AMERICAN SOCIETY FOR MICROBIOLOGY

VIRGINIA BRANCH 2013 ANNUAL MEETING

PROGRAM

UNIVERSITY OF VIRGINIA

School of Medicine Claude Moore Medical Education Building

HOSTS: ALISON CRISS AND IAN GLOMSKI

NOVEMBER 8-9, 2013

2013 VA Branch ASM

Welcome to the Virginia Branch ASM 2013 Annual Meeting

Virginia Branch ASM Officers 2013-2014

President – Janet Adams, Ph.D., J. Sargeant Reynolds Community College

President-Elect – David Gauthier, Ph.D., Old Dominion University

Secretary – Robyn Puffenbarger, Ph.D., Bridgewater College

Treasurer – Lynn Lewis, Ph.D., University of Mary Washington

Councilor – David Buckalew, Ph.D., Longwood University

Alternate Councilor – Laura Runyen-Janecky, Ph.D., University of Richmond

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2013 VA Branch ASM

VA Branch ASM

Gratefully Acknowledges our Sponsors

2013 VA Branch ASM

VIRGINIA BRANCH 2013 ANNUAL MEETING

FRIDAY, NOVEMBER 8, 2013

Check-in and on-site registration throughout the afternoon All sessions will be held in the School of Medicine Claude Moore Medical Education Auditorium, Room 3110

12:00 – 5:00 Registration Desk, Third Floor

1:15 – 1:30 ASM Welcome: Janet Adams, VA Branch President and Alison Criss and Ian Glomski,

UVA Hosts

Note: Names in bold print denote competitors for “Best Student Presentation”

Session I Moderator: Heath Damron, University of Virginia

1:30 – 1:45 Clostridium difficile toxins and pathogen associated molecular patterns synergistically

increase expression of the pathogenic cytokine IL-23

Carrie Cowardin, Sarah Kuehne, Erica Buonomo, Nigel Minton, and William A. Petri,

Jr. University of Virginia, Charlottesville, VA.

1:45 – 2:00 The Inhibitory Effects of Adenylate Cyclase Toxin (ACT) on Bordetella Biofilm

Formation

C. Hoffman (1), M. Gray (1), G. Donato (1), Y. Reyes (1), J. Eby (1), P. Cotter (2), R.

Osicka (3), P. Sebo (3), and E. Hewlett (1). (1) Division of Infectious Diseases and

International Health, Department of Medicine, University of Virginia, Charlottesville,

VA. (2) School of Medicine, University of North Carolina, Chapel Hill, NC. (3)

Academy of Sciences of the Czech Republic, Division of Cell and Molecular

Microbiology, Czech Republic.

2:00 – 2:15 A Francisella FopA mutant exhibits decreased attachment to host cells and delayed

phagosomal escape

G. Brett Moreau, Aiping Qin, and Barbara Mann. University of Virginia,

Charlottesville, VA.

2:15 – 2:30 Secretion of a nuclease by Neisseria gonorrhoeaeenhances escape from killing by

neutrophil extracellular traps

Richard A. Juneau and Alison K. Criss. University of Virginia, Charlottesville, VA.

2:30 – 2:45 Ethanolamine promotes expression of putative and characterized fimbriae in

enterohemorrhagic Escherichia coli O157:H7

Laura A. Gonyar and Melissa M. Kendall. University of Virginia, Charlottesville, VA.

2:45 – 3:00 The Sinorhizobium meliloti chemoreceptor McpU directly binds proline and mediates

chemotaxis towards host plant exudates

Benjamin A. Webb, Sherry Hildreth, Richard F. Helm, and Birgit E. Scharf. Virginia

Tech, Blacksburg, VA.

3:00 – 3:15 BREAK: refreshments

2013 VA Branch ASM

Session II Moderator: Katie Crump, Virginia Commonwealth University

3:15 – 3:30 OpaR Controls a Network of Downstream Transcription Factors in Vibrio

parahaemolyticus BB22

Alison Kernell-Burke, Leah T. C. Guthrie, Thero Modise, Guy Cormier, Roderick V.

Jensen, Linda L. McCarter, and Ann M. Stevens. Virginia Tech, Blacksburg, VA.

3:30 – 3:45 Factors Involved in Clostridium perfringens Gliding Motility on Agar Surfaces

Hualan Liu, Steve Melville, David Popham, Roderick Jensen. Virginia Tech,

Blacksburg, VA.

3:45 – 4:00 Identification of Proteases in Naegleria fowleri

Ishan Vyas, Melissa Jamerson, Jocilyn Budda, Francine Marciano-Cabral. Virginia

Commonwealth University, Richmond, VA.

4:00 – 4:15 Toxin Inhibition by Peptides and Small Molecules

Janis Doss and Dayle Daines. Old Dominion University, Norfolk, VA.

4:15 – 4:30 The role of LptA, an LOS-modifying enzyme, in gonococcal defense to human PMNs

Jonathan Handing and Alison Criss. University of Virginia, Charlottesville, VA.

4:30 – 4:45 BREAK

4:45 – 6:00 Introduction of ASMBL speaker: David Gauthier

6:00 – 6:15 Branch Photograph

6:15 – 8:30 Buffet Dinner and Poster Session

6:30 – 7:30 Poster Session 1: odd-numbered posters

7:30 – 8:30 Poster Session 2: even-numbered posters

Poster Sessions will be held in MR5, Rooms 1005 and 2005.

ASM BRANCH LECTURESHIP SPEAKER:

Dr. Chris Marx

Department of Organismic and Evolutionary Biology & FAS Center for Systems

Biology, Harvard University

The Math of Metabolism: Mutations That Don’t Like Each Other (and Species That Do)

2013 VA Branch ASM

VIRGINIA BRANCH 2013 ANNUAL MEETING

SATURDAY, NOVEMBER 9, 2013 8:00 am Breakfast sponsored by ThermoFisher Scientific

Coffee, juice, and bagels

Note: Names in bold print denote competitors for “Best Student Presentation”

Session III Moderator: Dwi Susanti, Virginia Tech

8:30 – 8:45 Localization of GSLEs in Bacillus anthracis Dormant and Germinating Spores using

PSICIC

Sean P. Mury and David L. Popham. Virginia Tech, Blacksburg, VA.

8:45 – 9:00 Quantitative Mass Spectrometry of Bacillus subtilis Germination Proteins

Yan Chen, W. Keith Ray, Richard F. Helm, Stephen B. Melville, and David L. Popham.

Virginia Tech, Blacksburg, VA.

9:00 – 9:15 Correlative Algorithm for Repeat Placement

Abhishek Biswas, David Gauthier, Desh Ranjan, and Mohammad Zubair. Old

Dominion University, Norfolk, VA.

9:15 – 9:30 Sex hormones differentially regulate HSV-1 gene expression during the early stages of

infection in vitro

Thanh Kim Nguyen and Patric Lundberg. Eastern Virginia Medical School, Norfolk,

VA.

9:30-9:45 Targeting Dengue Virus Dependent 3' - 5' Human Exoribonucleases

Krystal Haley and Dr. Daniel Engel. University of Virginia, Charlottesville, VA.

9:45 – 10:00 BREAK: refreshments

10:00 – 11:00 Safety in the Undergraduate Teaching Laboratory - Faculty Session (and interested

postdocs)

Guidelines for Biosafety in Teaching Laboratories (link):

http://www.asm.org/index.php/education-2/22-education/8308-new-version-available-

for-comment-guidelines-for-best-biosafety-practices-in-teaching-laboratories

10:00 – 11:00 Career Panel - Student and Postdoc Session

11:00 – 11:15 BREAK: refreshments

Session IV Moderator: Rick Juneau, University of Virginia

11:15 – 11:30 Protein Interaction Networks in Bacteria

Peter Uetz, Seesandra S Rajagopala, Roman Häuser, and Russell L. Finley. Virginia

Commonwealth University, Richmond, VA.

2013 VA Branch ASM

11:30 – 11:45 The Impact of Genome Reduction on Conservation of Microbial Protein Complexes and

Their Components

J. Harry Caufield and Peter Uetz. Virginia Commonwealth University, Richmond, VA.

11:45 – 12:00 Complement evasion strategies of oral treponemes associated with periodontal disease

Daniel P. Miller, John V. McDowell, Jessica K. Bell, J. Christopher Fenno, Richard T.

Marconi. Virginia Commonwealth University, Richmond, VA.

12:00 – 12:15 The Relationship of the Lipoprotein SsaB, Manganese, and Superoxide Dismutase in

Streptococcus sanguinis Virulence for Endocarditis

Katie E. Crump, Brian Bainbridge, Sarah Brusko, Lauren S. Turner, Xiuchun Ge,

Victoria Stone, Ping Xu, and Todd Kitten. Virginia Commonwealth University,

Richmond, VA.

12:15 – 12:30 Evolution of Thioredoxin (Trx) systems and the role of Trx in Methanocaldococcus

jannaschii

Dwi Susantia, Joshua H. Wong, William H. Vensel, Usha Loganathan, Rebecca

DeSantis, Ruth A. Schmitz, Monica Balsera, Bob B. Buchanan, and Biswarup

Mukhopadhyay. Virginia Tech, Blacksburg, VA.

12:30 – 12:45 Closing Remarks

12:45 – 1:45 Lunch & Business Meeting

VA Branch ASM Business Meeting Agenda: Nov 9, 2013

I. General Branch Information

II. Treasurer’s Report – Lynn Lewis

III. Secretary’s Report – Robyn Puffenbarger

IV. Councilor’s Report – Wade Bell

V. Upcoming Branch Meetings

a. 2014 – JMU

b. 2015 – volunteers?

VI. Branch Officer Elections

a. President-Elect: need interested candidates

b. Treasurer – Lynn Lewis

c. Secretary, 2015

VII. Uses for National Funds??

a. Student Branch funds – Jan 1, funds disbursed until gone

b. Region 2 funds – Nov 30, Career Panel, Student Travel Grants, etc.

c. National funds – Nov 30, collaborate with a regional meeting (does not

have to be another ASM Branch); increase membership

VIII. New Business

a. Undergraduate programming ideas

b. Other new business?

2013 VA Branch ASM

Abstracts for Oral Presentations

Session I Abstracts

Clostridium difficile toxins and pathogen associated molecular patterns synergistically increase

expression of the pathogenic cytokine IL-23

Carrie Cowardin, Sarah Kuehne, Erica Buonomo, Nigel Minton, and William A. Petri, Jr. University of

Virginia, Charlottesville, VA.

Clostridium difficile pathogenesis is primarily mediated by the Rho-glucosylating Toxins A and B, which

activate Nuclear Factor kappa B (NFκB) and the NLRP3 inflammasome. The pro-inflammatory cytokine

IL-23 is a pathogenic mediator in C. difficile infection, as 100% of IL-23p19-/- mice survive infection

while only 16.7% of WT mice survive (p=0.005). IL-23 is induced via Pattern Recognition Receptor

(PRR) and IL-1β receptor signaling. We hypothesized that the ability of toxins A and B to activate NFκB

would augment signals from PRRs to increase IL-23 expression from myeloid cells in the lamina propria.

To address this question, bone marrow derived dendritic cells were treated with purified toxin A and B as

well as toxin-containing supernatants from isogenic toxin mutant strains. We discovered that although

purified toxins were not sufficient to induce IL-23 (below detection), C. difficile toxins combined with

PAMPs exerted a combinatorial effect on IL-23 expression. Supernatants from the epidemic strain

R20291 induced 513 +/-123 pg/mL of IL-23 while an isogenic AB- toxin mutant strain induced only 14.8

+/-7.4 pg/mL (p value = 0.03). IL-1β was similarly increased in cells exposed to WT supernatant (2,737

+/-183 pg/mL) but not in supernatants without toxin (below detection) suggesting that IL-1β may play a

role in the induction of IL-23. Our data demonstrate that C. difficile is capable of inducing IL-23, and that

PRR signaling and IL-1β are likely necessary for this induction. These findings implicate toxin and PRR

activated signaling pathways as potential targets of therapeutic intervention to prevent pathogenic IL-23

production.

The Inhibitory Effects of Adenylate Cyclase Toxin (ACT) on Bordetella Biofilm Formation

C. Hoffman (1), M. Gray (1), G. Donato (1), Y. Reyes (1), J. Eby (1), P. Cotter (2), R. Osicka (3), P. Sebo

(3), and E. Hewlett (1). (1) Division of Infectious Diseases and International Health, Department of

Medicine, University of Virginia, Charlottesville, VA. (2) School of Medicine, University of North

Carolina, Chapel Hill, NC. (3) Academy of Sciences of the Czech Republic, Division of Cell and

Molecular Microbiology, Czech Republic.

Bordetellae are able to form biofilm but the mechanisms are not completely understood. Biofilm

formation is dependent on Bvg-regulated virulence factors; for example Filamentous Haemagglutinin

(FHA) has been shown to be required for biofilm formation. In addition, a strain lacking the cyaA gene,

encoding ACT, forms more biofilm than wild type (WT) bacteria, suggesting an inhibitory effect of ACT.

In the present studies, the ability of Bordetellae strains to form biofilm was evaluated and quantified by

the crystal violet assay, under a variety of conditions. To characterize the role of ACT, we examined the

effects of exogenously added B. pertussis ACT. As hypothesized, addition of ACT to any of the strains

capable of producing biofilm results in reduced biofilm accumulation. This inhibitory effect is

concentration dependent and does not require the enzymatic activity of ACT (production of cAMP from

ATP). We have shown previously that a physical interaction occurs between FHA and ACT. Collectively

these data suggest that the dependence of biofilm formation on FHA and the inhibition of this

phenomenon by ACT may reflect that interaction. To understand better the relationship between FHA and

ACT, and the possible role of their interaction in biofilm formation, strains with deletions in FHA and

ACT and the effects of mutations in ACT and FHA, are being characterized. To explore further this

interaction we are using flow cytometry with the Amnis ImageStream to examine and quantify surface-

associated molecules of Bordetellae, such as ACT and FHA.

2013 VA Branch ASM

A Francisella FopA mutant exhibits decreased attachment to host cells and delayed phagosomal

escape

G. Brett Moreau, Aiping Qin, and Barbara Mann. University of Virginia, Charlottesville, VA.

Francisella tularensis is the causative agent of the potentially life threatening disease tularemia. It is also

a significant biosecurity threat and is classified as a Tier 1 select agent because it is easily aerosolized and

has a low infectious dose. As an intracellular pathogen, attachment and internalization into host cells, as

well as rapid escape from the phagosome, are critical steps in Francisella virulence. However, the

bacterial factors involved in attachment and internalization and their downstream affects on intracellular

growth have not been well characterized. An in vitro competition assay between wildtype Francisella

novicida and individual Francisella novicida transposon mutants determined that a transposon mutant in

the fopA gene, which encodes an abundant outer membrane protein, was deficient in the ability to

competitively attach and internalize into a lung epithelial cell line. Further characterization of Tn:fopA

revealed that it was not attenuated in internalization into either epithelial or macrophage cell lines or

intracellular growth at 24 hours post-infection, but was deficient in attachment. These results were

recapitulated within the highly virulent Francisella tularensis ssp. tularensis Schu S4 strain. Interestingly,

a Schu S4 ∆fopA mutant exhibited a decrease in intracellular growth within both epithelial and

macrophage cell lines at 6 hours post-infection. Immunofluorescence analysis determined that ∆fopA

more readily colocalized with the phagosomal marker LAMP1 at 2 hours post-infection than wildtype,

indicating that there is likely a delay in phagosomal escape. These data suggest that FopA could play a

key role in Francisella virulence by enhancing phagosomal escape.

