variabilis and A. maculatum were capillary fed with either Rickettsia or delivery control and allowed to feed to repletion. Fitness-related metrics including engorgement weight, egg production index, nutrient index, and egg hatch percentage were then assessed. Subsamples of eggs, larvae, nymphs, and adults of the F1 generation were assessed for transovarial and transtadial transmission. Mammalian and tick cell lines were infected with R. parkeri wild-type, R. parkeri Δsca2, or R. parkeri ΔrickA. Rickettsia was visualized within each cell line at 15 and 30 minutes post inoculation, or 2, 8, 24, and 48 hours post inoculation. Results: Results show that rickettsial exposure had a minimal fitness effect in D. variabilis and transovarial transmission was observed for all groups except R. rickettsii. In contrast, rickettsial exposure influenced A. maculatum fitness and transovarial transmission of rickettsiae was demonstrated only for R. amblyommii and R. parkeri- exposed ticks. Sustained maintenance of rickettsiae via transstadial transmission was diminished from unfed larvae to unfed F1 adults. Experimentation with mutant strains of R. parkeri is still ongoing. Conclusions: The findings suggest transovarial transmission specificity may be tick species dependent and sustained vertical transmission is not common. Results of this study will lead to a better understanding of the ecology and epidemiology of rickettsial diseases.

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[10:15 a.m.] Rickettsial Interactions in the Amblyomma maculatum-Rickettsia System Andrea S. Varela-Stokes, Jung Keun Lee, Gail M. Moraru, Amanda Benton, John V. Stokes, Haley Parker, Kaitlin Graham, Keiko Rausch, Jacob Hughes Mississippi State University, Mississippi State, Mississippi

Background: Prior to the 21st century, Rocky Mountain spotted fever, caused by Rickettsia rickettsii, was the only spotted fever group rickettsiosis considered endemic to the United States. In 2010, the CDC relabeled this reporting category as “Spotted Fever Rickettsiosis,” highlighting a recent recognition of other domestic spotted fever agents. Among these other agents, Rickettsia parkeri, which is transmitted primarily by the Gulf Coast tick, currently holds the strongest association with human disease. The Gulf Coast tick, Amblyomma maculatum, may also harbor a spotted fever group rickettsia of unknown pathogenicity, “Candidatus Rickettsia andeanae,” and potential interactions between R. parkeri and “Ca. R. andeanae” during their natural maintenance among tick and vertebrate hosts are unknown. Methods: In order to approach our goal of understanding how within host and among host rickettsial interactions influence transmission of the known pathogen, R. parkeri, we first performed transmission studies using A. maculatum artificially infected with R. parkeri-GFPuv, “Ca. R. andeanae”, both rickettsiae, or uninfected, and evaluated rickettsial infection in tick tissues, pre- and post-engorgement on rabbit hosts. Results: In results from one of three feeding trials, we found a general decrease in rickettsial infection rates in tick tissues over the feeding period, as well as in progeny from treatment group. Tick tissues positive for rickettsial DNA are being analyzed by fluorescence and electron microscopy to confirm presence of rickettsiae, while serology and tissue testing from rabbits will indicate infection status of the vertebrate. Conclusions: The apparent decrease in rickettsial infection rates in tick tissues over the feeding period, as well as in progeny from treatment groups, may have implications on the role of the tick and vertebrate in serving as a rickettsial reservoir. Studies using naturally infected A. maculatum report high transovarial and transstadial transmission efficiencies for R. parkeri alone, suggesting all A. maculatum may eventually be expected to carry R. parkeri. However, the wide range of infection rates in natural tick populations suggest these transmission routes may be influenced by other factors. If analyses from the two addition trials demonstrate results consistent with the first trial, our data suggest inefficient vertical transmission in the tested system and that the tick vector may not always be a primary reservoir of these rickettsiae.

[10:40 a.m.] Vector Transmission Model of Monocytotropic Ehrlichiosis

Tais Berelli Saito University of Texas Medical Branch at Galveston, Texas

Background: Ehrlichioses are emerging infectious diseases affecting veterinary hosts and humans. Studies reveal potential pathogenic mechanisms of infection; however, little is known about the role of the vector on disease establishment, as demonstrated by a successful needle challenge vaccine that failed in field challenge by infected ticks. Methods: We have developed a tick transmission model for ehrlichial infection to study the vector-pathogen-host interaction. We infected mice by the intravenous (IV) and intradermal (ID) routes and compared the subsequent infection with tick transmission of the newly described E. muris-like agent. We collected samples during the course of infection to evaluate bacterial levels in tissues and antibody production to characterize the differences induced by the route of infection. Results: Our results showed bacterial levels and distribution patterns that were similar between mice experimentally infected by the ID and IV routes. Higher IgM antibody levels were observed in the early stage of infection in mice infected by tick transmission compared to the ID and IV routes. Moreover, IgG antibody levels in tick-transmitted ehrlichiosis were higher than in ID inoculated mice and much higher than in IV infection, which showed only low levels during both the early and later phases of infection. Conclusion: These initial results suggest important differences in host processing and presentation of ehrlichial antigen associated with the route and mode of infection, which will be further explored in vaccine development research.

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[11:05 a.m.] Studying temperature-dependent gene regulation in the relapsing fever spirochete, Borrelia turicatae Jon Blevins1, Jacob Latham1, Hannah Wilder2, Job Lopez2

1The University of Arkansas for Medical Sciences, Little Rock, Arkansas and 2Baylor College of Medicine, Texas Children’s Hospital, Houston, Texas

Background: Relapsing fever, caused by vector-borne spirochetes of the Borrelia species, are transmitted to humans via the bite of soft ticks or human body lice. During their enzootic cycle, vector-borne spirochetes exist in the two distinct niches found within the arthropod vector and vertebrate. It is well established that Lyme disease spirochetes undergo significant changes in global gene expression to allow them to adapt to these diverse environments, but the correlate that occurs in tick-borne relapsing fever spirochetes, such as Borrelia turicatae, remains undefined. Methods: We used large-scale proteomic analyses to define global regulatory changes that occur in B. turicatae in response to temperature alteration. To begin studying the molecular mechanisms contributing to temperature-dependent gene regulation, one temperature-responsive gene was selected for further characterization. Trans expression experiments and a luciferase-based transcriptional reporter were used to identify cis regulatory sequences contributing to temperature-dependent regulation in B. turicatae. Results: 223 proteins were identified as differentially produced between bacteria grown at 37°C and 23°C; the plasmid-encoded bta121 was one of these temperature-regulated genes. Trans expression studies showed that BTA121 was readily produced from a construct carrying bta121 expressed from its adjacent 95-bp upstream region, but BTA121 production was no longer responsive to temperature. Luciferase reporter experiments using fusions with the same 95-bp upstream region confirmed these findings. Conclusion: B. turicatae undergoes global changes in protein production in response to temperature, and expression studies suggest a region of DNA upstream of the bta121 promoter contains an operator site that represses transcription of this gene at 37°C. Experiments are underway with the luciferase reporter to precisely define the sequences involved in regulation of bta121. Once the site is identified, trans-acting factors contributing to bta121 regulation will be identified. We will also survey the B. turicatae genome to identify other temperature-responsive genes with the putative regulatory sequence.

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Invited Speaker Sessions Applied and Environmental Microbiology, Invited Speakers September 18, 9:00-11:30 a.m., Room 102 Chair: Dr. Fiona Crocker, Engineer Research and Development Center [9:00 a.m.] Field-Scale Demonstration of Aerobic Bioaugmentation of Explosive-Contaminated Groundwater

Fiona H. Crocker, Carina M. Jung, Mandy Michalsen, Dawn E. Hancock, and Karl J. Indest U.S. Army Corps of Engineers, Engineer Research and Development Center, Vicksburg, MS

Background: Groundwater at the Umatilla Chemical Depot (UMCD) in Hermiston, OR is contaminated with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) between 2 and 300 µg/L. A mixed culture that was composed of three RDX-degrading bacterial strains remained viable and able to degrade RDX for at least six months following inoculation into UMCD sediment columns. The objective was to demonstrate and evaluate groundwater bioaugmentation with Gordonia sp. KTR9 for the treatment of RDX-contaminated groundwater. Methods: Gordonia sp. KTR9 was injected into three test wells under natural gradient conditions along with periodic low concentration fructose amendments in order to stimulate the growth and activity of KTR9. A series of replicate push-pull tests were conducted to quantify the rate and extent of RDX transformation in these wells. Also, groundwater samples were periodically monitored for viable KTR9 cell numbers, and quantitative PCR analysis of an RDX biomarker gene. Results: Bioaugmentation increased RDX degradation rates compared to wells that were only stimulated with low carbon amendments. However, viable cells and xplA copy numbers, and rates of RDX degradation declined over time possibly due to the frequency of well testing. Conclusion: We have demonstrated that bioaugmentation with aerobic RDX-degrading bacteria has the potential to be a feasible option for groundwater remediation of dilute RDX plumes. [9:30 a.m.] Chemosynthetic microbial ecosystems at the bed of polar ice sheets

Brent Christner 1, Amanda Achberger 1, Erik Broemsen 1, Karen Cameron 2, Markus Dieser 1, Birgit Hagedorn 3, Karen Junge 2, Gary King 1, Alexander Michaud 4, Jill Mikucki 5, Andrew Mitchell 6, Mark Skidmore 7, Ron Sletten 8, John Priscu 4, & Trista Vick-Majors 4 1 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 2 University of Washington, Polar Science Center, Applied Physics Laboratory, 1013 NE 40th Street, Seattle, WA, 3 University of Alaska Anchorage, Environment and Natural Resources Institute, Applied Science Engineering and Technology Laboratory, 3211 Providence Drive, Anchorage, AK, 4 Department of Land Resources and Environmental Science, Montana State University, Bozeman, MT, 5 Department of Microbiology, University of Tennessee, Knoxville, TN, 6 Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth, UK 7 Department of Earth Science, Montana State University, Bozeman, MT, & 8 University of Washington, Department of Earth and Space Sciences, 4000 15th Avenue NE, Seattle, WA

Background: Active microbial ecosystems exist at the bed of polar ice sheets, function in the dark at subzero temperatures, and their biogeochemical activities could be of a magnitude sufficient to affect global elemental cycles. Methods: Subglacial Lake Whillans (SLW) lies beneath ~800 m of ice on the lower portion of the Whillans Ice Stream in West Antarctica and is part of an extensive and evolving subglacial drainage network. During January 2013, a hot water drilling system was used to create a ~0.6 m diameter borehole through the overlying ice sheet into SLW, allowing for physical measurements and the direct collection of water column and sediment samples. To investigate the ecology and biogeochemistry of a subglacial ecosystem at the western margin of the Greenland Ice Sheet, we analyzed subglacial water discharged during the summer of 2012 and 2013 from the Russell Glacier. Results: The ~2.2 m water column of SLW contained metabolically active microorganisms and was derived primarily from glacial ice melt with solute sources from lithogenic weathering and a minor seawater component. Rate experiments revealed that chemoautotrophic primary production in SLW was adequate to support heterotrophic metabolism in the subglacial ecosystem. The most abundant phylotypes in the SLW water column were most closely related to chemolithoautotrophic species that use reduced nitrogen, iron, or sulfur compounds as energy sources Molecular data from the Russell Glacier implied that the most abundant and active component of the subglacial microbial community at these locations were bacteria within the order Methylococcales. Dissolved methane in the subglacial waters ranged between 2.7 to 83 µM, the concentration was inversely correlated with dissolved oxygen while positively correlated with electrical conductivity, and rates of methane oxidation were estimated at 0.32 µM d-1. Conclusion: These new data indicate that environments beneath the world’s major ice sheets support viable ecosystems and corroborate previous reports suggesting that subglacial microbial communities can mobilize elements from the lithosphere, influence marine geochemical and biological systems, and significantly affect climate during deglaciation.

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[10:30 a.m.] The use of fungal cultures for biotransformation of drugs John B. Sutherland National Center for Toxicological Research, U. S. Food and Drug Administration, Jefferson, AR 72079

Fungi reduce the activity of many drugs, including the antibacterial fluoroquinolones. For instance, N-oxidation or N-acetylation by fungi inactivates ciprofloxacin by making it negatively charged and less able to enter a bacterial cell. In contrast, other fungal processes biotransform terpenoids, steroids, and flavonoids to serve as the precursors of new drugs and other high-value products. Unlike chemical transformations, many microbial biotransformations are regiospecific and stereospecific, producing unique metabolites as possible new drugs. Most microbial biotransformations of antibiotics reduce the antimicrobial activity, but compounds formed by microbial transformation followed by chemical synthesis may become new drugs with antimicrobial, antiparasitic, antiviral, and antitumor activity. The microbial transformation processes that are of greatest interest produce regiospecifically and stereospecifically hydroxylated derivatives of drug precursors in high yields. Novel steroids isolated from marine invertebrate sources and novel terpenoids, alkaloids, and flavonoids from little-known plants may be used as the starting materials for biotransformations for the development of new pharmaceutical drugs. [11:00 a.m.] Priming in the microbial landscape: metabolic stimulation of litter-associated microbial heterotrophs by periphytic

algae K. A. Kuehn1, S. N. Francoeur2, R.H. Findlay3 1 The University of Southern Mississippi, Hattiesburg, Mississippi, 2Eastern Michigan University, Ypsilanti, Michigan, 3 The University of Alabama, Tuscaloosa, Alabama

Background: In aquatic ecosystems, decaying plant detritus harbors a diverse community of autotrophic and heterotrophic microorganisms. We observed rapid metabolic responses of heterotrophic microbes to algal photosynthesis in detrital-periphyton communities, thus establishing the potential for algal priming of microbially-mediated litter decomposition. Methods: We grew natural periphyton communities (containing algae, bacteria, and fungi) on submerged Typha litter, and then manipulated photosynthesis by controlling light (0 or 400 µmol m-2 s-1 PAR) while simultaneously quantifying rates of algal (14C-bicarbonate), bacterial (3H-leucine) and fungal (14C-acetate) productivity. Results: Short-term incubation of natural detrital-periphyton under light in the laboratory (400 µmol m-2 s-1 PAR) significantly increased (p<0.01) the productivity of bacteria (3H-leucine) and fungi (14C-acetate), presumably as a result of algal photosynthesis. Experimental incubations of natural detrital-periphyton complexes with 14C- and 13C-bicarbonate confirmed that inorganic C was rapidly transferred and incorporated into fungal ergosterol and a wide range of heterotrophic microbial phospholipid fatty acids in the presence of light (PAR), respectively, but little to no incorporation was observed when incubated in the presence of light and the photosynthesis inhibitor DCMU. Conclusion: These results indicate that metabolic stimulation of, and carbon transfer to, detrital microbial heterotrophs is facilitated by active periphytic photosynthesis. Such microbial interactions may have important implications for our understanding of heterotrophic microbial decay processes and carbon flow pathways within aquatic ecosystems.

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Pathogenic Microbiology, Invited Speakers September 18, 1:00-3:30 p.m., Room 103C Chair: Dr. Gyan Sahukhal, The University of Southern Mississippi

[1:00 p.m.] Role of msaABCR operon in Biofilm Development in Staphylococcus aureus

Gyan S. Sahukhal and Mohamed O. Elasri Department of Biological Sciences, The University of Southern Mississippi

Background: Staphylococcus aureus is an important human pathogen that causes a wide range of acute to chronic nosocomial and 3community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. S. aureus also possesses a wide variety of virulence factors, the expression of which is carefully coordinated by a variety of regulators. Several virulence regulators have been well characterized, but others have yet to be thoroughly investigated. We have identified and characterized a global regulator that plays an important role in S. aureus biofilm development, virulence, and antibiotic resistance. Methods: We have used several approaches to characterize and evaluate the role of msaABCR operon in the USA300 LAC strain. We used SMARTerTM RACE cDNA amplification assay to find the transcription start and end of the msa transcript. We used northern blot and RT-qPCR to verify the results from RACE analysis. We also deleted the msaABCR operon in strain CA-MRSA USA300 to determine its role in virulence, antibiotic resistance, cell death and biofilm development. Finally, we examined the structure and composition of biofilm in the deletion mutant using confocal microscopy. Results: msaABCR is a four-gene operon, in which msaC, msaA and msaR are non-protein-coding RNAs that are essential for the function of the operon. MsaB is the only protein that is translated from msaB ORF. Deletion of the msaABCR operon resulted in a defective biofilm with increased localized cell death. We also found that increased protease activity in the mutant lead to increased processing of major autolysin, Atl, resulting in increased cell death and a defective biofilm. Furthermore, deletion of the msaABCR operon also affects several virulence factors and antibiotic resistance. Conclusion: These results show that msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm. This operon is also important in the regulation of several processes related to pathogenesis in S. aureus.

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[1:30 p.m.] Puma: A Critical Component of Innate Immunity Against Extracellular Pathogens Justin Thornton, Katie E. Heath, Katie Hill, Keun Seo, Tim Morgan, Jim Cooley. Mississippi State University. Mississippi State, MS.

Background: Cells of the innate immune system are the first line of defense against bacterial pathogens. Essential to the resolution of infection and inflammation is the effective recruitment and organized death of these important cells. Organized cell death (apoptosis) is triggered by a variety of stressors following infection and serves to clear immune cells from infected tissues after they have performed their function and to subsequently down-regulate inflammation. We previously identified that the pro-apoptotic Bcl-2 family member Puma is essential for surviving Streptococcus pneumoniae infection in a mouse pneumonia model. We hypothesized that reactive oxygen species (ROS) produced following phagocytosis induces DNA damage and subsequent Puma expression and that this process is essential for apoptosis and down-regulation of inflammation. We also sought to determine if Puma’s contribution to bacterial clearance extended beyond pneumococcal infection by utilizing a Staphylococcus aureus skin infection model. Methods: Phagocytosis assays were performed by exposing HL-60 human granulocytic cells to opsonized S. pneumoniae in vitro at a 1:1 bacteria to cell ratio for 1 h followed by plating aliquots on blood agar. DNA damage was quantitated by exposing cells to bacteria in the presence or absence of inhibitors and performing comet assay. Comet tail lengths were measured using ImageJ software (NIH). Production of ROS was quantitated by BioTek fluorescent plate reader following exposure of HL-60 cells to bacteria in the presence of 123 Dihydrorhodamine. Expression of puma was assessed by qRT-PCR on cDNA synthesized from RNA isolated from HL-60 cells exposed to pneumococcus for 1h and 3h. Apoptosis of HL-60 cells and murine neutrophils was assessed following bacterial exposure by AnnexinV/PI staining and flow cytometry. To determine the effect of Puma on S. aureus clearance, 10-12 week old mice were challenged intradermally with LAC strain for up to 7 days. Imaging of dermonecrosis, plate counts from tissues, and quantitation of immune cells was performed at 4d and 7d post-infection. Results: ROS production by HL-60 cells in the pneumococcal challenge model was found to independent of phagocytosis. However, DNA damage was increased when phagocytosis occurred. Interestingly, Puma induction was significantly greater in the absence of phagocytosis (7-fold vs 3-fold). Annexin V staining indicated increased apoptosis in the absence of phagocytosis in both HL-60 cells and bone marrow neutrophils. Puma-/- mice challenged with S. aureus demonstrated more severe dermonecrosis at both 4d and 7d post-infection and had greater numbers of S. aureus remaining in the tissue. Dramatic differences in the populations of immune cells were seen between Puma+/+ and Puma-/- mice following challenge. Conclusion: While Puma appears to be important for cell survival following bacterial challenge, changes in its expression does not appear to be dependent on phagocytosis-induced ROS production or DNA damage. While DNA damage may contribute to Puma’s role in innate immunity, other factors are likely involved including possibly ER stress and inflammatory signaling. Challenge of mice with S. aureus demonstrated differential recruitment or clearance of macrophages and neutrophils in Puma-/- mice. Together, these results indicate that during the innate immune response to bacterial infection, Puma regulates cell fates following cellular stress thereby preventing prolonged or ineffective resolution of inflammation.

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[2:30 p.m.] Analyses of Gene Expression Profiles in Mycobacterium marinum in an Activated Virulent State Lisa Shirreff1, Amrita Mallick1, Martin Cheramie1, Caroline Sciarrillo1, B. M. Fredrik Pettersson2, Leif Kirsenbom2 and Don G. Ennis1* 1Department of Biology, University of Louisiana, Lafayette, LA 70504, USA 2Department of Cell and Molecular Biology, Uppsala University, Uppsala, SE-751 24, Sweden.

Mycobacterium marinum (Mm) is an intracellular bacterial pathogen with greater than 85% nucleotide identity in more than 3,000 orthologous genes of Mycobacterium tuberculosis (Mtb). Mm is the leading cause of fish mycobacteriosis (or “fish TB”), which burdens wild fish stocks including hundreds of fish species in both fresh and saltwater systems. Like human Mtb infections, Mm survives and multiplies within host macrophages, produces granulomas, the hallmark lesion of human Mtb and mounts life-long chronic infections in many fish. Because of the similar disease presentation in fish and the close genetic relatedness between Mm and the Mtb complex, the faster-growing Mm has been used as a surrogate model to study disease progression for human TB. Mm infections of fish have been well characterized, yielding granulomatous lesions primarily carried within fish macrophages in several target organs, including spleen, liver and kidney. We have shown the small freshwater laboratory fish Japanese Medaka (Oryzias latipes), is a natural host of Mm, which we have employed as an experimental model host to study fish TB. Despite the high incidence of infection in wild fish stocks and outbreaks in laboratory and aquaculture fish colonies, costing billions annually, transmission of Mm was poorly understood. We have worked to elucidate routes of mycobacterial transmission amongst fish in nature and fish colonies, we have documented that a major route of Mm infection is by ingestion of infected food items. Efficient oral infections was shown to occur by ingestion of infected fish tissues or by ingestion of small prey carrying Mm, such as insect larvae or by paramecia. Mosquito larvae (and paramecia) routinely ingest aquatic bacteria, especially mycobacteria that are planktonic or in biofilms. We are characterizing the infectious transmission of Mm in fish by yellow fever mosquito larvae (Aedis aegypti) as a delivery vessel. We have shown that Mm could reside in the larval gut for days with little if any Mm killing. Interestingly, we discovered that passage of laboratory-cultured Mm through the larval gut increases virulence of this bacterial pathogen by some 100- to 1,000-fold. A similar activation of virulence was also observed for Mm when present in infected fish tissues and then fed to other fish.

The passage of Mm through either the caustic larval digestive tract or the stress within macrophages of infected tissues appear to induce an “activated infectious metabolic state” for virulence. We have previously speculated that either of these pretreatments likely induce suites of Mm genes that include virulence factors. To investigate this notion we are conducting global transcriptomic analyses of Mm obtained from both fish tissues and from larval gut and then compared to the expression profiles of bacteria grown in culture media. We have focused on sigma factors since they are known to have global control on gene expression and are implicated in the regulation of virulence genes during mycobacterial infections. Comparative genomic analyses have revealed that Mtb and Mm share twelve highly conserved sigma factors; however, Mm carries an additional five sigma factors, presumably important in the growth and survival outside a host in aquatic environments. We have focused on the transcript levels for all 17 Mm sigma genes during the infection of fish target organs or carried within the mosquito larval GI tract and then compared these profiles to the transcript levels measured in bacteria under different growth states in laboratory culture media and under different stressful conditions. These studies revealed that the Sigma transcript profiles for Mm during the infection of fish tissues was nearly identical as found in Mm residing in the larval GI tract, both were characterized by high SigB mRNA levels. Interestingly, the sigma expression profiles identified in fish tissues and larval gut were very similar to Mm that had been to heat-shock or hypoxia treatment. We propose that the increased virulence documented following passage in the larval gut is indeed due to induction of key virulence genes, specifically genes that are part of the large SigB stress-response regulon. We hypothesize that up-regulation of the SigB regulon when carried in larvae, establishes a metabolic state for Mm that helps to prepare the pathogen to mount fish infections by an oral route.

