Staphylococcus aureus is a Gram-positive bacterium that colonizes the nose and skin and causes a variety of infections in humans. The acquisition of resistance to many first line antibiotics has made S. aureus increasingly difficult to treat. Previous studies have shown that an S. aureus enzyme called nitric oxide synthase (NOS) is necessary for both colonization and infection in animal models. The goal of the present study is to determine the role of NOS in staphylococcal physiology. We treated the S. aureus strain COL and an isogenic mutant strain lacking NOS with several redox-active compounds including menadione, paraquat, and pyocyanin. The minimal inhibitory concentration (MIC) of each agent was measured in liquid culture. A NOS mutant was substantially more sensitive than wild-type S. aureus to menadione, and menadione-resistance was restored by treatment with exogenous nitric oxide. In contrast, NOS mutant and wild-type strains were equally sensitive to paraquat. As both menadione and paraquat are redox cycling reagents that generate superoxide radicals, this observation suggests that the menadione-sensitivity of the NOS mutant is unrelated to superoxide. The NOS mutant also appears sensitive to pyocyanin, which blocks the electron transport chain. We hypothesize that nitric oxide regulates electron flow through the electron transport chain, most likely by inhibiting terminal oxidases. Further studies will investigate the effect of electron transport inhibitors that target NADH-dehydrogenase (rotenone) or the F₀F₁-ATPase (DCCD), and the effect of mutations that impair cytochrome assembly or the regulation of redox metabolism, in wild-type or NOS mutant bacteria. ATP and NAD/NADH levels will be monitored in parallel to determine effects on energy generation and redox balance. These studies will reveal how endogenously generated nitric oxide maintains redox homeostasis in bacteria and may lead to novel strategies to treat or prevent S. aureus infections. 

‘Aigarchaeota’ is a candidate phylum of Archaea known only by 16S rRNA gene fragments from cultivation-independent microbial surveys and a single composite genome from Candidatus ‘Caldiarchaeum subterraneum’, an inhabitant of a subterranean gold mine in Japan. Gene sequences reported in various publications were found almost exclusively in geothermal settings, but a comprehensive assessment has not yet been performed. The purpose of this study was: (i) to rigorously define the phylum; (ii) to gain insight into the phylogenetic and potential taxonomic structure of the phylum; (iii) to assess the distribution of ‘Aigarchaeota’; and (iv) to design ‘Aigarchaeota’-specific 16S rRNA gene primers. Public databases were mined for 16S rRNA gene sequences related to known ‘Aigarchaeota’ and a combination of approaches were used to rigorously define the phylogenetic boundaries of the phylum and compile a neighbor-joining and maximum likelihood phylogenetic tree. Primer template gene sequences were aligned in ClustalW in order to locate regions that are suitable for targeting genus-level groups. ‘Aigarchaeota’-specific primers for the polymerase chain reaction (PCR) amplification of 16S rRNA genes were designed using sequence alignments and reviewed using the Ribosomal Database Project Probe Match tool. The analyses supported the proposed relationship between ‘Aigarchaeota’, Thaumarchaeota, Crenarchaeota, and Korarchaeota in the so-called ‘TACK’ superphylum, and identified ~300 16S rRNA genes and gene fragments affiliated with ‘Aigarchaeota’, including those recovered from terrestrial geothermal systems on several continents and marine geothermal and subsurface samples. ‘Aigarchaeota’ belonged to at least three family- to order-level groups and at least 13 genus-level groups which are represented in the resulting phylogenetic tree. All genus-level groups were recovered from geographically distant locations, suggesting a global distribution. The primers will be used to determine the presence and abundance of ‘Aigarchaeota’ in a wide variety of samples from terrestrial geothermal systems in the U.S. and Asia using quantitative real-time PCR.

