NW ASM main site

2016 Posters

Note to authors: Those wishing to present a poster at the conference should register your poster here. A $50 and $25 Amazon gift certificate will be awarded to the top poster and runner up. Tape will be provided to attach your poster to the wall. You may bring any size poster, typical sizes range from 3' x 3' to 4' x 4'. Please contact Derek Wood should you have questions. Thank you for your support of the NW ASM!

Novel High-throughput Screen Identifies Inhibitors of Sarcocystis neurona.
Gregory Bowden and Heather Fritz.
Department of Veterinary Microbiology and Pathology, Washington State University.

The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM), the most significant infectious progressive neurologic disease in horses. Many horses the U.S. are at risk of developing EPM; serologic studies estimate that 50% of all U.S. horses have been exposed to S. neurona and treatments for EPM are at best 60-70% effective. Advancement of treatment for EPM requires new technology to identify novel compounds. To address this insufficiency, we developed, validated, and implemented a novel high-throughput screen using 725 FDA-approved chemical compounds of the NIH clinical collections library. Our screen identified 18 novel compounds with confirmed inhibitory activity against S. neurona growth, with some compounds being active at concentrations in the nM range. Many of the inhibitory compounds identified have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that almost all compounds (15/18; 83%) have activity against one or more related apicomplexan. In addition, investigation of the recorded activity of library compounds on dopamine receptor subtypes identified many (10/15; 66%) inhibitory compounds with activity against dopamine receptors. These findings demonstrate the use of a robust new tool in discovering new chemotherapeutic agents for EPM and aid in the discovery of important biologic pathways required for successful S. neurona infection.
Post-replication roles of the Brucella VirB type IV secretion system.
Erin Smith, Cheryl Miller, Jennifer Cundiff, Jean Celli.
Paul G Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, WA.

Brucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, the Brucella-containing vacuole (BCV). Initially of endosomal origin (eBCV), BCVs are remodelled into replication-permissive organelles (rBCV) derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by the Brucella VirB type IV secretion system (T4SS). Following replication, rBCVs are converted into autophagic vacuoles (aBCVs) that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of the Brucella intracellular cycle. To address this question, we have generated a B. abortus strain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc)-dependent complementation of a deletion of the virB11 gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs) that early VirB expression is essential for optimal rBCV biogenesis and bacterial replication. Transient, expression of VirB11 prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication, but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these post- replication stages are also T4SS-dependent. Hence, our findings support additional, post-replication roles of Type IV secretion in the Brucella intracellular cycle.

Molecular Evolution of sRNA.
Austin Wright, Rahul Raghavan.
Department of Biology, Portland State University.

Many of the most common pathogenic bacteria have been treated using Polymyxin B, which changes the permeability of the outer membrane by binding to the lipopolysaccharide (LPS). In response to antimicrobial peptides, bacteria such as Salmonella and E. coli change the chemical structure of the lipid A molecule at the base of the LPS layer where Polymyxin B binds. The regulatory mechanism of this process has been studied in E. coli and Salmonella. A small non-coding RNA (sRNA), MgrR, inhibits the expression of eptB, which encodes a phosphethanolamine transferase that modifies the LPS. The modifications of the LPS happens when the concentration of Mg2+ is low, for e.g., inside a mammalian host, and cause the outer membrane to be more negatively charged and less sensitive to cationic peptides (e.g., defensin) released by the host. MgrR is conserved in all enteric bacteria, and is regulated by the PhoP/Q two-component system. However, mgrR in Escherichia fergusonii contains a 52-nucleotide insertion and the PhoP binding sequence is only partially conserved. The importance of this extra nucleotide loop is not understood, but when we deleted mgrR from E. fergusonii, the mutant strain became highly sensitive to Polymyxin B, whereas mgrR-deletion strains of E. coli are 10 times more resistant than the wild type to Polymyxin B. The goal of this project is to understand how a nucleotide insertion event has impacted the functions of mgrR in E. fergusonii. I hypothesize that PhoP/Q regulates MgrR expression in E. fergusonii, and due to the 52 bp insertion, MgrR has acquired new target mRNAs that contribute to Polymyxin B resistance.