Secretion of a nuclease by Neisseria gonorrhoeaeenhances escape from killing by neutrophil

extracellular traps

Richard A. Juneau and Alison K. Criss. University of Virginia, Charlottesville, VA.

Symptomatic infection by Neisseria gonorrhoeae, the gonococcus (Gc), results in a highly inflammatory

environment caused by a potent infiltration of neutrophils, which possess within their antimicrobial

arsenal both oxidative and non-oxidative components. These components can be directed to the

phagolysosome, or released by exocytosis, to kill microbes. Another approach used by neutrophils to trap

and kill microbes is the release of neutrophil extracellular traps (NETs). NETs are web-like structures

comprised of chromatin, decorated with antimicrobial proteins, and are released from neutrophils

undergoing a unique form of cell death. In spite of these robust activities, viable bacteria are recovered

from gonorrheal disease exudates. We seek to define the bacterial virulence factors that contribute to

gonococcal survival after exposure to neutrophils. The NG0969 open reading frame encodes a

thermonuclease (nuc) that contributes to gonococcal biofilm architecture and remodeling†. To examine

the potential role of nuc in Gc resistance to neutrophils and in NET association, we generated a nuc

insertion-deletion mutant in strain FA1090. While neutrophils infected with both parent and ∆nuc mutant

produce NETs, the overall area of extracellular DNA was significantly higher in ∆nuc mutant compared

to parent Gc infection. Additionally, purified Nuc protein cleaved human neutrophil DNA. When

exposed to neutrophils induced to undergo NET formation, the ∆nuc mutant survived less well than the

parent, even in the presence of the phagocytosis-inhibiting chemical cytochalasin D. DNAse I addition

prior to introduction of the bacteria partially rescued ∆nuc mutant survival. These studies suggest a

model in which Nuc enables Gc escape of NET trapping and killing during symptomatic infection.

†Steichen, et al. “The Neisseria gonorrhoeaeBiofilm Matrix Contains DNA, and an Endogenous

Nuclease Controls Its Incorporation.” Infection and Immunity. 1504-11. 79(4), April 2011.

Ethanolamine promotes expression of putative and characterized fimbriae in enterohemorrhagic

Escherichia coli O157:H7

Laura A. Gonyar and Melissa M. Kendall. University of Virginia, Charlottesville, VA.

2013 VA Branch ASM

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes severe bloody

diarrhea and hemolytic uremic syndrome. In order to colonize the human gastrointestinal (GI) tract and

cause disease, EHEC must be able to sense the host environment and promote expression of virulence

genes essential for adherence. Ethanolamine (EA) is a major component of both mammalian and bacterial

membranes, and due to the turnover of enterocytes, EA is present in the GI tract at concentrations that

support growth of EHEC. EA is not only an important metabolite for EHEC but also serves as a signal of

the host environment. We have determined that EA influences EHEC adherence to epithelial and bacterial

cells, and we hypothesized that these EA-dependent adhesion phenotypes were mediated through fimbrial

expression. EHEC encodes 16 fimbrial operons which are thought to be important for adhesion and early

interactions with host tissue; however, very little is known about the conditions that promote expression

of these fimbriae or their roles in host colonization. Using quantitative RT-PCR, we found that EA

promoted transcription of genes encoded in characterized and putative fimbrial operons. Moreover,

putative fimbrial structures were produced by EHEC cells grown with EA. These findings describe

transcription of several putative fimbrial loci in EHEC for the first time. Importantly, the biologically

relevant molecule EA, which is abundant in the GI tract, promoted expression of EHEC fimbriae.

The Sinorhizobium meliloti chemoreceptor McpU directly binds proline and mediates chemotaxis

towards host plant exudates

Benjamin A. Webb, Sherry Hildreth, Richard F. Helm, and Birgit E. Scharf. Virginia Tech, Blacksburg,

VA.

The soil bacterium Sinorhizobium meliloti and the agriculturally important legume, alfalfa can form an

agriculturally important mutualism inside the plant’s roots that leads to drastic increase in plant yield. A

prelude to this symbiosis is chemotaxis of S. meliloti through the rhizosphere toward the host plant. This

behavior is mediated by seed and root exudation of attractant compounds into the soil. Amongst the

multitude of compounds in the exudate, we identified the strong attractant, proline. Here we show that

methyl accepting chemotaxis protein U (McpU) of S. meliloti is important for chemotaxis toward proline

and that McpU binds proline directly. By testing single residue substitution mutants in capillary assays,

we found conserved aspartate residues are important for chemotaxis toward proline. These tests were

followed up with in vitro binding assays between proline and variant proteins of McpU. The results

indicate that proline binds in a conserved Cache domain of the periplasmic region of McpU and affects

chemotaxis to allow for cell movement up a gradient of proline. In all, chemotaxis to proline of a

germinating seed is likely a key step in the early establishment of the mutualism. Further studies on the

interaction between attractants and their chemoreceptors may aid in propagating a greater efficiency in

symbiosis.

Session II Abstracts

OpaR Controls a Network of Downstream Transcription Factors in Vibrio parahaemolyticus BB22

Alison Kernell-Burke, Leah T. C. Guthrie, Thero Modise, Guy Cormier, Roderick V. Jensen, Linda L.

McCarter, and Ann M. Stevens. Virginia Tech, Blacksburg, VA.

Vibrio parahaemolyticus is an emerging pathogen that is associated with food-borne gastroenteritis when

raw or undercooked seafood is consumed. The pathogenesis of this organism is controlled by the

phenomenon known as quorum sensing and the master regulator of quorum sensing in V.

parahaemolyticus is OpaR. OpaR controls the virulence of V. parahaemolyticus, as well as the colony

and cellular morphology associated with growth on a surface and biofilm formation. RNA-Seq, whole

transcriptome Next Generation sequencing, was utilized to determine the direct and indirect targets

controlled by OpaR in V. parahaemolyticus BB22. This work confirms but expands upon a previous

microarray analysis done with a V. parahaemolyticus RIMD chip. There was excellent correlation

2013 VA Branch ASM

between the microarray and RNA-Seq data. Eleven transcription factors under OpaR control have been

identified and further studied using qRT-PCR. A previously published position specific weighted matrix

(PSWM) was utilized to assist in identification of possible OpaR binding sites. Eight direct targets were

ascertained via in vitro electrophoretic mobility shift assays (EMSA) with purified hexahistidine tagged

OpaR. Discovery of the direct and indirect targets of OpaR, including sRNAs, will enable a network map

of regulatory interactions important for the switch between the nonpathogenic and pathogenic state to be

modeled.

Factors Involved in Clostridium perfringens Gliding Motility on Agar Surfaces

Hualan Liu, Steve Melville, David Popham,Roderick Jensen. Virginia Tech, Blacksburg, VA.

C. perfringens is a Gram-positive, anaerobic, spore-forming bacterium, which can cause several diseases

in humans, from mild food poisoning to life-threatening gas gangrene. C. perfringens possesses gliding

motility, which helps the bacteria to move across an agar surface. We isolated two natural mutants from

C. perfringens strain SM101, which is the causative pathogen for C. perfringens foodborne illness.

Compared to the wild type, those spontaneous mutants are hypermotile and form much larger and thinner

spreading colonies. Peptidoglycan structure analysis indicated those mutants have thinner peptidoglycan

layers. Video microscopy showed the mutants form long cells in chains during growth and have defects in

cell division. Whole genome sequencing found several mutations across the genomes of two hypermotile

isolates, one in minE and the other strain in ftsI. To determine whether they are the actual cause for

hypermotility, we are complementing each strain with a wild-type copy of the cell division gene they are

defective in. We also developed a random mariner transposon mutagenesis system in C. perfringens strain

13 and identified a peptidoglycan hydrolase homologue, SagA, as being required for gliding motility.

Since many pathogenic bacteria adopt motility mechanisms for host colonization, it would be very

valuable to understand the mechanism for forming those hypermotile strains, and also to evaluate the role

of hypermotility in the pathogenesis of C. perfringens.

Identification of Proteases in Naegleria fowleri

Ishan Vyas, Melissa Jamerson, Jocilyn Budda, and Francine Marciano-Cabral. Virginia Commonwealth

University, Richmond, VA.

Naegleria fowleri (N. fowleri), a free-living amoeboflagellate, is the etiologic agent of Primary Amoebic

Meningoencephalitis, a rapidly fatal disease of the central nervous system. Proteases have been shown to

be essential for invasion of host tissue by protozoan parasites and may be a major factor contributing to

the pathogenicity of N. fowleri. The purpose of the present study was to detect protease activity using

zymography. Whole cell lysates were used to detect protease activity in amoebae, while conditioned

medium was used to characterize secreted protease activity. In addition, invasion assays were performed

using matrigel as an ECM substrate to characterize invasion patterns and examine protease secretion over

time from virulent mouse-passaged versus weakly-pathogenic N. fowleri. A differential pattern of

protease activity was observed in the culture medium of highly-pathogenic versus weakly-pathogenic

amoebae. These results suggest that select proteases play a key role in amoebic invasion of the host ECM

and host invasion leading to damage of brain tissue. Studies are in progress to characterize these proteases

and to establish their functional relevance in the neuropathogenesis process.

Toxin Inhibition by Peptides and Small Molecules

Janis Doss and Dayle Daines. Old Dominion University, Norfolk, VA.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media and infections of the

respiratory tract in humans. Like other prokaryotes, NTHi contains several type II toxin-antitoxin (TA)

systems, in which an endoribonuclease toxin is bound to a labile antitoxin that inhibits the activity of the

toxin. The antitoxin is degraded during conditions of stress, allowing the toxin to inhibit the growth of

2013 VA Branch ASM

NTHi, which helps the organism evade antibiotics and the host immune system. Developing a more stable

molecule that could bind to and inhibit the toxin would prevent NTHi from entering this dormant state.

Our model for this study was the VapBC-1 TA system, in which VapC-1 is the toxin and VapB-1 is the

antitoxin. Initially, we used a phage display procedure to identify peptides from a commercial 7-mer

phage library that could bind to the VapC-1 toxin and performed fluorescence-quench RNase interference

assays to determine which bound peptide could interfere with the activity of the VapC-1 toxin. We also

tested a set of seven synthetic 15-mer overlapping peptides based upon the VapB-1 antitoxin sequence.

We then expanded our investigation to include the discovery of small molecules that could inhibit the

toxin. We are currently working with the National Center for Advancing Translational Sciences (NCATS)

to adapt our assay for high-throughput screening of their small molecule libraries to identify possible

chemical inhibitors of VapC-1.

The role of LptA, an LOS-modifying enzyme, in gonococcal defense to human PMNs

Jonathan Handing and Alison Criss. University of Virginia, Charlottesville, VA.

Infection with Neisseria gonorrhoeae(Gc) is marked by a rapid influx of polymorphonuclear leukocytes

(neutrophils or PMNs) to the site of infection. Despite a robust PMN immune response, viable Gc can be

recovered from PMN-rich gonorrheal secretions and from PMNs infected with Gc in vitro. We

hypothesize that Gc has defensive measures that help confer resistance to PMNs. Lipooligosaccharide

(LOS), an essential component of the bacterial outer membrane, has been implicated in gonococcal

defense but its role in interactions with human PMNs are underexplored. Of particular importance, Gc can

enzymatically modify the lipid A portion of LOS by the addition of phosphoethanolamine (PEA) moieties

to phosphate groups at the 1’ and 4’ positions. Loss of LptA, the enzyme catalyzing this reaction, leads to

an increased sensitivity of the bacteria to killing by human complement1 and cationic antimicrobial

peptides (CAMPs)2. We have demonstrated an lptA mutant in the FA1090 background shows a

significant survival defect in PMNs, compared to WT and an lptA complement (lptA+). Further

examination via Baclight viability dyes has demonstrated that the lptA mutant exhibits increased

susceptibility to PMN killing both intra- and extracellularly relative to WT and lptA+, however we

observed no difference in bacterial internalization. The observed survival defect could be due to altered

PMN responses to the lptA mutant and/or intrinsic sensitivity to PMN components. We have

demonstrated the lptA mutant is more sensitive to a purified PMN granule extract and select cationic

components indicating that the mutant survival defect is in part due to intrinsic sensitivity to components

of PMN arsenal.

1. L. Lewis et al. Infection and Immunity. 2013 Jan;81(1):33-42.

2. L. Lewis et al. Infection and Immunity. 2009 Mar;77(3):1112-20

Session III Abstracts

Localization of GSLEs in Bacillus anthracis Dormant and Germinating Spores using PSICIC

Sean P. Mury and David L. Popham. Virginia Tech, Blacksburg, VA.

During germination of Bacillus anthracis spores, the cortex peptidoglycan is degraded by germination-

specific lytic enzymes (GSLEs). Without the action of these critical enzymes, the germination process

will not be completed, and the spore will be unable to resume metabolism as a vegetative cell.

The limit of differentiation of two signals using light microscopy is approximately 250nm. Our project

involves the localization of GSLEs in the spore that may be separated by considerably less distance.

GSLE localization is made possible through the use of fluorescent proteins created by fusing mcherry to

genes encoding GSLEs and protein markers. The mCherry signal is analyzed and compared to that of

Mitotracker Green, a lipophilic membrane stain. The distance between these two signals will be analyzed

2013 VA Branch ASM

with the program Projected System of Internal Coordinates using Interpolated Contours (PSICIC).

Statistical analysis will elucidate the spatial relationship of GSLEs and marker proteins in relation to the

membrane. By arranging the relative locations of these signals, a distance weighted map will be created

in the dormant spore and changes will be tracked during cortex degradation in the germinating spore.

Knowledge of the localization of GSLEs can provide information that will be indispensible in

defining GSLE accessibility and function for intentional germination protocols. Compared with spores,

vegetative Bacillus anthracis cells are much more susceptible to heat, antibiotics, radiation, and chemical

treatment. Deliberate germination of spores by activating GSLEs will be crucial in designing cheaper,

more effective, decontamination protocols to counteract the use of Bacillus anthracis spores as a

bioweapon.

Quantitative Mass Spectrometry of Bacillus subtilis Germination Proteins

Yan Chen, W. Keith Ray, Richard F. Helm, Stephen B. Melville, and David L. Popham*. Virginia Tech,

Blacksburg, VA.

