Adapting to changes in environmental conditions is an essential process that ensures maximal fitness of an organism is maintained. For bacterial pathogens residing in the host, these changes include alterations in oxygen availability, nutrient abundance, and the presence of antimicrobials. Failure to meet these challenges limits the pathogenic potential of the bacterium, therefore, a better understanding of how bacterial pathogens respond to environmental changes will aid the development of treatment strategies to combat microbial infections. Staphylococcus aureus is an opportunistic pathogen that employs a diverse set of metabolic pathways to adapt to new host environments and stressors, which is driven by a branched aerobic respiratory chain and the ability to transition into a fermentative state of growth known as the small colony variant (SCV). The branched respiratory chain is powered by two terminal oxidases, CydAB and QoxABCD, and loss of either terminal oxidase leads to tissue specific colonization defects. Infections caused by SCVs are often resistant to antibiotic treatment, frequently leading to prolonged infections with worse clinical outcomes for the patient. In line with the goal of identifying novel therapeutic targets, this dissertation explores the pathways supporting the metabolic versatility of S. aureus including synthesis of lipoteichoic acid (LTA) and the isoprenoid biosynthetic pathway. LTA is a cell surface polymer that has been predicted to maintain surface ion homeostasis. Previous reports demonstrated that limiting the metabolic potential of S. aureus cells in which LTA synthesis is disrupted reduces viability. Accordingly, we sought to determine the mechanism driving this phenotype. The membrane embedded anchor on which LTA is synthesized is generated by YpfP, and inactivation of this enzyme results in LTA that is elongated and less abundant. Membrane potential of the ypfP mutant was maintained during aerobic growth, however, induction of SCV growth resulted in a loss of membrane potential and reduced viability. Suppressor mutants in the ypfP mutant background that displayed increased SCV viability were isolated and characterized. The suppressor mutants exhibited LTA that was more similar to the WT and anaerobic membrane potential was partially restored, demonstrating that LTA supports SCV viability via maintenance of ion homeostasis. Further investigation of pathways supporting metabolic versatility identified the isoprenoid biosynthetic pathway as essential for cellular respiration. Isoprenoids are molecules containing 5-carbon repeating units and are synthesized by prenyl diphosphate synthases (PDS). Farnesyl diphosphate (FPP) serves as a substrate for three downstream cellular processes: pigment production, cell envelope maintenance, and synthesis of respiratory cofactors. The only known PDS to synthesize FPP is IspA, however, ispA mutants still produce FPP, suggesting another FPP-producing PDS is present. Investigation of this pathway identified a high level of redundancy in which other PDSs not previously known to produce FPP are able to contribute to FPP dependent pathways. Studying the effects of isoprenoid synthesis disruption on respiration revealed a preference of the CydAB terminal oxidase for chain length of menaquinone, demonstrating the impact of isoprenoid synthesis on metabolic versatility. Lastly, a chemical library screen was performed to identify inhibitors of the fatty acid kinase (FAK) system. The FAK system is used to acquire exogenous fatty acids and limits the efficacy of antimicrobials that target endogenous fatty acid synthesis. Disruption of these pathways limits the production of the fatty acids necessary for cell membrane synthesis and thus inhibits growth of the cell. A putative FAK system inhibitor was identified and demonstrated to work synergistically with triclosan, a known inhibitor of endogenous fatty acid synthesis. This work demonstrates the feasibility of a dual therapy strategy for inhibition of cell membrane synthesis and lays the groundwork for expanding the toolkit available for treatment of S. aureus infections.
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