Pandemic Multidrug-Resistant E. coli strain ST131-H30 – does asymptomatic bacteriuria contribute to maintaining Gut colonization?.
Kendra Ferrier¹, Huxley Smart², Kim Riddell³, Delia Scholes⁴, Veronika Tchesnokova¹ and Evgeni Sokurenko¹.
¹Department of Microbiology, University of Washington
²ID Genomics Inc.
³Group Health Cooperative and Group Health Research Institute, Seattle, WA, USA

Background: Urinary Tract Infections (UTIs) are regularly caused by members of the normal gastrointestinal microbiota, with Escherichia coli being a primary uropathogen. The most successful and dangerous UTI-causing strain lineage of E. coli is the H30 sub-clone of the clonal group ST131. The H30 clone emerged in the late 1990s and is currently more than 90% resistant to Ciprofloxacin (Cipro) and is responsible for about half of all multidrug resistant E. coli infections around the globe.

Objective: We wanted to estimate (1) the prevalence of H30 clone carriage in the GI tract of healthy women, (2) the frequency of carriage of H30 clones from urine of women who had H30 clones in their gut, and (3) the persistence of H30 in the gut over a period of time.

Methods: In this cohort study, 1,031 primary fecal samples from a randomly-sampled group of healthy women enrolled in Group Health Cooperative (Seattle, WA) were supplied by mail. Presence of H30 clone was detected by qPCR [1]. Cipro-resistant E. coli were identified by culturing on ciprofloxacin-supplemented agar followed by SNP-based clonal type testing [2]. A follow-up urine sample was collected from a selected subset of 121 women and was analyzed for presence of H30 or other E. coli. Finally, a second fecal sample was collected and analyzed from 155 women, following 4 to 12 months after the primary sample collection.

Results: Of the primary fecal samples, only 4.6% of the E. coli positive samples were H30 clones. Other UTI-associated E. coli clonotypes had a much higher carriage in the gut, but were found 2-3 times less frequently in the urine of women with UTIs (P < .01) [1, 2]. Additionally, H30 clones constituted 48% of all gut Cip-R E. coli isolates, with the remaining half being split between 16 various E. coli clonotypes. Of the women who carried H30 in their gut, 30% had H30 isolates in their urine, which was approximately 3 times more frequent than other E. coli strains (P < .05), regardless of ciprofloxacin sensitivity. Furthermore, after analyzing the composition of the secondary fecal sample, the H30 clone was persistent in 72% of the original carriers, while other Cip-R strains were persistent only 25% of the time (P < .001). Interestingly, continued persistence of any Cip-R clones in the follow-up fecal sample was associated with isolation of the same strain in the previously collected urine sample (33% carriage in urine by persistent strains vs 13% in non-persistent strains, P = .045). In contrast, the Cipro-sensitive strains did not show such urine/fecal persistence association (8% carriage in urine in persistent cases vs 9% in non-persistent strains).

Conclusion: The pandemic multi-drug resistant E. coli strain H30 is carried by the healthy individuals at a much lower rate than it causes UTI, suggesting it has higher urovirulence relative to other UTI strains. H30 also has a tendency to cause bacteriuria much more frequently than other E. coli, which appears to be associated with H30 being a comparatively persistent member of the gut microbiota. The greater ability of the H30 clone to invade the urinary tract could be one of the reasons behind the astounding global success of this strain in the last two decades.

1. Tchesnokova VL, Ottley LL, Sakamoto K, Fierer J, Sokurenko E, Liss MA. Rapid Identification of Rectal Multidrug-resistant Escherichia coli Before Transrectal Prostate Biopsy. Urology 2015; 86:1200-5.
2. Tchesnokova V, Avagyan H, Billig M, et al. A Novel 7-Single Nucleotide Polymorphism-Based Clonotyping Test Allows Rapid Prediction of Antimicrobial Susceptibility of Extraintestinal Directly From Urine Specimens. Open Forum Infect Dis 2016; 3:ofw002.