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[3:00 p.m.] Ecology and Transmission Dynamics of Mycobacterium ulcerans M. Sanders1,7, J.L. Pechal2, P.L.C. Small3, E. Anagonou4, Y. Barogui5, G. Sopoh5, C. Johnson4,6, M.E. Benbow2, J.K. Tomberlin7, and H.R. Jordan1, 1 Mississippi State University, Starkville, MS; 2 Michigan State University, East Lansing, M; 3 University of Tennessee, Knoxville, TN; 4 University of Abomey Calavi, Abomey Calavi, Benin; 5National Buruli Ulcer Control Control Program, Ministry of Health, 6Cotonou, Benin; Foundation Raoul Follereau 7 Texas A&M University, College Station, TX;

Background: Mechanisms of Mycobacterium ulcerans transmission to humans remain undetermined and an important topic of investigation. Recent data from animal studies has shown that direct inoculation of M. ulcerans is necessary to establish infection and Buruli ulcer disease pathology. Several hypotheses for transmission of M. ulcerans have been proposed including insect vector, puncture from an M. ulcerans contaminated thorn, or puncture into M. ulcerans contaminated skin. Methods: We conducted a pilot study investigating whether M. ulcerans could be detected on human skin following exposure to M. ulcerans contaminated environments. Skin swabs were collected from investigators exposed to aquatic habitats historically M. ulcerans positive. Swabs were taken from hands and feet, and specific sampling site locations and investigator activities were recorded. Swabs were collected from up to 5 investigators on 4 different days and were preserved in ethanol for DNA analyses. Results: Quantitative PCR results showed selective positivity with positive DNA samples from three of the five investigators with a mean concentration of 5.67 x 106 genome units/sample. Further analysis showed that 4/5 positive swab samples were from investigators working in identical sampling locations, and VNTR profiling indicated these positive samples matched the M. ulcerans VNTR profile D. Additionally, a single positive skin swab sample was collected after working in a different sampling location and matched M. ulcerans profile C. Statistical analysis demonstrate a significant association of both location and investigator with positivity. Conclusion: These results raise questions regarding M. ulcerans transmission, and whether specific physical human characteristics or skin microbiome mediates M. ulcerans presence.

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Immunology and Virology, Invited Speakers September 19, 9:00-11:30 a.m., Room 103A Chair: Dr. Fengwei Bai, Assistant Professor, The University of Southern Mississippi [9:00 a.m.] IL-23-IL-17, a bridge links innate and adaptive immunity against West Nile virus infection

Fengwei Bai Ph.D Department of Biological Sciences The University of Southern Mississippi

We previously reported that an innate receptor, Toll-like receptor-7 (TLR7) mediates IL-23-dependent protective responses against WNV infection in mice. IL-23 is known as a prime regulator for stabilization and maintenance of CD4+ T helper-17 (Th17) cells, which are the major cell type secreting IL-17A. However, the role of IL-17A in the pathogenesis of West Nile virus (WNV) is not clear. Here we show that WNV induces IL-17A production in both mouse and human immune cells in vitro, and in plasma of WNV-infected mice. Despite unaltered inflammatory cytokine expression and antibody production, IL-17A deficient mice (Il17a-/-) are more susceptible to WNV infection and generate a higher viral burden in blood, liver, and brain after lethal WNV infection, when compared to wild-type (WT) mice. We detected a modest elevation of brain infiltrating leukocytes (including CD8+ T cells) in WNV-infected Il17a-/- mice, which is inconsistent with the previously reported function of IL-17A in recruiting leukocytes. Interestingly, splenic CD8+ T cells purified from Il17a-/- mice are less cytotoxic and express lower levels of cytotoxic mediator genes, suggesting a deficient cytotoxic function of CD8+ T cells in Il17a-/- mice. Importantly, exogenous supply of recombinant IL-17A restores expression of cytotoxic mediator genes in splenic CD8+ T cells isolated from WNV-infected Il17a-/- mice. In conclusion, this report identifies a novel function of IL-17A in facilitating WNV clearance by inducing expression of cytotoxic mediators to promote CD8+ T cell cytotoxicity. Thus, IL-23-IL-17 axis serves as a bridge links innate and adaptive immunity against West Nile virus infection. [9:30 a.m.] The battle between rotavirus and its host for control of innate cellular defenses

Michelle M. Arnold Department of Microbiology and Immunology LSU Heath Sciences Center – Shreveport

Cells have multiple ways by which they protect themselves from pathogens, and the production of interferon (IFN) is a critical early host defense mechanism against viral infections. Rotavirus causes severe diarrhea in infants and children, and globally is a leading cause of death due to diarrheal disease. The rotavirus nonstructural protein NSP1 suppresses IFN production by inducing the degradation of host proteins that are essential for the expression of IFN. NSP1 proteins can be broadly grouped by their principle target of degradation, either IFN regulatory factors (IRF3, IRF5, IRF7) or the β-transducin repeat containing protein (β-TrCP), which is a cellular protein required for NF-κB activation. In the laboratory, the SA11-4F strain of rotavirus has been a prototype for IRF degradation and the OSU strain has been a prototype for β-TrCP degradation. Although the targets vary among different NSP1 proteins, the mechanism by which degradation occurs is thought to be the same. Numerous NSP1 proteins from different virus strains have been characterized for their ability to induce degradation of one or more host proteins, but the impact of these NSP1s on IFN induction have typically not been examined. To gain a wider perspective on the effect of NSP1 activity on IFN promoter elements, we generated 293T cell lines stably expressing an IFN-stimulated response element (ISRE) or an NF-κB response element (NF-κB -RE) that drives transcription of a luciferase reporter gene. Each cell line was then transfected with plasmids expressing components of the IFN induction pathway. Luciferase activity in the NF-κB -RE cell line was strongly stimulated by the TLR agonist MyD88, and weakly stimulated by the constitutively active RIG-I 2 CARD domain. Luciferase activity in the ISRE cell line was strongly stimulated by the RIG-I 2 CARD domain and constitutively active IRF3. Cells were then co-transfected with plasmids expressing the stimulus of interest and various NSP1s. The prototype rotavirus NSP1 proteins functioned as expected: OSU blocked expression of the NF-κB -RE only, and SA11-4F blocked expression of the ISRE only. However, among the other NSP1s examined, most were able to prevent reporter expression from both the ISRE and NF-κB -RE. By analyzing the effects of NSP1 on both response elements in parallel, it appears that there are broader effects of NSP1 on inhibition of IFN production that require further exploration.

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[10:30 a.m.] A clinical perspective on the immunobiology of ovarian cancer Martin J Cannon, Department of Microbiology & Immunology, University of Arkansas for Medical Sciences, Little Rock, AR

The immune system plays an active and possibly critical role in the pathogenesis of ovarian cancer, disease progression, and overall survival. Of the positive parameters, CD3 T cell infiltration has been associated with prolonged survival in ovarian cancer, but the majority of studies point to mechanisms of immune suppression that correlate with poor patient outcomes. Regulatory T cell (Treg) infiltration has been widely noted as a negative correlate of clinical outcomes for many malignancies, and ovarian cancer is no exception. Expression of immune checkpoint molecules such as PD-L1 has also been associated with higher mortality in ovarian cancer, and has led to clinical interest in checkpoint inhibitors as novel therapeutics. Expression of indoleamine 2,3-dioxygenase (IDO) is a key inducer of Treg responses, and is also associated with increased mortality in ovarian cancer. In sharp contrast, pro-inflammatory Th17 T cell infiltration correlates with markedly improved overall survival, thus leading to the question of whether ovarian tumor antigen-specific Th17 responses could be induced for therapeutic benefit. Novel strategies for Th17-inducing dendritic cell vaccination have been effective in treatment of ovarian cancer in mouse models, and are currently being tested in a phase I clinical trial for patients with stage IIIC/IV ovarian cancer. [11:00 a.m.] CD8 T cells, functional diversity, and pathogen control

Shannon M. Kahan Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL

CD8 T cells are integral components of the overall immune response and function to ensure the clearance of acute infections, limit the reactivation of latent infections, control pathogen loads at steady state levels during chronic infections, and mediate tumor immunosurveillance. The activation of naïve CD8 T cells triggers a burst of cytokine production, which drives their proliferation and differentiation. Acute infections typically result in the formation of short-lived, but highly functional, terminally differentiated effector populations that clear the pathogen, as well as a set of phenotypically distinct memory precursors that are retained over time and mature to form the long-lived memory T cell pool. Notably, naïve, effector, and memory CD8 T cells display distinct patterns of IFN-γ and IL-2 production, but it is unclear why these functional disparities arise, how they regulate and forecast the developmental fate and protective efficacy of the ensuing response, and whether these patterns can be modified to improve host defense. To define how the intrinsic functional features of CD8 T cells regulate and predict their developmental choices and dictate short-term and long-term infection control, we harnessed innovative IL-2 and IFN-γ cytokine reporter systems. We discovered that recently activated naïve CD8 T cells that do not produce IL-2 preferentially develop effector traits, and conversely, subsets that do produce IL-2 predominately attain memory attributes. Furthermore, IL-2 producing effector cells transition into memory populations that proliferate vigorously upon secondary infection. Taken together these studies demonstrate that autocrine IL-2 production by CD8 T cells influences their fate decisions and ability to control infection.

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Oral Presentation Sessions Immunology & Virology, Oral Presentations Friday, September 18 @ 9:00 -11:30 a.m., Room 103A [9:00 a.m.] Efficiency of complement protein in saltwater hardhead catfish Ariopsis felis and freshwater yellow bullhead catfish

Ameiurus melas Abby L. Adams, Gary LaFleur, Rajkumar Nathaniel Nicholls State University, Thibodaux, Louisiana

Background: Complement is a family of innate immune system serum enzymes which lyse microbes through multiple pathways. Fish have well developed innate immune systems with less developed adaptive immune systems and endure high microbial stress. We selected hardhead catfish Ariopsis felis and yellow bullhead catfish Ameiurus melas for evaluation of serum complement. Methods: Antibacterial activity of catfish serum was tested by incubating serum with bacterial species for activation of putative complement proteins. Mixtures were plated on bacterial media, incubated overnight and colonies were enumerated. Hemolysis was tested by adding fish serum to rabbit red blood cells (RRBCs), and blocking hemolysis with ethylene diamine tetraacetic acid (EDTA). Results: Anti-bacterial activity of hardhead serum against E. coli and B. subtilis was found to be 97% and 29%, respectively, while that of yellow bullhead serum was found to be 89% and 38% respectively. Hemolysis of RRBCs by hardhead and yellow bullhead serum was found to be depleted by EDTA and was restored by the addition of ions. Conclusion: These results provide evidence of antibacterial activity present in hardhead catfish serum and yellow bullhead catfish serum. The hemolytic activity observed in both species indicates complement-like activity.

[9:20 a.m.] Monoclonal Antibodies CC34 and CC41 Bind Catfish Leukocyte Immune-Type Receptors

Laura E. Blackmon, Erin Taylor, Eva Bengten, Melanie Wilson The University of Mississippi Medical Center, Jackson, Mississippi

Background: Channel catfish, Ictalurus punctatus, is a model for studying teleost immune function. Two monoclonal antibodies (mAbs), CC34 and CC41, were developed by immunizing mice with a catfish natural killer (NK) cell line and cytotoxic T lymphocyte (CTL) cell line, respectively. Recently it was determined that both mAbs bind to a subset of catfish leukocyte immune-type receptors (LITRs) present on catfish cytotoxic cells. LITRs comprise a large, complex family of activating and inhibitory immune receptors of different size and domain composition. The membrane distal LITR domains, D1 and D2 are phylogenetically related to Fc-receptors, while the membrane proximal domains are more related to NK receptors. Previously, mAb CC41 was shown to reduce the cytotoxicity of effector cells in a catfish mixed leukocyte culture (MLC). Due to the structural complexity of the LITR family it is important to determine the binding sites of the CC34 and CC41 mAbs on LITRs. Methods: Different sections of the full length IpLITR1.1a cDNA were amplified by RT-PCR as regions encoding domains, D1-D3, D3-D5 and D5-D7. They were cloned into the mammalian expression vector, pSecTag2B, which incorporates an N-terminal 3X-FLAG-tag. The constructs were verified by sequencing and protein expression in 293T cells was confirmed by western blot. Expressed proteins were assayed for binding using immunoselection on CC34 or CC41-coupled magnetic beads. Results: CC41 binds to the recombinantly expressed IpLITR1.1a D1-D3 protein. Currently, expression constructs of individual domains are being generated to further define the CC41 binding site. Conclusions: Once the specificities of the CC34 and CC41 mAbs are determined, it will be possible to define the subset of these LITRs expressed on cytotoxic cells. This subsequently will allow for functional characterization of these receptors including their ligand specificities.

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[9:40 a.m.] Mechanism of HCMV-induced Monocyte-to-Macrophage Differentiation Stephen Cieply, Donna Collins, Emily Stevenson, Urska Cvek, Marjan Trutschl, Andrew Yurochko Louisiana State University Health Sciences Center in Shreveport

Background: Monocytes are highly plastic immune cells that exist in two distinct states: as short-lived cells circulating in the peripheral blood (monocytes) and as long-lived differentiated cells in tissue (macrophages). Human cytomegalovirus (HCMV) is believed to use monocytes as vehicles for viral spread and persistence. Highly mobile monocytes are infected by HCMV, an interaction which triggers extravasation into surrounding tissue and differentiation into long-lived macrophages that are productive for viral replication. Receptor-ligand interaction from the initial viral binding event induces signaling cascades that promote enhanced survival, macrophage differentiation, and polarization towards a proinflammatory state that appears to promote the viral persistence strategy. While these events have been established to occur during the first few days of infection, de novo viral gene expression is delayed nearly two weeks in monocytes following infection indicating that viral proteins do not play a role in mediating the pro-viral effects. Here we present a potential alternative mechanism for HCMV-induced monocyte-to-macrophage differentiation. Methods: Data were compiled from both laboratory-generated microarrays as well as data available in the published literature to identify the changes which occur in monocyte-to-macrophage differentiation induced by HCMV compared to alternate differentiation stimuli. From this set of data, a set of genes appeared to be strongly and uniquely regulated by a distinct set of transcription factors, which we then confirmed using biochemical techniques. Results: Through bioinformatics analysis of both generated data as well as comparison data based on I identified a set of genes which has been highly conserved throughout numerous tested points of infection and which are uniquely regulated by HCMV (their regulation is largely absent at such levels in other stimuli). This cluster of genes was identified as regulated primarily through the promotor region known as the Interferon Sensitive Response Element (ISREs), which our bioinformatics data reveals to be a combination of two transcription factors, STAT-1 and IRF-7. Current work has expanded upon these analyses to include biological studies aimed at uncovering the specific role of these two transcription factors in mediating differentiation (and potentially viral permissiveness) in HCMV-infected monocytes and macrophages. Conclusion: This study shows a specific, conserved set of genes are highly upregulated throughout the course of HCMV infection, primarily under the control of a common promotor region which is likely responsible for promoting monocyte-to-macrophage differentiation and, potentially, viral permissiveness. [10:30 a.m.] I Will Survive: HCMV Utilizes a Distinct Pro-Survival Mechanism in Infected Carrier Monocytes

Donna Collins-McMillen, Emily V. Stevenson, Joshua Caskey, Stephen J. Cieply, Maciej T. Nogalski, Gary C.T. Chan, Andrew D. Yurochko LSU Health - Shreveport, Shreveport, Louisiana

Background: The widespread dissemination of Human Cytomegalovirus (HCMV) to multiple host organ sites through manipulation of infected monocytes allows the virus to establish life-long persistence. However, HCMV infection of monocytes is not without significant biological obstacles that the virus must overcome; primarily that monocytes are naturally short lived cells with a biological lifespan of 48-72 h in circulation. Recent work from our laboratory suggests the existence of a critical 48 h monocyte viability checkpoint, consistent with the in vivo lifespan of these cells. We report that HCMV induces a distinct regulatory pattern of the cellular anti-apoptotic proteins, Mcl-1 and Bcl-2, to navigate the initial 48 h cell viability hurdle, and then to extend cell survival for weeks to months by combating the anti-viral pro-apoptotic response at later times post infection. Methods: We first analyzed expression levels of cellular survival proteins by Western blot and constructed a temporal profile detailing HCMV-induced expression of cellular Bcl-2 family proteins. Based on these data, we next used a combination of small molecule antagonists and small interfering RNAs to alter the expression or functional activity of these cellular proteins and evaluated the contribution of each protein to the long-term survival of HCMV-infected monocytes via MTT viability and TUNEL apoptosis assays. Results: We have previously shown that Mcl-1 controls early survival of infected monocytes; here we demonstrate that Bcl-2 becomes the primary anti-apoptotic player for long-term survival of these cells. Protein analyses demonstrate an up-regulation of Bcl-2 in HCMV-infected monocytes beginning at 48hpi. Treatment with a small molecule Bcl-2 antagonist, ABT-199, reduces the pro-survival effects of HCMV infection in target monocytes, consistent with a role for Bcl-2 in promoting the extended survival of these cells. With ABT-199 treatment, we do not observe a significant reduction in viability of infected cells until 48 hpi, further supporting our model that Mcl-1 controls infected monocyte survival prior to the 48 h viability gate and that Bcl-2 controls survival beyond 48 h. This regulation is in contrast to normal monocytes, which require both Mcl-1 and Bcl-2 for their survival prior to the 48 h viability checkpoint. Conclusion: Taken together, these data suggest that HCMV usurps cellular anti-apoptotic pathways and up-regulates Bcl-2 family proteins in a distinct multi-step process. This multiphasic survival strategy allows HCMV to negotiate multiple apoptotic checkpoints and is designed to favor infected cell survival throughout the course of infection. By reprogramming the biology of infected monocytes to favor long-term cellular survival, HCMV promotes viral carriage and persistent life-long infection.

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[10:50 a.m.] Outer-membrane vesicles derived from Burkholderia pseudomallei drive innate immune responses in vivo in populations of antigen-presenting cells Christopher JH Davitt, Hailey E. Petersen, Nicole Kikendall, Lisa A. Morici Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana

Background: Burkholderia pseudomallei (Bp) is a significant pathogen in Southeast Asia and Northern Australia and the causative agent of melioidosis. Due to the ease of its dissemination, the significant impact of the disease, and difficulty of treatment Bp has been classified as a Tier 1 select agent by the CDC. An effective vaccine is therefore a highly desirable alternative to current treatments. Our group has demonstrated that vaccination with purified outer membrane vesicles (OMVs) derived from Bp drive both humoral and cellular responses that were protective against challenge. However, despite the use of OMVs as vaccine antigens, little is known about the mechanism by which these achieve protection. Antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages (MΦs) act as the bridge between the innate and adaptive arms of the immune system and are a central component of vaccine efficacy. Considering OMVs contain a large repertoire of pathogen-associated molecular patterns (PAMPs), multiple antigens, and are nano-sized, we sought to determine the effects of OMVs on APC function and to elucidate the early innate events following OMV encounter in vivo. Methods: We investigated the mechanism of Bp OMV uptake, the effect on antigen-presentation machinery, enhancement of co-stimulatory molecules, and the production of cytokines by bone-marrow derived-DCs and MΦs in vitro using microscopy, flow cytometry, and cytokine-multiplex assay. We also investigated the ability of Bp OMVs to modulate APC function in vivo following intraperitoneal injection of mice. Finally we evaluated the ability of Bp OMVs to promote antigen-specific T cell responses following subcutaneous vaccination by flow cytometry. Results: We found Bp OMVs were taken up in vitro and in vivo by both DCs and MΦs. Mechanisms of uptake included both actin-mediated and cholesterol-dependent processes and led to the localization of antigens into both the lysosomal and cytoplasmic compartments. Following OMV uptake, we found enhanced expression of both major histocompatibility complex (MHC) class I and II molecules on the surface of DCs and MΦs. In addition, we saw potent up-regulation of the co-stimulatory molecules CD80 and CD86 on both APC populations both in vitro and in vivo. Moreover, Bp OMVs were shown to drive strong production of pro-inflammatory and T cell polarizing cytokines including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and IL-12p70 from DCs and MΦ in vitro and in vivo. Finally, following vaccination of mice, we demonstrated that Bp OMVs are capable of eliciting potent antigen-specific CD4+ and CD8+ T cell responses. Conclusion: We propose that Bp OMV uptake by APCs occurs via multiple mechanisms, resulting in the enhancement of both MHC class I and II expression, increased expression of co-stimulatory molecules on surface of APCs, and the production of cytokines, leading to the development of cellular immunity. These attributes reinforce the opportunity to utilize OMVs as a potent and versatile non-replicating vaccine platform for the development of vaccines against Bp and other gram-negative infections.

[11:10 a.m.] HPV16 utilizes vesicular transport to deliver the viral DNA to the nucleus during mitosis

Stephen DiGiuseppe, Wioleta Luszczek, Malgorzata Bienkowska-Haba, Timothy R. Keiffer, Lucile G. M. Guion, and Martin Sapp Department of Microbiology and Immunology, Center for Molecular Tumor Virology, Feist-Weiller Cancer Center, LSU Health Shreveport, Shreveport, Louisiana, USA.

Human papillomaviruses (HPVs) are non-enveloped DNA tumor viruses that infect skin and mucosal epithelial cells. Infection of HPV can induce hyperproliferative lesions of the infected tissue, which can progress to carcinomas. Infection of epithelial cells by HPV involves a complex series of events. Following primary attachment, the virion is internalized and uncoats within acidified endosomes. The major capsid protein, L1, is dissociated from the minor capsid protein, L2. The L2 protein remains in complex with the viral DNA and the complex is rescued from lysosomal degradation by the retromer complex and trafficked to the trans-Golgi network (TGN). The L2/DNA complex egresses from the TGN and enters the nucleus during mitosis, requiring nuclear envelope breakdown to establish infection. For HPV to successfully navigate these cellular pathways, the virus utilizes numerous host cell factors involved in retrograde transport. Currently, we have good understanding of the early trafficking events of an HPV infection; however, the detailed mechanism of how the viral genome navigates the late stages of nuclear entry is unknown. Herein, we provide evidence that the L2/DNA complex is released from the TGN during prophase and migrates by minus-end directed transport along cytoplasmic microtubules (MT) to the microtubule-organizing center. This seems to be followed by a switch in directionality to plus-end directed transport towards the kinetochores along spindle MTs in the later stages of mitosis, followed by association of the L2/DNA complex with condensed chromosomes. To our surprise, the viral genome is still residing in a vesicular compartment during the mitotic transport until after mitosis is completed and the nuclear envelope has reformed. At the same time, a large portion of the L2 molecule faces the cytosolic side of the vesicle. Taken together, these data suggest that, during an infection, the incoming HPV genome is protected within membranous vesicles until after the completion of mitosis. This is an exciting, novel strategy that HPV utilizes to protect and deliver the viral DNA to the nucleus during mitosis.

