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Salmonella enterica is a facultative intracellular pathogen capable of eliciting severe, systemic disease necessitating antibiotic intervention. Systemic infection is facilitated by intracellular replication within host immune cells, which is enabled by complex regulatory networks governed by two-component systems (TCSs). Intracellular-active TCSs sense antimicrobial chemical cues in the microenvironment and respond adaptively through transcriptional regulation to support intracellular survival. SsrA/SsrB and PhoQ/PhoP are two essential TCSs that elicit a robust defense against host immunity by regulating clusters of virulence genes and integrating novel targets to support regulon expansion and enhance pathogenicity. Metabolic adaptation is critical to bacterial survival and can initiate host-pathogen interactions that influence infection outcome. Further, mitigation of host immunity by manipulation of arginine metabolism has been documented in intracellular pathogens. Herein, I investigated TCS-mediated regulatory evolution pertaining to arginine metabolism, hypothesizing that adaptations to metabolic regulation might confer a fitness advantage to Salmonella replicating intracellularly. I explored intracellular regulation of de novo biosynthesis and extracellular import of arginine, establishing PhoP-mediated regulation of arginine transport. I determined that arginine transport contributes to bacterial fitness in macrophages and began to investigate the mechanism by which arginine importation enriches for intracellular replication. This work informs on evolutionary mechanisms that serve to enhance virulence in Salmonella and provides further insight into our understanding of the intracellular lifestyle of infection. / Thesis / Master of Science (MSc) / Salmonella enterica is an intestinal pathogen that survives within host immune cells and causes systemic disease. These bacteria replicate within antimicrobial cells by using sensory networks to detect harmful immune factors and respond adaptively by eliciting change in gene expression to defend against immune-based killing. The amino acid arginine is an important component of host immunity, as well as bacterial antimicrobial defenses; therefore, I hypothesized that bacterial metabolism might be adapted to the host immune cell environment in order to mitigate arginine-dependent antimicrobial activity. Here, I establish that arginine metabolism is controlled by intracellular-specific sensory networks, and demonstrate that this regulation is important for bacterial survival. This work provides evidence for the importance of this amino acid in Salmonella infection, which informs on our overall understanding of systemic disease.

Buruli ulcer disease (BUD) remains a ‘mysterious disease’ due to the unknown mode of M. ulcerans transmission and pathogenesis. To understand these, it is important to determine the reservoir of the organism in its natural environments, and stress response and interactions of M. ulcerans in its natural niche and during infection of a host. The major virulence factor of M. ulcerans is mycolactone, a lipid cytotoxin that is encoded on a giant plasmid pMUM001. Genetic analysis suggests that plasmid pMUM001 was acquired by M. ulcerans during evolution from its progenitor, M. marinum. Coincidental evolution of virulence hypothesis suggests that many microbes evolve to acquire traits to outcompete or overcome biotic and abiotic forces during their normal life cycle in the outside-host environment, which can confer virulence during infection of a human host. Hence in this study, we exposed M. ulcerans to selective abiotic forces such as UV, and dynamic oxygen and temperature conditions to determine their effect on M. ulcerans growth, and mycolactone and global gene expression. We also studied the role of mycolactone in determining polymicrobial interaction of M. ulcerans in its natural aquatic habitat by exposing mycolactone coated and uncoated slides in M. ulcerans endemic and non-endemic aquatic locations and determining differences in microbial community composition between them. Further, we studied quorum quenching ability of mycolactone against an opportunistic pathogen, S. aureus. The results obtained showed that exposure of M. ulcerans to abiotic stresses such as higher temperature and lower than optimal oxygen conditions modulate its global and mycolactone gene expression. Further, we also showed that mycolactone can impact overall microbial community structure in a polymicrobial environment in its natural, aquatic habitat. Mycolactone also effected virulence and quorum sensing in an opportunistic pathogen, S. aureus, without inhibiting its growth. These findings are important as they provide insight toward potential reservoirs or environmental niches which may harbor M. ulcerans and inform new potential mechanisms of pathogenesis. Further, our novel research of synergistic or antagonistic interactions within the complex polymicrobial communities colonizing skin and aquatic habitats is a powerful approach in determining M. ulcerans colonization efficiency, resiliency, and transmission mechanisms.