The majority of Bacillus subtilis dormant spores will germinate rapidly in response to nutrient germinants

with dramatic changes in the physical properties of the cells. However, a small subpopulation termed

superdormant spores are resistant to germination. The germinant valine was used to prepare rapidly-

germinating and superdormant spore populations. The purified superdormant spores (~1% of spore

population) germinated poorly with the germinant used for isolation. However, they germinated as well,

if not better, than the initial dormant spores in response to a germinant that targets a different germinant

receptor. A targeted LC-MS/MS method was developed using multiple reaction monitoring to quantify 11

membrane-associated germination-related proteins. Membrane-associated YpeB, GerD, PrkC, GerAC and

GerKC decreased 6.8, 3.5, 3.8, 1.9 and 2.4-fold, respectively, during spore germination. In contrast,

SpoVAD was 1.7-fold more abundant in germinated spore membranes than in those of dormant spores.

GerAC, GerKC, and GerD were the only proteins that were significantly less abundant in superdormant

spores than in the initial dormant spores, showing 3.4, 1.9, and 1.8-fold decreases, respectively.

Similarities in the protein-abundance differences between dormant spores and superdormant or

germinated spores suggest that the superdormant spore isolation procedure may have resulted in certain

early, non-committal germination steps in the superdormant spores, but that they were blocked at a

particular stage in the process. Our results suggest that, in addition to low levels of germinant receptor

proteins, a deficiency in the GerD lipoprotein can contribute to heterogeneity of spore germination.

Correlative Algorithm for Repeat Placement

Abhishek Biswas, David Gauthier, Desh Ranjan, and Mohammad Zubair. Old Dominion University,

Norfolk, VA.

De-novo genome assembly from DNA fragments is primarily based on sequence overlap information. In

addition mate-pair reads or paired-end reads provide linking information for joining gaps and bridging

repeat regions. Genome assemblers in general assemble long contiguous sequences (contigs) using both

overlapping reads and linked reads until the assembly runs into an ambiguous repeat region. These

contigs are further bridged into scaffolds using linked read information. However, errors can be made in

both phases of assembly due to high error threshold of overlap acceptance and linking based on too few

mate reads. Identical as well as similar repeat regions can often cause errors in overlap and mate-pair

evidence. Also, the problem of setting the correct threshold to minimize errors and optimize assembly of

reads is not trivial and often requires a time consuming trial and error process. Therefore, we propose a

novel scaffolding tool, Correlative Algorithm for Repeat Placement (CARP), capable of joining low error

contigs using mate pair reads, resolved repeat structures and verification of joins based on synteny with

one or more reference organisms. The CARP tool requires a set of long repeat sequences such as insertion

sequences that can be manually determined or found computationally. The tool is designed to match very

low error contigs with strong overlap using the ambiguous partial repeat sequence at the ends of the

2013 VA Branch ASM

contig. These matches are verified by synteny with reference to one or more related organisms. We show

that the CARP tool can be used to verify low mate pair evidence regions, independently find new joins

and significantly reduce the number of scaffolds.

Sex hormones differentially regulate HSV-1 gene expression during the early stages of infection in

vitro

Thanh Kim Nguyen and Patric Lundberg. Eastern Virginia Medical School, Norfolk, VA.

The sexual dimorphism of Herpes Simplex Virus-1 (HSV-1) pathogenesis has been demonstrated where

mortality and morbidity are more severe in male mice compared to female mice suggesting that gender

and/or sex hormones have a role in viral pathogenesis. We propose that sex hormones can alter HSV-1

gene expression thereby changing the pathogen life cycle and consequently pathogenesis. By

bioinformatic search, putative HREs were identified throughout the HSV-1 genome. Custom HSV-1

microarrays were performed to examine HSV-1 gene expression changes in the presence of E2 (b-

estradiol) or DHT (dihydroxytestosterone) in both sex hormone receptor neutral (CV-1) and positive

(MCF7) cell lines. In MCF7 cells, global HSV-1 gene expression is upregulated in the presence of DHT

and unchanged in the presence of E2 compared to the unstimulated control; no differences in viral gene

expression were noted in CV-1 cells. Additionally, qRT-PCR demonstrated that a key HSV-1 regulatory

gene ICP0 was upregulated more than 3-fold in the presence of DHT while no difference occurred in the

presence of E2. ChIP analysis confirmed the location of viral HREs within the ICP0 promoter. qPCR

demonstrated a functional change in viral replication when HSV copy number was drastically increased in

the presence DHT compared to E2 and the unstimulated control. We show that androgens provide an

environment where viral gene expression is enhanced and viral replication increased thus showing the

direct effect of sex hormones on HSV-1. To our knowledge, this is the first study to demonstrate sexual

dimorphism at the level of the pathogen.

Targeting Dengue Virus Dependent 3' - 5' Human Exoribonucleases

Krystal Haley and Dr. Daniel Engel. University of Virginia, Charlottesville, VA.

The World Health Organization reports that dengue fever is the fastest spreading mosquito-transmitted

disease in the world, with a 30-fold increase in incidence over the last 50 years. Correlatively, dengue-

associated morbidity and mortality are on the rise, causing a significant public health burden. Since

mosquito population control is currently the only form of disease prevention, therapeutic discovery is

critical. To date, dengue virus has eluded virus-targeting therapies, such as vaccines and antivirals, due in

part to serotype differences. An alternative treatment approach is targeting host cell factors, which are

required for each stage of the dengue virus life cycle. Recent studies have demonstrated that human 3’ –>

5’ exoribonucleases – EXD2, ERI3, and DIS3L2 – are necessary for dengue virus replication in cell

culture. Therefore, we aim to identify novel compounds that inhibit both virus replication and cellular

exoribonuclease activity. We expressed each exoribonuclease in S. cerevisiae, and each induced a slow

growth phenotype. We then used a high-throughput, toxicity suppression screen and identified small

molecule compounds that improved transformed S. cerevisiae growth. Verified compounds will be

selected for future experiments based on their low cellular toxicity and potent antiviral properties. We will

also investigate the role of each exoribonuclease during dengue virus replication. Lastly, we plan to

evaluate the potential use of these inhibitors in the treatment or prevention of dengue virus infection in

humans.

2013 VA Branch ASM

Session IV Abstracts

Protein Interaction Networks in Bacteria

Peter Uetz, Seesandra S Rajagopala, Roman Häuser, and Russell L. Finley. Virginia Commonwealth

University, Richmond, VA.

Protein-protein interactions (PPIs) are a powerful way to elucidate the functions of proteins, including

those of unknown function. However, PPIs have been neglected in the study of enzymes and enzymatic

activities, at least in large-scale studies. We have systematically mapped PPIs in bacteria using a novel

Yeast Two-Hybrid (Y2H) system that uses multiple bait and prey vectors [1].

We have conducted systematic Y2H screens in Treponema pallidum [2], Escherichia coli, Streptococcus

pneumoniae, and Helicobacter pylori that resulted in 3649, 2234, 2057, and 2089 PPIs, respectively.

Given the limitations of the Y2H we estimate that these numbers represent no more than a third of all

interactions. When combined with data from protein complex purifications and mass spectrometry data,

we were able to map another third of all interactions in protein complexes of E. coli, including multi-

protein enzyme complexes.

The E. coli genome encodes an estimated ~1,500 enzymes (i.e. proteins having an EC number in

Uniprot). Among the 2,234 binary PPIs that we found in E. coli 1,276 involve at least one enzyme.

Similarly, of the 1,400 proteins found to be in complexes [3] 655 are enzymes. We conclude that PPIs can

affect both regulatory and metabolic networks.

[1] Stellberger etc 2010, Proteome Sci. 8: 8

[2] Titz etc 2008, Plos ONE 3: e2292

[3] Hu etc 2009, Plos Biol. 7: e1000096

The Impact of Genome Reduction on Conservation of Microbial Protein Complexes and Their

Components

J. Harry Caufield and Peter Uetz. Virginia Commonwealth University, Richmond, VA.

Protein-protein interactions and protein complexes have been systematically studied for only a few

bacterial species. For instance, about 300 and 116 heteromeric complexes have been isolated from E. coli

and Mycoplasma pneumoniae, respectively. Although there are currently more than 20,000 binary

protein-protein interactions published for several species, they rarely capture the full extent to which

proteins participate in complexes. We have coupled the results of published mass spectrometry-

characterized protein complexes with databases of gene orthology, conservation and essentiality to better

characterize protein interactions. We used complexes isolated from E. coli to identify proteins that are

either conserved or lost in other species, shedding light on the essentiality of these proteins and

complexes. Complexes isolated from Mycoplasma pneumoniae, a model genome-reduced species, were

used as a minimal set of protein complexes for most bacterial species. Microbial protein complexes will

help us to understand the biological role of uncharacterized proteins and complexes, especially in less-

studied bacterial species.

Complement evasion strategies of oral treponemes associated with periodontal disease

Daniel P. Miller, John V. McDowell, Jessica K. Bell, J. Christopher Fenno, and Richard T. Marconi.

Virginia Commonwealth University, Richmond, VA.

Periodontal disease, the most prevalent disease of microbial etiology in humans, is a chronic

inflammatory disease that initiates with a shift in the bacterial flora of the subgingival crevice. Oral

spirochetes, which typically represent less than 1% of the total bacterial population in the healthy

subgingival crevice, account for 50% of the bacterial population in periodontal pockets. We have

2013 VA Branch ASM

demonstrated that T. denticola binds FH via its FhbB protein and exploits FH activity to downregulate

complement activation on the bacterial cell surface. We further demonstrate that T. denticola cleaves

bound FH using the dentilisin protease. It is our hypothesis that this activity results in the local depletion

of FH with subsequent damaging immune dysregulation. To better understand the FH-FhbB interaction

we have determined the molecular structure of FhbB (1.7A resolution), mapped FH-FhbB contact points,

and through gene deletion analyses, demonstrate the essential role of FhbB in serum resistance. To date

FhbB-FH studies have focused on T. denticola strain 35405. In this study we seek to define the potential

of other oral treponeme species and diverse T. denticola isolates to influence complement regulation. FH

binding, dentilisin activity and FH cleavage by each isolate was assessed and the influence of FhbB

sequence variation on these processes determined. Lastly, we conducted the first comprehensive analysis

of the serum sensitivity of oral treponemes. The data obtained from this study enhances our understanding

of complement manipulation by T. denticola and other oral treponemes and will facilitate efforts to

develop therapeutic and preventive strategies for this significant human health concern.

The Relationship of the Lipoprotein SsaB, Manganese, and Superoxide Dismutase in Streptococcus

sanguinis Virulence for Endocarditis

Katie E. Crump, Brian Bainbridge, Sarah Brusko, Lauren S. Turner, Xiuchun Ge, Victoria Stone, Ping

Xu, and Todd Kitten. Virginia Commonwealth University, Richmond, VA.

Streptococcus sanguinis is a normal inhabitant of the mouth and an important cause of infective

endocarditis. SsaB, a protein belonging to a family of orthologous metal-transport proteins termed

Lipoprotein Receptor Antigen I (LraI), is critical for endocarditis virulence in S. sanguinis. While LraI

proteins have been shown to transport manganese and additional metals, we are interested in the role of

metal homeostasis in oxygen tolerance. Using isogenic mutants lacking SsaB, the manganese-dependent

superoxide dismutase, SodA, or both, we addressed the specific role of manganese in oxygen tolerance

and virulence in S. sanguinis. The role of SsaB as a metal transporter was confirmed by reduction of

cellular manganese and iron levels in the ssaB mutant. In an animal model of endocarditis, all three

mutants exhibited reduced virulence, with the double mutant exhibiting the greatest defect and the sodA

mutant, the least. This reduction of virulence was not due to diminished resistance to phagocytic killing

or heart valve colonization in either single mutant. Furthermore, the relative virulence of wild-type and

mutant strains was reproduced by a growth assay employing only normal rabbit serum and physiological

levels of oxygen. Using these physiological conditions, SodA activity was decreased but not absent in the

ssaB mutant. Additionally, antioxidant studies suggested that increased damage by superoxide was

necessary for the ssaB mutant’s reduced-growth phenotype, but not sufficient, with additional

contributions by hydroxyl radicals likely. Together, these studies indicate that SsaB-mediated manganese

uptake enables virulence by providing resistance to oxidative stress through SodA-dependent and

independent mechanisms.

Evolution of Thioredoxin (Trx) systems and the role of Trx in Methanocaldococcus jannaschii

Dwi Susantia, Joshua H. Wong, William H. Vensel, Usha Loganathan, Rebecca DeSantis, Ruth A.

Schmitz, Monica Balsera, Bob B. Buchanan, and Biswarup Mukhopadhyay. Virginia Tech, Blacksburg,

VA.

Thioredoxin (Trx), a small redox protein, controls a spectrum of processes in Eukaryotes and Bacteria by

changing the thiol redox status (SH/S-S) of selected proteins. Some the notable examples of Trx-

controlled systems are photosynthesis in plant and cell death and aging in human. Although the Trx is

present in the Archaea, the role of this redox protein in the Archaea remained unknown.

We are studying the development of the Trx systems in a broad range of microorganisms and their

distribution and roles in methanogens. For the last item we focused on Methanocaldococcus jannaschii, a

deeply-rooted hyperthermophilic methanogen inhabiting a deep-sea hydrothermal vents, as the model.

2013 VA Branch ASM

The organism performs only hydrogenotrophic methanogenesis, one most ancient respiratory metabolism

of Earth.

A bioinformatics analysis suggested that the two known Trx systems, one (NTR) being dependent on a

flavin containing NADPH-Trx reductase (NTR) and other involving an iron-sulfur cluster containing

ferredoxin-thioredoxin reductase (FTR) arose independently. FTR was likely invented by the ancient

Bacteria and NTR system was likely developed by the Archaea beginning with an ancestral form (XTR)

that was not dependent on nicotinamides. Their original roles in the control of carbon dioxide fixation and

defense against oxidative damage apparently have been preserved through their evolutionary

development.

Trx was found to be nearly universal in methanogens. Deeply-rooted hydrogenotrophic methanogens,

which are predominantly thermophiles, carried two Trx homologs on average, whereas their more

evolved, nutritionally versatile counterparts carry 4-9 Trx homologs.

Methanocaldococcus jannaschii carries two Trx homologs: Trx1, a canonical Trx, reduces insulin and

accepts electrons from E. coli NADP-thioredoxin reductase. Trx2 is atypical. Proteomic analyses

identified 154 potential Trx1 targets in air-oxidized M. jannaschii cell extracts. These targets represented

a broad range of cellular processes, including methanogenesis, biosynthesis, transcription, translation, and

oxidative response. In enzyme assays, Trx1 activated partially deactivated forms of two of these targets,

F420-dependent methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and

sulfite detoxifying F420-dependent sulfite reductase, validating proteomics observations. These results

suggest that Trx assist methanogens in combating oxidative stress and synchronizing metabolic activities

with the availability of reductants such as H2, thereby influencing global carbon cycle and methane

emission.