Tuberculosis (TB), an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb), kills nearly 2 million people a year and is a major cause of morbidity and mortality worldwide. The increasing prevalence of multi-drug resistant (MDR) strains that fail to respond to all currently available drugs underscores the critical need for new therapeutic strategies. DNA topoisomerases (topos) are a class of enzymes responsible for maintaining the correct topological state of DNA molecules by modulating the degree of supercoiling through the breakage and reforming of covalent phosphodiester bonds in the DNA backbone. With the precedent-setting success of several anticancer and antibacterial drugs that target topos, focus in TB research has increasingly turned to these enzymes as new potential targets for chemotherapy. In this study, we isolated Mtb topoisomerase type IA (MtbTOPA), a subclass that only acts on single-stranded DNA (ssDNA), as our target of interest. At least one type IA topo activity must be present in every organism for resolving topological obstacles that require ssDNA cleavage, and fortuitously, Mtb happens to only encode one type IA, further validating MtbTOPA as a high-value drug target. We have cloned, expressed, and isolated highly purified recombinant MtbTOPA protein for enzymatic studies and work is currently underway to solve its crystal structure to aid in target-based approaches to drug development. In efforts to develop a high-throughput screen (HTS) for identification of small molecule inhibitors of MtbTOPA, we have collaborated with the Global Alliance for TB Drug Development (GATB) and leading structural genomics centers to gain access to large compound libraries as well as to accelerate the drug discovery process. We adopted a HTS method based on fluorescence resonance energy-transfer (FRET) and requisite MtbTOPA mutants were designed. It is our hope that the HTS will identify several hits that can be developed into novel TB drugs.

Most living systems have complex bacterial communities (microbiomes) associated with them that are essential to the host's wellbeing. Microbiomes provide their host with nutrients, protection against pathogens and stress, and influence host immunity. Microbiomes can consist of hundreds of different bacterial species making them difficult to study. However, the algal species Chrysochromulina sp. (referred to as P3) has a small microbiome consisting of less than 10 species, making it a prime candidate for studying interactions between a host and its microbiome. Chrysochromulina sp. is also of interest as a source of biofuels since it produces a variety of fatty acids, and it belongs to an algal taxa that plays a major role in global carbon sequestration. I hypothesize that the microbiome of P3 allows P3 to withstand periods of stress. I tested this by growing P3 and an axenic (bacteria-free) culture known as P5.5 in high salt medium, a known stressor, and determined cell counts by flow cytrometry and bacterial colony counts by culturing. I found the P3 grew better than P5.5, indicating the bacteria help the alga survive high salt conditions. We cultured and identified 8 different bacterial species associated with P3 by sequencing the 16S rDNA, which encodes ribosomal RNA and is unique to each species. Next I will determine which bacterial species protect P3 from high salt. I will further determine the protective role of the microbiome by growing P3 and P5.5 under other stress conditions (e.g. low nitrogen, low pH, low light, and temperature shifts) to understand how the microbiome protects Chrysochromulina sp. during periods of environmental challenge. This will allow insight into how to optimize Chrysochomulina sp. growth for biofuel production and the effects of global warming on this alga.

We are interested in the effects of naturally secreted defensins on viral replication. Defensins are small, cysteine-rich, antimicrobial peptides produced by the innate immune system. Although most studies of defensins have focused on their antimicrobial activity, we have shown in vitro that growth of enteric mouse adenovirus 2 (MAdV-2) is unexpectedly enhanced by defensins. Since this observation was made using purified defensins, we sought to determine if naturally secreted defensins also enhance enteric viral infection. Mouse defensins are exclusively produced by Paneth cells (PCs) in the small intestine. As isolated PCs are not culturable, we utilize a novel 3D small intestinal organoid model, which allows us to grow primary small intestinal epithelial cells in culture. The organoid lumen is topologically equivalent to the apical side of the small intestine. Thus, to mimic oral infection, I directly microinjected the virus into the organoid lumen. I first demonstrated that MAdV-2 replicates in wild-type organoids. Then, I compared the growth of MAdV-2 in wild-type organoids to growth in knockout organoids that do not produce functional defensins. I found that, consistent with the enhanced infection, replication was accelerated in the wild-type organoids as measured by plaque forming units. To confirm these results, and to examine any effect of defensins on viral tropism, I created and characterized a fluorescently tagged construct of MAdV-2, in which I fused green fluorescent protein (GFP) to a minor capsid protein. After microinjection, more green cells were observed in the wild-type organoids. Enteric viruses are routinely exposed to defensins during natural infection. Therefore, the observed enhancement of MAdV-2 may represent a novel mechanism by which enteric viruses hijack a host defense peptide and use it to augment their own infection. Future studies will look at enhancement of other enteric viruses and use additional models with altered levels of defensin expression.