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Applied & Environmental Microbiology, Oral Presentations Friday, September 18 @ 1:00-3:30 p.m., Room 102 [1:00 p.m.] Presence of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes in Raw Source Water and Treated Drinking

Water in Southeast Louisiana Scott Bergeron and Raj Boopathy Nicholls State University, Thibodaux, Louisiana

Background: Antibiotic resistance is becoming a very large problem throughout the world. The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment is a major public health issue. Aquatic ecosystem is a significant source for ARB and ARGs. The drinking water treatment system is designed specifically to eliminate bacteria and pathogens in drinking water. The presence of ARB and ARGs in source water and drinking water may affect public health and it is an emerging issue in drinking water industry. Therefore, this study was conducted to study the presence of ARB and ARGs in a source water, treated drinking water (finished water), and in the distribution line (tap water) in a rural water treatment plant in Louisiana. Methods: Monthly samples were collected from Bayou Lafourche, treated drinking water, and the distributed lines for a year. Samples were analyzed for the presence of antibiotic resistant bacteria by Kirby-Baur assay. Presence of ARGs was monitored by molecular method using various antibiotic resistance gene primers. Results: The results showed the presence of several ARB in the source water including, Enterobacter cloacae, Klebisella pneumonia, Escherichia coli, Pseudomonas, Enterococcus, Staphylococcus and Bacillus spp. However, the water treatment plant effectively removed these bacteria in the treated water as none of these bacteria were found in the tap water as well as in the finished water at the water treatment plant. Bacterial DNA including 16s rRNA and ARGs of sulfonamides and tetracycline antibiotics were observed in raw water. The presence of 16s rRNA was found consistently in every month of sampling in raw water, finished water, and tap water. Conclusion: This study suggests that the filtration system at the treatment plant was ineffective in filtering out small fragments of bacterial DNA. Also, the possibility of the presence of biofilms in the water pipeline exists, which may develop antibiotic resistance due to the selective pressure of chlorination in drinking water. [1:20 p.m.] Does pathogenic Rickettsia parkeri shape the native microbiota within the tick host?

Khemraj Budachetri, Christine Beck, and Shahid Karim Department of Biological Sciences, The University of Southern Mississippi

Introduction: The Gulf coast tick (Amblyomma maculatum) is a competent vector of Rickettsia parkeri, a pathogen of public health significance responsible for milder fever and generalized rashes in the victim. Beside pathogenic R. parkeri, A. maculatum harbors a range of bacterial consortia of non-pathogenic and unknown pathogenicity. The interaction between pathogenic R. parkeri and non-pathogenic native microbiota within the tick host has barely been investigated. In this study, we hypothesized that R. parkeri shapes the native microbial colonization by displacing non-pathogenic microbes within the tick host. Methods: We maintained R. parkeri infected and uninfected A. maculatum tick colonies. R. parkeri was quantified in embryonic, immature and mature tick developmental stages using qPCR assay and similarly a 16S rRNA qPCR assay was used to determine the total microbial load within the tick host. Results: Our results revealed a significant increase in pathogenic R. parkeri in immature and mature developmental stages. Total bacterial load in infected ticks was significantly higher as compared to uninfected stages developmental stages. Conclusions: Our results showed the transovarial and trans-stadial transmission of R. parkeri from embryonic, immature, and mature developmental stages. R. parkeri infection in the tick is instrumental in shaping native microbiota in A. maculatum. The further study on profiling of bacteria by 16S rRNA based on culture independent next generation method will provide impact of R. parkeri in community profile of non-pathogenic bacteria in the ticks.

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[1:40 p.m.] Regulation of Glycine Rich Proteins in Rickettsia parkeri infected ticks Rebekah Bullard, Khemraj BC, Shahid Karim University of Southern Mississippi

Introduction: The vector for Rickettsia parkeri, Amblyomma maculatum, has a geographical distribution including the entire southern region and has begun evading into the Mid-Atlantic coast region and the Plain States. R. parkeri is a Spotted Fever Group Rickettsia characterized by eschars on the skin and fever. Glycine Rich proteins (GRPs) have been identified as anti-microbial peptides in multiple species however previous publications demonstrate the differential expression of GRPs in the presence of a pathogen. Here, the regulation of various GRPs is discussed in connection with R. pakeri infected ticks. Methods: R. parkeri infected and uninfected ticks were fed on a sheep and the soft tissues were dissected from 5-day partially fed females. RNA extracted from these partially fed salivary glands was used to determine the differential regulation of GRPs in R. parkeri infected and uninfected samples by RNA-Seq. To validate the sialotranscriptome data, qRT-PCR is utilized for a more direct measurement of the selected GRPs. Results: Comparison of the sialotranscriptomes from R. parkeri infected and uninfected samples, GRP expression ranges from +5000 fold upregulation to 75 fold downregulation. A subset of the upregulated GRPs were selected for further validation using qRT-PCR. Sequences were also examined for homology or sequence repeats which could elucidate possible mechanism of function. Conclusions: Infection of R. parkeri does affect the differential expression of GRPs in the tick A. maculatum. GRPs can maintain a variety of functions including anti-microbial and stress responses which could explain the results obtained here. It is also unclear and requires further investigation to determine R. parkeri’s role in altering GRP expression. [2:30 p.m.] Engineering Bacillus subtilis Shows Biofilm Promotes Cellulose Degradation

Yijie Daniel Deng and Shiao Y. Wang The University of Southern Mississippi, Hattiesburg, Mississippi

Background: Bacterial degradation of cellulose in aqueous environments is thought to be mainly dependent on secreted extracellular cellulases. However, this traditional view is greatly simplified and ignores some fundamental problems. For example, how do bacteria benefit from their own cellulases and prevent the loss of cellulases into the surrounding milieu? Because cellulose is insoluble, it is not degraded by planktonic bacteria but instead by adherent bacteria. We hypothesize that biofilm facilitates cellulose degradation by concentrating bacteria and cellulases in close proximity with the insoluble substrate. Methods: Bacillus subtilis strains were genetically engineered as a model organism to study our hypothesis. A recombinant secretory cellulase gene was transformed into wild type B. subtlis and its biofilm-deficient mutants. Biofilm-deficient mutants were constructed by knocking out biofilm synthesis genes from the wild type strain. Either single or double knockouts of the extracellular polysaccharide (EPS) synthesis genes (epsA-O operon) and/or the tasA gene encoding the major biofilm protein were created. All engineered B. subtilis strains were grown in biofilm medium with cellulose filter paper as the substrate. Cellulase production among all strains was also measured. Mass loss of the filter paper was used to measure the rate of cellulose degradation. Results: Wild type B. subtilis degraded filter paper faster than did all the biofilm-deficient mutants. There was little difference in cellulase production between wild type and mutants. This suggests that the presence of biofilm facilitated cellulose degradation. The mutant strain without EPS degraded less filter paper than the mutant strain without TasA. This suggests that extracellular polysaccharide plays a more significant role than extracellular protein with regards to the function of biofilm in cellulose degradation. Conclusion: This study shows that biofilm is important in cellulose degradation by bacteria in aqueous environments. Our results support the hypothesis that biofilm promotes cellulose degradation by concentrating bacteria and secreted enzymes in close proximity to the substrate. This study brings new insight to the importance of biofilm in cellulose degradation and potentiates the development of biofilm-based technology to enhance cellulose degradation for biofuel production.

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[2:50 p.m.] Analyzing the Influence Oxygen Deprivation has on the Capability of Listeria monocytogenes to Induce Listeriosis in Gerbils Jillian Harris1, Justin A. Thornton1, Si Hong Park2, Sally J. White1, Daniel McClung1, Jessica Wilson1, Alicia K. Olivier3, Steven C. Ricke2, and Janet R. Donaldson1 1Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762 2Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, AR 72704 3Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762

Background: Listeria monocytogenes is a facultative anaerobic, foodborne pathogen capable of surviving harsh environments, including sites of varied osmolarity, pH, and oxygen concentrations. Recent work from our laboratory has indicated that anaerobic conditions increase the resistance capability of certain strains of L. monocytogenes to stressors. To determine if oxygen availability correlated to a change in virulence in vivo, a gerbil listeriosis model was developed where aerobic and anaerobic cultures of the serovar 4b strain of F2365 were evaluated. The hypothesis of this study was that anaerobic cultivation will induce expression of virulence factors that otherwise would not be expressed under aerobic conditions, thereby increasing the virulence of L. monocytogenes in vivo. Methods: Twenty gerbils (aged 9 weeks) were inoculated with one of 4 challenge doses by oral gavage: control of phosphate buffered saline (PBS), 5x106 CFU aerobic culture of L. monocytogenes F2365 (L_Aer), 5x106 anaerobic culture of F2365 (L_An), or 5x108 aerobic culture of F2365 (H_Aer). Body weights and fecal samples were collected daily. On d 6-post challenge, intestinal contents were collected for enumeration of L. monocytogenes and microbial community in the cecum and colon was investigated. Results: Body weights declined for gerbils that received the H_Aer dose. Feces collected daily indicated that gerbils provided the L_An inoculum of F2365 had a longer persistence of L. monocytogenes in the intestines than the L_Aer inoculum. Gerbils provided either the L_An or the H_Aer inoculum had nearly 3 Log10 F2365 present in fecal samples on d 6. This was in contrast to gerbils that received the L_Aer, as only 1 of the 5 gerbils was still positive by d 6. Cecums and colons collected from gerbils that received the L_An inoculum had greater concentrations of L. monocytogenes than those from gerbils receiving the L_Aer inoculum. Histological examinations of the livers indicated gerbils infected with F2365 had severe locally extensive areas of necrosis, though the extent of this damage differed between treatment groups. Conclusions: This study indicates that anaerobic conditions alter the ability of L. monocytogenes to transit and/or persist within the gastrointestinal tract. Further studies are needed to determine the impact of oxygen availability on the invasive potential of L. monocytogenes. [3:10 p.m.] Members of the Conserved DedA/Tvp38 Membrane Protein Family are required for growth of Escherichia coli at

alkaline pH. Sujeet Kumar1 and William T. Doerrler1

1Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 Background: Alkali-tolerance is important in epidemiology of pathogenic bacteria, for existence in marine environments and for industrial applications and bioremediation processes. Many bacterial species including E. coli have the ability to survive under alkaline pH conditions. In order to survive at alkaline pH, E. coli has to maintain a steady cytoplasmic pH of about 7.6 that is more acidic than the environment. Bacteria employ numerous mechanisms to survive alkaline environments; however cytoplasmic membrane cation/H+ antiporters play a key role by active inward transport of protons. To date, NhaA, NhaB, ChaA, MdfA and MdtM are known cation/H+ antiporters that reportedly function in alkaline pH homeostasis in E. coli. The DedA/Tvp38 membrane protein family is a highly conserved protein family present in most sequenced genomes with no amino acid identity to known transporter families. However, the function of this membrane protein family is not known. YqjA and YghB are E. coli DedA proteins with 62 % amino acid identity and partially redundant functions. Previously, we have shown that a strain of E. coli lacking these two genes cannot properly maintain the proton motive force (PMF) and is compromised in PMF dependent drug efflux and other PMF dependent functions. Membrane embedded acidic amino acids, a trademark of proton dependent transporters have been shown to be important for the function of YqjA and YghB. Methods: We have measured growth of an E. coli ∆yqjA mutant at different pH’s using a serial dilution technique to measure the alkaline pH sensitivity. Complementation of the mutant phenotype was carried out with a plasmid copy of the yqjA gene to show dependency upon sodium/potassium salts. We have also carried out a reporter assay to measure the expression level of the yqjA gene in response to pH and sodium and potassium. Results: Here, we show that the ∆yqjA mutant is not able to grow at alkaline pH (ranging from pH 8.5 to 9.5), unlike the parent E. coli strain. Overexpression of yqjA from a plasmid restores growth at alkaline pH but only when millimolar concentrations of sodium or potassium are present in the growth medium. The ∆yghB mutant is not alkaline sensitive but overexpression of yghB from a plasmid restores growth of ∆yqjA mutant up to pH 9. Using a reporter gene placed under the control of the yqjA promoter, we found that yqjA expression was higher at alkaline pH than at acidic and neutral pH. Furthermore, increased expression of yqjA at elevated pH also required sodium or potassium salts. Conclusion: These results show that DedA/Tvp38 members likely are a new family of membrane transporter and that YqjA is absolutely required for survival of E. coli at alkaline pH.

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Immunology and Virology, Oral Presentations Friday, September 18 @ 1:00-3:30 p.m., Room 103A [1:00 p.m.] Viral binding induced signaling drives a unique and extended intracellular trafficking pattern during infection of

primary blood monocytes Jung Heon Kim and Andrew D. Yurochko Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana

Background: Human cytomegalovirus (HCMV)-infected monocytes and macrophages play a critical role in the hematogenous dissemination of the virus, life long viral persistence, and in the progression of HCMV-mediated diseases. We have previously reported that HCMV-infected monocytes differentiate into proinflammatory macrophages that are characterized by the secretion of proinflammatory cytokines, enhanced survival and motility. We showed that HCMV utilizes a different signaling and entry pathway during infection of monocytes, when compared to infection of fibroblasts and other cell types. We also found that these changes in infected monocytes during HCMV entry are linked to the interaction between EGFR/integrins and the viral glycoproteins (gB or gH/gL/UL128-131 complex). These data strongly argue for distinct biological pathways being utilized by HCMV during infection of monocytes. Methods: Primary monocytes were isolated from peripheral blood and infected with various HCMV strains such as TB40/E, Towne/E, BADwt, and BADrUL131. We extracted DNA from the nuclear fraction of HCMV- and mock-infected monocytes and performed PCR to detect HCMV DNA in the nucleus at various time points. We examined the trafficking pattern of the virus in HCMV-infected monocytes using confocal microscopy and analyzed colocalization between various viral proteins and select cellular compartments. Results: To expand our understanding of the unique infection process in monocytes, we initiated experiments to examine infection of monocytes post entry. Our new data shows that HCMV DNA is initially detected in the nucleus beginning at 3 days post infection (d.p.i.) in monocytes, when compared to 30 minutes post infection (m.p.i.) in fibroblasts and endothelial cells. We now show that HCMV is initially retained in early endosomes, and then moves sequentially to the trans-golgi network (TGN) and recycling endosomes prior to nuclear translocation of the viral genome. From temporal analyses, our data show that HCMV is retained in early endosomes until around 1 h.p.i., and then the viral particle translocates to the TGN (~1 h.p.i.-24 h.p.i.), and to recycling endosomes, where it can be retained for up to 5 d.p.i. (prior to nuclear translocation). We also have evidence that gB and UL32 colocalize until 2 d.p.i., suggesting that HCMV is retained initially as a mature particle prior to uncoating around 2 d.p.i. in recycling endosomes. Interestingly, we found that only HCMV DNA is retained in cells when they are infected with BADrUL131, which has the key gH/gL/UL128-131 complex. In contrast, we could not detect viral DNA in BADwt-infected monocytes; BADwt has a mutation in UL131A preventing for maturation of the gH/gL/UL128-131 complex. This data strongly indicates that viral binding induced signaling drives a unique nuclear translocation pattern in HCMV-infected primary blood monocytes. Conclusion: Our new data suggest that HCMV infection of monocytes has many unique properties and that these differences in infection are likely due to the inherent biological challenges that exist during infection of this important cell type. Furthermore, our data indicates that we have uncovered an unexpectedly different pattern of infection of monocytes that likely directly contribute to severe viral-mediated disease.

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[1:20 p.m.] Determining the mechanism of rotavirus NSP1-induced degradation of host target proteins Lindy M. Lutz and Michelle M. Arnold Department of Microbiology and Immunology, LSU Health Sciences Center - Shreveport

Background: Rotavirus nonstructural protein 1 (NSP1) inhibits the production of interferon-β (IFN-β) in order to promote viral spread. NSP1 induces the degradation of key host proteins that are required for IFN-β production, including interferon regulatory factors (IRF3, IRF5, IRF7) or the β-transducin repeat containing protein (β-TrCP), a protein required for the activation of NF-κB. The pattern of IRF and β-TrCP degradation varies among different rotavirus strains, but in general, animal strains induce degradation of IRFs whereas human strains induce degradation of IRFs and β-TrCP. Treating infected cells with proteasome inhibitors prevents the degradation of IRF3 and β-TrCP, indicating that NSP1-induced protein degradation is mediated by the proteasome. The target binding site on NSP1 is located at the C terminus, which is also the region with the most sequence variability when comparing different strains. In contrast, the N terminus of NSP1 is well conserved and contains a Cys-rich domain that is predicted to form a RING domain. The Cys residues of this domain are critical for NSP1-induced degradation of IRF proteins. The available evidence suggests that NSP1 acts an E3 ubiquitin ligase, however the mechanism of NSP1-induced degradation of IRFs has not been demonstrated definitively. Methods: We constructed plasmids that express tagged NSP1 proteins from human and animal rotavirus strains. Plasmids were transfected into human 293T cells and lysates were used to pull-down NSP1 followed by mass spectrometry to determine what host proteins associated with NSP1. To confirm our mass spectrometry results, we performed additional pull-down assays using tagged NSP1 proteins to identify if NSP1 associated with components of the ubiquitin-proteasome machinery. Results: Our data from mass spectrometry analysis of NSP1 pull-down assays indicated that NSP1 associated with components of cullin-RING ligase (CRL) complexes, which function in the host ubiquitin-proteasome pathway. We confirmed our mass spectrometry results and determined which proteins within the CRL3 complex associated with NSP1 by pull-down. We found variability in association between the NSP1 proteins tested and the different components of CRL complexes. All NSP1 proteins tested were able to associate with the CRL scaffolding subunit Cul3, the E3 ubiquitin ligase component Rbx1, and the BTB adaptor protein KEAP1. In contrast, only some NSP1 proteins were able to associate with the BTB adaptor proteins KLHL12 and SPOP. Conclusion: Our results suggest that NSP1 is not substituting for E3 ubiquitin ligase component of CRLs, Rbx1. The observed differences in association with the BTB adaptor proteins suggest that KEAP1 is likely involved in NSP1 activity whereas KLHL12 and SPOP may only transiently interact with NSP1. In order to determine which components of CRL complexes interact with NSP1 within this complex, direct protein-protein interaction studies will be necessary. Further, in vitro ubiquitination assays need to be performed to determine if NSP1 functions as an E3 ubiquitin ligase. These data will provide insight into the mechanism by which NSP1 targets proteins of the IFN-β response for degradation. [1:40 p.m.] Growth Factor Signaling Pathway Regulation by HPV16 E5 Protein

Matthew L. Scott, James A. Cardelli, Jason M. Bodily Louisiana State University Health Sciences Center – Shreveport, LA 71130

Background: Human papillomaviruses (HPV) infect keratinocytes of squamous epithelia and cause benign hyperproliferation that may progress to malignancy. The cellular changes that promote virus persistence and progression to malignancy currently remain unclear. The stroma is an important source of growth factors that facilitate cancer growth, and hepatocyte growth factor (HGF) is one of these. The receptor for HGF, Met, is a tyrosine kinase receptor that is upregulated in many cancers. Oncogenic upregulation of Met results in increased motility, invasion and metastasis of tumor cells. Met upregulation has been observed within cervical intraepithelial lesions of patients infected with oncogenic HPVs. We decided to determine if HPV16 regulates Met expression within the host cell and to analyze the interaction between HPV16-containing cells and the surrounding stromal cells. Methods: We used luciferase reporters containing the MET promoter, immunoblotting and RT-qPCR. We also generated cell lines that stably express the E5 oncoprotein Results: We found that Met is upregulated in keratinocytes containing episomal HPV16 genomes as compared to uninfected human foreskin keratinocytes. Upregulation occurs at the mRNA level and requires the HPV oncogene E5, with the E6 oncogene playing a minor role. This upregulation of Met by E5 requires epidermal growth factor receptor (EGFR), which is also upregulated at the mRNA level in an E5-dependent manner. We found that HPV does not induce HGF expression in infected cells, but that HGF and several other growth factors are upregulated in neighboring fibroblasts in an E5-dependent manner. Furthermore, E5 was able to increase the expression of transforming growth factor-beta 2 (TGFβ2), an important factor for communication between cancer cells and the stroma. Conclusion: These results show a novel activity of the E5 protein in regulating the interaction between HPV-infected cells and the stroma, and suggest a mechanism by which E5 can promote the progression of benign lesions to malignancies

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[2:30 p.m.] Characterization of an Oral Vaccine for M. marinum: Examination of Both Mucosal Immunity and Systemic Immunity in a Fish Model Lisa Shirreff, Martin Cheramie, Dr. Don G. Ennis The University of Louisiana at Lafayette, Lafayette, LA

Mycobacterium marinum (Mm) shares at least 80% amino acid sequence identity with over 3,000 orthologous genes of Mycobacterium tuberculosis (Mtb) and is thus used as a surrogate pathogen for Mtb. Our laboratory investigates mycobacteriosis using Japanese medaka (Oryzias latipes) as an aquatic animal model. Mm disease presentation in medaka is similar to Mtb disease presentation in humans, including growth in macrophages, granulomatous lesions, and life-long chronic disease. We have previously shown that a major route of infection in fish is through an oral route and have thus developed methods to infect medaka with Mm utilizing mosquito larvae as vectors. Recently, our lab was able to show that Mm is able to cross the gut epithelia of medaka in a relatively short-time frame and travel to the underlying submucosa. Therefore, Mm must have the ability to attach to the gut mucosal layer and evade killing by GALT immune cells. Mm is apparently able to exploit macrophages of the mucosal immune system to transport the bacteria to target organs like the head kidney, liver, and spleen for a systemic infection. Utilizing an Mm strain engineered to carry a deletion in the RD-1 region, known to include a number of virulence genes, our lab has shown that mucosal immunity against Mm can be induced in medaka. We have shown that exposure to the mutant RD-1 strain offers some protection against a chronic wild-type oral challenge. We are currently investigating whether the sensitization by the mutant RD-1 strain offers protection against an acute wild-type challenge by an IP route. Preliminary results suggest that oral exposure to the mutant RD-1 strain offers only partial protection against this acute wild-type challenge.

[2:50 p.m.] The Regulation of Cellular and Viral Gene Expression by HPV16 E7

William K. Songock, Seongman Kim and Jason M. Bodily Center for Molecular and Tumor Virology, Department of Microbiology and Immunology Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932

Background: High-risk human papillomaviruses (HPVs) persistently infect keratinocytes of the squamous epithelia and cause benign lesions and several malignancies, including cervical cancer. Although much effort has gone into understanding how HPV can cause cancer, relatively little is understood about what happens during the normal viral life cycle in benign infections. In addition to inhibiting the transcriptional activity of TGFβ, HPV16 E7 has been shown to block negative regulation of cellular proliferation due to TGFβ. Most of these studies were done in transient transfections thus not accounting for how the regulation of TGFβ by E7 is critical for complete viral life cycle. Previous work in the lab has shown that E7 bind to the transcription regulator, cyclin dependent kinase 8 (CDK8), and F57A mutation on E7 resulted in reduced binding of E7 to CDK8 in transient transfection as well as in keratinocytes that maintain episomal HPV genomes. Methods: To better understand the significance of E7:CDK8 interaction, we generated stable cells lines maintaining episomal wild type HPV or F57A genomes. Microarray analysis was used to compare transcripts from F57A cells to those containing wild type HPV16. Additionally, relative mRNA of TGFβ and viral gene levels were measured using RT-qPCR. Chromatin Immunoprecipitations (ChIP) and Western blots were used to show various interactions of viral and cellular proteins. Results: We observed that the F57A mutation did not affect cell growth and episomal maintenance of HPV genome. However, the F57A mutant failed to downregulate TGFβ1 transcript levels suggesting that the interaction of E7 with CDK8 is critical for suppression of TGFβ activity thus allowing the virus to replicate. The viral late promoter was not activated resulting in a reduction of viral late gene transcripts in the F57A-containing cells. ChIP results showed reduced recruitment of CDK8 to the virus early and late promoters in F57A cells, further supporting the hypothesis that CDK8 is critical for the induction of TGFβ transcriptional activity. Microarray analysis showed a significant increase in TGFβ pathway genes in HPV cells containing the F57A mutation compared to the wild type. In addition, regulated pathways included all of the cellular pathways previously shown to be regulated by CDK8. Conclusions: Our study shows that episomal cells maintaining HPV16 genomes inhibit TGFβ activity through interaction with CDK8. Since CDK8 a positive regulator of TGFβ pathways it is possible that the interaction of CDK8 with E7 prevents CDK8 kinase activity which is critical for TGFβ transcriptional activation.

