Abstract
During infection of its host, Salmonella encounters diverse and rapidly changing conditions, including an arsenal of innate host defenses that are insurmountable by other pathogens. Salmonella responds to the stresses with a battery of its own defense mechanisms that have evolved for successful pathogenesis in mammalian hosts. Among the host defenses encountered by Salmonella are reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are of interest due to their potent cytotoxicity and their function as signaling mechanisms that enhance Salmonella virulence, especially within phagocytic immune cells. Salmonella mitigates the cytotoxic effects of the reactive molecules using well-characterized defenses and repair mechanisms, many of which rely on an appropriate redox balance within the cell. This is achieved through enzymatic reactions within the oxidative branch of the pentose phosphate pathway (oxPPP) that reduce NADP+ to NADPH. However, the role of this central metabolic pathway in Salmonella pathogenesis has yet to be investigated outside the context of redox homeostasis. Downstream of the oxPPP, the non-oxidative branch (non-oxPPP) serves as a metabolic hub that connects many metabolic pathways and generates intermediate metabolites that are precursors for diverse biosynthetic reactions including those for nucleotides, aromatic amino acids, and lipopolysaccharide. Here I show that Salmonella possesses three transketolase isoenzymes that function within the non-oxPPP to maintain carbon flux and that the isoenzymes are necessary for resisting host-derived ROS and RNS independently of maintaining the cellular redox state in a murine model of salmonellosis. I found that all three transketolase isoforms catalyze the canonical transketolase reactions with high efficiency, although each enzyme has unique kinetic properties that may be beneficial during the diverse conditions encountered by Salmonella. Further, I demonstrated that a library of transketolase-knockout Salmonella strains all have distinct susceptibilities to chemically-derived ROS and RNS, highlighting both the unique contributions of transketolase isoenzymes to pathogenesis, as well as their compensatory functions. To further elucidate the functions and contributions of individual transketolases, I describe the development and validation of a molecular-based technique for accurately quantifying bacterial strains. Individual strains were engineered with unique DNA barcodes that can be quantified in a background of highly similar strains using Droplet Digital PCR. This new technique facilitates direct, quantitative comparisons of fitness to be made between strains and is highly adaptable to various experimental designs. Ultimately, the work summarized in this dissertation highlights the importance of central metabolism in bacterial pathogenesis and demonstrates the utility of a novel approach to assess bacterial fitness that will facilitate future bacterial pathogenesis studies not only for Salmonella, but also for diverse microorganisms.