There is currently an estimated 350 million people worldwide chronically infected with the Hepatitis B virus (HBV). These individuals, over the course of their life, are at an increased risk of developing liver cancer. While there are currently antiviral drugs that can suppress HBV replication, latent HBV so far has proven difficult to eradicate. Homing endonucleases, enzymes that recognize long DNA sequences and induce DNA double strand breaks, could be a means by which latent HBV is targeted and inactivated. In particular, we aim to modify the recognition site of the wild type I-GzeII homing endonuclease (HE) into a HBV specific DNA targeting enzyme that will recognize specific sequences in the HBV genome and introduce double-strand DNA breaks. By exploiting the error prone nature of non-homologous end joining, the process cells undergo to repair double-strand breaks, these modified HEs will have a mutagenic effect on target sequences in the HBV genome. Repeated HE activity will eventually cripple the replicative ability of the latent virus. To selectively modify the structure of homing endonucleases, the amino acid residues that interact with the DNA substrate are identified and randomized. A library of variant forms of I-GzeII is generated. Variants that encoded for active forms with the appropriate HBV specific recognition site are selected for by in vitro compartmentalization. After several rounds of selection, bacterial selection assays and in vitro cleavage assays using a reporter cell line will be utilized to select for structurally stable variants that functions in vitro. The final step is to test the HE product on an in vitro HBV cell line. Through our research, we hope to produce a proof of concept that latent viruses can be targeted and inactivated through mutagenesis by homing endonucleases and that specificity and target of the enzyme can be modified and assessed through this protocol.

Tumor necrosis factor (TNF) is a key host factor in tuberculosis (TB). In the zebrafish model for TB, excess TNF triggers necrosis of infected macrophages which leads to exuberant extracellular bacterial growth and host susceptibility. In infected macrophages, excess TNF induces production of reactive oxygen species (ROS) which activate two death pathways one of which involves the mitochondrial transition pore, and the other leads to ceramide production and lysosomal permeabilization. In exploring the events downstream of ceramide production we found that the pro-apoptotic factor Bax is required for macrophage necrosis in excess TNF conditions. Bax has been well-characterized for its role during apoptosis, but this result suggested that Bax might also play a role in necrosis. To determine if this was the case, we engineered Bax mutants that lack its different functional domains. We studied the ability of each mutant protein to regulate apoptosis and necrosis in the zebrafish and found the BH3 domain, which is required for Bax oligomerization during apoptosis, not to be required for necrosis under excess TNF conditions. Rather, an C-terminal transmembrane helix was found to be required for necrosis. These findings demonstrate that the pro-apoptotic factor Bax also regulates necrosis but through a distinct functional domain. Since excess TNF-mediated necrosis has been implicated in human TB pathogenesis, this understanding of how Bax regulates necrosis may guide host-derived therapies.

Thirty-five million people worldwide currently live with HIV. Despite the availability of highly-active antiretroviral therapy (HAART) which may prolong the lifespan of HIV patients, HAART does not target the latent viral reservoir in infected cells, enabling viral rebound in the absence of treatment. Introducing genetically-engineered DNA cleavage enzymes (endonucleases) into HIV-infected cells may disrupt the integrated provirus. The HIV-directed site-specific DNA cutting activity of these endonucleases operating in conjunction with cellular error-prone DNA repair mechanisms can introduce targeted mutations in the integrated HIV genome that prevent viral reactivation. We are investigating three classes of engineered endonucleases: homing endonucleases (HEs), zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENS). While the DNA binding domains of these endonucleases allow for customization to a desired target sequence, delivering these enzymes to HIV-infected cells poses a challenge. TALENS in particular require a DNA coding sequence beyond the capacity of many viral vectors. Thus, a method of co-transduction must be pursued, whereby two halves of the endonuclease coding sequence are delivered by separate viral vectors and coordinately expressed in the target cell. My project aims to determine the optimal capsid serotype, promoter, and multiplicity of infection (MOI) for co-transducing HIV-infected cells using adeno-associated virus (AAV) vectors. I first determined AAV-1 to be the optimal serotype of AAV for infecting SupT1 cells, a cell line used as a model for HIV-infected T-cells. I then constructed two sets of AAV-1 viral vectors to express either Green Fluorescent Protein (GFP) or a red fluorescent protein (mCherry) as surrogates for endonuclease genes and infected SupT1 cells with both sets of vectors at variable MOIs, using flow cytometry to determine the rate of co-transduction. Through this method, I have successfully co-transduced cells with two AAV vectors. These results will have applications in future HIV research and clinical therapy.