2013 VA Branch ASM

Abstracts for Poster Presentations

Session A

1 Suppression of Dendritic Cell Maturation, Activation, and T Cell Stimulatory Capacity by

Melanoma-derived Factors

Johnathon Drake Bishop and Kristian Michael Hargadon. Hampden-Sydney College, Farmville, VA.

We have previously shown that melanoma-derived factors suppress the maturation/activation of the

murine dendritic cell (DC) line DC2.4. Importantly, the extent of this suppression is dependent upon

the tumorigenicity of the melanoma under study, with the highly aggressive B16-F1 melanoma

exhibiting greater suppressive effects than D5.1G4 melanoma, a mutated variant of B16 whose

growth and metastasis is immunologically controlled in a large percentage of hosts. We wished to

extend our previous studies with the in vitro DC2.4 cell culture system by exploring whether

melanoma-derived factors also suppress the maturation and activation of bona fide immature DC

isolated from the murine spleen. Here, we show in an ex vivo system that melanoma-derived factors

also differentially suppress the maturation and activation of DC, which in turn regulates the T cell

stimulatory capacity of these cells. Finally, we identify partial roles for both melanoma-derived

TGFbeta1 and VEGF-A in the suppression of DC function.

2 Antimicrobial Analysis of the Xylylene Series of Novel Amphiphiles

Tara Gallagher, Nicholas Minahan, Kyle Bonifer, Jade LaDow, Kevin Caran, Kevin Minbiole, and

Kyle Seifert. James Madison University, Harrisonburg, VA.

Antibiotic resistance is advancing ubiquitously via multiple modes of horizontal gene transfer (HGT),

and has given rise to highly resistance life-threatening human pathogens such as Mycobacterium

tuberculosis and methicillinresistance Staphylococcus aureus (MRSA). To date, our collective aim

has been to develop a new class of potent, broad-spectrum antimicrobials, based on novel series of

amphiphiles that will be difficult for organisms to counteract or to resist via mutation. In order to

determine the biological activity our amphiphiles, minimum inhibitory concentration (MIC) assays

and minimum bactericidal concentration (MBC) assays were performed for each compound to

determine the inhibitory efficacy of each compound against known bacterial pathogens. Notably, our

amphiphiles have shown greater inhibitory and bactericidal activity than control compounds DTAB,

CTAB and benzalkonium chloride (Lysol), falling within the low micromolar range. Although our

xylylene series (oX, mX, pX) amphiphiles do not match the inhibitory or bactericidal activity of our

mesitylene series (M) or paraquat series (PQ) amphiphiles, they achieve striking synergy in

combination against both Gram-positive and Gram-negative organisms. Synergy of our amphiphiles

is determined by the fractional inhibitory concentration (FIC) index, where an FIC index of ≤0.5

indicates synergy. For example, amphiphiles mX,14,14/mX-1,14 of the xylylene series have an FIC

index of 0.19 for Gram-negative Escherichia coli and 0.50 for Gram-positive Staphylococcus aureus.

All the while, our xylylene series amphiphiles maintain MIC and MBC values as low as 4μM for

Staphylococcus aureus and 8μM for Escherichia coli.

3 Phylogenetic Analyses of Streptococcus parauberis from Fish and Cattle

Keaira Thornton and Ashley Haines. Norfolk State University, Norfolk, VA.

Streptococcus parauberis is a gram-positive lactic acid bacterium that infects cattle and fish, which

can negatively impact dairy and aquaculture fisheries. Recently it has become an emerging pathogen

in these industries. In this work, nucleic and amino acid sequences of multiple housekeeping genes

from fish and cattle isolates were analyzed. These gene sequences were used to construct

phylogenetic trees using publicly available software. We hypothesized that analysis of multiple genes

2013 VA Branch ASM

will better predict the closest relative to S. parauberis than the previous single gene analyses. In

addition, amino acid sequences may further clarify our proposed phylogenies. Our data suggest that

S. parauberis may be more closely related to Streptococcus iniae (a fish pathogen) than to

Streptococcus uberis (a cattle pathogen), the two model species that are most closely related to S.

parauberis. These phylogenetic analyses improve our understanding of the evolutionary

relationships between related bacterial species from different animal hosts. Focusing on the

molecular epidemiology of S. parauberis will help explain how different host species are infected and

may help prevent future outbreaks.

4 Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial

survival inside neutrophils

M. Brittany Johnson and Alison K. Criss. University of Virginia, Charlottesville, VA.

Gonorrhea is a global health problem with 106 million cases each year worldwide. Symptomatic

infection with Neisseria gonorrhoeae(Gc) promotes inflammation featuring influx of

polymorphonuclear leukocytes (PMNs), yet some Gc survive PMN exposure during infection. We

demonstrate a novel mechanism of Gc resistance to PMNs: Gc phagosomes avoid maturation into

phagolysosomes by delayed fusion with primary granules, which contain antimicrobial components

including serine proteases. Reduced phagosome-primary granule fusion was observed via

immunofluorescence in gonorrheal exudates and human PMNs infected ex vivo. Using bacterial

viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated

with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to

PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These

results suggest that Gc surface characteristics, rather than active bacterial processes, influence

phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. In

support of this hypothesis, we observed that delayed phagosome-granule fusion could be overcome

by opsonizing Gc with immunoglobulin or by Gc surface expression of opacity-associated proteins

(Opa). The residence of Gc in mature phagosomes due to opsonization or Opa expression resulted in

decreased Gc viability, which was attributed in part to serine protease activity. We conclude that one

method for Gc to avoid PMN clearance in acute gonorrhea is by delaying primary granule-

phagosome fusion via Gc surface composition, thus preventing formation of a phagolysosome.

5 Stabilization of Vaccines against Heat – I

Samson S. Stratton, Jeffrey D. Raiford, Anthony W. Thomas, and Carl W. Vermeulen. Paul D. Camp

Community College, Franklin, VA.

As almost all vaccines require refrigeration, nearly 20% of the world (1 billion people and even more

livestock) has little or no access to their protections. In this Phase I, various enzyme models for

vaccines were straight-jacketted in pure dried starch and continuously subjected to temperatures up to

70c for many weeks with little if any loss of activity. Furthermore, dried starch is inherently its own

delivery device as thin "noodles" of it have the strength to penetrate intramuscularly through skin,

and the body's amylases degrade it within minutes releasing its vaccine content.

6 Identification of Fungi Associated with Rhizome Rot in the Medicinal Plant, Actaea racemosa

(Black Cohosh)

Michelle Donahoe, Lindi Hutchinson, Jasmine L. Vaughn, Christine Small, and Georgia A.

Hammond. Radford University, Radford, VA.

Black Cohosh (Actaea racemosa) is a widely known and used medicinal plant that is endemic to the

southeast region of the United States, more specifically, the Appalachian mountains of Virginia, West

Virginia, Tennessee, North Carolina, and Kentucky. Black Cohosh is in high demand because it is

2013 VA Branch ASM

internationally used as an alternative treatment for menopausal symptoms. A number of studies have

been devoted to transplantation of black cohosh rhizomes because there is severe danger of over-

harvest and extinction of the plant. During transplantation, Black Cohosh has been found to be

susceptible to fungal infections that are loosely referred to as rhizome rot. Rhizome rot severely limits

the success of transplantations. Our research focuses on identifying the fungi causing rhizome rot in

transplantations that are ongoing in southwestern Virginia, using ITSrRNA sequencing. The ITS

region is found in all fungi and it varies in term of sequence and there for can be used for

identification. We have isolated fungal DNA from infected rhizomes, as well as from soils from pre-

and post-transplantation sites. Our goal is to identify the various fungi that are associated with the

rhizome rot, to identify any fungi that could potentially be in the soil and to use these data to design

strategies to improve transplantation of this valuable native plant.

7 Identifying bacteria that live in high concentrations of arsenic

Jessica O'Grady, Lindi Hutchinson, and Dr. Georgia Hammond. Radford University, Radford, VA.

Microorganisms are exceedingly abundant and diverse in the soil, making it difficult for scientists to

characterize them. Some bacteria can thrive in extreme environmental conditions due to their genetic

makeup. Our focus is on bacteria capable of metabolizing arsenic, a heavy metal. We obtain soil

samples from a headwater stream at an abandoned arsenic mine in Floyd County, Virginia. Erosion at

this site has caused arsenic to leach out of the soil and into a headwater stream nearby, potentially

contaminating major river systems in Virginia. According to our previous research, bacteria isolated

at this site are able to live in arsenic rich environments because they contain arsenic resistance genes,

and these genes are expressed at high levels when pure lab cultures of bacteria from the site are

grown in arsenic. Our goal is to characterize these bacteria down to the genus and if possible, species

level using the 16s rRNA gene. This gene sequence is highly conserved among bacteria; however, it

contains variable regions with point mutations that can be used for identification purposes. To

accomplish this, we isolated DNA from pure cultures of the bacteria, amplified the 16s rRNA genes

with a PCR reaction, purified the PCR product, and sequenced the DNA. We then put the obtained

sequences into databases to compare our sequenced data with known ribosomal subunit sequences of

identified bacteria. Our results contribute to knowledge regarding bacteria capable of surviving in

environments containing the heavy metal, arsenic.

8 The quorum sensing regulator ExpR directly regulates symbiotically important Flp pili in

Sinorhizobium meliloti

Hardik Zatakia, Cassandra Nelson, Anjali Sharma, and Birgit Scharf. Virginia Tech, Blacksburg, VA.

Type IVb pili (Tfpb) are mostly studied in enteropathogens and have diverse functions in bacterial

aggregation, attachment, and microcolony formation. The Sinorhizobium meliloti genome contains

two pil gene clusters; one on the chromosome (pil1) and the other on the pSymA megaplasmid (pil2),

both of which code for the genes required for Tfpb synthesis. pilA1 in the pil1 gene cluster encodes

the putative pilin subunit belonging to the flp (fimbrial low-molecular-weight protein) family of

pilins. To establish the role of Tfpb in the symbiotic interaction of S. meliloti and its host Medicago

sativa, we conducted competitive nodulation assays. The S. meliloti pilA1 deletion strain was about

30% deficient in nodulation as compared to the wild type. Absence of pili in the pilA1 deletion strain

was confirmed using transmission electron microscopy. Transcriptional reporter gene assays

illustrated that the expression of pilA1 peaks at early stationary phase and is repressed by ExpR.

ExpR is a LuxR-type transcriptional regulator and part of the quorum sensing system in S. meliloti. It

regulates exopolysaccharides production and motility in S. meliloti. Direct binding of AHL-activated

ExpR to the pilA1 promoter region was confirmed with electrophoretic mobility shift assays. A 28-bp

protected region on the pilA1 promoter was defined using DNase I footprinting analyses. This

protected region comprised a 17-bp sequence that matches the consensus sequence for ExpR binding.

2013 VA Branch ASM

Thus, our studies show that S. meliloti coordinates temporal expression of Type IVb pili with other

cellular processes namely exopolysaccharide synthesis and motility for optimal interaction with its

host.

9 Integration of a new chemotaxis protein CheT in the chemosensory signaling chain of

Sinorhizobium meliloti

Umair Syed and Birgit E. Scharf. Virginia Tech, Blacksburg, VA.

Sensory signal transduction in bacterial chemotaxis is generally mediated through a two-component

regulatory system consisting of a histidine autokinase, CheA, and a response regulator, CheY. The

symbiotic alpha-proteobacterium Sinorhizobium meliloti employs an indirect phosphate sink

mechanism rather than direct dephosphorylation to inactivate the motor-binding response regulator,

phosphorylated CheY2. In this mechanism, phosphoryl groups are shuttled from CheY2-P back to

CheA, which in turn phosphorylates CheY1. While reported for the first time for S. meliloti, the

presence of more than one response regulator in the chemotactic signaling chain in bacterial species

outside the enterobacterial group suggests that a phosphate sink is a widespread mechanism for signal

termination. In addition to the canonical chemotaxis genes, the che1 operon contains two open

reading frames coding for novel proteins, CheS and CheT. We have recently shown that CheS

reduces CheY2-P levels by enhancing the binding of CheY1 to the kinase CheA. Behavioral assays of

DcheT cell populations showed that CheT has the opposite effect in promoting an increase in CheY2-

P levels by a yet unknown mechanism. Bacterial chemoreceptors prototypically assemble into polar

clusters where they form large sensory complexes that recruit cytoplasmic components of the

signaling pathway. The current study analyzes the localization of CheT fused to the fluorescent

proteins eGFP and mCHERRY by fluorescence microscopy. Furthermore, the interaction of CheT

with all eight chemotaxis proteins will be determined using individual deletion strains. Results of this

study will give insight into the placement of CheT in the signaling transduction chain of S. meliloti.

10 Towards an Understanding of the Role of Motility and Chemotaxis in Antitumor Efficacy of

Salmonella typhimurium VNP 20009

Katie Broadway and Dr. Birgit Scharf. Virginia Tech, Blacksburg, VA.

Several bacterial species have been found to be attracted to tissues ridden with cancerous cells. This

leads to colonization of the tissue, effective tumor shrinkage and host survival. Salmonella, a

facultative anaerobic, enteric, motile bacterial species, is tumor targeting. These bacteria

preferentially penetrate and replicate within tumors, suppressing their growth and metastasis. The

selected bacterial strain used in this project is VNP 20009. This is a mutagenized derivative of

Salmonella enterica serovar Typhimurium 14028, a species commonly studied in virulence and

immunology. The long term goal is, in a collaborative effort joining the fields of engineering and

microbiology, to assess the effect of motile VNP 20009 bacteria loaded with anticancer drug carrying

nanoparticles on tumor eradication. The aim of this project is to characterize and maximize the

positive chemotactic response VNP20009 possesses towards tumor cells. In previous work, it has

been shown that motility is critical for this strain to accumulate in tumor tissue. Various mutant

strains of the VNP 20009 were constructed by lambda-red genetic engineering, and their behavior

compared to the parental 14028 strain. In addition, the genome of VNP 20009 will be annotated and

the genes responsible for the tumor seeking characteristic will be investigated. With this knowledge,

it is anticipated that the bacteria can then be genetically modified to increase their affinity to

cancerous cells in host tissue. Through understanding and analyses of this strain's ability to colonize

tumors by the bacterial cell-drug delivery complex, cancerous cell death can be maximized.

11 Designing Tricistronic and Bicistronic Lenti-Viral Vectors using Scar-less DNA Assembly

Methods and Web-based Software to Study GRK4

2013 VA Branch ASM

Christophe Langouet-Astrie, Louise Temple, Dora Wang, John Gildea, and Robin Felder. James

Madison University, Harrisonburg, VA.