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[3:10 p.m.] Human Cytomegalovirus Exploits Clathrin-Mediated Endosomal Trafficking for Maturation and Egress Madeline A. Archer1, Leslie E. Davis1, Teal M. Brechtel1 and Ritesh Tandon1 1Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi

Background: Clathrin-mediated endocytosis is critical for cellular entry of several viruses; however, the role of clathrin in cellular trafficking of viruses beyond virus entry is only partially understood. Here, we utilized two laboratory strains (AD169 and Towne) of human cytomegalovirus (HCMV), which are known to use cell surface membrane fusion rather than clathrin-mediated endocytosis to enter fibroblasts, in order to study a post-entry role of clathrin in HCMV life cycle. Methods: We used pharmacological as well as biological inhibitors of clathrin-mediated endocytosis in primary human fibroblasts and measured the impact on viral entry, viral gene expression and localization of virus structural proteins in the cells. We also performed transmission electron micrography to study the morphogenesis of virus particles during inhibition of clathrin versus a mock-treated control. Results: Upon inhibition of dynamin-2, clathrin terminal domain (TD) ligand association, or pH decrease in endosomes, AD169 and Towne entered the cells successfully based on the expression of immediate early viral protein. There was no difference in early and late viral gene expression in these cells compared to mock-treated cells. However, all three inhibitions significantly reduced the growth rates and final virus yields without impacting the expression levels of early to late viral proteins. A dominant negative inhibitor of dynamin had similar impact on virus growth. Inhibition of dynamin prior to virus entry, at early times post infection or at late times post infection had a similar impact on virus replication and spread suggesting no impact on early replication events. Moreover, clathrin accumulated in the cytoplasmic virus assembly compartment (vAC) of infected cells co-localizing with virus tegument protein pp150. Transmission electron micrographs (TEM) of infected cells where dynamin-2 and clathrin TD ligand associations were blocked showed intact nuclear stages of nucleocapsids assembly but the cytoplasmic virus maturation was greatly compromised. Conclusion: The data presented here clearly indicate that host clathrin-mediated endocytic pathways are exploited by HCMV for maturation and egress.

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Pathogenic Microbiology, Oral Presentations Saturday, September 19 @ 9:00 -11:30 a.m., Room 103C [9:00 a.m.] The role of universal stress proteins in Edwardsiella ictaluri virulence

Ali Akgul, Seong Won Nho, Mark L. Lawrence, Attila Karsi College of Veterinary Medicine, Mississippi State University

Edwardsiella ictaluri is a Gram-negative bacterium that causes enteric septicemia of catfish (ESC). The universal stress proteins are present in archaea, bacteria, plants and fungi, but humans. Previous reports showed differential expression of USPs during invasion of several pathogens. However, the role of USPs in Edwardsiella is unknown. Bioinformatics analysis indicated that E. ictaluri genome has 9 USPs. We targeted these USPs and constructed mutants by in-frame deletion. Their attenuation and vaccine potential will be assessed in catfish. We expect that our study will explore the roles of universal stress proteins (USPs) in E. ictaluri pathogenesis and yieald development of live attenuated vaccines against ESC. [9:20 a.m.] The msaABCR Operon Activates Capsule Production in Staphylococcus aureus

Justin Batte and Mohamed O. Elasri The University of Southern Mississippi, Hattiesburg, Mississippi

Background: Capsule production in Staphylococcus aureus plays a major role in pathogenesis. Capsule allows S. aureus to evade phagocytosis during movement from one localized site to another in the course of an infection. S. aureus has developed a complex regulatory network that is responsible for regulating capsule production. We have identified and defined the msaABCR operon that regulates several virulence factors. In this study we show that the msaABCR operon regulates capsule production by activating the cap operon. Methods: To examine the role the msaABCR operon plays in the regulation of capsule, we deleted the msaABCR operon in two strains of S. aureus that produce two different capsule serotypes. We tested the effects of the mutation on various conditions including transcription of cap genes, total capsule polysaccharide (CP) production, and functional killing assays using human polymorphonuclear neutrophils or (PMNs). Additionally, we used the electrophoretic mobility shift assay or (EMSA) to determine if the regulation observed is directly related to a component of the msaABCR operon. Results: We observed in both strains tested that deletion of msaABCR significantly reduces the expression level of cap genes. Measurement of the crude capsule production showed that the reduction in cap transcription reduced total CP production to undetectable levels in the deletion mutants. We also observed that the mutants, in both strains tested, are significantly more susceptible to elimination by host immune components including PMNs. Additionally, we found that a protein product of the msaABCR transcript, MsaB, binds upstream of the promoter region of cap as an activator of CP production in a growth dependent manner specifically in the late and post-exponential growth phases. Conclusion: In this study we show that the msaABCR operon plays an important role in the regulation of CP production within S. aureus at the transcriptional level that significantly alters total CP production. Furthermore, we show that the msaABCR operon directly regulates CP production in S. aureus. We found that the MsaB protein binds upstream of the promoter region of the cap operon and prospectively serves as an activator of CP production. We are currently exploring this regulation observed by the msaABCR operon and specifically the MsaB protein in more detail.

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[9:40 a.m.] AliC and AliD Enhance Virulence of Nonencapsulated Streptococcus pneumoniae Jessica L. Bradshaw, Haley R. Pipkins, Lance E. Keller, Jessica M. Friley, and Larry S. McDaniel University of Mississippi Medical Center, Jackson, Mississippi

Background: Streptococcus pneumoniae (the pneumococcus) is a major pathogen that colonizes the human nasopharynx and causes a wide variety of diseases such as otitis media (OM), pneumonia, bacteremia, and meningitis following dissemination from the nasopharynx. A majority of pneumococcal clinical isolates express a polysaccharide capsule, which is the current target of licensed pneumococcal vaccines and is thought to be required for invasive pneumococcal disease (IPD). However, recent surveillance of IPD isolates has identified nonencapsulated S. pneumoniae (NESp) as etiological agents of IPD. AliC and AliD are proteins expressed by NESp that have been associated with IPD. This study examines the role of AliC and AliD in pneumococcal virulence. Methods: MNZ85 and MNZ41 are NESp carriage isolates that possess aliC and aliD, which replace the capsule polysaccharide synthesis (cps) locus. Isogenic mutants of MNZ85 and MNZ41 that lack aliC and aliD, LEK08 and JLB01 respectively, were constructed. Human epithelial cell lines Detroit 562 and A549 were utilized to assess pneumococcal adhesion and epithelial cell invasion. In vitro assays examining biofilm production and viability were also performed. Additionally, pneumococcal colonization was assessed in a mouse model following intranasal challenge. All strains were also investigated in a chinchilla OM infection model. Survival of wild-type and isogenic deletion mutant strains within blood was examined using whole chinchilla blood. Results: Significant differences in epithelial cell adhesion and invasion were strain dependent. Significant increases in biofilm viability and production were observed in deletion mutants. We also demonstrated that AliC and AliD enhanced murine nasopharyngeal colonization and were required for OM in a chinchilla model. Furthermore, AliC and AliD increased pneumococcal survival in chinchilla whole blood. Conclusion: This study reveals that the expression of AliC and AliD enhances virulence of NESp. Despite the increase in NESp isolates, little is known about this pneumococcal population. Thus, characterization of NESp virulence factors is essential to understanding how these isolates cause disease. [10:30 a.m.] Investigating the role of transition metals in Streptococcus pneumoniae colonization

Lindsey R. Brown1, Adam C. Coombs1, Jason W. Rosch2, and Justin A. Thornton1 1Mississippi State University, Mississippi State, MS 39762 2St. Jude Children’s Research Hospital, Memphis, TN 38105

Background: Streptococcus pneumoniae is commonly diagnosed as the causative agent of both otitis media and community acquired pneumonia. In order for pneumococcus to disseminate and cause invasive diseases such as pneumonia and septicemia, the organism must first colonize the host. It is known that transition metals like zinc and manganese are not only crucial for bacterial pathogenicity, but also host immune responses. In this study, we investigated the role of transition metals on early colonization by pneumococcus. Methods: We analyzed early colonization in vivo of mutants lacking a zinc-binding lipoprotein AdcAII, in a murine model. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to determine metal concentrations of bacterial cultivation media. Growth was analyzed in Chelex-treated Chemically Defined Media (CDM) with calcium, copper, iron, magnesium, manganese, and zinc supplementation to determine the effects of metal stress on S. pneumoniae. In vitro biofilm assays were analyzed via staining with 0.5% crystal violet to detect total biofilm mass, and plating to detect cell viability within biofilms. Results: This study shows that pneumococcal biofilm total mass increases as zinc concentrations increase, in a dose dependent manner (p<0.00001). However, viability of bacteria grown within the biofilms is unaltered despite growth in different zinc environments. Interestingly, we saw that pneumococcus is able to grow without the addition of any zinc back to the media, thus indicating that the organism is able to compensate for zinc stress by utilizing other transition metals such as manganese or calcium. Conclusion: The novel findings from this study indicate that changes in zinc concentration could significantly alter the ability of pneumococcus to form biofilms. Pneumococcus encounters a variety of metal concentrations as it traverses through the human body, and altering the capacity of the bacteria to acquire metals, thereby affecting bacterial colonization, could inhibit downstream progression to invasive disease. It is our hope that this knowledge could potentially lead to the development of alternative treatment strategies against pneumococcal disease.

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[10:50 a.m.] Determining the Impact of Bile Induced Damage on Listeria monocytogenes Oindrila Paul, Jessica G. Wilson, Amber Thompson, and Janet R. Donaldson Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762

Background: Listeria monocytogenes is a Gram positive, facultative intracellular organism responsible for the foodborne disease listeriosis. To cause disease in humans, L. monocytogenes must survive a variety of stressors encountered within the gastrointestinal (GI) tract, including variations in pH, oxygen availability, and bile. Though it is known that the oxidative stress response is expressed following exposure to bile under aerobic conditions, little is known about the response under anaerobic conditions. The hypothesis for this project is bile exposure under anaerobic conditions will differ from damage induced under aerobic conditions. Methods: L. monocytogenes strains F2365 (serovar 4b), 10403S (serovar 1/2a), and HCC23 (serovar 4a) were treated with porcine bile extract under aerobic or anaerobic conditions and membrane alterations were examined by transmission electron microscopy, gas chromatographic analysis of fatty acid methyl esters, and alterations in ratios of NADH:NAD+. Results: Microscopic observations indicated that following bile exposure, the average size of all strains under both aerobic and anaerobic conditions decreased; this difference was greater under aerobic conditions than in anaerobic conditions. No differences in cell envelope width were observed with HCC23 under aerobic or anaerobic conditions; 10403S increased under both conditions and F2365 width increased only under anaerobic conditions. Cultivation of these strains with the known antioxidant esculetin and bile did not improve survival under anaerobic conditions, suggesting that bile does not induce oxidative damage under anaerobic conditions. The fatty acid profiles of cell membranes also were altered following exposure to bile: saturated fatty acids palmitic acid and stearic acid increased under aerobic and anaerobic culture conditions, as well as the presence of unsaturated fatty acids linoleic acid and oleic acid. The content of branched chain fatty acids decreased when exposed to bile. These results suggest that modifications to the cell membrane in the proportion of palmitic acid, stearic acid, linoleic acid and oleic acid impacts bile resistance. To determine if reductive stress could be a possible cause of damage under anaerobic conditions in L. monocytogenes, the NADH:NAD+ ratios were analyzed in the presence of bile. Results indicated that ratios of NADH:NAD+ shift under anaerobic conditions, but the effect is limited to bile sensitive strains of L. monocytogenes. Conclusion: These results suggest that the response to bile induced membrane damage varies between strains of Listeria, and the response is dependent upon alterations at the cell membrane. Further research is needed to examine the direct proportion of lipid peroxides in aerobic and anaerobic treatments. [11:10 a.m.] Deletion of the Polyamine Transporter potABCD Attenuates the Virulence of Encapsulated but Not Nonencapsulated

Streptococcus pneumoniae H. R. Pipkins, J. L. Bradshaw, L. E. Keller, J. M. Friley, E. Swiatlo, and L.S. McDaniel University of Mississippi Medical Center, Jackson, MS

Background: Streptococcus pneumoniae (the pneumococcus) is a gram positive coccus that colonizes the nasopharynx of humans and can lead to pneumonia, otitis media (OM), sinusitis, and meningitis. Most pneumococcal infections are associated with the encapsulated form of the pneumococcus, making the capsular polysaccharide the target of licensed pneumococcal vaccines. However, due to selective pressure against the pneumococcal capsule, we are seeing an increased distribution of non-vaccine serotypes, including nonencapsulated S. pneumoniae (NESp). Both encapsulated and NESp possess the polyamine oligo-transport operon (potABCD). Previous research has shown that the inactivation of potD in encapsulated S. pneumoniae results in changes in protein expression. A significant reduction in pneumococcal murine colonization, as well as attenuated virulence in a mouse model of pneumococcal pneumonia, are also seen. However, the role of the pot operon in NESp has not yet been established. Methods: We examined potD in colonization and virulence of NESp. PotD deficient mutant (PIP01) was constructed in the virulent NESp MNZ67. A mouse model was used to examine the effect of potD on nasopharyngeal colonization, and a chinchilla model was used to assess its effect on the development of OM. Additionally, we assessed Pneumococcal surface protein K (PspK) expression, pneumolysin expression and activity, biofilm formation, and epithelial cell adhesion and invasion. Results: The absence of potD reduced pneumolysin production and activity, but PspK production and biofilm formation increased. Epithelial cell adhesion and invasion results varied depending on the epithelial cell source (nasopharyngeal or lung). Neither murine colonization nor chinchilla OM were attenuated in the absence of potD. Upon complementation of potD back into PIP01, pneumolysin was increased to levels significantly greater than wild type, while PspK was reduced to levels significantly lower than wild type. Conclusion: Our in vitro studies indicate that the pot operon in NESp is important for the regulation of protein expression. However, in contrast to encapsulated pneumococci, we conclude the pot operon is not required for full virulence of NESp in a model of OM.

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Applied & Environmental Microbiology, Oral Presentations Saturday, September 19, 9:00 -11:30 a.m., Room 102 [9:00 a.m.] An insight into the antioxidant responses to exogenous oxidative stress agents and a glimpse into the role of catalase

in the reproductive fitness of the Gulf-Coast tick (Amblyomma maculatum) Deepak Kumar and Shahid Karim Department of Biological Sciences, The University of Southern Mississippi

Background: Ticks are important ecto-parasites and blood is the only nutritious meal taken by ticks. Aside from dealing with the host’s hemostatic and immune mechanism, ticks must cope with the potentially toxic molecules in their large blood meal. Blood digestion may contribute to significant oxidative stress resulting from the release of heme, free iron radicals, H2O2, and other stress-inducing molecules. Severe and prolonged oxidative stress can trigger apoptosis and necrosis in ticks. To overcome the toxic effects of reactive oxygen species (ROS), ticks utilize a battery of antioxidant molecules available in their repertoire, including catalase. We hypothesized that the induction of endogenous and exogenous ROS activates the tick antioxidants machinery to mitigate oxidative stress. Methods: The relative gene expression of select antioxidant targets was determined by real time quantitative PCR in tick tissues after injecting them with H2O2 and Paraquat. A reverse genetics approach was used to silence the gene expression of tick catalase to assess the functional significance of cat in tick hematophagy, and reproductive fitness. Transcriptional gene expression and native microbiota quantification were also performed using qRT-PCR. Results: The selected tick antioxidants transcript level increased from 2-18 fold in the tick tissues injected with H2O2 and Paraquat. The gene knock-down of cat showed the depletion of transcript level and increase in reactive oxygen species level but it did not interfere with tick hematophagy or phenotype. The transcript levels of various tick antioxidants in cat knocked-down tissues were differentially regulated indicating the presence of alternate pathway. Interestingly, cocktail of cat dsRNA and cat inhibitor impaired the tick reproductive fitness. Conclusion: Our results support the presence of a robust antioxidant system and demonstrate the significance functional role of tick catalase in mitigating the reactive oxygen species generated during the prolonged tick feeding on the vertebrate host. [9:20 a.m.] E. coli PBP5 active site mutations alter cell shape

William MacCain, Suresh Kannan, Kevin Young University of Arkansas for Medical Sciences, Little Rock, Arkansas

Background: Bacterial morphology is determined by the overall structure of the semi-rigid peptidoglycan. Peptidoglycan is made up of sugar molecules linked together by short peptides, which is later synthesized and modified by enzymes called penicillin binding proteins (PBPs). In Escherichia coli, the major periplasmic DD-carboxypeptidase, PBP5, removes the terminal D-alanine from the pentapeptide side chains of peptidoglycan and plays a pivotal role in defining cell shape. PBP5 could perform this function either by regulating the amounts of pentapeptides in the cell wall or by interacting with other periplasmic proteins. Methods: To determine if cell shape is determined principally by the number of pentapeptides in the cell wall, we created three PBP5 variants having different rates of enzyme activity. Two variants, (PBP5 K213H and PBP5 S44G), exhibited zero DD-carboxypeptidase activity; the third variant (PBP5 H216S) was 25%. Results: Cells expressing K213H had drastically altered cell shapes – instead of being rod shaped, the cells were longer, wider and were no longer uniform, having numerous bulges and kinks. In contrast, cells expressing the H216S variant were normal. These results suggest that the DD-carboxypeptidase activity of PBP5 does affect cell morphology. Conclusion: PBP5 DD-carboxypeptidase activity does affect cell morphology and PBP5 enzymatic activity does not dictate midcell localization.

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[9:40 a.m.] Elucidating the Functional Role of the H2O2-Generating Dual Oxidase (Duox) in the Gulf Coast Tick, Amblyomma maculatum Virginia C. Meyers, Shahid Karim The University of Southern Mississippi, Hattiesburg, Mississippi

Background: In the absence of effective targeted control, the increased transmission of disease-causing microbes by the tick vector is of critical concern. At present, little is known about the relationship of tick molecules and their means of controlling their native microbiota on tissue level. Looking to literature of hematophagous arthropods, dual oxidase (Duox) has been implicated to be involved in the defense against pathogen invasion of epithelial tissues facing an external environment through H2O2 generation and formation of dityrosine crosslinks between extracellular proteins, such as within midgut lumen. For evaluation in the Rickettsia parkeri vector, Ambylomma maculatum, an RNA interference approach was used to assess the role of Duox in tick blood feeding and modulation of associated total microbiota. Methods: Duox-specific dsRNA was injected into adult female A. maculatum and blood fed on a sheep, followed by removal 5 days post-infestation for qRT-PCR analysis or fed to repletion and allowed to ovipostion. Confocal imaging targeting dityrosine was performed to demonstrate Duox role in formation of extracellular dityrosine luminal barriers in target tissues. Results: Physical weakening in cuticle integrity of partially-fed dsDuox ticks was observed when removed, resulting in tearing of the ticks and large sample loss. Those left on the sheep fed to repletion and showed significant increase in engorgement weight compared to controls. Additionally, replete dsDuox ticks developed a fungal infection that spread during ovipositioning, killing some ticks and spreading throughout the eggs; dsDuox injection wound sites also showed an impairment to heal compared to controls. Due to loss of samples, three trials were performed, with each producing similar physical and transcriptional expression results; qRT-PCR data was compared in a gene study with actin used as reference. Duox knockdown showed increase in total microbial load of dissected midgut, salivary gland, and ovaries. Compensatory mechanisms of antioxidant and selenoprotein expression were determined. Confocal imaging confirmed a decrease of dityrosine crosslinks within dsDuox cuticle compared to control. Conclusion: H2O2 produced by dual oxidase appears to have a role in the formation of dityrosine crosslinks that strengthen then growing cuticle during tick engorgement as well as a potential role in microbiome homeostasis while blood feeding. Additionally, Duox appears to be critical to the maintenance of wound healing and antifungal immunity by ticks during blood meal and preventing susceptibility while ovipositioning. [10:30 a.m.] Physiological and genetic characterization of lipid accumulation in Gordonia sp. KTR9.

Karl J. Indest, Jed O. Eberly, David B. Ringelberg, and Dawn E. Hancock. U.S. Army Engineer Research and Development Center, Vicksburg, MS.

Background: Actinomycetes may be ideal organisms for direct biodiesel synthesis because of their capacity to synthesize high levels of triacylglcerides (TAGs). We investigated TAG accumulation in Gordonia sp. KTR9, a strain that possesses a large number of genes dedicated to fatty acid and lipid biosynthesis. Methods: Total and neutral lipids were assayed via GC–MS via fatty acid methy esters. TAG content was determined by a colorimetric assay using an ABcam Triglyceride Quantification Kit. Gene knockouts targeting a putative lipase (KTR9_0186) and wax ester synthase/ acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; KTR9_3844) were constructed in KTR9 based on a conjugation strategy using a sacB counter selectable marker. Custom Gordonia sp. KTR9 expression microarrays were developed for downstream transcriptome studies. Results: Total lipid fatty acids content increased by 75 % and TAG content increased by 50 % under nitrogen starvation conditions in strain KTR9. Gene disruption of KTR9_0186 resulted in a twofold increase in TAG content in nitrogen starved cells. Lipase mutants subjected to carbon starvation, following nitrogen starvation, retained 75 % more TAGs. Transcriptome expression data confirmed the deletion of KTR9_0186 and identified the up-regulation of key genes involved in fatty acid degradation, a likely compensatory mechanism for reduced TAG mobilization. In vitro assays with purified KTR9_3844 demonstrated WS/DGAT activity with short chain alcohols and C16 and C18 fatty acid Co-As. Conclusions: Collectively, these results indicate that Gordonia sp. KTR9 has a suitable tractable genetic background for TAG production as well as the enzymatic capacity to catalyze fatty acid esters from short chain alcohols.