The high antigenic diversity HIV-1 presents an enormous challenge for vaccine design as effective vaccines must be broad and potent in order to recognize and effectively counteract diverse circulating HIV strains. The identification of broadly neutralizing antibodies to recognize and counteract these diverse strains provides optimism for the ability to elicit broad and potent responses against HIV via vaccination. PGT128 is one such antibody and has been shown, in combination with other broadly neutralizing antibodies, to improve neutralization coverage of both viruses transmitted heterosexually and vertically, i.e. from mother to infant. Thus it is crucial that we understand the molecular determinants of the virus that leads to its sensitivity to broadly neutralizing antibodies. We studied viruses from longitudinal time points, one at 21 days post infection and one from 560 days post infection, of one infected individual who displayed increasing sensitivity to PGT128. Using nested PCR to create chimeras between the two viruses, we found that the loss of a single potential N-linked glycosylation site in the V1 region of HIV envelope conferred sensitivity to PGT128. This emphasizes the importance of N-linked glycosylation sites in conferring PGT128 recognition.

With ~34 million people infected with human immunodeficiency virus (HIV), researchers are actively trying to decipher the evasive nature of the virus--primarily its high mutation rate that allows it to escape immune recognition. We are thus analyzing evolutionary distances between viral DNA sequences and applying them to other aspects of HIV pathogenesis such as the highly variable course of infection. There is typically an initial short period of flu-like symptoms as the virus begins to destroy CD4+ T-cells, which are crucial to the immune system, then seroconversion, when the body produces cytotoxic T-lymphocytes that suppress and stabilize viral levels, and virus-specific antibodies. An asymptomatic period that varies greatly in length then follows. However, viral levels eventually increase again and T-cell levels drop and/or the immune system can no longer control opportunistic infections, the patient is diagnosed with Acquired Immunodeficiency Syndrome (AIDS). However, the majority of research focuses on HIV-infected men, despite intriguing evidence suggesting that there are gender-specific differences in the progression to AIDS. In this comprehensive longitudinal study of HIV-1 in eight different women, we are investigating the patterns of viral genetic diversity and divergence, from the time of seroconversion to the development of AIDS or until disease progression was halted by antiretroviral drugs. We amplified the conserved group specific antigen (gag) gene and the variable envelope gene (env) of various time points of each infected woman by performing Multiplex Polymerase Chain Reactions. Sequencher and Geneious programs were used to determine and align the DNA sequences after they were sent out for Sanger sequencing. We will investigate the patterns of diversity and divergence over the time course of an infection in each patient using phylogenetic trees of their DNA sequences. Results from this work will be presented, as well as a comparison to similar measures from men.

Kaposi’s sarcoma-associated herpesvirus (KSHV), is the infectious agent that causes Kaposi’s sarcoma (KS)—a tumor that is the most prevalent cancer found in untreated HIV-infected individuals worldwide. KSHV establishes predominantly latent infection both in cells in vitro, as well as in KS tumors. KSHV, like all viruses, lacks an independent metabolism, and it has been shown to dramatically alter host cellular metabolism, particularly in carbon utilization pathways. Similar to cancer cells, latent KSHV-infected cells require increases in the most abundant carbon sources, glucose and glutamine for survival. However, it is still unknown how glutamine is utilized during latent KSHV infection. Glutathione (GSH) is the most abundant antioxidant in eukaryotic cells. GSH is essential for cell survival because it functions to stabilize reactive oxygen species (ROS), which are deleterious to the cell. I hypothesized that latent KSHV-infected cells require glutamine to produce GSH and to stabilize ROS in infected cells. KSHV-infected cells have been shown to be under chronic oxidative stress, and increased levels of GSH could explain how latent KSHV-infected cells inactivate excess ROS to avoid cell death. To test my hypothesis, I will determine the abundance of GSH in uninfected samples compared to that of KSHV-infected samples. I will use a fluorescent GSH detection reagent and measure GSH absorbance through a spectrophotometer or flow cytometry. Secondly, to determine if glutamine is the necessary precursor for GSH production, I will compare GSH levels between KSHV-infected samples in the presence of all carbon sources and KSHV-infected samples starved of glutamine. If glutamine is the required carbon source, I expect to see decreased GSH levels in KSHV-infected cells in the absence of glutamine. This finding could reveal important drug targets for future strategies to inhibit and treat latent-KSHV infection and ultimately KS tumors.