Heterogeneous production of c-di-GMP during early stages of P. aeruginosa biofilm formation.
Catherine R. Armbruster¹, Jessica Parker-Gilham¹, Hannah Ledvina¹, Lucas R. Hoffman^1,2, and Matthew R. Parsek¹.
¹Department of Microbiology, University of Washington.
²Department of Pediatrics, University of Washington.

Biofilms are surface-associated aggregates of bacteria encased in a protective, extracellular matrix. Biofilm bacteria resist antibiotic treatment and killing by the host immune response, leading to persistence in a variety of chronic infections. Pseudomonas aeruginosa is an opportunistically pathogenic bacterium and a model organism for studying biofilm formation. One major factor that drives biofilm formation in P. aeruginosa and other bacteria is the intracellular second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP). Elevated c-di-GMP levels promote cell surface adhesiveness by up-regulating production of biofilm extracellular matrix components and down-regulating motility genes. In contrast, planktonic P. aeruginosa cells are known to have comparatively lower c-di-GMP levels than their biofilm counterparts. While factors contributing to the formation of mature biofilms have been well-characterized, early biofilm formation, when a bacterium first senses a surface and transitions from a planktonic state to a surface-attached state, remains largely understudied. Using a fluorescent reporter of intracellular c-di-GMP and confocal microscopy, we have monitored the dynamics of c-di-GMP production at the single cell level and followed the downstream fate of cells during early surface attachment. Our major finding is that c-di-GMP levels are elevated in only a subpopulation of P. aeruginosa cells during this time. We have used flow cytometry to separate surface-attached cells with high and low c-di-GMP, in order to examine ways in which these populations are physiologically distinct. We have found that surface-associated cells with high intracellular c-di-GMP are more likely to produce polysaccharide than their surface-associated, but low c-di-GMP counterparts. We hypothesize that the heterogeneity in c-di-GMP observed during P. aeruginosa surface sensing represents a specialization among the genetically homogenous population into subpopulations of non-motile, early polysaccharide producers and motile, surface-exploratory cells, both of which contribute to downstream biofilm maturation.

ESBL-producing Escherichia coli are targeting younger children.
David Schroeder¹, Lydia Larson¹, Scott Wiseman², Veronika Tchesnokova², Evgeni Sokurenko², Hov Avagyan³.
¹Department of Microbiology, University of Washington.
²Seattle Children's Hospital

Background: Escherichia coli is a major causative agent of extraintestinal infections in both children and adults. Antibiotic resistance among E. coli is rising at an alarming rate. One of the most dangerous types of resistance in E. coli is associated with strains producing extended spectrum beta lactamases (ESBL) that are resistant to all beta-lactam antibiotics, including 3rd generation cephalosporins, which are prescribed among young children at an especially high frequency.
Objective: In this study, we wanted to see whether E. coli isolated from children three years old and under differed in antibiotic susceptibility compared to the E. coli seen in older children, especially in regard to ESBL production. We also wanted to see whether the resistance rates are associated with the prescription patterns and how the resistance is driven by individual ‘superbug’ strains of E. coli.
Methods: E. coli isolates in this study were obtained from clinical lab at Seattle Children’s Hospital in the time period from 2010 to 2016: 264 isolates from patients 3 years or younger and 183 isolates from older children. The resistance of isolates to a panel of antibiotics was determined using the Kirby-Bauer disk diffusion susceptibility test. Clonal identity of isolates was determined using a SNP-based qPCR test [1]. Prescription at the index day was abstracted from EMR and could be analyzed for 84 younger and 123 older children.
Results: Resistance to third-generation cephalosporins and production of ESBLs was over 10 times higher in E. coli from younger children than in older children (6.4% vs 0.5%, P < .001). This was in sharp contrast to the resistance to other antibiotics, with no difference between the age groups in the resistance to ampicillin (55% vs 44%, P = .03), ampicillin/clavulanate (26% vs 17%, P < .001), trimethoprim-sulfamethoxazole (30% vs 22%, P < .001), and fluoroquinolones (11% vs 13%, P = .088). Moreover, resistance in the young children was lower than in the older children to nitrofurantoin (3.8% vs. 11%, P < .001) and first generation cephalosporins (15% vs 20%, P = .033). In the younger group, first and third generation cephalosporins were used in 39% and 45% of the cases, which was significantly more frequent than in older children (20% and 17%, respectively; P<.01 for both). In contrast, fluoroquinolone and trimethoprim-sulfamethoxazole was more often used in older children (15% and 32%, respectively) than in younger children (0% and 5%, respectively; P<.01 for both). Nitrofurantoin was used at a negligible rate in both younger (1%) and older (2%) children. The pandemic H30 subclone of the ST131 clonal group of E. coli comprised 41% of all ESBL-producing isolates in younger children, with the rest split between 6 smaller clonotypes. H30 also comprised 38% and 33% of fluoroquinolone-resistant isolates in younger and older children, respectively.
Conclusion: ESBL frequency in younger children is significantly higher than in older children, which contrasts with resistance to any other group of antibiotics. It is unclear to what extent this could be due to the increased prescription rate of third generation cephalosporins in the former group. The H30 strain of E. coli is the primary contributor of resistance at the clonal level.