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[10:50 a.m.] Metagenomic study of bacteriophage diversity from the gut of the Formosan subterranean termite, Coptotermes formosanus Shiraki Chinmay Tikhe, Claudia Husseneder Department of Entomology, LSU Agricultural Center, Baton Rouge, Louisiana

Background: Formosan subterranean termite workers harbor a complex community of bacteria and protozoa in their guts. The presence of a diverse bacterial community makes the termite gut a perfect niche for bacteriophages. In a previous study, we isolated three novel bacteriophages from the termite gut. However, the difficulty to culture the majority of termite gut bacteria limits our ability to identify the total phage diversity in the termite gut by conventional isolation techniques. Therefore, we employed culture-independent next-generation sequencing of the viral metagenome to study the phage community in the termite gut. Methods: We dissected workers from three different termite colonies and purified phage DNA from their guts. The DNA was sequenced using the Illumina miseq platform. DNA reads were assembled using Spades genome assembler and were annotated using the Metavir web server. Results: Approximately 22-24 % of the sequences could be assigned to virus related genes using the NCBI database of completely sequenced viral genomes. Sequences related to the bacteriophage families Siphoviridae, Myoviridae, Podoviridae and Microviridae were present in all the termite colonies. Apart from bacteriophages, sequences related to large eukaryotic viruses and Circoviruses were also present in all the termite colonies. Conclusion: The study shows the presence of a diverse virus population in the termite gut. Diversity and distribution of virus related sequences suggests the presence of a colony specific as well as a core virome in the termite gut. Further, we intend to use the termite gut as a model system to study the tripartite relationship between bacteria, phages and the host. [11:10 a.m.] Ehrlichia chaffeensis –Vector-Host-Pathogen Interactions

Jaclyn Williams, Paige Allen, Shahid Karim The University of Southern Mississippi, Hattiesburg, Mississippi

Background: Ticks are ectoparasites best known for their role in human disease. Amblyomma americanum is a vector of multiple pathogens, including Ehrlichia chaffeensis, the causative agent of Human Monocyte Ehrlichiosis. This pathogen is transmitted by the salivary glands, thus we investigated the molecular mechanisms of tick salivary reprolysin metalloproteases (MP’s) in tick feeding and pathogen maintenance to gain a better insight into this relationship. Methods: Utilizing both an in vitro and in vivo infection studies, female ticks were infected with E. chaffeensis at varying life stages to better understand the differential transcriptional expression within the tick. Using q-PCR, differential expression of reprolysin MP’s were assessed across various time points in both clean and infected samples. Results: Using both nested VLPT primers and a probe based q-RT PCR assay, ticks were successfully infected with E. chaffeensis using both infection models (in vivo and in vitro). Additionally, infected salivary glands demonstrated a significantly higher transcriptional expression of salivary reprolysin MP’s when compared to clean salivary glands. Conclusions: This is the first report of in vitro membrane feeding used to infect hard ticks with a pathogen. There are multiple implications with this success, as it could allow for a better understanding of the dynamics between pathogen-tick interactions. E. chaffeensis is an intracellular pathogen and likely utilizes and modulates a variety of tick salivary proteins. The differential expression of MP’s gives insight into that phenomenon.

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Poster Session September 18, 3:00-5:00 p.m. Applied & Environmental Microbiology, Poster Session 1.1 Dissemination of Ehrlichia chaffeensis using an Artificial Membrane Feeding System

Paige Allen, Jaclyn Williams, Rebekah Bullard, Shahid Karim The University of Southern Mississippi, Hattiesburg, Mississippi

Background: Amblyomma americanum ticks are ectoparasitic arthropods that have medical/veterinary importance. In this study, E. chaffeensis infected A. americanum ticks were fed on an artificial membrane feeding system to determine the dissemination of E. chaffeensis. Methods: An artificial membrane feeding system was constructed of a silicone covered, reinforced membrane attached to an acrylic chamber. Ticks were fed on blood collected from an abattoir. A. americanum males infected with E. chaffeensis were fed in the chambers, and blood was collected from each well during the infection transmission study. RNA isolated from male and female tissues was subjected to qRT-PCR for gene expression and verification of the presence of E. chaffeensis. Results: The gene expression from the artificial membrane feeding system is compared by weight and by feeding intervals to in vivo fed ticks. The expression for both approaches is similar with few significant changes (11% of the samples tested). PCR of collected tick exposed blood showed the transmission of E. chaffeensis within the first 48 hours of feeding. Conclusion: This study shows that the Amblyomma species are able to feed on an artificial membrane feeding system that mimics that of the in vivo system. Comparison of tick weights indicates a lag in blood uptake efficiency. However, this novel approach allows for a more complete analysis of E. chaffeensis transmission. 1.2 Presence of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes in Different Salinity Gradients in Water from

Bayou Petit Caillou in Southern Louisiana Ryan Brown, Justin Homer, Scott Bergeron, and Raj Boopathy Nicholls State University, Thibodaux, Louisiana

Background: The growing bacterial resistance to antibiotics has been a large concern in clinical settings. The monitoring of antibiotic resistant bacteria (ARB) and their antibiotic resistance genes (ARGs) in the environment has become an important area of study for public health. Bayou Petit Caillou is a distributary from Bayou Lafourche that travels through the small town of Chauvin, and the bayou is commonly used for recreational activities such as swimming, fishing, and boating. This study was conducted to assess the antibiotic resistance of bacteria, and look for the presence of six different ARGs present at differing salinity levels on Bayou Petit Caillou. Methods: Monthly samples were collected in duplicate from three sites on Bayou Petit Caillou. The salinity ranges from 0 to 9 parts per thousand (ppt). Bacteria of interest were identified using BIOLOG™ and standard identification methods. Kirby-Bauer assays were used determine antibiotic resistance of identified bacteria. PCR and gel electrophoresis was used with primers erm (B), sul1, tet(A), tet(W), tet(X), and mec A for erythromycin, sulfonamides, tetracycline and methicillin respectively. Results: Multiple ARBs were found in all three sites, including the Enterococci spp. and E. coli in all three sites. Species such as Enterobacter cloacae/aerogenes and various Vibrio spp .were found in two of the three sites. The molecular data shows that tet (A) was present in all three sites but appeared less often in site 3. Erm (B) and tet (W) was present during one sampling event in sites 2 and 3, but not site 1. Tet (X) was present one month in site 1 but never in any other sites. Conclusion: The antibiotic resistance of identified bacteria was pretty similar among all three sites. The molecular results show the presence of multiple ARGs in Sites 2 and 3 with site 2 having the most appearances of ARGs studied. Site 1 also showed the presence of tet (A) which was the most abundant in all sites, and was the only site that contained tet (x).

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1.3 The Microbial Clock: Tracking Bacterial Translocation through Successional Host Decomposition Z.M. Burcham1, J.L. Pechal2, Jeffrey Bose3, C. J. Schmidt4 M.E. Benbow2, and H.R. Jordan1 1Department of Biological Sciences, Mississippi State University, MS; 2 Department of Entomology and Department of Osteopathic Medical Specialties, Michigan State University, MI; 3 Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS; 4Department of Pathology, University of Michigan, MI

Background: Microbially mediated mechanisms of human decomposition begin immediately after death and are a driving force for conversion of a once living organism to a resource of energy and nutrients. Little is known about postmortem microbiology in cadavers, particularly the microbial structure of microflora residing within the human ecosystem, and their associations with decomposition stages. Recent work suggests that these bacterial communities are surprisingly dynamic during the postmortem interval. In this study, we describe how the microbiome of a living host changes and translocates within the body after death. Methods: Immunocompetent mice were inoculated nasally with fluorescently labeled Staphylococcus aureus-RFP and Clostridium perfringens. A subset of mice was immediately surface sterilized with 10% bleach solution following sacrifice and compared to non-surface sterilized in order to determine the influence of external microbiota. S. aureus-RFP was tracked using in vivo and in vitro imaging to determine colonization routes. Dissection and organ extractions took place at various time points for DNA/RNA analyses, and plated on Mannitol Salt Agar and Reinforced Clostridial Medium. Results: Fluorescent whole body imaging demonstrates notable S. aureus-RFP proliferation along the pharynx and lungs within the first 5 hours of decomposition. S. aureus-RFP was also present from organ tissue plated on MSA showing the translocation of S. aureus-RFP from the inoculation site to once sterile organs. Conclusion: Early results suggest that S. aureus-RFP is able to translocate to organs surrounding the pharynx and lungs in as early as a few hours after host death. As more information is gathered from this study, the tracking of postmortem microbial communities may help to provide a more accurate indication of decomposition stage and ultimately time of death when coupled with existing forensic techniques. 1.4 Effect of Diet and Geography on the Horse Gut Microbiota and the Potential for Rapid Growth of ‘Bloom’ Taxa in Fecal

Samples. Kalie F. Beckers, Christopher J. Schulz, and Gary W. Childers Department of Biological Sciences. Southeastern Louisiana University, Hammond, LA

Background: Horses are hindgut fermenters that rely on their gut microbiota for converting plant material into substrates that are usable, such as short chain fatty acids. Additionally, sudden changes in horse diet can results in laminitis or colitis, which has been associated with rapid changes in gut microbiota in some instances. Previous research of the equine gut microbiota has examined the influence of diet on gut microbiota, but did not examine effect of location and co-habitation on gut microbiota. In this study 24 horse fecal samples from three Louisiana horse stables were collected and 16S rRNA amplicon sequencing was performed to examine geographical and diet effects simultaneously. Methods: Fecal samples were obtained from 24 horses on three different concentration diets and two different forage diets, including grass and Alfalfa hay from three different stables (Hammond, Loranger, and Folsom). Fecal samples were collected and stored on ice for transport to lab. DNA was extracted from samples using MoBio Powersoil DNA extraction kits (MoBio, Carlsbad, CA). Bacteria and Archaea 16S rRNA genes were sequenced using 515F/806R fusion primers targeting the V4 region. Operational Taxonomic Units (OTUs) were assigned using 97% similarity clustering and were assigned to the Greengenes taxonomy. Results: Nine phyla were detected in all 24 samples, including: Firmicutes, Bacteroidetes, Fibrobacteres, Spirochaetes, Actinobacteria, Tenericutes, Verrucomicrobia, Proteobacteria, and Euryarchaeota. Community-level composition was not diet-dependent in these samples, but did vary by site. In particular, the Hammond and Loranger samples were highly similar. The Hammond fecal samples harbored significantly more Treponema spp. and less Clostridium, Psuedobutyrivibrio, and Blautia spp. than the Loranger samples. The Folsom samples were significantly different at the community-level. The Folsom samples harbored the significantly more Firmicutes and Proteobacteria than the other sites (as well as many other taxa abundance shifts). The taxa most abundant at the Folsom site included Bacillus and other taxa that have previously been associated with extra-intestinal ‘bloom’ species (i.e. growth of the taxa in feces outside of the host gut) in the American Gut Project samples. Conclusion: The presence of potential ‘bloom’ taxa overwhelmed the Folsom site making any conclusions about diet or geographical effects from this study limited. The other sites did not contain potential ‘bloom’ taxa, but did suggest some geographical effects on horse gut microbiota. Current work includes sampling of fresh horse fecal samples in a time dependent manner to test for growth of bloom species, and to develop rapid field preservation techniques for horse fecal microbiota sampling.

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1.5 Superoxide dismutases modulate the native microbiota of Amblyomma maculatum, a vector of Rickettsia parkeri. Gary Crispell, Khemraj BC, Shahid Karim The University of Southern Mississippi, Hattiesburg, Mississippi

Background: The Gulf Coast tick (Amblyomma maculatum) is an obligate blood-feeding ectoparasite and carrier of Rickettsia parkeri, a pathogen related to Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Ticks possess the unique ability to ingest huge blood meals, which requires robust anti-hemostatic strategies; one of which is an important anti-oxidative mechanism against blood meal derived oxidative stress. We hypothesized that tick superoxide dismutase (mitochondrial and cytoplasmic) enzymes are critical in combating stress caused by radical oxygen species during the blood-meal cycle, both on and off the host. Methods: An RNAi-mediated gene silencing approach was used to assess the functional role of target genes in tick blood-feeding to determine enzymatic activity, oxidative stress damage, and the regulation of associated microbial communities in Rickettsia parkeri infected and uninfected ticks. Total microbial load and compensatory mechanisms were assessed with qRT-PCR. Superoxide dismutase enzymatic activity and malondialdehyde (MDA) lipid peroxidation were used to assess the redox states. Results: Knockdown of one SOD isozyme led to the upregulation of the other SOD isozyme in our experiments. Successful knockdown of superoxide dismutase genes led to an increase in malondialdehyde (MDA) lipid peroxidation and a decrease in superoxide dismutase enzymatic activity in the ticks. Mn-SOD knockdown appeared to cause microbial overgrowth, whereas Cu/Zn-SOD depletions caused increases in the bacterial load of the guts and reductions in the salivary glands. Conclusion: Superoxide dismutase plays a functional role not only in the regulation of microbial communities, but also as a defense mechanism from damage caused by reactive oxygen species (ROS) within the tick. Knocking down of superoxide dismutase increased total oxidative stress in ticks shows the interplay between multiple SOD isozymes, and results in the transcriptional upregulation of the other isozyme. 1.6 From Microbes to Mosquitoes: An Interdisciplinary, Multi-Institutional Approach to Student Engagement in the Ecological

and Microbiological Sciences William H. Dees, Christopher G. Struchtemeyer, Caroline E. Hennigan, Caleb M. Ardizzone and Janet R. Woolman McNeese State University, Lake Charles, Louisiana

Background: The Microbes to Mosquitoes (MtM) Project is a three-pronged interdisciplinary, multi-institutional approach to student immersion into the agricultural, biological, chemical, environmental, and mathematical sciences, including the principles of innovation. This experiential learning project provides undergraduate students the opportunity to: 1) participate in undergraduate research, 2) visit facilities supporting scientific operations and to hear presentations about opportunities in a variety of science fields/disciplines, and 3) participate in local-global networking opportunities through attendance and participation at local, state and regional scientific and entrepreneur-based conferences. Methods: Through the MtM Project, we recruit freshman/sophomore level science majors who are struggling academically and who may be at risk of changing majors or leaving their academic pursuits altogether. During their time in the project, undergraduate students have opportunities to serve as field/laboratory research assistants and participate in a variety of professional scientific engagements (e.g., seminars, presentations by science professionals outside academe, and off-campus tours). Results: The outcomes of the MtM Project include: 1) retaining, advancing and placing students into professional scientific fields, and 2) nurturing a scientifically educated citizenry who will serve as the basis on which population growth, healthcare, safety and economic development (industry, government and business) in southwest Louisiana and beyond can flourish. Conclusion: The MtM Project will increase current collaborative arrangements with the local-global science community and allow students to apply course content from their undergraduate education. The project provides freshmen and first-semester sophomores with opportunities to personally engage with, learn about, experience, and access the world of science beyond the classroom.

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1.7 Presence of Vibrio vulnificus and V. parahaemolyticus in Louisiana Seafood. Richard Grabert and Raj Boopathy Nicholls State University, Thibodaux, Louisiana

Background: Louisiana seafood is a multi-million dollar industry. Every year there are few Vibrio associated outbreaks in Oyster or crab. Vibrio vulnificus and V. parahaemolyticus are the common gastroenteritis causing pathogen in estuarine environment. Vibro species are gram-negative halophilic bacteria that are commonly associated with bivalve such as oyster. Ingestion of undercooked shellfish or wound water exposure can result in gastroenteritis, wound infection, and septicemia. Louisiana state public health laboratories conduct routine surveillance for oysters for the presence of V. vulnificus, in water samples where oysters are grown, but monitoring of oysters and other seafood are not done. Global warming contributes to increase in water temperature, which promotes the growth of vibrio population in the Gulf of Mexico. There seems to be no continuous monitoring of various Vibrio species in Louisiana seafood such as crab, oyster, fish, and shrimp. Methods: This study was conducted in order to monitor vibrio pathogens in various Louisiana seafood. Various vibrio bacteria including V. vulnificus, V. parahaemolyticus, V. cholerae, and V. harveryi were monitored every week in various local seafood for nine months using vibrio specific chrom agar medium and by molecular method using various vibrio primers. Results: The results showed the presence of V. vulnificus and V. parahaemolyticus in oysters in every sampling period. V. cholerae was observed on specific occasions when the water was polluted with sewage and high nutrients. V. harveyi was present in many fish samples on different sampling events. Conclusion: This study shows the presence of various Vibrio species in Louisiana seafood. Routine monitoring of seafood is necessary to protect public health. The proper cooking of seafood will avoid any vibrio infection. 1.8 An Assessment of the Microbiological Impacts of Aerobic Septic Systems in Southwest Louisiana

Brittany Joseph, Daniel Gary, Tanner Trouth, Benjamin Clements, and Christopher G. Struchtemeyer. McNeese State University, Lake Charles, Louisiana

Background: Aerobic septic systems are commonly used to treat residential wastewater in many rural areas of the country. In spite of their importance and widespread use, very little is known about the microbial quality of the effluent from these systems. In this study, the microbial quality of effluent from eighteen aerobic septic systems in southwest Louisiana was evaluated by quantifying numbers of E. coli, fecal coliforms, and total bacteria. Methods: E. coli and fecal coliforms were quantified in effluent samples using ChromAgar ECC medium. The numbers of total bacteria in effluent samples were quantified using R2A agar. Results: The numbers of E. coli, fecal coliforms, and total bacteria varied from system to system and ranged from 1.8 x 101 to 6.3 x103 CFU/ml, 1.1 x 101 to 1.2 x 104 CFU/ml, and 1.8 x 103 to >1.5 x 105 CFU/ml, respectively. Conclusion: The results of this study clearly show that aerobic septic systems are releasing bacteria into the environment. The E. coli and fecal coliform concentrations in all effluent samples exceeded federal and state regulations that are commonly used to evaluate the quality of treated wastewater. Thus, it appears likely that aerobic septic systems negatively impact the environment and surrounding communities. 1.9 Comparison of antibiotic resistance in bacteria isolated from Atlantic Bottlenose Dolphins inhabiting Barataria Bay, LA,

and Sarasota Bay, FL Shuo Shen The University of Southern Mississippi, Gulf Coast Research Laboratory, Ocean Springs, Mississippi

Background: In the past few years, increasing numbers of marine pollution events have been reported. As a result, the habitat of marine life is under serious threat and marine mammals may be a good indicator for contaminant levels in marine waters and potential health effects on humans. In this study, we analyzed antibiotic resistance of 350 bacteria from wild bottlenose dolphins (Tursiops truncates). Widespread resistance was observed. Methods: Samples were collected in the capture-release dolphin health assessment conducted by National Oceanic and Atmospheric Administration, National Ocean Service scientists at Barataria Bay (BB), LA and Sarasota Bay (SB), FL. We used both culture based and molecular methods to identify the bacterial isolates. Twelve clinically relevant antibiotics were tested for their resistance with the Kirby-Bauer Disk diffusion method. We also used WITEK 2 AST to test the MIC for each bacteria. Results: After being tested with 12 clinically relevant antibiotics, widespread resistance was observed. In comparison, samples from BB exhibited a higher rate of resistance than that of SB. Also, there were more multi-resistant strains in the collections from BB. Conclusion: This study shows widespread antibiotic resistance of the sampling sites. Isolates from BB had obviously higher rate of antibiotic resistance than that from SB. The results in this study support a former research considering the bottlenose dolphin health condition difference between these two sampling sites. However, the causes for the differences observed need further elucidation.

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1.10 Antimicrobial Evaluation of Essential Oils Encapsulated in Latex Nanoparticles D. N. Amato1, D. V. Amato1, S. Walley1, O. Mavrodi2, D. Mavrodi2, and D. Patton1 (1) School of Polymers and High Performance Materials, The University of Southern Mississippi, Hattiesburg, USA; (2) Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS

Background: Essential oils have shown to have antimicrobial activity against a wide range of bacteria through a multitude of different mechanisms. In this research, two essential oils (EOs), thymol and carvacrol, were encapsulated in nanoparticles made via miniemulsion thiol-ene photopolymerization. Methods: We developed a synthetic methodology to tailor the antimicrobial efficacy of the encapsulated EO-nanoparticles. The antimicrobial activity of the EO-nanoparticles was evaluated against Escherichia coli O157-H7, E. coli ATCC 25922, Staphylococcus aureus RN 6390, Burkholderia cenocepacia K-56 and Bacillus subtilis ATCC 6633 through a combination of well diffusion, minimum inhibitory concentration (MIC) and time-kill assays. Results: Nanoparticles loaded with a combination of carvacrol and thymol showed greater antimicrobial efficacy than carvacrol-containing nanoparticles. The MIC values were consistent across all strains of bacteria except B. subtilis, which exhibited highest sensitive to the EO-nanoparticles. Conclusion: This study shows that the emulsion polymerization can be used as a methodology to create antimicrobial particles with encapsulated EO. The results show that nanoparticles loaded with a mixture of carvacrol and thymol are effective against both gram-positive and gram-negative bacteria. 1.11 Analysis of Rhizosphere Microbial Communities from the Verkhnekamsk Salt Mining Region of Russia

Ekaterina Korsakova1, Olga Mavrodi2, Alexey Nazarov1, Vitaly Demakov1, Elena Plotnikova1, and Dmitri Mavrodi2 (1) Institute of Ecology and Genetics of Microorganisms, Russian Academy of Sciences, Perm, Russia; (2) Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS

Background: To date, the impact of soil salinization on indigenous microbiota has been addressed in only a few studies focused on the microorganisms of bulk soil or macrotidal estruaries. The impact of soil salinization on rhizobacteria remains largely unexplored. This project focused on rhizosphere microbial communities associated with plants growing in areas of Russia that are polluted with potash mine tailings and industrial waste. Methods: We geotagged and sampled five polluted sites near town of Solikamsk, which is home to two mines owned by the world’s largest potash producer “Uralkali”. The effect of soil salinization and pollution on the rhizosphere microbial communities was characterized by conventional microbiological techniques and culture-independent approaches (Illumina sequencing of 16S rRNA tags and qPCR). Results: Results of the ongoing study revealed that plants growing in control areas and areas affected by potash mine tailings harbor similar amounts of heterotrophic rhizobacteria. However, the composition of rhizosphere microbial communities was very distinct and affected by potash mining waste. Rhizobacteria recovered near potash mines harbored genes for degradation of polycyclic aromatic hydrocarbons, which suggests their contribution to the natural remediation of polluted Solikamsk soils. Conclusion: Our results reveal the impact of soil salinization on rhizosphere microbial communities and the role rhizobacteria play in phytoremediation of areas affected by potash mining activities. 1.12 Biosynthesis of phenazine metabolites in Burkholderia spp.

Samuel Hendry, Olga Mavrodi, Alex Flynt, Dmitri Mavrodi Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS

Background: Phenazines (Phz) represent a large class of metabolites that are produced by diverse bacteria and exhibit unique redox properties and broad-spectrum antibiotic activity. In fluorescent pseudomonads, phenazines contribute to the development of surface biofilms and to the virulence or competitiveness of producing strains. Our study is focused on the biological functions of phenazines produced by species of the Burkholderia cepacia complex (Bcc). Members of the Bcc are ubiquitous in the environment and contain non-pathogenic species, as well as species that cause hospital-acquired infections and colonize the lungs of individuals with cystic fibrosis. Methods: We have assembled and characterized a collection of phenazine-producing Burkholderia strains, and identified Burkholderia lata as a model for further study. To date, we have generated several isogenic Phz mutants and characterized phenazine pathway in strain 383 using analytical techniques. Results: We demonstrated that in Burkholderia phz biosynthesis genes reside on IS composite transposons and form compact operons. Our functional analysis demonstrated that B. lata produces several different phenazines including 4,9-dihydroxyphenazine-1,6-dicarboxylic acid dimethylester. We evaluated the contribution of phenazine production to pathogenicity of Phz+ Burkholderia using onions and Drosophila melanogaster, and are currently testing the importance of phenazines for biofilm formation. Conclusion: Our results will help to better understand the role of phenazine compounds in an important group of bacterial pathogens, and will aid in the development of strategies to inhibit the production of phenazines and to neutralize their toxicity.