Due to rapid mutation of the Hemagglutinin (HA) antigen, the current influenza vaccine only induces strain specific protection, requiring people to get vaccinated yearly. Hemagglutinin is the main glycoprotein on the surface of the influenza virus and consists of two main components, the variable head and the conserved stem. Previous studies have shown that vaccination with the conserved stem region of the HA antigen induces the production of broadly neutralizing antibodies, which are able to neutralize a broad spectrum of influenza sub-types. Therefore we designed a vaccine regimen in order to increase the effectiveness of our current DNA vaccination delivered by a gene gun to induce antibodies against the stem. We hypothesized that priming mice with the conserved stem region of the HA antigen followed by a boost with the full-length HA would lead to an increase in antibodies against the stem and the production of broadly neutralizing antibodies. Groups of mice were primed with either the conserved stem or the full-length HA and were boosted 6 weeks later with either full-length HA or the conserved stem. Two weeks after the boost, the mice were challenged with a lethal dose of a homologous or heterologous strain of influenza. Weight loss was monitored and blood was collected throughout the experiment. Neutralization and binding antibody assays were performed in order to compare the relative ability of each immunization regimen to induce antibodies against the HA head vs. stem.

Glucose and glutamine are primary carbon sources for cells to produce biomolecules and energy. Increased aerobic glycolysis, de novo fatty acid synthesis (FAS), and glutaminolysis are hallmarks of cancer cell metabolism and many tumors are dependent upon these for their survival. Likewise, virally infected cells also have considerably altered metabolic requirements. Our lab studies Kaposi’s Sarcoma Herpes Virus (KSHV), the etiologic agent of Kaposi’s Sarcoma (KS). KS is the most common tumor found in AIDS patients. Previous work from our lab showed that latent KSHV infected endothelial cells also require increased aerobic glycolysis and FAS. Our lab also published a global metabolic analysis of latent KSHV infection, revealing that latent KSHV infection also induces increased glutamine levels. Exogenous glutamine is metabolized through Glutaminolysis; glutamine is converted to glutamate and alpha-ketoglurarate (αKG) through two key enzymes, glutaminase (GLS) and glutamate dehydrogenase (GDH). Further, αKG can enter the tricarboxylic acid cycle (TCA) and support ATP production. Cancer cells utilize glutamine to replenish the TCA cycle when glucose is primarily used to produce lactate and fatty acids. Preliminary data shows that exogenous glutamine is required for latent KSHV infection through starving infected endothelial cells of glutamine. While glutamine starvation does not reduce viability of mock-infected endothelial cells, the absence of glutamine leads to significant cell death in KSHV infected cells. I hypothesize that glutamine is required during latent KSHV infection to replenish the TCA cycle. To study this, I inhibited glutaminolysis using a compound called BPTES, an inhibitor of GLS. Inhibiting glutaminolysis also leads to significant cell death of KSHV infected cells. This data indicates that glutamine is required during latent KSHV infection to replenish the TCA cycle. In the future I will add back TCA cycle intermediates to rescue cell death. Through our metabolomics studies of latent KSHV infection, we are identifying potential targets for KS therapy.

 Waste water and run-off from hospitals, sewage treatment plants, and homes discharge human byproducts to aquatic systems all over the world. These anthropogenic byproducts range from toxic compounds and personal care product chemicals to biological and genetic waste. When examining the microbial component of these byproducts, biomarkers such as antibiotic resistance genes can be identified by genetic techniques. Extended spectrum beta-lactam (ESBL) antibiotics are valuable in treating bacterial infections, and increased multi-ESBL resistance by carbapenemase-producing Enterobacteriaceae has been detected in waste water. The Klebsiella pneumoniae carbapenemase (KPC) enzyme was identified approximately 20 years ago and the resistance gene has spread to 37 states in the US. Discovered in 2008, NDM-1 is a member of metallo-beta-lactamase family revealed in K. pneumoniae and Escherichia coli, and found world-wide. Moreover, ESBL resistance genes, such as KPC and NDM-1, have been shown to move to environmental bacteria, most likely by horizontal transfer. Given the recent discovery of NDM-1 and national spread of KPC, we wished to assess for the presence of KPC and NDM-1 in nearshore pelagic waters of Puget Sound. DNA for examination was isolated from filtered seawater collected in six of the seven oceanographic basins of Puget Sound, including areas near waste water treatment plant outfalls. Analysis via quantitative PCR for KPC and NDM-1 were performed using positive controls of meropenem- and cefotaxime-resistant strains of K. pneumoniae bearing KPC or NDM-1 as assay standards. Results for these resistance markers were compared to companion analyses for human-origin and bovine-origin fecal markers, water quality parameters, and potential sources. These results can provide insight into additional stressors introduced into Puget Sound by human activities.