In this study, the goal is to develop a diagnostic tool for salt sensitivity that involves detection of

specific protein, in this case G protein-coupled receptor kinase 4 (GRK4). To measure this protein’s

activity and production, two issues arise: constructing a delivery system to introduce the DNA in a

timely and cost-effective manner and quantifying the amount of protein produced in live cells. For the

former issue, novel gene cloning techniques known as scar-less multipart DNA assembly methods

have been created that have been developed to optimize cost and time effectiveness. Due to the

difficulty in optimized protocols for these methods, a web-based software known as j5 contains

programs that aid in creating a protocol. For the latter issue, these assembly techniques and the web-

based software are used to construct lenti-viral delivery system containing bicistronic and tricistronic

vectors. Bicistronic vectors are commonly used for measuring protein synthesis my indirect

measurement of a fluorescence protein. Development of tricistronic vectors allow for introduction of

one protein with two modes of measurement i.e. microscopy and antibiotic selection. Two bicistronic

vectors were constructed containing GRK4 and Zeocin resistance in one vector and an AT1 receptor

and a CYPHR protein in the other. In addition, a tricistronic vector is being constructed containing

GRK4, a Zeocin resistance, and a fluorescence protein with self-cleaving peptide sequences and

internal ribosomal entry sites. These tools will help measure GRK4 production and activity in order

to help develop an assay for detecting salt sensitivity in humans.

12 Identifying Environmental Bacillus

Vincent A. Gentilcore, Dylan S. McKnight, and Georgia A. Hammond. Radford University, Radford,

VA.

The Bacillus genus is one of a large group of bacteria found in nature. Many environmental Bacillus

species can’t be identified using the Small Subunit rDNA sequence. Our study focuses on Small

Acid-Soluble proteins (SASPs), which are unique to endospore-forming bacteria including Bacillus.

SASP proteins in Bacillus species are unique relative to SASP proteins found in other genera of

endospore formers such as Clostridium. Using DNA sequences for two major SASP proteins (SASP-

A and SASP-B) from Bacillus subtilis, we have developed PCR primers to use in PCR reactions that

examine the DNA of bacteria that we collect from the environment. Our objective is to use these

primers in a PCR reaction that uses a gradient of annealing temperatures to detect major SASP genes

in unknown species. Detection of a PCR product would be very good evidence that the unknown

bacterium was a member of the genus Bacillus.

13 Antibiotic resistance and putative virulence factors of clinical and environmental strains of

Vibrio vulnificus

James Conrad, Pradeep Vasudevan, and Joanna Mott. James Madison University, Harrisonburg, VA.

Antibiotic resistance in the pathogen, Vibrio vulnificus, is a major concern for the medical community

as well as aquaculture and saltwater aquaria hobbyists. Infections progress rapidly and effective

treatment is crucial. V. vulnificus is found in warm brackish coastal waters, sediments and associated

with organisms such as oysters and fish. Several virulence factors have been correlated with clinical

isolates, including mannitol fermentation, salicin metabolism, and presence of pilF. Previously 253 V.

vulnificus isolates were collected from infected patients, oysters, and water samples from several

states. Each isolate was characterized based on its pilF gene and D-mannitol fermentation. In this

study, a subsample of these isolates was subjected to antibiotic resistance analysis (ARA) to

determine any correlations between antibiotic resistance and clinical or environmental origin.

Additional isolates were collected from residential and commercial saltwater aquaria. Each isolate

was confirmed as V. vulnificus by amplification of the vvhA gene and then subjected to ARA. A drug

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panel was selected based on CLSI M45-A2 recommendations as well as antibiotics frequently used to

test V. vulnificus. At least one antibiotic from each class in CLSI M45-A2 was included in the drug

panel. Isolates were then grouped based on their ARA, pilF, and D-mannitol fermentation profiles to

determine any correlations between putative virulence factors, ARA patterns, and origin. This data

will provide information on the current levels of antibiotic resistance in V. vulnificus from different

sources.

14 Antimicrobial Analysis of the Meta-Pyridine series of Novel Ampiphiles

Brandi Volkers and Kyle Seifert. James Madison University, Harrisonburg, VA.

In the US, approximately 2 million patients acquire nosocomial infection each year during their stay

in health care facilities, resulting in over 100,000 deaths. Multi-drug resistant organisms initiate many

of these infections, making the development of novel effective antimicrobials paramount in hospital

settings. As a continuation of our previous research on bicephalic amphiphiles, which correlated

antibacterial activity with head group arrangement and chain length, the antimicrobial activity of

three distinct series of amphiphiles were investigated. The first series, gemini, is characterized by a

bi-substituted arene core with identical head groups each with hydrocarbon tails of the same length

(14 carbons). The second series, bicephalic, is also characterized by a bi-substituted arene core with a

pyridine group in place of a head group and hydrocarbon tail. The final series, mesitylene, contains

an arene core substituted with two identical head groups each with hydrocarbon tails of the same

length (10-16 carbons) and a non-substituted pyridine head group. The series of amphiphiles were

assayed against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Bacillus

subtilis, Streptococcus agalactiae and Escherichia coli, by performing standard minimum inhibitory

concentration (MIC) and minimum bactericidal concentration (MBC) assays. MIC and MBC values

for these amphiphiles were in the low micromolar range. Specifically, the compound M-P,12,12 from

the mesitylene series, “meta-pyridine,12 carbon chain, 12 carbon chain”, showed greater inhibition

and bactericidal activity than control compounds, DTAB, CTAB and Lysol. Synergy assays

performed against E. coli and S. aureus and biofilm disruption assays against P. aeruginosaalso

yielded positive results, bringing biofilm levels down significantly in some cases.

15 Mutational Analysis of Disulfide Bond Oxidoreductase DsbA2 Function in Legionella

pneumophila

Zegbeh Z. Kpadeh, Shandra R. Day, and Paul S. Hoffman. Division of Infectious Diseases and

International Health, Department of Medicine, Department of Microbiology, Immunology and

Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA.

Background: Legionella pneumophila (Lpn) is a Gram negative, intracellular parasite of free-living

protozoa that when aerosolized causes Legionnaires’ disease, an occasionally fatal pneumonia. The

Dot/Icm type IVb secretion system (T4SS) is the main virulence mechanism and is dependent on

correct disulfide bond (DSB) formation catalyzed by a novel and essential DSB oxidoreductase

DsbA2 and not by DsbA1, a nonessential DSB oxidoreductase. DsbA2 is a bifunctional enzyme

containing a highly conserved N-terminal dimerization domain enabling the protein to form a

homodimer. Deletion of the dimerization domain produced the monomer (DsbA2N), which no longer

exhibits protein disulfide isomerase activity, but complements DsbA activity in an E. coli dsbA

mutant.

Methods: A cis-proline mutant of DsbA2N (P198T) was expressed in Lpn AA100 strain to assess the

effect on virulence. Intracellular growth in Acanthamoeba castellanii, attachment and invasion of

HeLa cells and T4SS contact-dependent hemolysis of erythrocytes was compared to empty vector

control. The interacting DSB partners of DsbA2N were identified in an E. coli background using a

soft agar motility assay.

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Results: A measurable defect in intracellular growth by expressing DsbA2NP198T in A. castellanii

was seen at 48 and 72 hours, and attachment and invasion of HeLa cells was significantly reduced

(p=0.005); suggesting DsbA2 is important for pathogenesis. Erythrocyte hemolysis was significantly

attenuated by expression of DsbA2NP198T (p<0.0001), demonstrating the importance of DsbA2 for

T4SS function. Soft agar motility assay determined DsbA2N interacts with both DsbBs of Lpn

(DsbB1 20% and DsbB2 (LidJ) 40% complementation).

Conclusions: DsbA2NP198T produces a dominant negative effect on DsbA2 function, resulting in a

decrease in infectivity, intracellular replication and function of the Dot/Icm T4SS. DsbA2N now

interacts with DsbB2 (LidJ). These results suggest that the oxidized DsbA2N out competes DsbA2

for substrates, affecting T4SS assembly and function and Lpn virulence.

16 Two Lytic Transglycosylases are Important for Neisseria gonorrhoeaeSurvival from Human

Neutrophils

Stephanie Ragland and Alison Criss. University of Virginia, Charlottesville, VA.

Symptomatic infection by the obligate human pathogen, Neisseria gonorrhoeae(Gc), induces a potent

inflammatory response involving a purulent, neutrophil-rich exudate. Despite the neutrophils’

plethora of mechanisms to neutralize bacteria, some Gc are viable in their presence. This neutrophil

recruitment may be influenced by the extracellular release of peptidoglycan (PG) during periods of

bacterial growth and cell wall remodeling, stimulating the intracellular pattern recognition receptor

NOD1. PG is comprised of chains of repeating disaccharide subunits cross-linked by each subunit’s

attached peptide stem. Lytic transglycosylases A (LtgA) and D (LtgD) cut the β-1,4-linkage between

the disaccharide subunits of PG. Consequently, one disaccharide subunit with its attached peptide

stem, a monomer of PG, is released (3). Most extracellularly released monomers are NOD1 agonists

and alone can recapitulate the in vivo tissue damage caused by Gc infection on fallopian tube

epithelium (2). Monomer influence on neutrophils, however, has not been investigated. We found an

ltgA and ltgD double mutant, shown to abrogate release of NOD1 agonists (1), survives less well than

wild type when challenged with primary human neutrophils. The ltgA ltgD survival defect requires

mutations in both genes and is not caused by a differential internalization of the mutant within

neutrophils. These findings suggest cell wall remodeling may contribute to the ability of Gc to

stimulate yet survive the human inflammatory response in gonorrhea. We are testing two non-

exclusive hypotheses to explain the mutant defect: (1) differential activation of neutrophils and/or (2)

increased sensitivity to neutrophil antimicrobial components due to changes in cell wall architecture.

1. Cloud-Hansen K, Hackett KT, Garcia DL, and Dillard JP. “Neisseria gonorrhoeaeUses Two Lytic

Transglycosylases To Produce Cytotoxic Peptidoglycan Monomers.” Journal of Bacteriology. 190.17

(2008), 5989-5994.

2. Melly AM, McGee ZA, and Rosenthal RS. “Ability of Monomeric Peptidoglycan Fragments to

Damage Human Fallopian-Tube Mucosa.” The Journal of Infectious Diseases. 149.3 (1984), 378-

386.

3. Yolande CA, Hackett KT, and Dillard JP. “The Lytic Transglycosylases of Neisseria

gonorrhoeae.” Microbial Drug Resistance. 18.3 (2012), 271-279.

17 The Effect of Harmonic Frequencies on the Disruption of a Bacterial Biofilm

Amarilis A. Dyer, Jarrod Cath, Onur Bilgen, and Dayle A. Daines. Old Dominion University,

Norfolk, VA.

Biofilms are composed of microorganisms that adhere to a surface using a self-produced matrix of

extracellular polymeric substances. Biofilms that form on biotic or abiotic surfaces are often

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antibiotic resistant. This resistance is due in part to the failure of an antimicrobial agent to penetrate

the full depth of the biofilm. We hypothesized that a biofilm could be physically removed from an

abiotic surface if subjected to harmonic vibrations. More specifically, the disruption of biofilms in

tympanostomy tubes and catheters were of primary interest. The overall goal of this study was to

identify the characteristics of the harmonic excitation, namely the frequency, which would be most

effective to de-laminate biofilms from an abiotic surface. In order to achieve this goal, we first tested

the static biofilm formation capabilities of five different bacteria, two Gram-positive (Staphylococcus

epidermidis and Micrococcus luteus), and three Gram-negative (Escherichia coli, Serratia

marcescens, and Enterobacter cloacae). The organisms were allowed to form static biofilms in 96-

well plates for 24 and 48 hours. Each biofilm was then quantitated via the crystal violet assay. It was

determined that E. cloacae formed the maximum biofilm of all strains tested, and that a 24 hour time

point was sufficient. Accordingly, we subjected 24 hour E. cloacae biofilm plates to various

frequencies of harmonic excitation and measured each for their biofilm disruption capabilities

compared to an untreated control plate. We conclude that 1931 Hertz was the optimal frequency to

de-laminate E. cloacae biofilms.

18 Internalization of non-opsonized, opacity protein-negative Neisseria gonorrhoeae by human

neutrophils

Kylene Daily, Asya Smirnov, Brittany Johnson, and Alison Criss. University of Virginia,

Charlottesville, VA.

Neisseria gonorrhoeae, the gonococcus or Gc, is a gram-negative diploccocal bacterium responsible

for the sexually transmitted infection gonorrhea. We seek to understand how polymorphonuclear cells

or neutrophils (PMNs), the most abundant white blood cells in acute gonorrhea, phagocytose

unopsonized Gc. Previously, the only accepted mechanisms of Gc internalization in PMNs were via

opsonic receptors, for antibodies or complement, or via CEACAM receptors for Opas, the opacity-

associated proteins, which are a major surface component of Gc. However, we reported that piliated,

unopsonized, Opa-negative Gc of strain FA1090 (Opaless) is internalized by adherent PMNs (Ball

and Criss, Journal of Bacteriology 195:2982, 2013). Here, we found that when PMNs were treated

with an antibody against the CD11b component of complement receptor 3 (CR3), Gc internalization

was significantly reduced compared to untreated PMNs. CD11b was found to cluster at sites of

Opaless Gc internalization. These results indicate a role for CR3 in non-opsonized Opaless Gc

internalization, but the mechanism remains unknown. We are exploring three possibilities for how

CR3 contributes to Gc internalization by PMNs: 1) PMNs release complement to opsonize Gc, 2)

CR3 directly interacts with Gc via bacterial pili and porin surface components, as previously shown

to occur in epithelial cells, and/or 3) signaling through CR3 enhances macropinocytosis or other

phagocytic mechanisms by PMNs. These findings will shed light on how Gc survives the

inflammatory response in its human hosts to cause persistent infection.

19 Examination of Secreted Cyclic AMP Levels in Mycobacterium smegmatis

Blakley Sproles and Dr. Michaela Gazdik. Ferrum College, Ferrum, VA.

This research analyzed the produced and secreted levels of cyclic AMP in Mycobacterium smegmatis.

This species was used because it can serve as a model for Mycobacterium tuberculosis, the bacteria

responsible for the tuberculosis infection. Mycobacterium tuberculosis (Mtb) contains a total of

fifteen adenylate cyclases, an abnormally large number for a bacterium. Adenylate cyclases are

responsible for the production of cyclic AMP, which plays an important role in the tuberculosis

infection. Additionally, it has been shown that secreted cyclic AMP plays a role in Mtb virulence.

