Methylobacterium extorquens AM1 is a methylotrophic bacteria ubiquitous in the environment and in particular on the undersides of leaves. This organism is able to metabolize both single carbon compounds like methanol and multi-carbon compounds such as succinate and pyruvate. M. extorquens has been studied for decades because of its potential for not only creating value added compounds from methanol, but important regulatory systems including the regulatory pathway of methanol metabolism, which has yet to be fully elaborated. Our laboratory focuses on single carbon compound metabolism and the regulation of the pathways required for growth. The first step in methylotrophic growth is the oxidation of methanol via methanol dehydrogenase encoded by the mxaFI genes. Promoter fusion data suggests a complex regulatory system involving two two-component systems MxbDM and MxcQE, two homologues of the main methanol dehydrogenase XoxF12, and an unknown transcriptional repressor. In order to elucidate this process, the direct binding of a transcriptional regulator to the mxaF promoter region must be demonstrated. Unfortunately all putative regulators have historically failed to show activity in vitro. In order to isolate direct regulators, whole cell extract will be washed over an abundance of the mxa promoter region that is bound to magnetic beads. Then using a magnet, the promoter region can be removed along with proteins that have bound from the cell extract, any non-specific binding proteins can be washed off using a low salt buffer, and specific binding proteins removed using a high salt buffer. The elutions containing the proteins of interest can be run out on an SDS PAGE gel, and the unique bands of interest excised and sent for mass spectrometry analysis for identification. Our work will define transcriptional regulators of the mxa operand, determining which specific proteins are binding the promoter region of that operand.

 The effect of viral evolution on the duration of latency in Human Immunodeficiency Virus Type 1 (HIV-1) infection has been studied extensively in men. The asymptomatic latency period, after HIV-1 infection and before the onset of Acquired Immune Deficiency Syndrome (AIDS), is characterized by high viral variability. In many cases, a viral phenotype that utilizes CXCR4 and/or CCR5 receptors on target host cells to advance HIV-1 infection progress also emerges during this period. Despite known sex-dependent variation in the progression of HIV-1, the effect of viral evolution and CXCR4/CCR5 utilizing virus (X4 virus) prevalence on HIV-1 latency has not yet been studied in women. I am investigating trends in viral evolution in 9 women from seroconversion until initiation of antiretroviral therapy. I will then compare my results to previously studied male viral evolutionary trends. Ultimately, a total of 72 blood plasma samples, an average of 8 samples from each woman collected progressively during the course of infection, will be used to generate 25-30 HIV-1 sequences per time point. These sequences will then be evaluated through 1) viral divergence from a founder strain 2) viral population diversity and 3) emergence and prevalence of X4 viruses. I will then analyze the relationship between viral evolutionary trends and X4 virus prevalence, and the duration of HIV-1 infection before the onset of AIDS. Ultimately, a comparison between the sexes will be made. Furthering the scientific communities’ knowledge of sex-related HIV-1 infection variation could lead to more effective sex-specific treatment and vaccine development. The important biological question of whether sex can influence HIV-1’s evolutionary path will be addressed. This project is currently in the preliminary stage of sequence generation. Trend analysis and conclusions will take place in Fall 2012.

Quorum sensing is cell density-dependent communication that regulates many functions in bacteria. Signal synthases (I) produce acyl-homoserine lactones, which diffuse into the environment and activate signal receptors (R) when they reach a critical concentration. Synthases and receptors often form cognate pairs. The soil bacterium Burkholderia thailandensis encodes three I-R pairs. The I-R1 system controls cell-cell aggregation and activates many putative aggregation factors including the bce genes that likely encode an exopolysaccharide. The I-R2 system controls antibiotic production. The antibiotics are members of the bactobolin family and are effective against both Gram-positive and Gram-negative bacteria. Both aggregation and antibiotics are believed to promote competitiveness in the diverse soil communities where B. thailandensis is found. We have developed genetic screens to learn more about quorum-controlled antibiotics and aggregation in B. thailandensis. The first screen identified bactobolin-resistant isolates of the soil bacterium Bacillis subtilis, which is normally sensitive to bactobolin. We submitted these mutants for whole genome sequencing to investigate the mutations that confer resistance, and with our results we hope to gain insight into how bactobolin acts on target cells. The second screen was designed to isolate variants that overproduce aggregation factors. Quorum sensing mutants do not wrinkle but variants arise that produce aggregation factors by other means. We are isolating such variants and identifying the underlying mutations to understand how they compensate for the loss of quorum sensing regulation. This may reveal the elements involved in regulating aggregation. These variants will also be useful to learn about the advantages of aggregation in different contexts. Through our two screens we hope to better understand quorum-controlled aggregation and antibiotic phenotypes. Our findings will be useful in further studies of the importance of each of these in competition.