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1.13 Pseudomonas/Brachypodium as a Model System for Understanding Rhizosphere Plant-Microbe Interactions under Abiotic Stress Janiece Rawalt1, Olga Mavrodi1, Nicholas Rinderer1, Liam Elbourne2, Ian Paulsen2, David Weller3, Linda Thomashow3, and Dmitri Mavrodi1 (1) Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS; (2) Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia; (3) USDA-Agricultural Research Service, Pullman, WA

Background: Plant roots host bacterial communities that supply plants with nutrients, defend them against pathogens, and contribute to the plants’ ability to survive under abiotic stress. Stressed plants actively recruit and shape their beneficial microbiome, but molecular details of this process are still poorly defined. Here, we report the establishment of a new Pseudomonas/Brachypodium model system for studying plant-microbe communication in the rhizosphere under abiotic stress. Methods: We used greenhouse assays to evaluate the capacity of P. synxantha 2-79 to colonize roots of B. distachyon Bd21. We used Illumina and PacBio sequencing to produce a high-quality genome assembly of 2-79, and identified microbial pathways that may help 2-79 to persist in the rhizosphere under conditions of hypoxia and drought stress. Results: Analysis of plants colonized by 2-79 revealed rhizosphere populations in excess of log 6 CFU/g root, thus indicating that B. distachyon Bd21 is an excellent host plant for P. synxantha. The analysis of the 2-79 genome revealed a 59.3%-GC circular chromosome of 6.43 Mbp and encodes 5,729 proteins, 69 tRNAs, and 6 rRNA operons. We identified multiple traits that may help 2-79 to persist in the rhizosphere under conditions of water stress and hypoxia. Among these are pathways that are involved in the uptake of osmoprotectants, aggregation and formation of biofilms and production of redox-active phenazines. The strain also carries multiple traits that favor plant growth by modulating stress phytohormone levels and stimulating nutrient uptake and organ development. Conclusion: Our findings suggest that P. synxantha 2-79 and the model monocot plant Brachypodium represent a promising model system for studying molecular dialogue that enables plants and their associated microbes to cope with major abiotic stresses.

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Immunology & Virology, Poster Session 2.1 The Effect of Route and Anatomical Location On Antigen-Specific Immune Responses in the Respiratory Tract Following

Parenteral Immunization Sarah M. Baker, John D. Clements, Lisa A. Morici Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana

Background: Respiratory infections are responsible for 3.1 million deaths annually and are the leading cause of mortality in children under five, partially due to the lack of effective vaccines. It is currently unclear how immunization route and anatomical location influence protective immunity in mucosal tissues, such as the lung. Recent work by Clements and colleagues suggests that it may be possible to direct a protective immune response following parenteral immunization to mucosal tissue by inclusion of adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT). Therefore, the objective of this work was to examine the impact of route and location of immunization on the humoral and cell-mediated immune responses in the lung and draining lymph nodes using dmLT and tetanus toxoid. Methods: Groups of five C57BL/6 mice were given tetanus toxoid + dmLT or saline alone either intradermally at four different locations, intramuscularly on the hind leg, or subcutaneously between the shoulder blades. Tetanus toxoid was chosen as a model antigen. Doses were administered three times at three-week intervals. Two weeks after the final immunization, spleen, lymph nodes (inguinal, mediastinal, popliteal, cervical and axillary), bronchoalveolar lavage fluid (BALF), and serum were collected to assess antigen-specific antibody responses by ELISA and antigen-specific CD4+ T cells by intracellular cytokine staining (ICS) and flow cytometry. Results: Subcutaneous immunization and intradermal immunization on the right flank or the lower ventral surface induced the production of antigen-specific IFN-g and IL-17A-producing CD4+ T cells in the mediastinal lymph nodes. Intradermal immunization on the ventral surface of the mouse induced the production of significantly more IFN-γ-producing CD4+ T-cells in the spleen than other immunization routes and induced the highest concentration of antigen-specific serum IgG. Intradermal immunization, but not subcutaneous or intramuscular immunization, induced the production of antigen-specific IgG in the BALF. Conclusion: This study suggests that both immunization route and location should be evaluated when designing vaccination strategies against respiratory pathogens. Specifically, intradermal immunization on the ventral surface or right flank as opposed to other locations or routes may better stimulate protective cellular and humoral immune responses in the pulmonary tissue, as seen by an increase in antigen-specific cytokine-producing CD4+ T cells and BALF IgG. Future intradermal immunization studies with the adjuvant dmLT and protective antigens from the respiratory pathogen, Pseudomonas aeruginosa, will evaluate the utility of this vaccination strategy in achieving a protective immune response in the lung.

2.2 Improving the Annotation of Bovine herpesvirus-1 Genome Using Experimental Data Kaley Barber1, Joseph Reddy2, Allen Shak2, Allison Martin2, Bindu Nanduri2, Mariola J. Edelmann2 and Florencia Meyer1 (1) Department of Biochemistry & Molecular Biology, Entomology & Plant Pathology (2) Department of Basic Sciences, College of Veterinary medicine Mississippi State University

Background: Until recently the genome of Bovine Herpes Virus Type -1 (BHV-1) was a conglomeration of several different strains. In 2013, the genome was re-sequenced using only the Cooper strain of BHV-1. While more cohesive than the genome previously available, the new annotation was completed using traditional algorithms for the prediction of open reading frames (ORF) without incorporating experimental data. Using only computational methods when annotating a genome may be problematic as some ORFs which may encode for a viral protein, could be overlooked or terminated prematurely due to the limits of the specified parameters. The overall objective of this project is to either confirm or amend the annotation of the new Cooper strain genome of BHV-1 using viral proteins directly derived from infected cells. Methods: We have infected cells with BHV-1 with different lengths of time in order to determine what viral proteins are being expressed at different parts of the infection in the cell. These samples were sent to mass spectrometry in order to identify the expressed proteins. The proteins seen in the samples will provide experimental evidence of the proteins that make up the BHV-1 proteome and improve the annotation of BHV-1. Additionally, we have perfected the technique for producing a pure virus sample that is free of host proteins, to determine the identity of the structural proteins that make up the viral particle. Results: Viral peptides derived from 8 and 16 hours post infection cultured were obtained and mapped onto the currently annotated Cooper strain genome. Preliminary analysis shows a variety of peptides that map to known viral proteins as well as a few novel peptides. A purified virus sample that contains little cellular debris has been produced and will be analyzed by mass spectrometry soon along with a control Data from mass spectrometry was analyzed along with the current annotated genome using bioinformatics software such as Scaffold Artemis. Conclusion: Overall, we are able to contribute to the better understanding of the BHV-1 genome. Preliminary results suggest that we are able to confirm the existence of many previously known viral proteins. Our results also suggest the existence of peptides that map to intergenic regions on the BHV-1 genome.

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2.3 HCMV pUL93 Interacts with pUL77 to Determine Late Events in Virus Maturation Bernadette DeRussy1, Molly Boland2, James Conway3, and Ritesh Tandon1

University of Mississippi Medical Center, Jackson, Mississippi1

University of Alabama at Birmingham, Birmingham, Alabama2

The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania3

Background: Human cytomegalovirus (HCMV) pUL93, a putative tegument protein, and pUL77, a putative capsid protein, are both essential for virus growth, but their precise functions in the virus life cycle are unknown. Homologs of pUL93 and pUL77 in herpes simplex virus (HSV) interact to form a complex at capsid vertices known as the capsid vertex-specific component (CVSC), which likely stabilizes nucleocapsids during virus maturation and also aids in nuclear egress. Methods and Results: Kinetic analysis of eGFP-tagged pUL93 engineered in recombinant HCMV (UL93-eGFP-TB40/E-BAC) demonstrates that pUL93 expression begins as early as six hours post-infection and maintains steady state levels throughout infection. Immunofluorescence data indicates that pUL93 localizes to the nucleus of infected cells, the site of nucleocapsid assembly in herpesviruses. Cells infected with a UL93 stop mutant virus (UL93st-TB40/E-BAC) express both early and late viral proteins; however, only empty (A-) capsids and scaffold-containing (B-) capsids are present in the nucleus of infected cells based on transmission electron microscopy data. Viral genome containing (C-) capsids and mature virions are not detected in these cells. We also show that pUL93 directly interacts with pUL77 and this interaction is mediated by multiple regions in pUL93. High-resolution cryo-EM studies to localize pUL93 on nucleocapsids are in progress, and preliminary data show the presence of a CVSC like structure in HCMV capsids, similar to HSV capsids. Conclusions: Targeting of essential structural viral proteins holds great promise for the development of antivirals that would be highly specific, effective, and also less susceptible to the development of resistance because the mutation of a structural protein would likely compromise capsid assembly and robustness. Characterization of pUL93 and pUL77 will aid in the development of these promising antivirals in addition to advancing our understanding of HCMV maturation events, including nucleocapsid assembly, nuclear egress, tegument acquisition, envelopment, and egress. 2.4 Expression of platelet-derived growth factor receptor alpha in the embryonic bursa of Fabricius

Claire Fellman, Nikhil Nuthalapati, G. Todd Pharr Mississippi State University, Mississippi State, Mississippi

Background: The bursa of Fabricius is the primary lymphoid tissue responsible for the development of B cells in avian species. Bursal epithelium appears around embryonic day 4.5-7 and separates the mesenchyme from the lumen. Subsequently, CD45 expressing hematopoietic cells are recruited to the bursal epithelium and initiate the formation of epithelial buds which develop into lymphoid follicles that support B cell maturation. Signaling events triggering differentiation of the bursal epithelium and subsequent recruitment of CD45 expressing hematopoietic cells in the embryonic bursa are not well understood. Epithelial-mesenchymal interactions are important for the differentiation of epithelium during organogenesis, and receptor tyrosine kinases are known to have roles in cell migration, differentiation, and survival. Previous work in embryonic day 15 and 18 chicken bursas showed that platelet-derived growth factor receptor alpha (PDGFRα) had the highest expression of the 12 receptor kinases identified. Therefore, the hypothesis of the present research was that PDGFRα is expressed in the bursal mesenchyme, and that platelet-derived growth factor (PDGF) may be responsible for epithelial-mesenchymal interactions. Methods: Initially, western blotting was used to confirm the presence of PDGFRα in embryonic day 15, day 20, and 4 week post-hatch bursa extracts. Immunohistochemistry was then used to determine the location of PDGFRα expression at embryonic day 14, 15, and 20. Tissue sections were colabeled with antibodies specific for PDGFRα and either vimentin or cytokeratin. Results: Similar degrees of PDGFRα expression were found in embryonic day 15, 20 and 4 week post-hatch bursa extracts. Using immunohistochemistry, PDGFRα colocalized with vimentin, and was most strongly expressed in the mesenchyme surrounding the epithelial bud at embryonic day 14 and developing bursal follicle at embryonic day 20. Conclusion: This work suggests a role for PDGFRα in lymphoid follicle formation in the embryonic bursa of Fabricius. Future work will investigate sources of PDGF, mesenchymal genes affected by increased PDGF signaling, and the onset and duration of PDGFRα receptor expression.

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2.5 Host Cell Splicing Factors hnRNP A/B and hnRNP H3 are induced and re-localized after infection with Bovine Herpesvirus type 1. Kathryn Foster and Florencia Meyer Department of Biochemistry & Molecular Biology, Entomology & Plant Pathology Mississippi State University

Background: Bovine herpesvirus 1 (BHV-1) infection is one of the major contributing factors in the bovine respiratory disease. Once BHV-1 is established within the host, it causes a lifelong latent infection that can be reactivated during stressful conditions. The active infection leaves the host more susceptible to secondary bacterial infections which can lead to pneumonia, and can also cause abortions. The BHV-1 immediate early protein bICP0 is necessary for establishing an efficient active infection. Earlier work showed that bICP0 interacts with certain host cell proteins in infected cells, including hnRNP A/B and hnRNP H3. These two proteins are splicing factors that participate in mRNA metabolism and affect mRNA transport. Our objective is to understand what happens to these proteins after infection to determine the role they may play during productive infection. Methods: SDS-PAGE followed by Western Blot Analysis was used in order to visualize changes in protein concentration during infection with BHV-1. In addition, we used confocal microscopy to visualize changes in distribution of these cellular proteins. Results: Western Blot analysis showed a strong upregulation of both hnRNP A/B and hnRNP H3 at later times during infection. Confocal microscopy suggests a dramatic change in the distribution of hnRNP A/B as infection progresses that corresponds to a mostly nuclear distribution of bICP0. Conclusion: This study suggests that the BHV-1 immediate early protein bICP0 interacts with the host cell splicing factors hnRNP A/B and hnRNP H3 in order to establish an active infection. Further confocal microscopy experiments and differential lysis experiments will be performed in order to define more precisely the time and location of the movement of these cellular proteins. 2.6 Investigating the role of the minor capsid protein L2 in delivery of HPV16 genome to PML nuclear bodies

Lucile G.M. Guion, Malgorzata Bienkowska-Haba, Stephen A. DiGiuseppe, Wioleta Luszczek, Timothy R. Keiffer, and Martin Sapp. Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, and Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA.

Human Papillomaviruses (HPVs) are small, non-enveloped DNA tumor viruses associated with benign and malignant lesions of the skin and mucosa, specifically cervical, anogenital, and oropharyngeal carcinomas. Our laboratory focuses on the entry and trafficking of the oncogenic HPV type 16 (HPV16) in the target cells and how it establishes infection. Upon primary attachment and internalization, the major capsid protein L1 dissociates from the viral genome, which remains in complex with the minor capsid protein L2 within acidified endosomes and is transported to the trans-Golgi network (TGN) via retromer complexes. Following mitosis, the L2/viral DNA complex is released from the TGN, enters the nucleus, and associates with promyelocytic leukaemia nuclear bodies (PML NBs). Our data suggests that the viral genome is contained in a lipid vesicle with the majority of the L2 protein exposed on the cytosolic side of the membrane, allowing for interactions with numerous cytosolic host cell factors to facilitate these events. However, the exact mechanism of how the L2/viral DNA complex enters the nucleus is still unknown. PML protein and its SUMOylation are required for PML NBs to form. PML protein has been shown to be important for the expression of viral genomes delivered by HPV particles in a promoter-dependent fashion. The L2 protein displays several features that make it a putative substrate for post-translational modifications such as SUMOylation. Previously characterized motifs on the L2 protein include a SUMOylation site at Lys35, a SUMOylation interacting motif (SIM) at aa284-289, and a region downstream of the SIM at aa291-309. Previously published data and our preliminary findings suggest that these regions of L2 are required for infection. We hypothesized that the L2 protein associates with PML NBs, possibly through at least one of these domains, resulting in the retention of the viral DNA in the nucleus following mitosis. To test this hypothesis, pseudovirions, generated with L2 protein harboring mutations at these specific residues and encapsidating a reporter plasmid, will be used to determine the localization of the L2/viral DNA complex within the nucleus and its association with PML NBs during infection.

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2.7 Novel bioconjugated hybrid gold-graphene oxide nanoparticles for the treatment of cytomegalovirus infection Bernadette Derussy1, Madeline A. Archer1, Mohammad H. Hasan1, Karen Stokes2, Sudarson S. Sinha3, Paresh C. Ray3

and Ritesh Tandon1* 1Department of Microbiology and Immunology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA. 2 Department of Molecular and Cellular Physiology, Center for Cardiovascular Disease and Sciences, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130. 3Department of Chemistry and Biochemistry, Jackson State University, 1400 J.R Lynch Street, Jackson, MS 39217, USA.

Background: Human cytomegalovirus (HCMV) causes major health problems in neonates as well as in immunocompromised individuals. At present, a vaccine is not available for HCMV infection and the available antiviral drugs suffer from issues related to poor efficacy, side effects and antiviral resistance. Earlier, we reported the efficacy of bioconjugated gold nanoparticles (GNP) as an antiviral against HCMV (Derussy et al, 2014). Here, we report the synthesis and application of bioconjugated second-generation hybrid gold-graphene oxide nanoparticles (GOP) for the inhibition of mouse cytomegalovirus (MCMV) infection and the assessment of their safety as well as antiviral efficacy in mice. The GOP are superior to GNP because of high yield and low cost production, improved sensitivity to detection and enhanced photothermal cytotoxic abilities. Moreover, GOP are expected to be inert in vivo because carbon is their major constituent. Methods: We conjugated anti M55 (gB) monoclonal mouse antibody with the popcorn shaped GOP to produce M55-GOPop. MCMV-infected NIH-3T3 cells were treated with these conjugated nanoparticles to evaluate inhibition of viral replication. We also injected M55-GOP in BALB/c mice and compared with saline injected mice up to 14 days post infection to assess any adverse effect by comparing weight loss and health status. Then MCMV-infected mice were treated with M55-GOPop and weight was monitored over a period of 14 days and compared with mock treated mice to study the viral inhibition properties of M55-GOPop. Results: M55-GOPop conjugated nanoparticles block MCMV replication, virus-induced cytopathogenic effects and virus spread in cell culture without inducing cytotoxicity. M55-GOPop are tolerated well in BALB/c mice, as indicated by the comparable weight and health status of the M55-GOPop injected mice and the saline injected mice. MCMV-infected mice that are mock treated loose significant weight over a period of 14 days post infection; however, M55-GOPop treated mice continue to gain weight over this period indicating promising viral inhibition properties of M55-GOPop in vivo. Conclusion: In this study, we have characterized a potential antiviral strategy that specifically blocks cytomegalovirus infection in cell culture and has shown promising results in a mouse model. 2.8 Partial characterization of a chicken B-cell antigen recognized by monoclonal antibody EIV-E12

Nikhil Nuthalapati, Navatha Alugubelly, B. Felfoldi, I. Bódi, N. Fejszák, G. Todd Pharr, Imre Olah Mississippi State University, Mississippi State, Mississippi

Background: The antigen recognized by the EIV-E12 monoclonal antibody (mAb) is first detected in the mesenchyme of the chick embryo bursa of Fabricius on the precursor to the bursal secretory dendritic cell. Later in bursal embryonic development the B-cell precursors (prebursal stem cells) which enter the developing follicle express the antigen recognized by mAb EIV-E12. The goal of this project is to characterize the antigen recognized by mAb EIV-E12. Methods: Initially we evaluated the reactivity of mAb EIV-E12 with immunohistochemistry staining of primary and secondary lymphoid tissues of posthatch chickens. Western blotting with bursal protein extracts incubated with a mixture of protein deglycosylases was then used to determine if the B-cell antigen recognized by mAb EIV-E12 is glycoprotein. Results: The antigen recognized by mAb EIV-E12 is expressed by developing B-cells in the posthatch bursa and peripheral B-cells which colonize the spleen and the thymus. In the thymic cortex stellate – shaped cells were also recognized by mAb EIV-E12. The deglycosylated bursal proteins showed reduced reactivity to mAb EIV-E12, suggesting that the epitope recognized by mAb EIV-E12 may consist of a carbohydrate group. Conclusion: The 200 kDa antigen recognized by mAb EIV-E12 is a glycoprotein and possibly represents a novel chicken pan - B-cell antigen that may be shared with non-lymphoid cells in the thymic cortex.

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2.9 Evaluating vector, route, and dose effects on the adjuvant capacity and safety of flagellin when encoded in DNA or adenovirus vaccines Hamada F. Rady, Guixiang Dai, and Alistair J. Ramsay Departments of Microbiology, Immunology and Parasitology and the Louisiana Vaccine Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA.

Background: We have previously evaluated the adjuvant capacity of flagellin when encoded in heterologous gene-based DNA/Ad prime-boost immunization and found that flagellin has differential and route-dependent for both T and B cell responses, particularly it was included only in the DNA vaccine prime, but toxic side effects were observed when delivered in adenovirus (Ad) boosting vectors, particularly via the intranasal (IN) route. Here, we wanted to study the mechanisms underlying the route-dependent and toxic effects of flagellin when delivered in Ad vaccine in comparison to DNA-encoded flagellin. Methods and Results: Intramuscular (IM) co-immunization with Ad-Ag85B and serial dilutions of Ad-flagellin led to enhanced Ag85B-specific CD4+ and CD8+ T cell responses compared to immunization with Ad-Ag85B alone. In contrast, IN co-immunization led to decreases in mucosal CD4+ and CD8+ T cell responses. By either route, flagellin induced a dose-dependent transient weight loss and strongly increased inflammatory mediators in the circulation at 24hr post-immunization. Levels of IL-12p40 were higher when Ad-flagellin was given IM but significantly lower when it was given IN compared to immunization with Ad-Ag85B alone. In addition, IN immunization with Ad-flagellin strongly increased pulmonary inflammatory responses and caused lung injury. DNA-encoded flagellin given to mice via the IM route enhanced antigen-specific splenic CD4+ and CD8+ T cell responses without causing any noticeable morbidity. In footpad immunization model, DNA-flagellin mediated potent increases in early innate inflammatory mediators measured in the popliteal LNs, while Ad-flagellin induced robust responses at earlier time point which could underpin the morbidity seen when this vector was given alone or as a booster via either IM or IN route. Conclusion: Downregulation of IL-12p40 along with the strong systemic and pulmonary inflammation may explain the reduced T cell immunity seen when Ad-flagellin was given IN. On the other hand, DNA-flagellin was capable of inducing potent, but not robust, innate inflammatory and adaptive responses without causing any toxic effects. This may explain the superior cellular and humoral responses induced safely when DNA-flagellin was used as a prime for subsequent boosting with Ad vector encoding the same vaccine antigen without flagellin. 2.10 Identification and Characterization of Interleukin-6 in Channel Catfish, Ictalurus punctatus

David Spencer, Erin Taylor, Melanie Wilson, and Eva Bengtén University of Mississippi Medical Center

Background: Cytokines are small (5-30 kDa) soluble proteins responsible for immune cell to cell communications. Interleukin-6 (IL-6) is an important pro-inflammatory cytokine in mammals and has been identified in teleost fish, including rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), but has yet to be functionally characterized in teleosts. Here we identify and report preliminary functional studies of IL-6 in channel catfish. Methods: An IL-6 catfish homolog was identified by NCBI’s BLAST searches of the catfish EST databases using previously published teleost sequences. Cell line expression of IL-6 was analyzed using RT-PCR. In order to perform functional studies, the putative catfish IL-6 sequence was amplified by RT-PCR, cloned into the pET100 vector that incorporates a N-terminal 6X-histidine tag, and expressed as recombinant (r) protein in E. coli. Purified rIL-6 was treated for endotoxin removal, which was confirmed by limulus assays. Catfish peripheral blood leukocytes (PBL) were stimulated with purified rIL-6 and analyzed by flow cytometry for immunoglobulin cell surface expression. Results: A putative IL-6 transcript was identified that encodes a mature protein of 213 amino acids and contains six instability motifs in the 3' untranslated region (UTR). The predicted protein contains four characteristic alpha helices, possesses the distinct IL-6 family consensus pattern (C-x(9)-C-x(6)-G-L-x(2)-[F/Y]-x(3)-L), and contains two conserved cysteine residues that in mammals form the disulfide bond necessary for cytokine function. Expression of IL-6 was detected in catfish mixed leukocyte cultures, but not in clonal B, T, or macrophage cell lines. Functional studies are ongoing. Conclusion: Interleukin-6 is present in channel catfish and likely plays a role in B cell activation. Functional studies may reveal IL-6 to promote antibody-mediated immune responses in teleosts.