This research was performed to gain a better understanding of when cyclic AMP production and

secretion levels are highest throughout the life cycle of the bacteria. The obtained results suggest that

Escherichia coli and Mycobacterium smegmatis have differing levels of intracellular and extracellular

2013 VA Branch ASM

cyclic AMP throughout their growth. These levels included a peak of intracellular cyclic AMP

concentration during the transition from log phase to stationary phase and a peak of extracellular

cyclic AMP concentration at both the lag and log phases. Mutants of Mycobacterium smegmatis are

currently being generated through the use of transposon mutagenesis. An analysis of these mutants

can allow for differences in cyclic AMP secretion to be detected. If changes are found, the transposon

can be located within the genome of the mutants and any key proteins that are involved with the

secretion of cyclic AMP can be identified.

20 Exploring Mycobacteriophage Host Range in Mycobacterium ulcerans Using Genomic

Comparisons

Anisa C. Tracy, Nicole M. Roberto, and Dr. Steven G. Cresawn. James Madison University,

Harrisonburg, VA.

The genus Mycobacterium is diverse, ranging from slow growing human pathogens such as M.

tuberculosis to fast-growing saprophytes such as M. smegmatis. M. ulcerans is the causative agent of

the neglected disease known as Buruli ulcer, an emerging pathogen first described in Buruli County,

Uganda. Antibiotic resistance among Mycobacterial pathogens is growing at an alarming rate and is

creating an urgent need for novel diagnostic and therapeutic approaches. Elucidating the details of

virus/host interactions among the mycobacteria and their phages thus has the potential to affect how

we think about the role of these bacteria in both clinical and environmental settings.

Toward this end, we are analyzing the complete genome sequence of 461 mycobacteriophages

isolated on M. smegmatismc2155. Based on the average pairwise nucleotide identity and phamily

composition, the genomes have been divided into 20 clusters, with some clusters further subdivided

into 43 subclusters. These phages were isolated by a variety of institutions involved with the Howard

Hughes Medical Institute (HHMI)-sponsored SEA-PHAGES program and by the HHMI-sponsored

PHIRE program at the University of Pittsburgh.

Among those tested, the ability of phages to infect M. ulcerans appears to be restricted to a subset of

those phages in just two clusters. To broaden the diversity of our collection, we have isolated seven

mycobacteriophages from Ugandan soil and are determining their ability to infect M. ulcerans. We

anticipate that soil in an M. ulcerans-endemic region will be likely to contain the greatest number of

phages for which this is a permissive host.

21 Tools for membrane protein investigations: integrating bacterial outer membrane proteins to

functionalized proteoliposomes

Jennifer N. Martin, Louise M. Ball, Tsega Solomon, Alison K. Criss, and Linda Columbus.

University of Virginia, Charlottesville, VA.

Bacterial pathogens have evolved to gain entry into target cells using ligands specific to host

receptors. In the case of Gram negative pathogens, these are often outer membrane proteins.

Neisseria gonorrhoeae(Gc) is the causative agent of the human sexually transmitted infection

gonorrhoea and highly evolved to causing disease in humans. Gc possesses a family of outer

membrane proteins - opacity-associated (Opa) proteins. Opa proteins are eight stranded β-barrel

proteins, and are involved in bacterial engulfment into human host cells. We are taking an approach

to integrate these proteins into a biological toolbox in order to improve understanding of structure and

interaction with the host receptors, independent of the biological cells. In order to study Opa proteins

independent of the cell system, they need to remain in a membrane like environment. Liposomes,

vesicles formed by lipid bilayers, provide the membrane-like structure and are commonly used for

biochemical research, including cell tracking and drug delivery (1). A variety of OpaCEA proteins,

which bind to human Carcinoembryonic Antigen-related Cell Adhesion Molecules (CEACAMs), and

Opa proteins that are non-CEACAM binding were expressed in Gc and expressed to the outer

membrane Escherichia coli. Opa proteins were also overexpressed in E. coli, purified and refolded

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into liposomes. Interactions of these Opa proteins with CEACAMs 1 and 3 were investigated and

assessed using pull-down assays and immunoblotting. We observed similar binding behavior between

all investigated Opa systems, suggesting a conservation of Opa structure outside of Gc.

1. Csiszar, Agnes et al. Bioconjugate Chemistry 21, no.3 (2010).

22 Pseudomonas Culture Supernatants as a Novel Antiviral and Mosquito Larvicidal Agent

Nate Frisch and Justin Anderson. Radford University, Radford, VA.

Pseudomonas species secrete extracellular pigments, such as pyocyanin, that are known to have

antibiotic properties. Our goal was to investigate the possibility of antiviral capabilities of these

extracellular molecules. To test for larvicidal activity, 25 3rd instar Aedes albopictus mosquito larvae

were added to a beaker with 50% Pseudomonas culture supernatant in water. Mortality was observed

at 24 hours and 48 hours. Thirteen pigments have been tested to date, with mortality ranging from 0%

in most cases to 100% in two isolates. Most tested cultures were isolated from mosquito midguts or

diverticula and have not yet been identified; they tended to cause little to no mortality. Pseudomonas

aeruginosa and P. chlororaphis culture supernatants demonstrated 100% larvicidal activity at 50%

dilutions. To test for antiviral activity, we used the mosquito-borne La Crosse virus (LACV) as our

model. La Crosse virus was incubated with serially diluted Pseudomonas supernatants and then

quantified by plaque assay in Vero cells. Pseudomonas aeruginosa demonstrated 100% viral

inhibition at concentrations as low as 22%, and purified pyocyanin also showed strong inhibition at

all concentrations tested. One mosquito midgut isolate (AaMG7) demonstrated potent anti-LACV

activity at high concentrations, but only minimal larvicidal activity, and this selectivity is especially

interesting. We believe these bacteria may thus be used to control mosquito-borne virus transmission,

pending isolation and characterization of the active compounds.

23 Characterization of von Willebrand Factor A-Domain Containing Protein in Clostridium

perfringens

Sarah Nikraftar, Stephen Melville, and Andrea Hartman. Virginia Polytechnic Institute and State

University, Blacksburg, VA.

Clostridium perfringens is an anaerobic Gram-positive bacterium which causes gas gangrene and

food poisoning. Although commonly found in gram negative bacteria, recently Type IV pili (TFP)

were discovered in C. perfringens which contributes to its gliding motility. In previous studies on

pilin subunits, the secretome of a pilA3 deletion mutant was missing the product of gene cpe0517, a

71 kD protein characterized as a von Willebrand factor type A domain-containing (vWA) protein.

vWA from C. perfringens has 30% homology to the eukaryotic von Willebrand Factor which is

involved in protein-protein interactions. Due to similarities between TFP and Type 2 Secretion

System apparatus (T2S), we hypothesize that vWA protein is secreted by a T2S. Our goal is to

characteristize the role of this protein in C. perfringens pathogenesis. So far, we have been able to

purify the vWA protein using nickel affinity chromatography and tested it on mouse myoblasts for

toxicity but no effect was seen. We have also performed binding assays using fluorescently labelled

vWA to study its ability to bind to eukaryotic cells and preliminary results suggest that it does so. An

in-frame deletion mutant of vWA in strain HN13 was also constructed to study the phenotype. We

also want to determine the structure of the vWA protein by X-ray crystallography because of its

predicted structural homology to the RagA protein located on the tip of Streptoccus pyogenes

fimbrae.

24 Determining Ixodes scapularis salivary proteins that could be designed for tick vaccines.

Ryan Hart, Noble Egekwu, and Daniel Sonenshine. Old Dominion University, Norfolk, VA.

2013 VA Branch ASM

Acaricides are currently used to prevent tick attachment and transmission of tick-borne pathogens to

livestock, but their application can be detrimental. Alternatively, vaccines against ticks can be used to

reduce the cost of protecting livestock and the probability of tick resistance, prevent health risks, and

are environmentally safe. Ixodes scapularis, a “black legged tick” is known to form a cement cone

during attachment. The cement cone is formed by proteins produced in the salivary glands, and most

of these proteins are unknown. Determining I. scapularis salivary proteins’ responsible for the

formation of the cement cone could be used to design tick vaccines that prevent attachment to host.

Through the dissection of I. scapularis ticks, we obtain multiple samples of the salivary gland and

then use NanoDrop spectrophotometry to determine the concentration of the proteins in each sample.

Using either Coomassie blue stain gel or Silver stain gel protein assays, we determine the type of

proteins in the sample. By using Liquid Chromatography Mass Spectrometry (LC/MS), we could

then determine the specific salivary protein obtained from the completed stained gel. From these

results, we could determine the proteins found in a cement cone formed on a host. These salivary

proteins could then be matched and used as a tick vaccine designed to reject tick attachment and

prevent the transmission of tick-borne pathogens.

25 Antibiotic resistance in Vibrio vulnificus isolated from the Chesapeake Bay during the summers

of 2006 and 2013.

Amanda L. Laverty, Despoina S. Lymperopoulou, Silvia Rondón-Delgado, and Fred C. Dobbs. Old

Dominion University, Department of Ocean, Earth & Atmospheric Sciences, Norfolk, VA.

Vibrio vulnificusis a human pathogen found in estuaries, brackish ponds, and coastal areas. This

bacterium accounts for 95% of seafood-related deaths in the United States, mostly in Florida and Gulf

Coast states. Scientists predict that as sea-surface temperatures increase, concentrations of this

bacterium also will increase and spread northward. Compounding the concern with V. vulnicificus’

anticipated northward expansion is the issue of antibiotic resistance. The principal objective of this

study was to assess whether antibiotic resistance in V. vulnificus isolated from the lower Chesapeake

Bay has changed over time. Antibiotic-resistance profiles of strains collected and cryo-preserved in

2006 were compared with profiles of strains isolated in summer 2013. In both years, samples were

collected from the water column and the eastern oyster, Crassostrea virginica. Resistance to

streptomycin was evident in both studies, but intensities of resistance differed. A greater percentage

of isolates from 2013 were resistant to streptomycin, along with increased intensities of resistance to

the antibiotic. These results suggest that V. vulnificus experiences changes in antibiotic resistance

over time. More studies will increase understanding of temporal changes in antibiotic resistance in

this deadly human pathogen.

26 Speciation and antibiotic resistance of Enterococcus spp. isolated from turkey litter

Steven McBride, Ben Holland, Pradeep Vasudevan, and Joanna Mott. James Madison University,

Harrisonburg, VA.

The use of antibiotics in agriculture and animal husbandry has been associated with an increase in

antibiotic resistance in microorganisms. Extensive research has addressed the prevalence of resistant

bacteria in livestock wastes such as pig manure, cattle manure, and broiler litter; however, little work

has been conducted on their prevalence in turkey litter. Enterococcus spp., a fecal bacteria group, has

been found in high levels in turkey litter and was chosen for the present study. Turkey litter was

collected at the end of the rearing period from a poultry house in Shenandoah County, VA.

Presumptive enterococci were isolated using membrane-Enterococcus Indoxyl-β-D-Glucoside agar.

The Biolog MicrologTM system was used to speciate isolates, and antibiotic resistance was

determined by the Kirby-Bauer disc diffusion test with automated image analysis using a BiomicTM

plate reader. We expect these results to add to the understanding of antibiotic resistance within the

farm-to-fork paradigm.

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27 Effect of rainfall on fecal indicator bacteria levels in urban retention ponds

Hilary Kurland, Marc Carpenter, and Dr. Joanna Mott. James Madison University, Harrisonburg, VA.

Fecal contamination remains a growing problem within urban water systems, and the presence of

fecal indicator bacteria (FIB) is used as a means of quantifying overall water quality. The Edith J.

Carrier Arboretum at James Madison University contains two separate retention ponds that overflow

and drain through a creek into Newman Lake on the opposite side of campus following rainfall.

Previous studies on Newman Lake revealed high levels of fecal indicator bacteria from undetermined

source(s). During rain events, the Lake overflows and the run-off drains into Black’s Run, the nearby

creek system that runs through the city of Harrisonburg, VA. The two ponds in the Arboretum were

sampled biweekly and immediately after rainfall (>1.00in) and the fecal indicator bacteria

Escherichia coli and Enterococcus spp. were enumerated through standard membrane filtration

methods. Quantification of fecal indicator bacteria levels after rainfall revealed an increase in both

E.coli and Enterococcus levels at all of the sampling sites, both entering and leaving the ponds, with

no discernible trend between sites. The increase in fecal indicator bacteria levels following rainfall

indicates the run-off from the Arboretum may be a source of fecal contamination in Newman Lake.

As such, this poses a potential problem for water quality downstream through Black’s Run and into

the city of Harrisonburg.

28 Effect of Lipomannan from M. smegmatis on RAW 264.7 Cells

Stephanie DeRonde and David Freier. Lynchburg College, Lynchburg, VA.

The dose response activation of RAW 264.7 murine macrophages by M.smegmatis lipomannan (LM),

a TLR2 ligand, and lipopolysaccharide from E. coli (055:B5), a TLR4 ligand, will be compared.

Concentrations ranging from 10 ng/mL to 3 ug/mL are used. Activation measures include nitric oxide

(NO) production as measured by the Greiss reaction, western blot analysis of MAPK p38, and ELISA

for TNF-α production in culture supernatants. Initial experiments examining 10ng/ml and 3µg/ml LM

concentrations in comparison to 100 ng/ml of LPS (from E.coli O55:B5) demonstrated that the

3µg/ml concentration of LM produced a response of 60 µM nitrite, while the 100 ng/ml concentration

of LPS induced a response of 108 µM of nitrite. This effect was seen in cells grown in DMEM, but

not ones grown in RPMI 1640. This is an unexpected and potentially novel observation. The broad

purpose of this research is to elucidate mechanisms by which Mycobacterium activate innate immune

/ inflammatory responses in order to provide a better understanding of Mycobacterium infection

through comparison and analysis of the role of the TLR-2 pathway relative to the bioactive lipid and

signaling molecule sphingosine-1-phosphate (S1P).

29 Characterizing the Invasion Domain of Anaplasma phagocytophilum Outer Membrane Protein

A

Kathryn S. Hebert*, David Seidman, and Jason A. Carlyon. Department of Microbiology and

Immunology, Virginia Commonwealth University, Richmond, VA.

Obligate intracellular bacteria use outer surface proteins called invasins to enter eukaryotic host cells.

Infection by these organisms can be prevented or cured simply by blocking internalization. Thus, it is

desirable to characterize invasins of obligate intracellular bacteria, specifically the domains of these

proteins that are critical for entry into host cells. Anaplasma phagocytophilum is a tick-transmitted

obligate intracellular rickettsial pathogen that causes the emerging disease, human granulocytic

anaplasmosis (HGA). Our lab previously determined that the A. phagocytophilum outer membrane

protein A (OmpA) is critical for invasion of mammalian host cells and its invasion domain lies within

its N-terminus. Here, we generated a series of OmpA N-terminal peptide-specific antisera and

assessed their efficacies at blocking A. phagocytophilum infection. Antiserum targeting OmpA amino

2013 VA Branch ASM

acids 59 to 74 (OmpA59-74) significantly inhibited bacterial binding to the OmpA receptor, α2,3-

sialylated PSGL-1 and infection of host cells. Recombinant OmpA proteins carrying insertions of 5-

amino acids in the same region as that targeted by the blocking antiserum, but not any other region,

were unable to bind host cells or competitively inhibit A. phagocytophilum infection. Using alanine

substitutions, we pinpointed glycine 61 and lysine 64 as being critical for OmpA cellular adherence.