1. Tchesnokova V, Avagyan H, Billig M, et al. A Novel 7-Single Nucleotide Polymorphism-Based Clonotyping Test Allows Rapid Prediction of Antimicrobial Susceptibility of Extraintestinal Directly From Urine Specimens. Open Forum Infect Dis 2016; 3:ofw002.

Characterization and Comparative Genomics of a Novel Archaeal Virus : Sulfolobus Spindle-shaped Virus Lassen (SSV-L/SSV-10).
David Goodman, Kenneth M. Stedman.
Center for Live in Extreme Environments, Department of Biology, Portland State University.

Viruses that infect extremophiles are unique in both their structure and genomic sequences. The lemon-shaped fuselloviruses are some of the most ubiquitous and best studied viruses of the thermoacidophilic Archaea. Fuselloviruses infect members of the order Sulfolobales that grow optimally at 80ºC and pH 3. All Sulfolobus Spindle-shaped virus (SSV) genomes share a highly conserved core set of open reading frames (ORFs). About 90% of all SSV ORFs have no homologs outside of the fuselloviridae family. Moreover, the best studied fusellovirus, SSV-1, has recently been shown to be surprisingly tolerant to mutation, with ~50% of ORFs tolerating mutations without completely abrogating activity. This research reports the complete genome of a novel fusellovirus, its ability to withstand mutation, and its phylogenetic relationship relative to other fuselloviridae. Results indicate that the SSV-L genome has similar tolerance to mutations as SSV-1, with mutants generated in ~85% of ORFs. Phylogenetic and genomic analyses highlight significant homology with both SSV-8 and SSV-9, as well as four ORFs with no homology within or outside of the fuselloviridae.

Anti-activation in bacterial quorum sensing gene regulation.
Kyle L. Asfahl and Martin Schuster.
Department of Microbiology, Oregon State University, Corvallis OR, United States.

Many bacteria coordinate population-wide changes in gene expression using a communication circuit consisting of a diffusible signal and a cognate receptor-regulator in a process termed quorum sensing (QS). Frequently, these QS circuits regulate costly cooperative behaviors in a tight balance of fitness tradeoffs that allows increased inclusive fitness of a population. Canonical QS is generally understood to function by the relative diffusion and accumulation of signal, with QS regulatory machinery transitioning to activated state at a critical threshold signal concentration. However, it is unclear what mechanisms allow the precise tuning of this threshold within the cell. In the well-studied model Pseudomonas aeruginosa, three proteins are known to affect QS gene expression through anti-activation: QteE, QscR, and QslA. Each of these factors individually represses QS activation, effectively increasing the signal threshold necessary to activate the QS regulatory network according to ecological conditions. Here we use genetic analysis, transcriptomics, co-culture experiments, and phenotypic observations to demonstrate a concerted anti-activation of P. aeruginosa QS allows the precise tuning of gene expression necessary for cooperative growth. Using RNAseq, we intend to show QteE-, QscR-, and QslA-based repression of QS ensures tight regulation of costly secreted factors is restricted to ecologically critical growth stages. The tight stoichiometric balance of anti-activator and QS componentry as determined by qRT-PCR, and observations of the dynamic production of costly cooperative secretions without anti-activation will bolster our results. Our study solidifies the importance of anti-activation components in the fitness-enhancing effects of QS gene regulation in bacteria.

