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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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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)

Usutu virus (USUV) is an emerging zoonotic virus within the Flaviviridae family that can cause neurological disease in humans and wild birds. USUV is maintained in an enzootic cycle between wild birds, primarily passerine species, and ornithophilic mosquitoes, predominantly Culex spp. mosquitoes. Since its first isolation in 1959 in South Africa, USUV has spread throughout sub-Saharan Africa and Europe. Its emergence into Europe was marked by large die-offs, or epizootics, of the Eurasian blackbird (Turdus merula), as well as an increase in human cases. This dissertation sought to understand whether USUV has evolved to become more pathogenic in humans or transmissible in birds. We compared the pathogenesis of five different USUV isolates, four recent isolates: Spain 2009, Netherlands 2016, Senegal 2003, Uganda 2012, and South Africa 1959, in an interferon α/β receptor knockout (Ifnar-/-) mouse model. We observed significant mortality, high viral levels in serum and tissues in all USUV strains except for the Netherlands 2016 strain. Eighteen non-synonymous mutations were identified throughout the genome of Netherlands 2016 strain compared to the other USUV isolates. To further understand USUV infection in wild birds, we developed a physiologically relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more pathogenesis and higher viral titers in tissues compared to the African strains. This work established the first viremic bird model of USUV infection. Passerine birds have been suggested to be important for USUV maintenance, however a species competent for transmission has not been identified. We first determined that wild-caught house sparrows (Passer domesticus) and Culex quinquefasciatus mosquitoes were susceptible to Netherlands 2016 and Uganda 2012 USUV strains. Following an infectious feed to assess enzootic transmission, house sparrows were able to transmit both USUV strains to Cx. quinquefasciatus mosquitoes, with the Netherlands 2016 strain being more infectious compared to the Uganda 2012 strain. The collection of these chapters provides great insights on the pathogenesis of distinct USUV strains, disease presentation in birds, and enzootic transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is entirely feasible. / Ph.D. / Usutu virus (USUV) is a mosquito-borne virus that can cause neurological disease in humans and wild birds. It is in the same virus family as West Nile virus and St. Louis encephalitis virus, both of which already occur in the United States. USUV is typically maintained by spreading back and forth between wild birds, typically songbird species, and mosquitoes with an affinity for biting birds, such as Culex spp. mosquitoes. USUV was first found in South Africa in 1959 and has since spread throughout sub-Saharan Africa and Europe. Upon emergence in Europe, USUV caused massive die-offs in Eurasian blackbirds (Turdus merula) as well as an increase in human cases. My work sought to understand whether USUV has evolved to cause more disease in humans or transmissibility in wild birds. We first assessed the disease severity of African and European USUV strains in an immune compromised mouse model. We showed that African strains of USUV caused more disease and developed higher viral loads than European strains in mice that lack full immunity. To further understand USUV infection in wild birds, we developed a relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more disease and higher viral loads in tissues compared to the African strains. This work established the first bird model of USUV infection where birds developed infectious virus that is detectable in blood. Finally, to identify possible reservoir hosts of USUV, we assessed USUV transmission between house sparrows (Passer domesticus) and Cx. quinquefasciatus mosquitoes. We found that house sparrows were able to transmit a European and African USUV strain to biting Cx. quinquefasciatus mosquitoes, with the European strain being more infectious to these mosquitoes. This indicates that house sparrows are competent hosts for USUV transmission and thus a potential reservoir species. The collection of these chapters provides great insights on the severity of distinct USUV strains, disease presentation in birds, and transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is a very real possibility.