Bacterial conjugation is a form of horizontal gene transfer that serves as an important source of genetic plasticity and diversity in both Gram positive and Gram negative bacteria. It has also been proved to be advantageous for dissemination of antibiotic resistance. Conjugation is a plasmid-mediated mechanism that allows single stranded DNA to be transferred across cell membranes from a donor to a recipient cell. In Escherichia coli, the conjugative F (fertility) plasmid encodes all necessary proteins for assembling the transfer apparatus thus allowing transfer and propagation of DNA into a recipient bacterial cell. Previous conflicting studies of bacterial conjugation in E. coli have shown DNA transfer to occur between donor and recipient pairs in intimate cell-cell contact or between distant donor and recipient pairs, connected only by the pilus, a filamentous protein extended from the donor cell. Using various fluorescent protein constructs to differentially mark donor and recipient cells and F plasmid DNA itself, we visualize conjugation in real time with single-cell resolution by fluorescent microscopy. Here we show that conjugation occurs only when donor and recipient cells are in intimate and close physical contact with each other. In addition, there appears to be no preference for cellular orientation for transfer. Furthermore, we propose a simple rate equation to model DNA transfer between bacteria and measure the rate of DNA transfer per unit time per cell contact. This quantitative model is a tool to elucidate conjugation at a single-cell scale and will provide a deeper understanding into the mechanism of bacterial conjugation.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis. Components of the outer and inner membrane, characteristic of Gram-negative bacteria, contribute to Salmonella’s mechanisms to avoid detection and killing by the host immune system. The anionic bacterial cell surface is disrupted by cationic antimicrobial proteins (CAMP) that increase outer membrane (OM) permeability and access to the inner membrane. To resist CAMP killing Salmonella activates the PhoPQ two-component system, which is essential for pathogenesis. Because many PhoPQ-regulated enzymes are dispensable during infection, we conducted a forward genetic screen that identified new components of the PhoPQ regulon involved in maintaining the OM permeability barrier. Sequencing revealed mutations in genes encoding a variety of membrane proteins including transporters, phospholipid and lipopolysaccharide biosynthetic enzymes and efflux pumps, as well as regulatory proteins. Amongst the results, yejM mutants expressed the highest levels of membrane damage. YejM is an inner membrane protein with a periplasmic enzymatic domain that is critically involved in maintaining the OM permeability barrier. Structural alignments suggest that YejM is a member of the alkaline phosphatase superfamily. Attempts at full length deletions of yejM were unsuccessful suggesting the transmembrane domain is essential for viability, whereas periplasmic deletion mutants were viable but expressed varied levels of OM damage. Complementation with periplasmic deletion mutants and plasmids carrying the periplasmic domain rescued the OM permeability defect. Using structural models generated by a Protein Homolog/analogy Recognition Engine (PHYRE), we located putative metal binding sites involved in catalysis. To investigate the enzymatic mechanism, specific metal-binding residues were mutated via Polymerase Chain Reaction. We aim to determine if these residues contribute to the OM permeability defect of the yejM mutant Salmonella.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), a proliferative and angiogenic tumor commonly associated with AIDS. The predominant cell type in KS tumors is the latently infected spindle cell, which expresses markers of lymphatic and blood endothelium. KSHV infection dramatically alters a number of host pathways, including angiogenesis and lymphangiogenesis. KSHV induces angiogenesis, but the viral gene or genes responsible are unknown. Also, KSHV infection of blood endothelium induces expression of lymphatic endothelium markers. Our data indicate that the viral gene vIL-6 is sufficient, but not necessary, to induce blood to lymphatic endothelial cell reprogramming. The goal of my project is to identify the latent viral gene or genes responsible for both the induction of the angiogenesis and blood to lymphatic endothelial cell reprogramming. My first aim was to determine if any single latent gene is sufficient to induce angiogenesis, through increased expression of the integrin alphaVbeta3 expression, or induce blood to lymphatic differentiation, as indicated by expression of prox-1, the master regulator of lymphatic endothelium. The results indicated that no single latent gene was sufficient to induce either change in expression. My second aim is to express the entire latency locus in endothelial cells to determine whether it is sufficient to induce angiogenesis-related phenotypes and/or lymphatic reprogramming. The KSHV latency locus will be cloned into an adenoviral vector and transfected into blood endothelial cells, which will be evaluated for alphaVbeta3 and prox-1 expression. If the latency locus is sufficient for induction of prox-1 or increased expression of alphaVbeta3, I will next silence each latent gene using siRNA to determine if any of the latent genes are necessary for the phenotypic alteration. My studies will help elucidate mechanisms by which KSHV induces activation of endothelium, and ultimately causes KS.