Pathways by which bacteria interact likely have a large effect on the bacterial communities that are present in the lungs of cystic fibrosis (CF) patients. CF is a genetic disorder that leads to an inability to clear out viscous secretions in the lungs, allowing bacteria to thrive. Pseudomonas aeruginosa (P. aeruginosa) is one of the most detrimental opportunistic pathogens infecting CF patients. The genome of P. aeruginosa encodes the type VI secretion system (T6SS), a toxin delivery system widely found in Gram-negative bacteria. P. aeruginosa outcompetes other organisms using the T6SS. Interestingly, P. aeruginosa targets more efficiently when the other organism has an active T6SS as well, suggesting that P. aeruginosa has a mechanism for sensing the activity of the T6SS in other organisms. The goal of this project is to find if this phenomenon is unique only to P. aeruginosa and whether it is dependent on the organism it is targeting. Another laboratory found that the threonine phosphorylation pathway (TPP), which is known to regulate the T6SS activity in P. aeruginosa, is required for T6SS-sensing. The TPP is only found in a subset of organisms with a T6SS, and I am characterizing which of these organisms exhibit this neighbor sensing behaviour. I hypothesize that this phenomenon is found in the group of organisms that have a TPP. Burkholderia cenocepacia, another bacterial species with a T6SS, is lacking the TPP, and I have found that it does not have T6SS-sensing ability. This suggests that not all T6SS positive organisms exhibit the sensing phenomenon. Additionally, I am investigating whether P. aeruginosa T6SS-sensing occurs with all recipient species. Since the T6SS is conserved among many Gram-negative bacteria, it is important to determine how widespread T6SS-sensing is, ultimately allowing us to better understand targeting among microbes in CF and other polymicrobial infections.

 The Gram negative, opportunistically pathogenic Gammaproteobacterium Pseudomonas aeruginosa has a propensity to form biofilms. Biofilms are surface associated or aggregated bacterial communities, embedded in a self-derived, extracellular matrix (EPS). One major component of the P. aeruginosa biofilm EPS is the polysaccharide Psl. Psl plays a structural and protective role in P. aeruginosa biofilms by mediating attachment to abiotic surfaces and mammalian cells, conferring protection from components of the host immune response and a variety of antimicrobials, as well as promoting cell-cell adhesion. More recently, Psl has also been shown to function as a cell-cell signaling molecule that activates two diguanylate cyclases through an unknown mechanism, which results in an increase in intracellular levels of c-di-GMP. The secondary messenger c-di-GMP is central to the P. aeruginosa virulence regulatory network, and promotes biofilm formation, in part, by inducing the production of EPS components, including Psl. Thus, presence of environmental Psl appears to act as a feed-forward signaling molecule that enhances biofilm formation. In order to further understand this function, this investigation will employ mini-Tn5 mutagenesis to define the signal transduction pathway(s) responsible for activating the diguanylate cyclases through a novel feed-forward mechanism of Psl-dependent signaling in P. aeruginosa.