Francisella endosymbionts of -cks evolved from mammalian pathogens.
Jonathan Gerhart, Rahul Raghavan.
Department of Biology and Center for Life in Extreme Environments, Portland State University.

Ticks subsist on a diet exclusively of mammalian or avian blood, which is a poor source of certain amino acids and vitamins that are necessary for healthy growth and reproduction. Endosymbiotic bacteria persist inside tick cells and synthesize or recycle these vitamins and amino acids, providing a significant boost to the tick’s fitness. Endosymbionts from many genera of bacteria have been described, most endosymbionts share a close relationship to tick borne pathogens.We sequenced complete genomes of two Francisella endosymbionts, FLE-Am and FLE-Om, and we show that Francisella endosymbionts evolved from mammalian pathogens such as Francisella tularensis.

Development of Smart Cycler-based Multiplex qPCR for Simultaneous Identification of Campylobacter jejuni, C. coli, and C. lari.
Kun C. Liu, Karen C. Jinneman, Jason Neal-McKinney, Wen-Hsin Wu and Daniel H. Rice
Applied Technology Center, U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Northwest, Bothell, WA 98012

Campylobacter is the most common bacterial pathogen causing diarrhea, with 2.4 million cases each year in the US. Foodborne Campylobacter infections are associated with consumption of poultry, unpasteurized dairy, raw shellfish, and contaminated produce or water. Among the 25 currently classified species, C. jejuni, C. coli and C. lari contribute to up to 90%, 5%, and 3.2% of total human infections, respectively. Traditional microbiological methods for Campylobacter identification are labor-intensive, time-consuming and with narrow differentiation spectrum, posing challenges to FDA laboratory testing. Molecular methods such as qPCR can be incorporated into food analyses for the detection and characterization of specific foodborne pathogens.
Here we report the development of a Smart Cycler-based multiplex qPCR for simultaneously identification of C. jejuni, C. coli, and C. lari. Three sets of published primers were combined and optimized for compatibility, and an internal control was designed based on gfp gene. Campylobacter strains were cultured following BAM guidance. Genomic DNA was extracted with a QIAcube and tested in duplicate qPCR. Thirty-one ATCC strains from non-Campylobacter genera and seven from non-target Campylobacter species were assessed for method exclusivity. Fourteen target ATCC strains and eighteen previously characterized field strains were examined for inclusivity. This method yielded 100% species specificity with qPCR efficiency of 90% or more for all targets. The linear regression coefficients were greater than 0.99 in simplex and multiplex qPCR across four-log magnitude. Limit of detection was as low as 2 genome copies for C. jejuni, 3 copies for C. coli, and 2 copies for C. lari. All three target species in one tube using a ratio of 1:1:1 artificially mixed DNA were simultaneously detected. More food and environmental strains will be characterized. This method could eventually facilitate the detection and confirmation of C. jejuni, C. coli, and C. lari from contaminated food products.

Evaluation of VITEK MS automated system for rapid identification of four major pathogenic Campylobacter species.
Kun C. Liu, Michael C. Brown, Kristopher Stanya, Wen-Hsin Wu, Lisa Newberry and Daniel H. Rice
FDA ORA Pacific Regional Laboratory Northwest, 22201 23rd Drive SE, Bothell, WA 98012