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No description available.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets. An uncharted area of antibiotic discovery can be explored by inhibiting nutrient biosynthesis. Herein, we investigate the potential of inhibiting biotin biosynthesis in monotherapy and combination therapy approaches to treat multidrug-resistant Gram-negative pathogens. In chapter 2, we validate biotin biosynthesis as a viable target for Gram-negative pathogens. Historically, biotin biosynthesis was overlooked as a target in Gram-negative pathogens as there was no observed fitness cost associated with its inhibition in standard mouse infection models. We discovered traditional mouse models do not accurately represent the biotin levels in humans. We developed an innovative mouse model to account for this discrepancy, validating biotin biosynthesis as an antimicrobial target in the presence of human-mimicking levels of biotin. Exploiting this sensitivity, we show that an inhibitor of biotin biosynthesis, MAC13772, is efficacious against Acinetobacter baumannii in a systemic murine infection model. In chapter 3, we continue to investigate the potential of targeting biotin biosynthesis in a combination therapy approach. In this work, we identify the ability of MAC13772 to synergize with colistin exclusively against colistin-resistant pathogens. The first committed step of fatty acid biosynthesis requires biotin as a cofactor; therefore, it is indirectly inhibited through the action of MAC13772. We propose that the inhibition of fatty acid biosynthesis leads to changes in membrane fluidity and phospholipid composition, restoring colistin sensitivity. The combination of a fatty acid biosynthesis inhibitor and colistin proved superior to either treatment alone against mcr-1 expressing Klebsiella pneumoniae and colistin-resistant Escherichia coli murine infection models. Together, these data suggest that biotin biosynthesis is a robust antibiotic target for further development in monotherapy and combination therapy approaches. / Thesis / Doctor of Philosophy (PhD)

Salmonella is a Gram-negative intracellular pathogen that causes gastroenteritis and typhoid fever in humans. Salmonella can survive and replicate within host cells and has adapted several mechanisms to evade host immune defenses. The innate immune system plays an important role as a first-line of defense against pathogens such as Salmonella, and is mediated in part by toll-like receptors (TLRs). TLRs recognize fundamental components of pathogenic microorganisms and activation of TLRs leads to downstream signaling cascades eventually resulting in the expression of pro-inflammatory cytokines (4) and also has a role in activating adaptive immunity through presentation of antigens to lymphocytes (86). There are several lines of evidence that suggest that TLR activation may have therapeutic potential in therapies against infectious disease and several TLR agonists have been shown to protect against both bacterial and viral infection in mice (7; 8; 38; 66; 75; 84; 89; 121). To understand how TLR-agonist treatment of host cells affects Salmonella pathogenesis, RAW 264.7 murine macrophages were treated with the TLR agonists liposaccharide (LPS), poly(I:C), peptidoglycan, and CpG-ODN. Treatment of macrophages with all TLR-agonists results in increased phagocytosis of Salmonella compared to control-treated macrophages. These increases in phagocytic activity, however, do not enhance macrophage anti-microbial activity, since Salmonella infection of TLR-treated macrophages results in increased intracellular replication compared to control-treated cells. Infection with Salmonella mutants indicates that increased intracellular replication of Salmonella in TLR-treated macrophages is dependent on a functional SPI-2 type III secretion system. This also indicates that there was not a generalized defect in macrophage anti-bacterial function. These data exemplify how interactions between macrophage defense mechanisms and bacterial virulence factors can result in evasion of the innate immune response. Studying how TLR-agonist treatment affects Salmonella pathogenesis will give us a better understanding of the host-pathogen relationship and may provide insight into novel strategies to fight intracellular microorganisms. / Thesis / Master of Science (MSc)

No / The inner retinal complex is a well-defined layer in spectral-domain OCT scans of the retina. The central edge of this layer at the fovea provides anatomical landmarks that can be observed in serial OCT scans of developing full-thickness macular holes (FTMH). Measurement of the movement of these points may clarify the mechanism of FTMH formation. This is a retrospective study of primary FTMH that had a sequence of two OCT scans showing progression of the hole. Measurements were made of the dimensions of the hole, including measurements using the central edge of the inner retinal complex (CEIRC) as markers. The inner retinal separation (distance between the CEIRC across the centre of the fovea) and the Height-IRS (average height of CEIRC above the retinal pigment epithelium) were measured. Eighteen cases were identified in 17 patients. The average increase in the base diameter (368 microns) and the average increase in minimum linear dimension (187 microns) were much larger than the average increase in the inner retinal separation (73 microns). The average increase in Height-IRS was 103 microns. The tangential separation of the outer retina to produce the macular hole is much larger than the tangential separation of the inner retinal layers. A model based on the histology of the Muller cells at the fovea is proposed to explain the findings of this study.