Simian immunodeficiency viruses (SIVs) naturally infect over 40 species of African primates. From these SIV strains, only two have crossed to humans giving rise to HIV-1 and HIV-2 which have now infected over 40 million people worldwide leading to several million AIDS-related deaths per year. The goal of this project is to understand what determines the ability of the SIVs to cross between hosts and to evaluate the risk of future SIV cross-species transmissions to humans. To efficiently transmit between species, an SIV needs to be able to overcome the multiple immunological barriers imposed by a new host or rapidly adapt to gain this ability. Several of these barriers are encoded by the antiviral proteins that limit virus replication and thus constitute an innate immunological response against lentiviral infection. In this study, we studied the evolution and adaptation of SIVs during cross-species transmission by identifying the amino acid mutations that the viral protein, Vif, needs to undergo to antagonize the human antiviral protein APOBEC3G (A3G). Since, A3G has been described as one of the major barriers for cross-species transmission its degradation by the virus might facilitate SIV transmission to humans. In order to identify the adaptive mutations required to gain anti-human A3G activity, a virus encoding for an HIV-1 backbone and a SIV Vif protein was serially passaged in a human CD4+T cell line expressing human A3G. Preliminary data suggests that in short-term infection the virus is not able to overcome human A3G. We will continue to passage the virus to determine whether or not viral replication is rescued which would indicate that Vif acquired resistance to A3G. The acquired resistance then will be mapped to specific amino acid mutations through sequencing. To test the anti-human A3G activity of these mutations, molecular clones of the virus containing the mutations will be generated using site-directed mutagenesis and tested in specific A3G degradation assays.

 Bacteria are encased by a cell wall made of peptidoglycan (PG). This macromolecular structure is essential for viability and defines the specific shape of each bacterium. Helicobacter pylori (H. pylori) is a helical-shaped, bacterial pathogen estimated to infect half of the worldwide population. It has long been postulated that H. pylori’s unique cell shape plays an important role in its pathogenesis; however, a longstanding question in the field has been how this helical cell shape is generated. My project focuses on studying essential genes involved in PG synthesis. The goal of my project is to determine whether the PG synthesis machinery overlaps with that which is responsible for cell shape. H. pylori displays efficient natural competence, meaning that it is able to take up extracellular DNA from its environment and recombine it into the genome (a process called transformation). I will introduce mutated copies of the essential gene by transformation at a second chromosomal locus making it possible to knockout the native copy if the mutated copy retains at least partial function. To date I have developed a plasmid vector that can direct integration at a unique position on the chromosome and shown that expression of cell wall synthesis genes of interest from this locus alone confers normal cell growth and cell shape. I have been working on increasing the transformation efficiency of my plasmid vector into H. pylori to generate a large number of mutants. While most of these mutants will have the helical phenotype associated with wild-type H. pylori, I expect a portion of these mutants to have interesting shape phenotypes. These studies will begin to address how PG synthases contribute helical cell morphology and provide a platform to study other essential processes in H. pylori.

HIV-1 positive patients on antiretroviral therapy (ART) who agreed to donate their bodies to science are the sampling population for this study. All patients had low or undetectable virus levels in the blood at or near time of death. DNA was extracted from each of the patient tissues and then using nested PCR, envelope and polymerase genes from single viral templates were amplified and the sequences determined. We used this data to determine whether any tissues harbored virus reservoir- or compartment-like qualities. This is the study of patient HV00103 who had been treated ART for the last 4.5 years of his life and had a viral load of less than 50 cps/ml, indicating successful suppression of virus. Despite this control, he died of multiple organ failure. We found that large intestine seemed to exhibit reservoir like properties but more samples would need to be examined before any conclusive claims can be made. We also found that 80% of his HIV-infected cells were derived from the proliferation of a single cell, as evidenced by the identical viral sequences found throughout his tissues. This represents the most extreme example to date that infected cell proliferation is an important contributor to the persisting population of infected cells under ART.

The type VI secretion system (T6SS) is a molecular export pathway that is widely conserved in Gram-negative bacteria. It mediates interbacterial competition by translocating antibacterial effector proteins from the donor bacterial cell to a neighboring bacterial cell in a contact-dependent manner. The T6 amidase effectors (Taes) are a superfamily of lytic enzymes that degrade the bacterial cell wall by cleaving amide bonds in the peptidoglycan (PG) layer. Taes also have cognate T6 amidase immunity proteins (Tais), which prevent intraspecies intoxication by binding and inhibiting Taes in the periplasm. We hypothesize that this pathway can be exploited for an antibacterial strategy. Specifically, we aim to engineer a P. aeruginosa strain that harbors a novel Tae/Tai pair that can escape recognition by native Tai proteins and predict that this strain will be able to outcompete its wild-type counterpart through the T6 pathway. Towards this end, our group previously generated a mutant library of Tae from P. aeruginosa and screened for variants that escape inhibition by Tai (Tsi1) in E. coli. I am currently experimentally validating several potential escape variants (Tae*) identified by this screen by testing Tae* toxicity in the presence of Tai1 in an in vitro bacterial lysis assay. I will also measure potential changes in in vitro Tai1-binding affinity by isothermal titration calorimetry (ITC).