These studies represent the most detailed characterization of any rickettsial invasin to date.

30 Orientia tsutsugamushi ankyrin repeat-containing proteins, Ank8 and Ank15, traffic to the

lumen of host cell endoplasmic reticulum

Sean Evans, Andrea R. Beyer, Lauren VieBrock, Smita Singh, and Jason A. Carlyon. Virginia

Commonwealth University, Richmond, VA.

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus, a

potentially deadly disease that afflicts one million persons annually in the Asia-Pacific region. The

virulence factors that this understudied pathogen uses to take over eukaryotic host cells are poorly

defined. An emerging theme among intracellular bacterial pathogens is the delivery of eukaryotic-like

ankyrin-repeat containing effectors (Anks) into host cells. Ankyrin repeats are 33-amino acid tandem

motifs that mediate protein-protein interactions. Prokaryotic Anks interact with host cell proteins to

mimic, alter, or disrupt eukaryotic cellular functions and thereby promote pathogen survival. We

previously confirmed that O. tsutsugamushi expresses 21 different anks during infection. As a first

step in functionally characterizing the Anks, we monitored the subcellular localization of ectopically

expressed Anks in host cells. Several were observed to traffic to distinct locales within the

endoplasmic reticulum (ER). Two such ER-tropic Anks are Ank8 and Ank15. Using confocal

miscroscopy, we observed that both Ank proteins colocalize with the ER lumenal marker, protein

disulfide isomerase but not with ER membrane markers. Ank8, but not Ank15, also carries a second

motif that mimics the F-box, which functions in eukaryotes to nucleate polyubiquitination machinery.

Using a GST-pulldown assay, we demonstrated that Ank8 interacts with polyubiquitination

machinery in an F-box dependent manner. These data suggest that Ank8 facilitates proteasomal

degradation of an ER protein component and that both Ank8 and Ank15 could hijack the host cell

secretory pathway to promote O. tsutsugamushi survival inside the host cell.

31 Acute Dose Response Effects of γ-HCH (lindane) on Measures of Innate and Adaptive Immune

Function in Female Swiss Mice

Roslyn Alexander and David Freier. Lynchburg College, Lynchburg, VA.

The primary functions of macrophages are defense against bacterial pathogens. To assess macrophage

function, levels of nitric oxide production can be observed following the stimulation by bacterial

lipopolysaccharides. In vivo, peritoneal exudate cells (PEC) can serve as a source of macrophages.

Elicitation of PEC by 4% TG (thioglycolate) will be examined in comparison to the antibody forming

(AFC) cell response, following treatment with γ Hexachlorocyclohexane (lindane). Five groups of

female Swiss mice, four in each group will be treated with: Saline Vehicle (gavage), 10 mg/kg

Lindane (gavage), 50 mg/kg Lindane (gavage), 100 mg/kg lindane (gavage) for 10 days, or 200

mg/kg Cyclophosphamide (IP) given 4 days prior to assay. Two mice per treatment will be used for

elicitation of PEC by IP injection of sterile 4% TG. For the AFC response, each mouse receives an IP

injection of 1x108 sheep erythrocytes 4 days prior to assay. The AFC response and PEC response to

bacterial LPS are done in a separate series of experiments. Mice are euthanized by CO2 inhalation at

day 0, and either peritoneal exudate cells or a single cell suspension of splenocytes will be prepared

for evaluation of immune responses and the acute immunotoxicity of lindane. Previous literature has

suggested a biphasic effect on T-dependent antibody responses, but no effect on macrophage

phagocytic activity.

2013 VA Branch ASM

32 The Bacterial Hyaluronan Synthase

Caitlin Hubbard and Jochen Zimmer. Department of Molecular Physiology and Biological Physics,

University of Virginia, Charlottesville, VA.

Hyaluronan (HA) is an extracellular, linear polysaccharide consisting of alternating residues of

glucuronic acid and N-acetyl-glucosamine. It is synthesized by the membrane integrated Hyaluronan

Synthase (HAS), an enzyme that is highly conserved and ubiquitously expressed among vertebrates,

where it forms a major component of the extracellular matrix. In addition to performing many

physiological and structural roles in the body, HA is implicated in cancer, rheumatoid arthritis and

wound healing, as well as the virulence of some bacterial species. Only a limited number of

pathogenic prokaryotes produce HA. In the group A Streptococcal bacterium Streptococcus

equisimilis, and Streptococcus pyrogenes, HA is extruded and surrounds the bacterial cell with a thick

capsule of non-immunogenic HA to avoid an immune response by its host. Additionally, acquisition

of HAS by Bacillus cereus allows the bacterium to cause an anthrax-like disease.

While HA is synthesized inside the cell, it performs its biological function on the outside of the cell.

Our aim is to understanding how the HA polymer is translocated across biological membranes. We

established an in vitro assay for HA translocation from purified components to address whether HAS

is sufficient for HA synthesis and membrane translocation. To this end, we purify HAS in a detergent

solubilized system and reconstitute the protein into proteoliposomes that are then used for in vitro

activity and translocation assays. Polymer translocation across the vesicle membrane can be assayed

with the help of hyaluronidases, which leave translocated HA intact. This analysis demonstrates that

HAS is necessary and sufficient for HA synthesis and translocation.

33 Analysis of plasmids from Streptococcus parauberis

Armonee Avent and Ian Copeland. Norfolk State University, Norfolk, VA.

Streptococcus parauberis is a gram-positive lactic acid bacterium that can be found in fish and dairy

cows. This bacterium infects the kidneys and brain causing inflammation and necrosis of tissue,

which can lead to death in certain fish species. It has been found in aquacultured fish in Spain and

Korea and recently in striped bass in the Chesapeake Bay. In these fish strains of S. parauberis, a

plasmid was identified recently. A plasmid is a genetic structure that can replicate independent of the

chromosomes. As part of this project, the genes of this plasmid are being sequenced. This will assist

in the identification of antibiotic resistance genes, virulence genes, and host-specificity genes.

Another goal includes the characterization of this plasmid using a recently proposed plasmid

classification system. Understanding the types of plasmids as well as any antibiotic resistance genes

found in these pathogenic strains should facilitate the effective treatment of infected aquaculture

fishes. These data may also contribute towards future development of a vaccine for this bacterium.

Together these efforts should improve the profits of fishing industries and limit the emergence of this

pathogen in wild populations, such as here in the Chesapeake Bay.

34 Protein interaction networks, complexes, and domains in bacteria

J. Harry Caufield, Christopher Wimble, Marco Abreu, Norman Goodacre, and Peter Uetz. Virginia

Commonwealth University, Richmond, VA.

Conservation of protein binary interaction networks. We have recently produced binary protein

interaction data for several species of bacteria, including Escherichia coli, Helicobacter pylori,

Treponema pallidum, and Streptococcus pneumoniae. We have analyzed the conservation and

evolution of protein-protein interactions across these and other species. This data will also assist in

the identification of protein function for those previously uncharacterized (around 30% of all proteins

in each proteome is currently of unknown function). Our preliminary analysis suggests that only few

interactions are conserved, which is not surprising, given that only a fraction of all proteins are

2013 VA Branch ASM

conserved.

Conservation of protein complexes across bacteria. Large-scale screens for microbial protein-protein

interactions rarely capture the extent to which proteins participate in complexes. We have coupled the

results of published mass spectrometry-characterized protein complexes with databases of gene

orthology and essentiality to better characterize the extent to which proteins interact, the complexes

they form, and how this is related to physiological traits.

Comparative analysis of protein interaction domains in bacteria. Protein domains are conserved

structural and functional elements of proteins, suggesting that domain-domain interactions will be

preserved across species. We are analyzing protein domains and their interactions in multiple

bacterial species (including E. coli, Treponema pallidum, and others) to find out to what extent there

interactions and thus functions are conserved. This should lead to a better understanding of domains

of unknown function (DUFs), uncharacterized proteins, and previously studied proteins.

35 Protein interaction networks in microbes and ecosystems

Neha Sakhawalkar, Jitender Mehla, Andrey V. Matveyev, and Peter Uetz. Virginia Commonwealth

University, Richmond, VA.

Various methods and techniques are used to study protein-protein interactions, including the Yeast

Two Hybrid system (Y2H) and affinity purification combined with mass spectrometry (AP/MS).

However, none of them is truly a high-throughput method that allows to capture all interactions in a

cell or organism at reasonable cost in a short time.

We are developing a new methodology that allows us to map whole interactomes in a single

experiment using a modified Y2H and next-generation sequencing. The technology could also be

applied to meta-interactomes in an ecosystem, e.g. to map all interactions among the proteins in a

microbiome, including interactions among species (e.g. virus-human, phage-bacteria etc.).

In parallel, we are working to adapt a bacterial two-hybrid system (B2H) to next-gen sequencing. The

Bacterial Adenylate Cyclase Two Hybrid (BACTH) system has been developed to study PPIs

between both cytoplasmic and as well as membrane proteins. Furthermore, the existence of Gateway

vectors and cloning should enable high throughput screening of interactions, in order to describe

microbial interactomes as well as meta-interactomes (e.g. between viruses and their hosts).

36 Comparison of the Nitric Oxide Response of RAW 264.7 Murine Macrophages Grown in RPMI

1640 and DMEM

Diana Spangler and David Freier. Lynchburg College, Lynchburg, VA.

Research literature in which RAW 264.7 murine macrophages are used to study inflammatory

activity vary significantly in factors such as cell number, duration of stimulus, and basic culture

conditions. This study is designed to compare the two most common culture media; RPMI 1640 and

DMEM. Both of these are supplemented with 10% fetal bovine serum, sodium bicarbonate, 100 IU

Penicillin Streptomycin 100mg, and 25mM HEPES. The most significant difference is that DMEM

contains 4.5g/l glucose where RPMI has 2 g/l. This comparison examines RAW 264.7 stimulation by

a dose response of bacterial LPS and the production of nitric oxide. RAW 264.7 cells were incubated

for 24 hours in a 5%CO2/ 37˚C environment to lipopolysaccharide from E. coli (serotype O55:B5) at

concentrations from 1-100 ng/mL. Cells are kept in continuous culture with passage every 4 days and

are at a limit of 25 passages or 3 months from thaw. Experiments are conducted on a 24-well plate

500ul volume at 4x105 cells/well. After stimulation, supernatants are collected to evaluate nitric

oxide response by the Greiss reaction. Experiments suggest that cells grown in DMEM have a nitric

oxide response that is 4-5 times greater than that of cells grown inRPMI1640. Both DMEM and

RPMI grown cells show a dose response to LPS, but at a concentration of 100 ng/mL DMEM cells

2013 VA Branch ASM

produce a 100uM concentration of nitrite, while RPMI grown cells produce only 17uM. This

difference in responsiveness of RAW 264.7 cells could significantly change interpretation of current

and past primary scientific literature.

37 Utilizing Coding Sequences to Increase Phylogenetic Resolution of Pilobolaceae

Heaven Cerritos and Dale Beach. Longwood University, Farmville, VA.

The family of Pilobolaceae is a collection of fairly ubiquitous fungal species associated with the

Mucorales, and best known as “Shotgun Fungi” capable of shooting sporangia great distances. The

Pilobolaceae typically are found in the feces of grazing animals such as horses, cows, deer, and

antelope. Animals eat the fungal spores that can be found attached to grass shoots. Once the spores

are re-deposited in the field, small shoots grow and provide a launching platform to disperse the

spores away from the dung. Continued study of these organisms is complicated since they are

difficult to maintain long term cultures in the lab, and traditional methods of identifying samples to

the level of species relies on difficult and ambiguous measurements of spore morphology. Such

phenotypic characteristics of spores alone are difficult due to overlapping characteristics across

genera. Recent application of phylogenetic methods has focused on ribosomal RNA sequences to

successfully differentiate genera within the family of Pilobolaceae. We are extending this work by

developing select protein coding sequences to classify isolates to species level. Using stored genomic

DNA samples from type strains as well as novel isolates we are determining the Tubulin gene

sequence to serve as a molecular “barcode” to differentiate samples and evaluate phylogenetic

relationships. In addition, we have begun the full-genome sequencing and assembly of a novel

Pilobolus sp. isolate from Prince Edward County, Va.

38 Quantification of Enterococcus spp. in litter from an organic turkey farm

Ben Holland, Steven McBride, Pradeep Vasudevan, and Joanna Mott. James Madison University,

Harrisonburg, VA.

Enterococcus, a Gram-positive gut commensal bacterial group, routinely contaminate waterways

through runoff from agriculture, and can present health risks to humans and animals who come in

contact with them. Their importance as indicators of fecal contamination has increased in recent

decades because of their growing antimicrobial resistance, high levels within fecal wastes, and ability

to survive in a wide range of conditions. Assessment of fecal contamination is an important step

towards keeping humans and animals safe from infections in environmental and clinical settings. In

this study, Enterococcus levels in turkey litter were assessed and species prevalence was analyzed.

Over one hundred presumptive Enterococcus isolates were collected from the litter of a single flock

of turkey poults during their first six weeks of rearing at an organic “No Antibiotic Use,” turkey farm

in Shenandoah Co., VA. These isolates were first isolated using membrane-Enterococcus Indoxyl-β-

D-Glucoside agar, and then speciated using the Biolog MicrologTM system. The results of this study

will provide information on levels of specific species of Enterococcus from agricultural settings that

could potentially become sources of environmental and waterway contamination.

39 Identification of the Staphylococcal Cassette Chromosome mec (SCCmec) element in an

Environmental Bacteriophage Population

Amber Brooke Sauder, Emily Pelto, and Dr. Louise Temple. James Madison University,

Harrisonburg, VA.

Methicillin-resistant Staphylococcus aureus has routinely been viewed as the responsible pathogen

for many nosocomial infections, but in the past decade, more disease has occurred outside hospitals,

indicating the possible spread of methicillin resistance primarily in agricultural settings. The genetic

element that carries the methicillin-resistance gene, mecA, is the SCCmec element. Because SCCmec

2013 VA Branch ASM

is a mobile genetic element, it could be transferred in three ways - conjugation, transformation, or

transduction. The method of interest to this study is transduction. In order to determine if

bacteriophages are transferring the SCCmec element between bacteria, a protocol was developed to

isolate the phage population and use PCR and sequencing technologies to examine the genetic

information contained within the bacteriophages. The developed protocol consists of sample filtration

and sterilization, phage precipitation, DNA extraction, PCR, agarose-gel electrophoresis, and

sequencing. This protocol was developed around positive controls, spiking samples, and general

troubleshooting. The protocol was altered several times to extract as much phage DNA as possible,

prevent the degradation of DNA, and deal with possible PCR inhibitors. The current protocol has

produced one sample that showed a PCR product of the right size for a gene in the SCCmec element

and was confirmed positive by sequencing. The sequence shows that the element is type I.2. Future

goals of this experiment include: collecting and processing more samples, sequencing more PCR

products, whole sample sequencing using PacBio technology, and possible chromosome walking to

determine the extent of the SCCmec element carried by the bacteriophages.