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2.11 Regulation of Paracrine Signaling Factors by HPV16 Brittany Woodby, Jason Bodily LSU Health Sciences, Shreveport, Louisiana

Background: In addition to their role as a source of growth factors for the epithelium, fibroblasts also participate in the stromal innate immune response by producing interferons (IFNs) and other cytokines. Human papillomavirus type 16 (HPV16) is known to regulate IFNs and interferon-stimulated genes (ISGs) within infected keratinocytes, but it is unknown how HPV affects other cell types in the stromal microenvironment, such as fibroblasts, that could affect persistence of the virus. Methods: We used organotypic raft culture and RT-qPCR, as wells as recombinant growth factor treatment of human keratinocytes. Results: Viral transcripts were suppressed in organtopyic epidermal cultures containing human fibroblasts compared to mouse fibroblasts. IFN production, specifically IFN-κ, in human foreskin keratinocytes (HFKs) and HPV16 rafts containing human fibroblasts was increased as compared to rafts containing mouse fibroblasts. ISG production in keratinocytes grown in raft culture followed the same trends in upregulation as those of IFN-κ but not other IFN types. Previous data generated in the lab showed that HPV upregulates growth factors in stromal cells. To determine if there was a connection between the upregulation of growth factors by the virus and downregulation of IFNs, we tested the effects of various growth factors on IFNs and observed that hepatocyte growth factor (HGF), a factor that regulates wound healing and cell movement, could downregulate levels of IFN-κ transcripts in a dose-dependent manner. We examined differences in HGF production between human and mouse fibroblasts co-cultured with HPV16 rafts and found that HGF was upregulated by HPV in the mouse fibroblasts but inhibited in the human fibroblasts. Conclusion: Our findings suggest that HPV regulates communication between keratinocytes and fibroblasts by altering expression of paracrine signaling factors, presumably to increase the likelihood of a persistent infection. 2.12 The Delta Peptide region of Ebola Virus Soluble Glycoprotein (sGP) is a Potent Viroporin.

Jing He1, Lilia Melnik2, Charlie G. Starr1, Taylor Fuselier1, Greg Wiedman3, Kalina Hristova3, William Gallaher4, Robert Garry2 and William Wimley1 1Biochemistry and Molecular Biology and 2Microbiology and Immunology, Tulane University Medical Center, New Orleans, LA; 3Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland; and 4Mockingbird Nature Research Group, Pearl River, Louisiana

Background: Ebola virus in humans, the Zaire type (EBOV), now in West Africa, has 60% mortality and diarrhea as a significant factor. The Reston type (RESTV) does not cause human disease. The basis of virulence is unknown, though localized in the viral glycoprotein gene. In EBOV, we noted in the Delta peptide of sGP a peptide with high similarity to a peptide in the diarrhea-inducing viroporin of Rotavirus. Methods: Synthetic peptides derived from Delta peptides of EBOV and RESTV were tested for pore formation in model membranes and mammalian cells using dye mobility of CAMBRO (red) and SYTOX (green) by fluorescence spectroscopy, and small ion electrochemical impedance spectroscopy. Results: A 23 amino acid sequence derived from EBOV Delta peptide permeabilized model membranes or mammalian cells to larger (300 Da) vital dyes with 50% endpoint of 25 uM, while that of RESTV did not. Permeability to small ions (K+, Na+) was induced on contact, by peptides of both EBOV and RESTV at the 50% endpoint of 10 uM. Delta peptide of both Ebola strains alters permeability to small ions, but only the peptide derived from virulent Ebola appears to cause larger, cytotoxic pores. Conclusion: Ebola virus Delta peptide contains a potent viroporin, permeabilizing nucleated mammalian cells, and potently increasing ion permeability across synthetic lipid bilayers. Like other enterotoxic viroporins, it likely disrupts cells by dysregulation of ion permeability.

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Pathogenic Microbiology, Poster Session 3.1 Evaluation of Baron Based Molecules in Listeria Monocytogenes LapB Protein

Ali Akgul, Nawar Al-Janabi, Mark L. Lawrence, Attila Karsi College of Veterinary Medicine, Mississippi State University, Mississippi

Listeria monocytogenes is a Gram-positive food-borne pathogen and the causative wall surface anchor protein is present only in high-risk serovars 4b, 1/2a, 1/2b, 1/2c. Effects of LapB gene on collagen binding and virulence have been evaluated by using a mouse model, and the mutant had attenuated virulence in the host. Here, we present the role of LapB protein in catfish fillet attachment using boron based small molecules targeting active site of LapB protein. To achieve this, we developed a fillet attachment model and tested fillet attachment characteristics of wild type (LmF2365) and mutant (LmF2365ΔlapB) L. monocytogenes. Later, attachment experiments in presence of small molecules were conducted to block wild type listeria attachment to catfish fillet. The three experimental groups were muscle + LmF2365 + small molecules (treatment), muscle + LmF2365 (positive control), and muscle + LmF2365ΔlapB (negative control). Results indicated that deletion of LapB protein attenuated fillet binding capacity of LmF2365ΔlapB in catfish fillet compared to LmF2365. Seven different small molecules reduced attachment of LmF2365 to catfish fillet. Interestingly, four of these small molecules blocked growth of LmF2365ΔlapB. 3.2 The use of bacteriophage to control biofilm formation of Vibrio parahaemolyticus in aquaculture

Asheligh Aubin, Corey Melancon, Angela Corbin Nicholls State University, Thibodaux, LA

Background: With an ever increasing demand for fresh seafood, shrimp aquaculture has become an important industry. Extensive measures are taken to farm the shrimp for human consumption. No exchange of water and the use of probiotics are techniques used to help prevent infections in aquaculture tanks. Shrimp feed is used to develop biofilm and biofloc for shrimp as addition nutrient sources to increase productivity. In September 2014, a Vibriosis outbreak forced an aquaculture facility to harvest their shrimp weeks early due to high shrimp mortality. . Our objective was to inhibit the biofilm growth of Vibrio parahaemolyticus in a pure culture using bacteriophage. Methods: The bacterial pathogen was previously isolated from morbid shrimp hemolymph. We isolated species specific bacteriophage from oyster juice in media at 30m PPT, the salinity used at the aquaculture facility. Over timed intervals pathogen inoculated broth with and without bacteriophage were tested for biofilm and biofloc mass using a colorimetric method. Results: There was a statistical difference between the biomass of the untreated and bacteriophage treated biofilm production over a 72 hour trial. Conclusion: The use of the phage significantly decreased the amount of biofilm that was produced by the organism throughout the experiment. This finding suggests that the use of phage could possibly be used as a method to controlling biofilm. 3.3 Observing The Mold-Specific Gene, M46 Expression during the Morphological Shift from Mold to Yeast and Yeast to

Mold in Histoplasma capsulatum Ashly Claiborne¹,Glen Shearer²,Davida Crossley³, ¹Alcorn State University, Alcorn State MS, ²The University of Southern Mississippi, Hattiesburg, MS

Background: Histoplasma capsulatum (Hc) is a dimorphic fungus that is found in contaminated soils. It is found in bird and bat excrements. In the soil, it exists mold at 25 ˚C. Once the soil is disturbed, and the Hc spores are released and inhaled, it is converted into yeast at 37˚C. It is the yeast form, which causes the upper respiratory infection histoplasmosis. This study focuses on the mold-specific M46 gene. Mold specific genes are genes that are only expressed in the mold phase and not the yeast phase. The objective of this study is to determine when the M46 gene is expressed when Hc cells are converting from mold to yeast, and yeast to mold. Methods: Total RNA was extracted from M46 expressing strain G186AS as the Hc cells were shifting from mold to yeast, and yeast to mold for ten days. Before each extraction, images of the morphological shift were taken via microscope (MicroMaster Microscope). Results: Northern blot revealed a decrease of expression when Hc cells are converting from mold to yeast and an increase of expression when cells are converting from yeast to mold. These results confirmed that M46 is mold specific and does not express when the cells are completely yeast. The function of M46 is currently unknown. The time in which M46 is expressed further aids in characterizing the gene. Conclusion: This study focuses on the mold specific M46 gene. This project objective is to determine when M46 expresses as cells are shifting from mold to yeast, and yeast to mold. The results show that as Hc cells are shifting from mold to yeast M46 expression decreases, and as cells are shifting from yeast to mold M46 expression increases. Future work will include confirming the northern blot results with qRT-PCR.

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3.4 Determining the Role of Alterations to the Cell Membrane Lipid Components in Improving Survival Against Bile Induced damage in Listeria monocytogenes Dominique N. Clark, Oindrila Paul, Jessica G. Wilson, and Janet R. Donaldson Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762

Background: Listeria monocytogenes is a gram-positive foodborne pathogen responsible for the disease listeriosis, which is the third leading cause of foodborne death in the United States. Following consumption of contaminated food, the pathogen resists stressors encountered within the gastrointestinal (GI) tract and migrates across the intestinal, blood-brain, and feto-placental barrier. One of the stressors encountered within the GI tract is bile. Bile is mostly composed of bile salts, which certain strains of Listeria are resistant. Bile induces damage to the cell membrane and DNA. Previous work from our laboratory indicated alterations in the composition of the membrane phospholipids occurred following bile exposure. Therefore, the hypothesis of this project was that certain strains of L. monocytogenes incorporate exogenous lipids and this alteration of the cell membrane improves resistance against bile. Methods: Strains of L. monocytogenes used in the study were F2365 (serovar 4b), 10403S (serovar 1/2a), and HCC23 (serovar 4a). Strains were cultured under either aerobic or anaerobic conditions to mid-logarithmic phase, then pre-treated with a lipid mixture (Sigma Aldrich; commercial mix contains palmitic acid, oleic acid, stearic acid, linoleic acid and others) for 1 h at 37°C. Cells were then exposed to either 0% or 5% porcine bile for 1 h and viability was assessed by plate counts. A minimum of three independent replicates was performed. Results: When exposed to 5% bile, HCC23 and 10403S strains pre-exposed to the lipid mix had increase in survival, whereas F2365 remained unaffected under aerobic conditions. Interestingly, this same impact on survival was not observed under anaerobic conditions. Pre-exposure to the lipid mix did not improve survival of F2365, 10403S, or HCC23 following bile exposure. Conclusions: Alterations to the cell membrane through incorporation of exogenous lipids improves bile survival only under aerobic conditions, and does so in a strain dependent manner. Additional research is needed to determine the mechanism by L. monocytogenes is resistant to bile induced membrane damage under anaerobic conditions. 3.5 The Contribution of Pyruvate Oxidase to Pneumolysin Release in Streptococcus pneumoniae

Ridge C. Dabbs1, Joseph C. Bryant1, Jason W. Rosch2, Larry S. McDaniel3, Justin A. Thornton1 1Mississippi State University. Mississippi State, MS. 2St. Jude Children’s Research Hospital. Memphis, TN. 3University of Mississippi Medical Center, Jackson, MS.

Background: Streptococcus pneumoniae (pneumococcus) is an important human pathogen causing infections such as pneumonia, meningitis, and otitis media, affecting primarily young children and the elderly worldwide. Pneumococcus produces a pore-forming cytotoxin, pneumolysin (PLY), which is one of its key virulence factors. Despite being a catalase negative organism, the pneumococcus produces up to millimolar concentrations of hydrogen peroxide through the activity of the enzyme pyruvate oxidase (SpxB). SpxB is considered a virulence factor of the organism, as mutants lacking the gene display attenuated virulence in vivo. The goal of this study is to investigate the correlation between PLY release and the activity of SpxB. Methods: We used a colorimetric hydrogen peroxide assay to quantitate the amount of H2O2 produced by strains T4R, WU2, AW267, and T4 and isogenic mutants lacking spxB (ΔSpxB) at a mid-log phase of growth. ∆SpxB mutants were complemented using a pNE-1 pneumococcal shuttle vector. Western blotting, along with peroxide values, were used to compare the relative amount peroxide produced and PLY released between wild-type and ΔSpxB mutants. Dot blotting was used to quantitate PLY in the supernatants of 15 clinical isolates at a mid-log phase of growth, these PLY values were graphed against H2O2 values quantitated via colorimetric assay. A549 human epithelial cells were exposed to sterile supernatants of wild type, ΔSpxB mutants, and a complemented ∆spxB mutant. The cells were assessed for loss of viability by propidium iodide staining as quantitated using flow cytometry. A hemolytic assay was used as a second method of assessing PLY release. Results: When exposed to sterile wild-type supernatant, a significant loss of cell viability was seen in A549 cells, whereas ∆SpxB supernatant failed to affect viability. Supernatant of the complemented mutant replicated the loss of viability observed with the wild-type. Deletion of spxB almost entirely eliminated H2O2 production and complementation restored H2O2 production. We observed a significant reduction in the amount of PLY in the supernatant observed by western blot upon deletion of spxB in AW267, WU2, and T4 (p<0.005), as well as a significant reduction in PLY release in T4R (p<0.05). Furthermore, we observed a significant reduction in the amount of PLY released when WU2 was treated with 10µg of exogenous catalase (p<0.05). A significant correlation was observed between H2O2 production and PLY released in a panel of clinical isolates (p<0.05, r2=0.3167). Finally, complementation of spxB to T4R∆spxB and T4∆spxB yielded a significant increase in PLY released into the supernatant versus the mutant alone (p<0.005 and p<0.0005, respectively). Conclusion: Based on our findings, an apparent connection exists between the activity of SpxB and the ability of the pneumococcus to release PLY into the extracellular space. We demonstrated that loss of SpxB reduces PLY release. Furthermore, complementation of the gene restores PLY release. SpxB-induced PLY release correlates with the increased cell toxicity. We hypothesize that this release could be related to the production of H2O2 alone, but could also be related to other byproducts of SpxB, such as acetate. Additionally, sxpB utilization could be a niche-oriented method of modulating PLY release depending on the oxygen availability of infection site. Finally, it is possible that this mechanism could be generalized for other cryptically secreted proteins in the pneumococcus.

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3.6 Emerging Infectious Diseases and Entomological Intelligence: A Biosurveillance Initiative

William H. Dees1, Desmond H. Foley2, David B. Pecor2, Douglas A. Burkett2, Leopoldo M. Rueda2, Richard C. Wilkerson2, and Caleb M. Ardizzone1

1McNeese State University, Lake Charles, Louisiana, and 2Walter Reed Biosystematics Unit/Smithsonian Institution, Washington, DC

Background: With world-wide vector biosurveillance efforts by governments, institutions and citizen scientists to monitor and forecast vector-borne disease risks, there is a need for an easily accessible spatial data repository enabling users to dynamically view the factors influencing risks of vector-borne diseases. VectorMap (www.vectormap.org) is an online repository for biosurveillance data that houses data management tools for uploading and managing surveillance information as well as spatial data critical for assessing disease risks. Methods: VectorMap is a web-based resource for reviewing and depositing collection records of mosquitoes, sand flies, ticks, fleas, mites, animal hosts, and disease pathogens from around the world. This resource contains distribution models of many components associated with vector-borne diseases, including ecological niche and disease risk models related to vector ecology. Results: Users have access to a plethora of information including 450-plus ecological niche models for vector species world-wide, climate data, slide presentations on current vector-borne diseases, and other resources, including links to the Centers for Disease Control and Prevention, World Health Organization, and Walter Reed Biosystematics Unit (WRBU)/Smithsonian Institution. Conclusion: The VectorMap Team at WRBU provides tools and support for publishing accurate and precise location data, vector identification and associated environmental data, and pathogen testing results, and contributes to global knowledge of vector-borne disease threats by collaborating with individual researchers and institutions around the world. For questions regarding VectorMap or if you wish to have your surveillance data (georeferenced/non-georeferenced data) posted to VectorMap, please email the VectorMap Team at mosquitomap@si.edu. 3.7 The Emerging Pathogen Nonencapsulated Streptococcus pneuominae: the Case of a 2-year-old with Chronic

Adenoiditis Cheshil Dixit, Lance E. Keller, Larry S. McDaniel The University of Mississippi Medical Center, Jackson, Mississippi

Background: Streptococcus pneumoniae is an important human pathogen. To cause disease, it must first colonize the nasopharynx. The widespread use of pneumococcal-conjugate vaccines which target the capsular polysaccharide has led to decreased nasopharyngeal carriage of vaccine serotypes, but a concomitant increase in carriage of non-vaccine serotypes and nonencapsulated Streptococcus pneumoniae (NESp). Some NESp express Pneumococcal Surface Protein K (PspK), a virulence factor previously shown to contribute to nasopharyngeal adherence. Here we present the case of a 2-year-old child with recurrent adenoiditis caused by a PspK+ NESp. Methods: We examined the strain, C144.66, isolated from the patient’s adenoid. We characterized the antibiotic resistance displayed by the strain. Via polymerase chain reaction, the presence of genotypic PspK was accessed. Expression of PspK was analyzed through Western blotting and FACS analysis. Multi-locus sequence typing was used for pneumococcal genotyping. Results: C144.66 was found to be resistant to erythromycin and displayed intermediate resistance to penicillin and trimethoprim/sulfamethoxazole. Genomic C144.66 was found to have PspK in place of the capsule locus. Additionally, PspK expression was confirmed by Western blotting. Conclusion: NESp are a growing concern as an emerging human pathogen, as current pneumococcal vaccines do not confer immunity against them. This inability to vaccinate against NESp may result in increased carriage and associated pathology.

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3.8 Effect of Pneumococcal H2O2 on Epithelial Cell Gene Expression Karien N. Dixon1, Percus Z. Mody3, Jason W. Rosch2, and Justin A. Thornton3 1Tougaloo College, Tougaloo, Mississippi 2 St. Jude Children’s Research Hospital, Memphis, TN 3Mississippi State University, Mississippi State, MS

Background: Streptococcus pneumoniae (pneumococcus) is a bacteria responsible for invasive and noninvasive diseases such as pneumonia, meningitis, and otitis media. This pathogen produces hydrogen peroxide via pyruvate oxidase (SpxB), which is known to be a virulence factor. Pneumococcal hydrogen peroxide is known to act as a messenger, setting in motion intracellular signals, sequentially, activating and upregulating certain genes. In this study, we quantitate changes in stress gene expressions to wild type or SpxB-negative strains. Methods: We exposed A549 cells to either media alone, a wild type strain (T4R), or an isogenic mutant lacking pyruvate oxidase (ΔSpxB) for 30 min or 1 hr at 37C. Total RNA was then isolated and used for microarray analysis, and results were verified by qRT-PCR. Beta actin served as an internal housekeeping gene for qRT-PCR. Results: The microarray identified several genes that were differentially expressed in response to production of hydrogen peroxide. The genes FOS, IL-8, N4RA2, and EGR-1 were found to be up-regulated. Quantitative PCR verified that the A549 cells that were exposed to the wild type strain had greater gene expression than those cells exposed to the strain lacking pyruvate oxidase at both 30 min and 1hr post-exposure. The effect of peroxide on gene expression was time-dependent with greater fold-changes seen at 1hr for both the wild-type and mutant strain. The fold changes for cells exposed to bacteria for 30 min were: Fos (10.7-fold vs 5.3-fold), IL-8 (1.3-fold vs 1-fold), EGR1 (5.9-fold vs 2.9-fold), and NR4A2 (7.2-fold vs 4.2-fold) for T4R and ΔSpxB, respectively. The fold changes for cells exposed to bacteria for 1 hr were: Fos (17.2-fold vs 9-fold), IL-8 (4.2-fold vs 2.9-fold), EGR1 (12.3-fold vs 6.4-fold), NR4A2 (14.4-fold vs 8.6-fold) for T4R and ΔSpxB, respectively. Conclusion: This study shows that pneumococcal hydrogen peroxide causes a greater expression of stress response genes than hydrogen peroxide negative strains. While it is known that production of hydrogen peroxide by pneumococcus is detrimental to the host, identifying processes involved in the response to this virulence factor is essential. 3.9 Role of msaABCR in Antibiotic Susceptibility during Biofilm Development in Staphylococcus aureus

Latoyia Downs, Bina L. Jayana, Gyan S. Sahukhal and Mohamed O. Elasri Department of Biological Sciences, The University of Southern Mississippi

Backgrounds: Community-acquired, methicillin-resistant Staphylococcus aureus strains cause severe infections among healthy individuals. Many of these infections are recalcitrant to antimicrobial therapy. Formation of biofilm within the host tissue and indwelling medical devices is one major contributing factor for their resistance to treatment. Biofilm formation is a complicated processes that is regulated by several global regulators. One of those global regulators is the msaABCR operon that is involved in biofilm development, antibiotic resistance and cell death in S. aureus. In this study we tested the impact of msaABCR deletion in the susceptibility of several antibiotics using three different biofilm assays. Method: We used minimum biofilm eradication concentration (MBEC) assay, in-vitro catheter based assay, and microfluidics BioFlux system to monitor the effect different antibiotics that are mainly used to treat the biofilm associated infections in S. aureus. We used daptomycin, linezolids, clindamycin, vancomycin, rifampicin and gentamycin. We also tested the combination of rifampicin and gentamycin with daptomycin, linezolids and vancomycin to make this study more relevant to clinical settings. Results: Deletion of msaABCR had no impact on the daptomycin, linezolids, clindamycin, and gentamycin susceptibility in planktonic conditions, but showed at least two fold reduction in sensitivity to rifampicin and vancomyin. However, all these antibiotics alone or in combination showed significant effect on biofilm formation in the msaABCR deletion mutant. The MBEC assay showed statistically significant effect on the mutant’s biofilm (18 fold) compared to the wild type in the presence of 40 µg/ml of daptomycin. Daptomycin concentrations above 40 µg/ml reduced the mutant’s biofilm to an undetectable level, whereas concentrations up to 160 µg/ml did not affect the wild type biofilm. None of the in vitro catheters colonized with WT strain were cleared even after continuous exposure of daptomycin (40 µg/ml) for 4 days, whereas the mutant’s biofilm was completely cleared by day 4. Both wild type and mutant strains showed increased susceptibility to antibiotics when used in combination. The wild type strain showed 32-fold and 8-fold increased susceptibility in daptomycin and rifampicin when used in combination relative to individual use. The mutant showed an even higher increase with 80-fold for daptomycin and 16-fold for rifampicin. Using microfluidic bioflux system, we found that the mutant biofilm was effectively cleared in the presence of 5 µg/ml daptomycin and 0.3 µg/ml rifampicin, which is clinically relevant in terms of treatment using these antibiotics. Likewise, the mutant’s biofilm was significantly reduced to an undetectable level, when treated with daptomycin-gentamycin, linezolid-gentamycin, and linezolid-rifampicin. Conclusion: In conclusion, since deletion of msaABCR operon limits the biofilm formation in S. aureus, this limitation was correlated with the increased susceptibility with different antibiotics treatment using all in-vitro models of biofilm formation. These findings will help with the development of future anti-biofilm therapeutics.