Our goal is to use human adenovirus to investigate how human defensins interact with viral capsids to neutralize infection. Defensins are small antimicrobial peptides of the innate immune system that prevent bacteria and viruses from infecting their host, but the mechanisms by which defensins neutralize viruses are not yet well known. For human adenovirus (HAdV), we have shown that infection by serotype 5 (HAdV-5) is completely sensitive to defensins while that of HAdV-19c is resistant. Adenovirus is a non-enveloped DNA virus with an icosahedral capsid comprised primarily of the hexon protein. Fiber proteins attached to penton base proteins at the icosahedral vertices interact with host cells during cell entry. Previous studies showed that penton base and fiber proteins contain specific sequences important for conferring sensitivity to defensins. In order to identify these sequences in the HAdV-5 penton base protein, we constructed a number of chimeric viruses in which parts of the HAdV-5 penton base are replaced with the HAdV-19c penton base. Replacing the last half of HAdV-5 penton base, but not just the third or fourth quarters, with the corresponding sequence from the HAdV-19c penton base resulted in significant resistance to defensins. Based on these results, we created new chimeras by replacing the entire third quarter in combination with specific residues in the fourth quarter of the HAdV-5 penton base in order to determine exactly which residues confer resistance to defensins. After identifying these residues, we will create new chimeras by replacing the fourth quarter and mutating specific residues in the third quarter of the penton base. Together with quantitative binding studies to measure the impact of these changes on the defensin-adenovirus interaction, these studies will provide insight into the molecular mechanism of defensin-mediated neutralization of adenovirus that may be generalized to other non-enveloped viruses that are sensitive to defensins.

In any niche occupied by multiple species, competition is inevitable; whether through killing opponents, or depleting resources first. Ecological theory posits the existence of r- and K-selected competitors. r-selected organisms colonize well and reproduce quickly (often at the expense of individual survival). K-selected organisms succeed in stable, dense environments and are typically larger and live longer; typically investing in self defense mechanisms. This observed quantity vs quality competition is the foundation for the r/K selection theory. One limitation of existing r and K models is that factors causing organisms to be either r or K selected are not clearly defined. Release of toxic substances (i.e., interference competition) is an example of a factor that could allow for a species to be good at excluding others near the carrying capacity. To study this, we created a bacterial warfare model with two antibiotic producing bacteria, Chromobacterium violaceum (Cv) and Burkholderia thailandensis (Bt). These species were competed in at a 1:1 ratio and were disturbed (re-plated via coring) at varying intervals. Both bacteria produce their antibiotics when cell density is high. Cv behaves like an r-selected organism in that it grows quickly but has a weak antibiotic. Bt shows characteristics of K-selected organisms, growing slower, but creating antibiotics that rapidly kill Cv. Our initial observations show that Cv succeeds in frequently disturbed environments by not allowing Bt to effectively reach quorum, while Bt wins in more static environments where its ability to produce potent antibiotics to pay off. Also, controls indicate while Cv's antibiotic provides an edge, its growth rate appears to be the determining factor for success. These results provide evidence that disturbance frequency acts as a primary selective force in the antibiotic mediated competition, and may provide parallels and contrasts to other systems where r and K selected organisms compete.