Tuberculosis (TB) is a bacterial infection that leads to 3 million deaths per year worldwide. Proper control of TB requires the ability of patrolling macrophages to detect, engulf and destroy invading bacteria and it is currently understood that macrophages play a key role in responding to early Mycobacterial infection. A forward genetic screen using the zebrafish larval model for host determinants of immunity to the bacterium Mycobacterium marinum found a hypersusceptible mutant that contains a nonsense mutation in the mTor (mechanistic target of rapamycin) gene. mTor has been shown in other systems to be crucial to protein synthesis, cell growth, and survival. Currently, our work has shown that mTor mutant fish have fewer macrophages and neutrophils than their wild type siblings. We have also shown that mTor mutants display hypersusceptibility to M. marinum infection, with bacteria displaying exuberant extracellular growth in the fish.. Treatment of fish with Rapamycin, an mTor inhibitor, phenocopies the macrophage deficiency and hypersusceptibility observed in the mutant. We hypothesized that hypersusceptibility to M. marinum in mTor mutant zebrafish is primarily due to the deficiency of macrophage immune cells. To test this hypothesis, we depleted macrophages in wild-type and mutant fish using lipoclodronate. Preliminary data has shown that without macrophages, mTor mutants still exhibit increased infection burden compared to wild type siblings also treated with lipoclodronate. This data provides us with exciting new avenues to continue searching for the cause of mTor’s hypersusceptibility. We are pursuing mTor’s role in neutrophil and cytokine induction as additional explanations for the mTor mutant hypersusceptibility phenotype. This research will help define the innate immune response to Mycobacterial infection and clarify the respective functions of mTor in host immunity.

Burkholderia thailandensis uses acyl homoserine lactones (AHLs) as quorum sensing (QS) signals. Broadly, QS is a communication system that, at high cell densities, allows bacteria to coordinate group activities. Recent work from our lab has shown that in B. thailandensis, QS controls contact dependent growth inhibition (CDI). CDI is an intraspecies bacterial competition system  where CDI-proficient bacteria (CDI+) contain a three-protein system of CdiA, CdiB and CdiI. CdiA is a toxic protein displayed by CdiB, and CdiI neutralizes the toxic activity of CdiA. We predict that QS-control of CDI is a means of eliminating QS mutants from the population. B. thailandensis has three QS-systems; QS-1, QS-2 and QS-3, which each comprise of a specific transcription factor and a cognate AHL signal synthase that produces a specific AHL. A B. thailandensis mutant in all three synthase genes (btaI1-3) is defective at growth, inhibiting a cdiAIB mutant. However, we do not know the specific contribution of each AHL signal synthase. Thus, we sought to test the CDI activity of synthase mutants against cdiAIB mutants within a competition. We compared the starting and ending ratios of the different strains tested in each competition. Preliminary results have demonstrated that the btaI1-3 mutant is CDI defective, relative to wild type. However, signal synthase mutants are not defective at CDI. QS is required for WT levels of CDI. Additionally, we are investigating the dynamics of how CDI controls the growth advantage of QS mutants. A btaR1 mutant has a growth advantage when co-cultured with wild type in liquid but not during growth on a solid surface, which allows for CDI to occur. Thus, we are evaluating if CDI is responsible for restraining the growth advantage of the btaR1 mutant during growth on a solid surface.

Alberta’s Athabasca oil sands are one of the world’s largest petroleum reserves. Oil sands comprise sand, clay, and bitumen – a viscous form of petroleum which is more difficult to extract than conventional crude. Current bitumen extraction practices such as surface mining and steam-assisted gravity drainage are energy-intensive. This independent project investigated the potential of employing microbial biosurfactants to reduce the energy required for bitumen recovery in oil sands. Biosurfactants are employed in the industrial practice of microbial enhanced oil recovery (MEOR), which is viable in the conventional oil industry, but has not been applied to the Athabasca oil sands. In this study, a biosurfactant-producing bacterial strain was isolated from an environmental sample and its biosurfactant compound was characterized. Biofilm formation under simulated oil sands reservoir conditions was analyzed by scanning electron microscopy, and bitumen extraction was tested in a small-scale model. Potential microbial applications in oil sands extraction, upgrading, and bioremediation were explored in a literature review.