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3.10 Virulence of the novel species Streptococcus tigurinus in a Galleria mellonella model Amal Fadil, Nathan A Tullos Mississippi College, Clinton, Mississippi

Background: Streptococcus tigurinus has recently been reclassified and separated as a novel species from Streptococcus mitis. Though S. tigurinus has been shown to cause fatal bacteremic human disease little is known about its virulence or pathogenesis. Using a Galleria mellonella, wax moth model we have characterized its lethality, growth and induction immune response during systemic infection. Methods: Fifth instar Galleria mellonella larva were inoculated with 5, 6, and 7 LogCFU/Larva of S. tigurinus (ATCC 15914) and observed over 24 hours. Bacterial virulence and growth were determined by lethal dose 50 (LD50) and colony forming unit (CFU) analysis at 24 hours p.i. The innate immune response was measured through determining the activation of the melanin deposition pathway by two assays. First the degree of larval pigmentation was determined using gray scale value photographic analysis. Second, the activity of hemolymph phenoloxidase, a key enzyme in the melanin pathway, was calculated at 24 hours p.i. Results: The LD50 for S. tigurinus at 24 hours p.i. was calculated to be 3.97x105 per larva. CFU analysis revealed growth of bacteria over 24 hours with larva infected with 6 LogCFU/Larva reaching 8.96 LogCFU/Larva (P<0.05). All three groups showed significant increase in pigmentation according to mean gray scale value analysis (P<0.05). Additionally, all three infection groups showed significant reduction in phenoloxidase activity compared to mock infected larva, indicating immune disruption. Conclusion: This study is the first to examine S. tigurinus virulence and pathogenesis in a G. mellonella model, and only the second study examining S. tigurinus virulence in vivo. We have demonstrated the ability of S. tigurinus to grow rapidly and cause mortality, as well as induce powerful innate response in this systemic model of infection. This study adds to the small but growing body of work related to this newly emerging human pathogen. 3.11 Susceptibility of Neisseria gonorrhoeae to the Plant Compound Neem In Vitro and In Vivo

Cathryn A. Frey1, Afrin Begum2, Jatinder Singh2, William H. Dees1, and Ann E. Jerse2 1McNeese State University, Lake Charles, Louisiana, and 2Uniformed Services University of the Health Sciences, Bethesda, Maryland

Background: Gonorrhea is the second most frequent notifiable infection in the United States and can impact reproductive and neonatal health. Due to the rapid emergence of antibiotic resistance in Neisseria gonorrhoeae, novel treatments, such as natural remedies, have become a main focus of research. We tested the effect of an extract from the neem tree (Azadirachta indica) that has been studied for years due to its ability to kill pathogens. Methods: We conducted in vitro tests to determine the effect of the neem extract against antibiotic-resistant and sensitive strains of Neisseria gonorrhoeae. For in vivo tests, we administered neem, its vehicle (negative control) or nonoxynol-9 (positive control) vaginally to female BALB/c mice prior to inoculation with N. gonorrhoeae strain FA1090. Vaginal swabs were cultured at days 1, 2, and 5 post-inoculation. Results: Neisseria gonorrhoeae strains MS11, FA1090, HO41 and F89 were sensitive to the neem extract by agar dilution and broth culture-based assays. Results from in vivo studies showed nonoxynol-9 effectively prevented gonococcal colonization; neem showed some evidence of effectiveness in that 2 of 9 mice were culture-negative on day 1 post-inoculation and the numbers of gonococci recovered were lower than that seen in mice treated with the vehicle alone. Conclusion: This study shows that N. gonorrhoeae is sensitive to the neem extract in vitro. Results from the mouse infection model show that the neem extract may reduce colonization. The use of neem as an antimicrobial against N. gonorrhoeae warrants further study. A repeat study is planned in which a higher dose of neem will be used; testing of individual factors within the neem extract also will be conducted.

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3.13 The Overexpression of the Mold Specific Gene, MS88, in the Pathogenic Dimorphic Fungi Histoplasma capsulatum

Mariah Lloyd¹, Glenmore Shearer Jr.², Davida Crossley², Center of Biotechnology, Alcorn State University, Lorman, MS¹ Biological Sciences, University of Southern Mississippi, Hattiesburg, MS²

Background: Histoplasma capsulatum (Hc) is the causative agent of the upper respitory infection, histoplasmosis. This organism can exist as a multicellular mold at 25˚C, or as a unicellular yeast at 37˚C. The yeast is the phase that causes histoplasmosis. Because the yeast morphology is pathogenic, it is often studied, therefore the mold is often overlooked. This study focuses on the investigation of the mold specific gene, MS88. We have fused the H2B promoter in frame to the MS88 open reading frame to force expression in the yeast phase. This will determine if the gene is involved in dimorphism. Methods: An NCBI BLAST(p) www.ncbi.nlm.nih.gov domain search was conducted on the MS88 gene to characterize the sequence. The overexpression construct was made by fusing the H2B promoter to the MS88 open reading frame via fusion PCR. The construct will then be electroporated into an Ms88 expressing strain of Hc. Results: A NCBI BLAST(p) search found that MS88 was classified in the CFEM family that suggests that the gene plays a role in pathogenesis and is cysteine rich. Conclusion: The Ms88 gene could be involved in pathogenesis due to the NCBI BLAST (p) domain search. 3.14 Rapid detection using tdh, trh-induced hemolysis on human erythrocytes and urease production as biomarkers for

screening pathogenic Vibrio parahemolyticus strains Maryam Mohammadi, Taejo Kim Mississippi State University, Starkville, Mississippi

Background: Rapid detection of pathogenic Vibrio parahaemolyticus (Vp) strains is an ongoing challenge for the seafood industry. Research suggests a link between pathogenicity and the expression of two hemolytic proteins: thermostable-direct hemolysin (tdh) and thermostable-related hemolysin (trh). Tdh and trh induced hemolysis could be used as a biomarker for rapidly screening virulent strains of Vp. The objective of this study is to observe how tdh and trh influence hemolysis when cultivated on Wagatsuma media and its relationship to urease production. Methods: V. parahaemolyticus strains were cultivated on Wagatsuma media formulated with human erythrocytes. At 24 and 48 hours incubation time the degree of hemolysis and growth was recorded. Sample colonies were collected for Western blot analysis to compare levels of tdh and trh expression to degree of hemolysis. Results: At 24 hours highly pathogenic strains exhibited higher levels of hemolytic activity than moderately pathogenic strains and non-pathogenic strains did not express tdh or trh, did not produce urea, and did not induce tdh, trh mediated hemolysis. Most strains expressing tdh and trh exhibited hemolysis at 24 hours while urease negative and tdh, trh negative samples exhibited hemolysis after incubating for 48 hours. Conclusion: This study shows that urease production is positively correlated with tdh, trh expression but is unreliable for detecting virulent strains of V. parahaemolyticus. Hemolysin and urease negative strains lysed erythrocytes at 48 hours because all V. parahaemolyticus strains express thermolabile hemolysin (tlh), which is not related to pathogenicity. Therefore, incubating longer than 24 is unreliable for detecting pathogenic strains.

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3.15 Early innate immune response to a polyamine transport deficient pneumococcus in a mouse model of pneumococcal pneumonia. Aswathy N. Rai1, John Stokes1, Justin A. Thornton3, Edwin Swiatlo2, Imran Sunesara2, and Bindu Nanduri1. 1 Dept. of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA 39762. 2 Dept. of Infectious Diseases, University of Mississippi Medical Center, Jackson, MS, USA 39216. 3 Dept. of Biological Sciences, Mississippi State University, Mississippi State, MS, USA 39762.

Polyamines are ubiquitous small cationic molecules that are important for growth and virulence of human pathogens, including pneumococcus. Polyamines such as spermidine, putrescine and cadaverine are synthesized in bacteria using precursor amino acids and intermediates which are eventually converted into functional polyamines. In addition, polyamine specific transport systems allow the uptake of polyamines from the extracellular environment. In pneumococci polyamine transport of putrescine/spermidine is carried out by a single transport system, potABCD. We generated an isogenic deletion of polyamine transport operon in S. pneumoniae TIGR4 ∆potABCD. Our results indicated that deletion of polyamine transport led to attenuation in a mouse model of pneumococcal pneumonia. The aim of this study is to identify specific host innate immune mechanisms that contribute to enhanced resistance in mice challenged with polyamine deficient pneumococci. We inoculated C57BL/6 mice (n=5) intranasally with 1 x 107 CFU of wild type TIGR4, ∆potABCD. Mice were euthanized 4h, 12h and 24h post infection (p.i.) and lungs were harvested. We obtained bacterial CFU, evaluated immune cell infiltration, and measured the concentration of cytokines/chemokines with lung homogenate. Our results showed that there was a significant difference in the in vivo bacterial burden with WT and ∆potABCD at all the time points measured. The mutant was cleared by the host immune system by 24h. Consistent with this observation we found significant differences in the infiltration of neutrophils in the lung tissue of mice challenged with ∆potABCD. Concentration of G-CSF, GM-CSF, IL-5, and MIP-1a were higher with the polyamine transport deficient strain. Taken together, these results demonstrate that polyamine transport in the pneumococcus is essential to evade early innate immune responses. 3.16 Enterotoxin Production in Clostridium perfringens is affected by antimicrobial agents and bile acids

Fatemeh Rafii and Miseon Park Division of Microbiology, National Center for Toxicological Research, FDA, Jefferson, Arkansas 72079

Background: Clostridium perfringens is the second most common cause of bacterial foodborne illness in the United States, with nearly a million cases each year. C. perfringens enterotoxin (CPE) produced during sporulation damages intestinal epithelial cells by pore formation, which results in watery diarrhea. The effects of antimicrobial agents, preservatives and bile acids on toxin production were investigated. Method: The vegetative cells of some field isolates of C. perfringens and strains SM101 and F4969, which carry chromosomal or plasmid-borne enterotoxin genes, respectively, were exposed to bile acids, nisin and subinhibitory concentrations of antimicrobial agents in a sporulation medium before a heat treatment to induce release of spores. The spores were enumerated by plating on BHI agar. Amplification by PCR primers was used to detect the presence of enterotoxin genes. ELISA with a specific antibody for enterotoxin was used to measure the toxin produced by different strains. Results: The presence of C. perfringens cpe in the field isolates was verified by PCR. The strains differed in the efficiency of sporulation and toxin production. Different antimicrobial agents, bile acids and nisin affected sporulation efficiency differently. ELISA showed that production of toxin by strains with the chromosomal and plasmid-coded genes also was affected differently. In general, exposure to norfloxacin, bile acids, and nisin resulted in the enhancement of enterotoxin in some strains regardless of the location of enterotoxin genes. Subinhibitory concentrations of β-lactams either decreased or did not affect sporulation and enterotoxin production of the strains tested. Conclusion: Our results indicate that exposure to nisin, bile acids and some antibiotics may alter the expression, production or release of enterotoxin in C. perfringens.

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3.17 Characterizing the Extensive Heterogeneity of Mycobacteriosis Disease Progression in Controlled Animal Model Caroline Y. Sciarrillo, Gregory W. Broussard, Lisa M. Shirreff, Amrita Mallick and Don G. Ennis Department of Biology, PO Box 42451, University of Louisiana, Lafayette 70504

Mycobacterium tuberculosis (Mtb) is the etiological agent for TB, and it is considered one of the most successful pathogens. It is estimated approximately one third of the world’s population is infected with Mtb. Mtb is a slow growing pathogen and requires special precautions with researchers; hence a surrogate pathogen that grows much faster with less risk is useful. Mycobacterium marinum (Mm) is the closest related species to Mtb complex, and it establishes TB-like chronic infections in fish. Similar to TB infections, Mm survives and multiples within host macrophages and produces granulomas. We use a M. marinum - Japanese Medaka (Oryizas latipes) model in order to establish a chronic TB-like infection. We have employed a See Through line of Medaka that is devoid of major pigments to study real-time disease progression. We are able to track bacterial colonization and spread to target organs and other tissues in the intact animals by using an M. marinum strain expressing fluorescent protein (e.g. Gfp). We have observed substantial variability in organ colonization and disease presentation in the Medaka-M. marinum infection model. Several factors can be attributed to the heterogeneity seen in humans such as: infectious dose, virulence of the strain, and the general health of the person. However, in the laboratory all of these parameters are controlled, yet great variability in colonization levels are observed in fish. In one set of experiments, Medaka were infected with M. marinum through IP injections, and were photographed throughout an 8 week time course. Photographs of the See Through line of fish were taken prior to infection (0 weeks), 2 weeks post infection, 4 weeks post infection, and 8 weeks post infection. We observed at 8 weeks post infection a large amount of heterogeneity of the total amount of fluorescence in each animal, which is often seen in TB infections in humans. In addition to highly variable colony counts, we are able to support these findings by quantification of fluorescent images of infected fish at the end stages of an infection. We used Image J in order to analyze the mean fluorescent, area, and integrated density of a set of RGB images (24-bit). Image J is able to convert each pixel of a RGB image to grayscale, and then default weighting factors are applied to convert the image to YUV.

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3.18 Understanding the Regulation of MsaB production from msaABCR operon in Staphylococcus aureus Sarah Simmons, Megan Mullis, Gyan S. Sahukhal and Mohamed O. Elasri Department of Biological Sciences, The University of Southern Mississippi

Backgrounds: Staphylococcus aureus causes a wide range of acute and chronic infections including severe invasive biofilm-associated infections in humans. Several global regulators control virulence of these stains. Yet, we do not fully understand the regulation of virulence factors involved in staphylococcal infections. The msaABCR operon regulates biofilm development, antibiotic resistance and virulence. Transcription of msaABCR generates several sub-transcripts including one that translates into the MsaB protein. However, the regulation mechanism of this operon and the role of the sub-transcripts is not yet understood. In this study, we investigate the role of 5’ end and 3’ end of the msaABCR operon in the regulation of production of MsaB. We also investigate mRNA stability and its role in biofilm development and virulence. Methods: We constructed a series of truncated msaABCR operon constructs (TC-1 to TC-12) from both the 5’ end and the 3’ end to study the role of 5’ end and 3’ end in transcript stability, regulation of MsaB production, proteases production, and biofilm development. We also performed the mutagenesis experiments to study the interaction between 5’ end and 3’ end of the msaABCR operon transcript and its role in mRNA stability and MsaB production. Results: Full msaABCR operon transcript with its complete intergenic region complemented to the wild type level in terms of pigmentation, protease production, biofilm development, and MsaB production. Two constructs, TC-5 and TC-9, complement the msaABCR deletion mutant and result in overexpression of MsaB. The constructs TC-1, TC-2, TC-3 and TC-4 did not complement the msaABCR deletion mutant and did not produce MsaB. Interestingly, TC-3 and TC-4 complemented biofilm formation suggesting a role for the 3’ end in biofilm formation that does not require MsaB. These result also suggest that the 5’ end and the 3’ end of the transcript interact and play a role in the production of MsaB and biofilm development. Conclusion: This study defines the regulatory functions of the 5’ and 3’ ends of the msaABCR transcript in the production of MsaB and Biofilm development. This study will allow us to eventually identify the environmental and/or host stimuli that control the functions and role of the msaABCR operon. 3.19 The Regulatory Relationship Between Transcriptional Regulators CodY and MsaB in Staphylococcus aureus

Brittany L. Trunell, Justin L. Batte and Mohamed O. Elasri The University of Southern Mississippi, Hattiesburg, Mississippi

Background: There are many newly described transcriptional regulators that been found in pathogenic Staphylococcus aureus or S. aureus. Many of these regulators have been found to be essential for the organism’s ability to switch from commensal form to the virulent pathogenic form. One of these main regulators that have been identified to be a direct link between metabolism and virulence is CodY. This regulator has been shown to be responsive to nutrient availability during the different phases of growth. Additionally, we have recently found that MsaB the only protein coding ORF of the msaABCR operon has close interactions with CodY during the regulation of capsule production in S. aureus. Methods: To examine the regulatory relationship between MsaB and CodY we produced mutations of the msaABCR operon the codY gene individually and a double msaABCR/codY mutantion. We tested the effects of the mutations made on regulation of capsule production. We also cloned and expressed the codY ORF into an expression vector that we will use to express and purify functional CodY protein. We will use this CodY protein in combination with purified MsaB protein to perform competitive binding assays to the cap promoter region as well as perform protein-protein interaction studies. Results: We observed that both MsaB and CodY bind to the cap promoter region in very close proximity. CodY binds to this region in early phases of growth as a repressor of cap transcription and MsaB binds to this region in the later phase of growth as an activator of cap transcription. Other groups have shown that CodY responds directly to nutrient availability. Using chemically defined medium or CDM we have also shown that MsaB is also sensing nutrient availability Conclusions: In this study we show that the transcriptional regulator MsaB protein has very close interactions with another known DNA binding transcriptional regulator CodY in S. aureus. These interactions have been found to be very important in the regulation of sensing environmental nutrients and regulating capsule production in S. aureus. We are currently exploring the interactions and regulation between MsaB and CodY and how they together may an undefined link between basic metabolism and virulence in S. aureus.

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3.20 The role of GenF in the asymmetric distribution of the virulence protein, IcsA in Shigella flexneri Birendra Sharma and L.D. Brandon, PhD. Mississippi University for Women, Columbus, Mississippi

Background: Shigella spp. are obligate intracellular pathogens and causative agents of shigellosis. The pathogenesis of Shigella involves the invasion of colonic epithelial cells. Once the bacteria have entered a cell they recruit actin filaments for directional movement within these cells and for subsequent invasion of adjacent cells. The Shigella outer membrane protein, IcsA is the sole bacterial protein required for the recruitment of actin filaments. It is essential to Shigella pathogenesis in that it is targeted and restricted to the old pole of the bacterium and in that the polar distribution of IcsA is directly correlated with directional movement of Shigella within colonic epithelial cells and its efficient dissemination to uninfected cells. In this study we show that GenF plays a role in the proper surface distribution of IcsA in Shigella. Methods: We are interested in identifying and characterizing mutations that affect the polar targeting of IcsA in Shigella. To this end, we chose to study an uncharacterized protein that is assymetrically associated with the inner membrane of Gram negative bacteria. We employed pcr to displace the corresponding gene (genF) with natA (nourseothricin acetyl-transferase). Such mutant was subjected to surface labeling experiments using antibodies to IcsA to examine the surface distribution of IcsA on intact bacteria. Results: We found that IcsA was associated with each bacterium in a circumferential fashion as compared to wild type strains where IcsA was located at the old pole of each bacterium. Conclusion: This study shows that GenF plays a role in the surface distribution of IcsA. We are interested in determining whether GenF represents a putative polar target for IcsA polar targeting and subsequent secretion to the surface of a given bacterium or whether it plays a role in the maintenance of IcsA at the bacterial pole after targeting and secretion.

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RECENT MEETINGS OF THE SOUTH CENTRAL BRANCH OF THE AMERICAN SOCIETY FOR MICROBIOLOGY

1983 Louisiana State University, Baton Rouge, LA 1984 University of Arkansas for Medical Sciences, Little Rock, AR 1985 McNeese State University, Lake Charles, LA 1986 Louisiana State University Medical Center, Shreveport, LA 1987 Southeastern Louisiana University, Hammond, LA 1988 Louisiana State University, Baton Rouge, LA 1989 Perdido Beach, Florida (Inter-Branch Meeting) 1990 University of Arkansas for Medical Sciences, Little Rock, AR 1991 University of Mississippi Medical Center, Jackson, MS 1992 Mississippi State University, University, MS 1993 Louisiana State University, Baton Rouge, LA 1994 Louisiana State University Medical Center, Shreveport, LA 1995 University of Arkansas for Medical Sciences, Little Rock, AR 1996 Tulane University School of Medicine, New Orleans, LA 1997 University of Mississippi Medical Center, Jackson, MS 1998 University of Alabama, Montgomery, AL 1999 Louisiana State University Health Science Center, New Orleans, LA 2000 University of Arkansas for Medical Sciences, Little Rock, AR 2001 Louisiana State University Veterinary School, Baton Rouge, LA 2002 University of Mississippi Medical Center, Jackson, MS 2003 Tulane University Health Science Center, New Orleans, LA 2004 Mississippi State University, Starkville, MS 2005 University of Louisiana at Lafayette, Lafayette, LA 2006 Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 2007 University of Arkansas at Little Rock, AR 2008 University of Texas, Austin, TX 2009 Nicholls State University, Thibodaux, LA 2010 The University of Southern Mississippi, Hattiesburg, MS 2011 The University of Louisiana at Monroe, Monroe, LA 2012 Mississippi State University, Starkville, MS 2013 Joint Meeting with Texas Branch at Tulane University, New Orleans, LA 2014 University of Arkansas, Fayetteville, AR 2015 The University of Southern Mississippi, Hattiesburg, MS

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Charles C. Randall Lectureship Past Recipients: 1991 Dr. Jan Bly, University of Mississippi Medical Center, Jackson, MS

1992 Dr. Daniel J.J. Carr, LSU Medical Center, New Orleans, LA

1993 Dr. R. Martin Roop, LSU Medical Center, Shreveport, LA

1994 Dr. John Battista, LSU, Baton Rouge, LA

1995 Dr. Bentley A. Fane, University of Arkansas, Fayetteville, AR

1996 Dr. Mark S. Smeltzer, University of Arkansas for Med. Sciences, Little Rock, AR

1997 Dr. Jiang Dong Chen, LSU Medical Center, New Orleans, LA

1998 Dr. Shouguang Jin, University of Arkansas for Med. Sciences, Little Rock, AR

1999 Dr. Henri van der Heyde, LSU Medical Center, Shreveport, LA

2000 Dr. Cheryl A. Nickerson, Tulane University, New Orleans, LA

2001 Dr. Andrew D. Yurochko, LSU Medical Center, Shreveport, LA

2002 Dr. William B. Klimstra, LSU Medical Center, Shreveport, LA

2003 Dr. William P. Hallford, Tulane University, New Orleans, LA

2004 Dr. Katherine D. Ryman, LSU Medical Center, Shreveport, LA

2005 Dr. Christopher A. Elkins, National Venter for Toxicological Research, Jefferson, AR

2006 Dr. Stephania Cormier, LSU, Baton Rouge, LA

2007 Dr. Mohamed O. Elasri, University of Southern Mississippi, Hattiesburg, MS

2008 Dr. Mary Marquart, University of Mississippi Medical Center, Jackson, MS

2009 Dr. James Smith, Mississippi State University, Starkville, MS

2010 Dr. Brent C. Christner, Louisiana State University, Baton Rouge, LA

2011 Dr. Janet R. Donaldson, Mississippi State University, Starkville, MS

2012 Dr. Jon Blevins, University of Arkansas Medical Sciences, Little Rock, AR

2013 Dr. Daniel Voth, University of Arkansas for Medical Sciences, Fayetteville, AR

2014 Dr. Ritish Tandon, The University of Mississippi Medical Center, Jackson, MS

2015 Dr. Jeremy Kamil, LSU Health Science Center, Shreveport, LA

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Sponsors

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www.fi shersci.com

15-2419 American Society for Microbiology Program.indd 1 9/11/2015 4:00:58 PM

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Acknowledgements We would like to acknowledge the following people and exhibitors for their enthusiasm and support. The University of Southern Mississippi has fully embraced this conference. We are also particularly grateful for the significant support of the Mississippi INBRE. And of course, THANK YOU for coming…

The University of Southern Mississippi Dr. Rodney Bennett, President

Dr. Gordon Cannon, Vice President for Research

Dr. David Hayhurst, Dean of the College of Science and Technology

Dr. Tom Burke, Interim Vice President for Student Affairs

Dr. Shiao Wang, Interim Chair, Department of Biological Sciences

Mississippi INBRE

Dr. Mohamed O. Elasri, Director

Dr. Glen Shearer, Program Coordinator

Mary Ann McRaney, Network Coordinator

Jamie Lott, Outreach Director

Conference Staff

Caroline Meyers, Rebekah Bullard, Paige Allen

Dr. Gyan Sahukhal, Justin Batte, Johnathan Hill

The Randall Lectureship committee The invited speakers and the session chairs

Oral session chairs Student award judges

The American Society for Microbiology Branch Lectureships (ASMBL) committee The Hattiesburg Visitors Center

Fisher Scientific Meyer Instruments

EMD Chemicals