Helicobacter pylori is a bacterium that infects the stomach of about half the world’s population. While usually asymptomatic, H. pylori infection is the main cause of peptic ulceration, gastric lymphoma, and gastric adenocarcinoma. This suggests variability within populations of H. pylori in their ability to cause disease. I am researching the effect of a mutation in the gene cagY on the ability of H. pylori to cause disease. This gene produces CagY, a structural protein of the bacteria’s Type IV Secretion System, which is involved in causing disease. H. pylori transports different materials across the Type IV Secretion System, including the protein CagA. Host cells receiving this protein elongate and narrow (a change called the hummingbird phenotype) and produce interleukin-8, a chemical messenger of the immune system. In host cells, the protein CagA is phosphorylated (a process by which a phosphate group is added to a molecule). To test the effects of the cagY mutation on the bacteria’s Type IV Secretion System, I will infect human stomach cancer cells with the cagY mutant. To detect changes in these cells caused by the infection, I will use two techniques that detect specific proteins, Western Blot and ELISA. The Western Blot will detect the presence of phosphorylated CagA, while the ELISA will detect interleukin-8. The length of infected cells will be measured to detect the hummingbird phenotype. These tests will measure the function of the Type IV Secretion System in the cagY mutant. The absence of a functioning Type IV Secretion System in the cagY mutant will suggest a change in bacteria and host interaction and a potentially decreased ability for the bacterium to cause disease.

Chronic infections with Pseudomonas aeruginosa are a significant cause of deterioration of lung function in patients with cystic fibrosis (CF), and have substantial clinical importance due to their marked resistance to treatment with antibiotics. Furthermore, existing laboratory methods for testing bacterial susceptibility to antibiotics are poor indicators of clinical response to therapy, which further increase the difficulty associated with treating these infections. Current research shows that phenotypically diverse P. aeruginosa subpopulations evolve within a single patient during prolonged infection, and that clinical antibiotic therapy causes some of these subpopulations to change significantly in relative abundance. Observed changes in abundance following antibiotic treatment signify varying degrees of antibiotic susceptibility within each patient’s P. aeruginosa population as a whole, with an increase in a particular subpopulation abundance indicating resistance, and a decrease in a particular subpopulation abundance indicating sensitivity. As previous studies have only evaluated P. aeruginosa antibiotic sensitivity prior to antibiotic treatment, we attempted to demonstrate the correlation between an increase subpopulation abundance following clinical treatment and the degree of resistance to antibiotics in laboratory testing. This was done by evaluating the antibiotic minimum inhibitory concentration (MIC) of a range of isolates collected from CF patients who had undergone antibiotic therapy, using antibiotics which had been given to each patient in clinical treatment. Our results demonstrate a marked lack of correlation between relative subpopulation abundance and antibiotic MIC, with clinically resistant subpopulations showing the same or lower MIC than subpopulations which were clinically sensitive treatment. We conclude that isolates resisting treatment do not have a different MIC than isolates responding to treatment, suggesting that mechanisms other than classic antibiotic resistance are responsible for treatment resistance in the CF lung. Further studies to evaluate these mechanisms, such as biofilm formation and formation of persister cells, are currently under way.

The type VI secretion system (T6SS) is a large protein complex utilized by bacteria to transport proteins into other cells. Initially implicated in virulence, the T6SS also participates in interbacterial interactions. A T6SS found in the opportunistic pathogen Pseudomonas aeruginosa, the hemolysin coregulated protein secretion island I T6SS (H1-T6SS), has been found to deliver a set of toxins to other bacteria. This provides P. aeruginosa a fitness advantage over competing bacteria. The H1-T6SS can target neighboring P. aeruginosa, which protects itself with cognate immunity proteins. Deleting these immunity proteins leaves P. aeruginosa susceptible to self-attack. However, the H1-T6SS as well as other T6SSs can target other bacteria, specifically they only target certain species. One of the goals of this project is determine the nature of such specificity. These findings would implicate new functions for the H1-T6SS. To further this, I am seeking to extend the scope in which the T6SS is viewed, both mechanistically and genetically. In doing so I am searching for new genetic loci that participate in T6SS-dependent interactions. Taken together, this work provides insights into a new aspect of type VI secretion and may change the genetic composition of T6SSs.