40 Temperature Study of Mycobacterial Infections in Striped Bass

Smith, D.E., Vogelbein, W.K., Korngiebel-Rosique, M., and Gauthier, D.T. Old Dominion

University, Norfolk, VA.

Striped bass (Morone saxatilis) are the most popular recreational sportfish in Chesapeake Bay.

Mycobacterial infections within these fish are present at very high levels and are an area of concern

within the fishing community. Mycobacterium shottsii and Mycobacterium pseudoshottsii appear to

be the major agents associated with disease in this system. With increased eutrophication, the deeper

waters of Chesapeake Bay have become increasingly hypoxic and it is speculated that striped bass are

subject to a “thermal oxygen squeeze”; hypoxia in the deeper waters pushes the fish into shallower,

warmer waters, potentially leading to increased susceptibility to infection. In this study, we examined

the effect of temperature on infection with M. shottsii and M. pseudoshottsii in striped bass. Striped

bass were experimentally infected with 10⁶ colony-forming units of M. shottsii or M. pseudoshottsii,

then held at 20˚C. Quantitative PCR and bacteriology were used to determine splenic bacterial

densities at 4 weeks post-infection. Water temperatures were then changed to 25˚ or 30˚C, or kept

constant at 20˚C for an additional 4 wk. Prior to the temperature change, there were no significant

differences in M. pseudoshottsii or M. shottsii densities between systems. Four weeks after the

temperature change, there were significant differences in density for both bacteria across

temperatures. The highest densities for both bacteria were observed at 20˚C, and the lowest densities

were observed at 30˚C. Our data therefore reveal an inverse relationship between temperature and

infection; which is inconsistent with the thermal oxygen squeeze hypothesis.

41 Genomic and Phenotypic Analysis of Bacillus pumilus Bacteriophage Lysis Cassette

Carly Starke, Nathaniel Burkholder, Dr. Ron Raab, and Dr. Louise Temple. James Madison

University, Harrisonburg, VA.

A novel lysis cassette in bacteriophage Pappano, isolated from Bacillus pumilus strain BL8, was

identified from whole genome sequence annotation. The lysis cassette consists of genes encoding

hydrolase and holin-like products predicted to be capable of lysing B. pumilus cells. Another gene

upstream of the hydrolase and holin genes encoding a peptidase was also speculated to play a role in

cell lysis. Various lysis cassette combinations were PCR amplified, ligated into broad host range

vector pTYB2 downstream of an IPTG inducible promoter, and transformed into E. coli. Cultures

containing the desired constructs were grown to mid-log phase and induced with IPTG.

Approximately one hour post induction of the entire cassette, a steep lysis curve was observed. It was

observed that the holin when expressed alone was capable of inducing cell lysis at a slightly lower

rate compared to the full cassette, but cells expressing the hydrolase alone did not undergo lysis.

2013 VA Branch ASM

There did not appear to be a difference in the rate of lysis in cultures with or without the peptidase

gene. Lysis of BL8 infected with Pappano at a multiplicity of infection (MOI) of 3 was observed

within five minutes post inoculation. Even though the lysis cassette exists in the genome of BL8 with

its hydrolase and holin genes in opposite orientation than typically found in other phages, we have

shown that it is a functional, novel lysis cassette. We have also shown that this cassette is capable of

lysing gram-negative cells that have a different cell wall composition.

42 Francisella tularensis interspecies communication

Scott N. Dean, Collette Marchesseault, and Monique van Hoek. George Mason University, Fairfax,

VA.

The goal of this study is to determine the interspecies communication of Francisella tularensis in the

context of the natural polymicrobial environment in the soil. These results will enhance our

knowledge of the persistence of this organism in its natural habitat. In this study we focused on

Francisella and its interaction with Gram-negatives, Pseudomonas aeruginosa and Burkholderia sp.,

known pathogens present in the soil. P. aeruginosa and produces quinolines (such as HQNO) and

monounsaturated fatty acids signals that cause significant effects on the metabolism, growth, and

biofilm formation of F. novicida. We hypothesize that small soluble factors produced by

Pseudomonas and Burkholderia may have specific effects on F. tularensis lifestyle through

dysregulation of Francisella gene expression. In our studies, we have shown the effect of 100 μg/mL

HQNO and P. aeruginosa conditioned media on static F. tularensis novicida biofilm, and that these

small soluble signals significantly decrease biofilm mass (p < 0.01) as quantified by COMSTAT

(without affecting bacterial growth). The metabolism and growth of Francisella was also studied in

response to P. aeruginosa signaling molecules. We have shown that P. aeruginosa produces signals

that significantly alter F. novicida metabolism, growth, and biofilm. In future work, we will assess

the effect of HQNO and other small signaling molecules on Francisella virulence and gene

expression. These studies may lead to a greater knowledge of F. tularensis persistence in the natural

polymicrobial environment. This work was supported by HDTRA1-12-1-030 Francisella interspecies

interaction with Pseudomonas, a soil microbe.

43 Birds as Vectors for Rickettsia parkeri

Jessica Vincent, Chelsea Wright, Erin Heller, Dr. David Gauthier, Dr. Eric Walters, and Dr. Holly

Gaff. Old Dominion University, Norfolk, VA.

Rickettsia parkeri is a bacterial pathogen carried by Amblyomma maculatum, the Gulf Coast tick. In

2004, R. parkeri was identified as a cause of disease in humans, which was called Tidewater spotted

fever because of its initial diagnosis in the Tidewater area of Virginia and similarity to Rocky

Mountain spotted fever. Increasing diagnoses of Tidewater spotted fever in the eastern United States

have raised concerns in endemic areas. Gulf Coast ticks are concomitantly spreading north in a rapid

and punctuated pattern. This dispersal is atypical of terrestrial animal vectors, and implicates birds as

likely dispersal agents. In order to help determine whether birds have played a role in the spread of A.

maculatum and, in turn, Tidewater spotted fever, we are attempting to detect Rickettsia parkeri in

wild avian blood samples. We use a TaqMan assay designed for Rickettsia spp., and intend to test

positive samples for species-level identification. Currently, out of the 250+ samples tested, none have

been positive. If the pathogen is detected in avian blood, this will be a transformative finding in our

understanding of tick ecology. If not, this may indicate that the pathogen is not detectable in the bird

blood or that birds do not play a vector role in the transmission of R. parkeri. To differentiate

between the two possibilities, we plan on testing A. maculatum collected from birds for Rickettsia

parkeri .

2013 VA Branch ASM

44 Genome-Wide Association Study of Mycobacteriophage Host Preference

Hayley Norian and Steven G. Cresawn. James Madison University, Harrisonburg, VA.

Mycobacteriophages are bacteriophages that infect the genus of bacteria Mycobacterium, which

includes pathogens such as Mycobacterium tuberculosis and Mycobacterium ulcerans. Full genome

sequences of 461 mycobacteriophages are currently available and have been grouped into clusters of

related genome sequences. Host range information, or whether or not the phages infect or do not

infect a particular host, is available. In a recent paper, some 220 phages were isolated on

Mycobacterium smegmatis strain mc2155, sequenced and placed into 42 groups of related genome

sequences. When testing host range, three different levels of infectivity were observed: phages that

were incapable of infecting the host, phages that were capable of infecting the host, and some that

were at a plating efficiency less than one relative to mc2155. The phages that are capable of infecting

a particular host are of particular interest. Using software to compare genome sequences, we can

observe subtle differences in the genomes that might play a role in whether or not the phage can

infect a particular host. Identifying crucial genes in phages to infecting bacterial hosts could be

potentially useful in the future in terms of therapeutics and treatments.

45 Identification and Characterization of Staphylococcus Spp. In Stream Water and Sediment

Michael Partin, Kevin Libuit, and Dr. Jim Herrick. James Madison University, Harrisonburg, VA.

Staphylococcus is comprised of 41 known species, of which 18 can colonize humans. Despite the

prevalence of infectious Staphylococcus within hospital settings and agriculture, there are few reports

of Staphylococcus in natural bodies of water. A recent study by the US FDA found substantial

contamination of poultry and other meats with Staphylococcus. We hypothesized that intensive

farming of poultry adjacent to streams would result in contaminated runoff, resulting in at least

transient occurrence of Staphylococcus spp. in streams waters and sediments. This study intends to

determine whether Staphylococcus occurs and persists within agriculturally impacted streams in the

central Shenandoah Valley. In preliminary work, large numbers of Staphylococcus spp. were detected

in water from Muddy Creek, which runs adjacent to a poultry processing plant. Mannitol Salt Agar

(MSA) was used to isolate 11Staphylococcus spp. from the Muddy Creek site. As the occurrence of

Staphylococcus in Muddy Creek seems to be transient and highly variable, perhaps depending upon

runoff and season, continuing efforts are underway to determine the temporal and spatial variability

in its occurrence in both water and sediment. Future studies will include determining the

susceptibility of Staphylococcus isolates to a range of relevant antibiotics – including oxacillin, which

would identify methicillin resistance – and whether resistance is transmissible and/or plasmid borne.

Overall, this study will provide insight into the occurrence of staphylococci in freshwater and a

possible link between the microflora of agricultural animals and that of native streams.

46 Brain-specific angiogenesis inhibitor-1 (BAI1) regulates macrophage bactericidal activity and

cytokine responses to Gram-negative bacteria Emily A. Billings, Soumita Das, Chang Sup Lee, Kodi S. Ravichandran, and James E. Casanova.

University of Virginia, Charlottesville, VA.

The detection of a pathogen and initiation of an immune response occurs through a limited set of

germ-line encoded receptors called pattern recognition receptors (PRR). The coordinated actions of

these receptors result in microbicidal activity, antigen presentation, cytokine and chemokine

production, and loss of these receptors can have devastating consequences on immunity. Brain-

specific angiogenesis inhibitor-1 (BAI1) has recently been characterized as a phagocytic receptor that

drives uptake of Gram-negative bacteria. Here we investigate the role of BAI1 in the cellular

response to Gram-negative bacteria, including crosstalk with other PRRs. We find that BAI1-

deficient macrophages exhibit both impaired uptake of and microbicidal activity against a model

2013 VA Branch ASM

Gram-negative species, E. coli K-12. BAI1 ligation stimulates the production of reactive oxygen

species (ROS), suggesting that BAI1-mediated microbicidal activity depends on the ROS burst. ROS

induction may be enhanced through BAI1-driven activation of Rac2. We have shown that BAI1

binds lipopolysaccharide (LPS), however TLR4 is the main immunostimulatory receptor during LPS

recognition. Interestingly, we find that TLR4 associates with BAI1, suggesting the formation of a

signaling complex, and in fact BAI1-deficient macrophages exhibit attenuated inflammatory

signaling. While activation of NFkB appears normal in BAI1-deficient cells, activation of IRF3 and

induction of TRIF-dependent cytokines and chemokines is significantly impaired. Collectively, these

results suggest that BAI1 may regulate the clearance of microbes through enhanced phagocytosis and

microbicidal activity and may also drive the immune response by influencing the cytokine milieu

during infection. Future studies will investigate the importance of BAI1 in-vivo using a peritoneal

infection model.

47 Manipulation of host signaling by the Salmonella translocon/effector protein SipC

Carissa B. Meyer, Adam Greene, and James E. Casanova. University of Virginia, Charlottesville,

VA.

Salmonella enterica are a genus of Gram-negative bacteria that cause a range of human diseases and

present a significant global health burden. To invade cells and evade host defenses, Salmonella

injects ‘effector’ proteins into the host cell through a ‘molecular syringe’ called a Type III secretion

system (T3SS). Bacterial effectors hijack numerous host signaling pathways to stimulate bacterial

uptake and create an intracellular niche for replication, the Salmonella containing vacuole (SCV).

SipC is an essential component of the invasion-associated T3SS that also functions as an effector.

Our previous work identified eukaryotic SipC-interacting proteins through a yeast-two-hybrid screen

using the SipC C-terminal tail as bait, including proteins involved in modulating actin, membrane

trafficking and signaling. Here we investigate the function of several ‘hits’ during Salmonella

infection of non-phagocytic cells. We have verified that filamin A, MAP4K4, PICALM, and Bax

interact with SipC-C in HeLa cell extracts. MAP4K4 is a mitogen-activated serine/threonine kinase

that regulates cortical actin architecture and JNK activity through multiple targets. Depletion of

MAP4K4 enhanced Salmonella invasion, suggesting that SipC co-opts MAP4K4 to limit bacterial

uptake. Bax is a pro-apoptotic Bcl2 family protein whose activity is inhibited by Akt. We find that

SipC-C also interacts with Akt in HeLa cell extracts, suggesting that SipC may influence apoptosis

through the Akt/Bax signaling axis. Finally, we show that SipC localizes to the SCV and persists on

many SCVs for up to four hours post-invasion. Collectively, our data suggests that SipC influences

bacterial invasion and intracellular survival by hijacking diverse cellular pathways.

48 Integrated Phylogenomic Approaches Toward Pinpointing the Origin of Mitochondria

Zhang Wang and Martin Wu. Department of Biology, University of Virginia, Charlottesville, VA.

"Overwhelming evidence supports the endosymbiosis theory that mitochondria originated once from

within α-proteobacteria. However, exactly when it happened remains highly debated. In this study,

we took advantage of integrated phylogenomic approaches to pinpoint the origin of mitochondria. In

particular, we 1) filled the gaps in the mitochondrial tree of life by sequencing genomes of 19 α-

proteobacteria that represent a broad phylogenetic diversity, 2) identified a number of mitochondrial-

derived nuclear genes as “well-behaved” phylogenetic markers with lower evolutionary rates and less

compositional bias, and 3) applied more realistic phylogenetic models that better account for LBA

and sequence compositional bias. With cutting-edge phylogenetic methods and more and better data,

our integrated phylogenomic approaches revealed an interesting tree topology. On the one hand, the

mitochondria were grouped with Rickettsiales, which is consistent with the well-accepted topology.

On the other hand, Pelagibacter, the smallest free-living bacteria previously claimed to be the sister

clade of mitochondria, were found to be distantly related to the mitochondria. In addition,

2013 VA Branch ASM

reconstruction of genome content of mitochondrial ancestor suggested it lacked a hydrogen-

producing machinery but likely possessed a complete aerobic pathway. Our results therefore

indicated a lack of evidence for the “hydrogen hypothesis” as opposed to the alternative “oxygen

scavenger hypothesis” for the origin of mitochondria.

2013 VA Branch ASM

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