Campylobacter is the most common bacterial cause of acute gastroenteritis. Each year there are over 2.4 million people infected by Campylobacter in the United States. C. jejuni, C. coli, C. lari and C. fetus are the major pathogenic species in this genus, responsible for up to 90%, 5%, 3.2% and 2.4% of total infections, respectively. Foodborne Campylobacter infections are often associated with consumption of poultry, unpasteurized dairy, raw shellfish, and contaminated produce or water. FDA BAM methods for Campylobacter identification rely completely on traditional microbiological and biochemical assays, which are labor-intensive, time-consuming, and have a narrow differentiation spectrum among species.
Here we evaluate the application of MALDI-TOF based VITEK MS system for rapid identification of four major pathogenic Campylobacter species using ATCC strains and previously characterized field strains. Campylobacter were cultured on commercial selective agar under microaerophilic conditions at 37°C to 42 °C for two days. Forgoing sub-culture steps used in BAM, two independent single colonies were picked from the primary plate of each strain, and directly tested by VITEK MS under the IVD mode following manufacturer’s guidance. Our results showed that VITEK MS method was able to correctly identify reference strains obtained from ATCC or a previous study, including 21 C. jejuni, four C. coli, four C. lari, four C. fetus, one C. hyointestinalis, and one C. upsaliensis.
In the initial phase of method evaluation, we achieved satisfactory specificity for target Campylobacter species with VITEK MS IVD mode using a total of 35 reference strains. This procedure does not require subculture, greatly reducing media cost, analytical labor and turnaround time in FDA laboratories. We will continue to develop a Campylobacter panel with more characterized strains to fully evaluate VITEK MS for identification of C. jejuni, C. coli, C. lari and C. fetus.

Troubleshooting FISH Probes in Gastropod Gut Microbiomes.
Parker Smith, Michael Baltzley, and Sarah Boomer.
Department of Biology, Western Oregon University

The gut microbiome plays an important role in the host organism’s well-being, contributing to the host’s immunity and metabolism. The goal of this project is to establish a protocol for the analysis of the gut microbiome of the land snail Cornu aspersum (formerly Helix aspersa) using FISH microscopy on samples of C. aspersum feces. A preliminary look at the feces of C. aspersum under a fluorescence microscope showed high levels of auto-fluorescence from plant tissue debris in the feces. Three different approaches have been taken to limit the effects of auto-fluorescence on the FISH microscopy results. We first attempted to quench the auto-fluorescence by incubating the feces samples in alcoholic 6% H2O2 after fixation, a method that had been shown effective in insect tissues. After eight weeks of treatment, however, we still observed auto-fluorescence from plant tissue in emission spectrum of Alexa 546, the fluorescent label on our GAM42a probe. We then looked at avoiding auto-fluorescence, by determining which wavelengths had the lowest amounts of auto-fluorescence. We found that there was very little auto-fluorescence from plant tissue in far red wavelengths. Since the plant debris is much larger than the bacteria present in the samples we are currently working on filtering out the auto-fluorescent plant debris. In conclusion, we are finding that the filtering of plant debris lowers the background auto-fluorescence enough to allow us to use fluorescent dyes with emission spectra that coincide with the wavelengths of auto-fluorescence from the plant debris. Or goal is to use these protocols to study the fluctuations of the gut microbiome in response to changes in diet, environmental stressors, and antibiotics

Functional Relatedness in the Inv/Mxi-Spa Type III Secretion System Family. Jessica A. Klein¹, Biren M. Dave², Amogelang R. Raphenya², Andrew G. McArthur², Leigh A. Knodler¹.
¹Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, WA, USA
²M.G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada

The Type III Secretion System (T3SS) is a common virulence factor among Gram-negative pathogens uniquely adapted for transkingdom communication between bacteria and eukaryotic hosts. Phylogenetically, T3SSs are divided into several families of structurally conserved nanomachines functioning as molecular syringes for translocation of type III effector proteins that modulate host cell signal transduction pathways. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs: one of which belongs to the Inv/Mxi-Spa family. The Inv/Mxi-Spa T3SSs are often involved in facilitating bacterial invasion of eukaryotic hosts, a process dependent on multiple proteins, including four that are transcribed from the translocator operon: a cytosolic chaperone, two integral membrane translocator proteins comprising the translocon, and a scaffold (“tip complex”). To study functional conservation of these proteins, translocator orthologs identified from an extensive phylogenetic analysis of Inv/Mxi-Spa pathogens were expressed in invasion-deficient S. Typhimurium mutants and assessed for type III effector translocation and host cell invasion. We identified several Inv/Mxi-Spa orthologs that can functionally substitute for S. Typhimurium chaperone, translocator or tip complex proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies between proteins of the translocator operon. This study is the first in-depth investigation of functional relatedness within the Inv/Mxi-Spa T3SS family.