 A small percentage of the HIV infected population remains asymptomatic for years to decades. Some of these persons, termed late progressors (LP), eventually progress to symptomatic infection, characterized by rapidly rising viral RNA levels and dramatic decreases in CD4+ cells, the main targets of HIV infection. This transition suggests significant changes in the virus-host relationship allowing viral replication to occur. To assess three possible hypotheses as to why the transition to progressive infection may occur, we examined blood plasma samples from four LPs, taken at multiple time points preceding the onset of disease progression. The gag and env genes of HIV isolates were amplified by the polymerase chain reaction (PCR), sequenced, and analyzed for viral population characteristics typical of either 1) superinfection with a second (less controllable) virus strain, 2) a change in the co-receptor used by the virus to infect CD4+ cells, or 3) viral escape, a phenomenon in which HIV-specific immune responses can no longer recognize, neutralize, or prevent replication of virus. Identification of superinfection consisted of locating subsets of virus exhibiting extreme levels of evolutionary divergence from the total virus population. Analysis for co-receptor usage focused on the env gene sequences, while viral escape was indicated by mutations at highly conserved amino acid sites and/or neighboring regions previously targetable by the host immune response. Any of these three hypotheses could potentially result in the sudden enhanced viral replication and disease progression observed in LPs. Examination of this data may clarify patterns of viral evolution in response to immune pressure, adding significantly to the understanding of late progression, viral escape and fitness, and overall HIV-host interactions. Thus, this study may potentially inform both timing of antiretroviral therapy for predicted late progressors and design of drugs and vaccines specific to identified critical regions of the HIV genome.

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium indigenous to estuarine and marine environments worldwide. A minority of strains are responsible for causing severe gastroenteritis in humans, often associated with the consumption of raw or undercooked molluscan bivalves, especially oysters. Pathogenic strains are normally detected by screening environmental samples for the presence of the virulence-associated thermostable direct hemolysin (tdh). In Washington State, the utility of tdh is questionable given the prevalence of tdh+ strains (25/28, 89.3%) among environmental multilocus sequence types and the recent emergence of two tdh- clinical sequence types. In light of this public health challenge, we are currently validating the utility of new candidate virulence markers, which were discovered via comparative genomic analysis of 13 clinical and 6 environmental strains. We identified a unique α-hemolysin (605 amino acids) in the two emergent tdh- sequence types having homology (83% identity) to the hlyA of the RTX toxin cluster in the uropathogenic E. coli 536. To investigate the utility of this α-hemolysin as a new virulence marker, PCR primers targeting this gene were validated against a collection of clinical and environmental strains isolated from WA State. To date, 11/97 (11.3%) of the strains tested harbored this gene. Notably, this gene was present in 5/5 (100%) of the strains representing the two tdh- clinical sequence types. Current efforts are focused on the validation of this candidate virulence marker against a larger strain collection as well as the investigation of additional candidate virulence markers. Future efforts will focus on whether this hemolysin represents a newly described mechanism of V. parahaemolyticus pathogenesis.

The secretion of signals or toxins is a vital aspect of bacterial communication with its environment. We recently found that type VI secretion systems (T6SSs) in Gram-negative bacteria mediate interbacterial interactions by translocating protein effectors into neighboring bacterial cells, providing a large fitness advantage. One protein exported by the T6SS is a type VI-exported (Tse) cell wall amidase that degrades peptidoglycan, and secretion of this effector, Tse1, results in target cell lysis. Bacteria can also self-intoxicate by T6S, potentially posing a problem for the survival of a given species. However, bacteria encode cognate immunity (Tsi) proteins that bind and inactivate their Tse partners. As we identify more T6 effectors, it is becoming clear that Tse–Tsi pairs are part of a widespread protein–protein recognition network underlying bacterial diversity and community structure. This study sheds light on how this network influences interbacterial interactions by uncovering the mechanism of Tsi1-dependent inhibition of Tse1 and defining the molecular determinants of this recognition. I have reconstituted the complex in vitro by using recombinant proteins to probe for direct interaction. Then, by mapping conserved residues onto the recently solved structure of Tse1, I have identified and mutationally analyzed candidate surface residues that may be involved in Tsi1 recognition. Further understanding the determinants of this interaction may allow us to predict which bacterial species are vulnerable to Tse1 or provide a template for the design of novel effector-immunity complexes that escape recognition in naturally occurring bacteria as an antibacterial strategy.

In developing countries nearly 80% of all diseases are linked to water and sanitation issues; globally, over two million people die annually from water-born diarrheal disease. It has long been known that some metals can exhibit powerful anti-bacterial properties. Copper has been used for hundreds of years as a means to purify drinking water but only recently have scientists begun to elucidate the mode of action for copper induced microbial death. In 2008 the U.S. Environmental Protection Agency officially registered copper alloys as biocidal agents. Now these alloys need to be studied to determine efficacy at different concentrations and with a variety of disease-causing microbes. This study investigates the powerful bactericidal effects of copper as a low cost accessible method to purify drinking water of pathogenic waterborne microorganisms. More specifically, E. coli was used as a model organism to test the efficacy of solid copper at killing microorganisms in drinking water over variable amounts of time. Efficacy of cell death was assessed with viable plate counts. Results show that ≥48cm2 of solid copper in 100 mL of water can kill E. coli (at an average concentration of 3.1x105 CFU/mL) within 24 hours. Further research is needed to determine the optimal copper to water volume ratio as well as the precise timing of cell death. It is also essential that the copper ion concentrations remain below EPA limits of 1.3 mg/L; preliminary findings produced inconsistent values. The results of this research could be beneficial to those living in locations where safe potable water is not readily available, or for those travelling to remote or foreign locations as well as hikers and campers.

Angiogenesis is the development of new blood vessels from pre-existing vasculature and is a hallmark of Kaposi’s sarcoma (KS), a highly vascularized neoplasm prevalent among immunosuppressed individuals. The etiological agent of KS, Kaposi’s sarcoma herpesvirus (KSHV), induces angiogenic phenotypes in endothelial cells during latent infection. Interestingly, KSHV infection leads to down-regulation of transforming growth factor-beta 2 (TGF-beta2), an anti-angiogenic cytokine. In addition to six viral genes, KSHV also encodes 17 recently discovered microRNAs (miRNAs) during latent infection that appear to directly contribute to pathogenesis and angiogenesis by targeting host gene expression.  These endogenously encoded RNAs of 19-23 nucleotides post-transcriptionally regulate gene expression through basepairing interactions with the 3’ untranslated region (UTR) of target messenger RNAs (mRNAs) and either mark them for degradation or prevent ribosomal binding. We have screened the miRNAs of KSHV to determine if any are involved in the downregulation of TGF-beta2 by KSHV. We found that two KSHV miRNAs, miK-K3 and miR-K8, are sufficient to downregulate TGF-beta2. To determine if these miRNAs act directly on the mRNA of TGF-beta2 as opposed to altering genes that affect its expression, I proposed two aims: (1) use quantitative RT-PCR to determine the levels of TGF-beta2 expression during KSHV infection when miR-K3 and miR-K8 are inhibited by antagomirs, small antisense inhibitors of miRNAs, and (2) determine if miK-K3 and miR-K8 directly bind to the 3ʼ UTR of TGF-beta2 mRNA using luciferase reporter constructs. By identifying specific targets of KSHVʼs miRNAs such as TGF-beta2, we can improve our understanding of how KSHV is able to maintain latent infection and disrupt the normal regulation of cell proliferation. These studies are the first steps to better understanding KSHV pathogenesis and to developing targets that disrupt KSHV replication.