The Effector BspB Modulates Golgi Trafficking to Promote Brucella intracellular replication.
Cheryl N. Miller¹, Erin P. Smith¹, Jennifer A. Cundiff¹, Vladimir Lupashin², and Jean Celli¹.
¹Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, WA, USA.
²Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

The bacterial pathogen Brucella abortus exploits functions of the host secretory pathway to grow within cells by generating a replicative Brucella Containing Vacuole (rBCV). The VirB Type IV secretion system (T4SS) is necessary for Brucella to establish infection and grow intracellularly, by delivering effector proteins into the cell. We identified a Brucella T4SS effector, BspB, which inhibits host protein secretion, and is required for Brucella growth in macrophages. BspB is necessary and sufficient to redistributes the Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC) to the Golgi. We show that BspB interacts with the host Conserved Oligomeric Golgi (COG) complex, which orchestrates the tethering and fusion of Golgi transport vesicles. COG1 or COG8 knockout cells reduced Brucella replication comparable to the ΔbspB mutant. The N-terminal transmembrane domain of BspB is necessary for Golgi localization, interaction with the COG complex, and for inhibiting the secretory pathway. These findings characterize the interaction between the Brucella effector protein BspB and the host-target the COG complex, which is required for Brucella pathogenesis.

Endophyte-assisted Phytoremediation of Arsenic.
Shruti Parikh, Robert Tournay, Dominic Sivitilli, Sharon Doty.
School of Environment and Forest Sciences, University of Washington.

Arsenic, a known carcinogen, has been linked to numerous cancers, and its toxicity and persistence in the environment at low levels impact both human health and ecosystem integrity. Due to natural and anthropogenic factors, the concentration of arsenic has elevated in many areas. Phytoremediation is a cost-effective, eco-friendly technology that uses the natural ability of plants to remediate pollutants, such as arsenic, from contaminated soils. Endophytes, mutualistic microbes colonizing the internal tissues of plants, have been shown to increase host-plant tolerance to a wide range of environmental stressors, and we hypothesize that arsenic-tolerant endophytes may improve the tolerance of their host plants to arsenic. In this study, we tested two endophyte isolates to see whether they grow and remove arsenic from solution in aerobic conditions and screened various isolates for exopolysaccharide (EPS) production with the presence of arsenic in order to test whether bacterial EPS could be promoting tolerance to arsenic. This will be explored further in future EPS and phytoremediation experiments.

Hot Tales of T4's Transition from Host to Phage Metabolism.
Georgia Ray, Vladimir Eryomin, Daniel Bryan, and Elizabeth Kutter.
The Evergreen State College.

Numerous facts about phage-host interactions were originally discovered by introduction of radioactively labeled molecules into phage-infected bacteria. While biologists today have access to tools like RNAseq, the old standby of radiolabeling is still a reliable technique that is uniquely useful in exploring details of the phage infection process. Particular opportunities include exploring how phage behave in conditions more similar to nature such as in stationary phase, in different phage and bacterial strains, and answering open questions from previous explorations in radiolabeling.

Epigenetics of Host-Pathogen Interaction: Agrobacterium rhizogenes C58.
Genny Roberts¹ and Derek Wood^1,2.
¹Department of Biology, Seattle Pacific University.
²Department of Microbiology, University of Washington.

Bacterial virulence is a powerful force and is well documented, though not always well understood. The past two decades have illuminated that many bacteria use epigenetic modification to change their genetic expression as needed, though understanding which genes are under this control and the environmental response mechanism have just begun to be explored. Agrobacterium tumefaciens C58 is a plant pathogen that turns from a avirulent soil-dwelling bacteria to a plant infecting and killing microbe at the response of the plant wound signal, Acetosyringone. In this research, I’ve demonstrated that bacteria genomes sequenced in the presence of this chemical lose epigenetic modification of close to 100 genes, suggesting these genes are expressed solely for virulence purposes. These findings will be examined and mechanisms will be explored to better understand which genes are involved in these virulence processes.