The age of antimicrobial resistance has driven a sharp demand for new routes that inhibit opportunistic pathogens such as Staphylococcus aureus. In response to this has been a steady rise in research characterizing pathways essential to pathogen proliferation, such as those relating to essential nutrient acquisition and metabolism. S. aureus employs a flexible metabolism that promotes colonization throughout most mammalian tissues. This characteristic, combined with an ability to develop antibiotic resistance, incites nosocomial infections that range from skin and soft tissue infection to osteomyelitis. Regarding essential nutrients, the foundation for S. aureus metabolism for the critical element sulfur is still being molded. The current thesis provides a series of investigations that expand our understanding of how S. aureus engages sulfur metabolism to meet nutritional demands.Expression of the machinery involved with sulfur acquisition and catabolism is under control of the transcriptional repressor, CymR. We investigate the transcriptional response of S. aureus to varying states of sulfur supplementation in vitro with a special focus on the sulfur regulon. We note that during sulfur starvation there is both a CymR- dependent and -independent response that occurs; notably, the majority of differentially expressed genes during sulfur depletion are influenced by the presence of CymR. Found within the sulfur starvation response is the upregulation of iron metabolism and oxidative stress response genes. Glutathione was used to exemplify this observed connection between sulfur, iron, and oxidative stress—the S. aureus sulfur source also protects against toxic levels of heme (iron source) and the oxidative stressor, H2O2. We further show that S. aureus expresses unique responses when exposed to organic (cysteine, reduced and oxidized glutathione) and inorganic (thiosulfate) sulfur sources. With this series of transcriptomics, the thiosulfate uptake protein A (TsuA) is shown as the sole importer associated with growth on thiosulfate as a sulfur source. Interestingly, each sulfur source induced upregulation of sulfur-associated transporters, suggesting that regulatory actions within the sulfur regulon are more complex than previously appreciated.Upon examination of the sulfur regulon, we next focused on the redundancy surrounding transportation mechanisms of S. aureus sulfur sources. Glutathione is one example, having at least two associated transporters in this pathogen. We identify that the di-tripeptide transporter, DtpT, supports proliferation of S. aureus on nutritional glutathione. This line of inquiry also describes cysteinyl-glycine, the glutathione breakdown product, as a DtpT-mediated sulfur source for S. aureus. DtpT contributions to S. aureus physiology is likewise demonstrated through its impact on colonization of the murine liver.We also initiated a new field of study that characterizes the distribution of host sulfur sources using matrix-assisted laser desorption/ionization imaging mass spectrometry. With this technique we confirm the presence of several known sulfur sources during S. aureus infection of the murine kidney, including reduced and oxidize glutathione as well as cysteinyl-glycine. An additional metabolite, cysteine-glutathione mixed disulfide, was found to be highly abundant in the S. aureus-infected kidney. We establish that this compound is a sulfur source for this organism and that growth on cysteine-glutathione disulfide is associated with three transporters: the glutathione import system (GisABCD) as well as the cysteine/cystine transporters, TcyABC and TcyP. This work is concluded with thoughts surrounding future research which, alongside the data presented here, will further enhance our understanding of the nutritional sulfur interface between the host and pathogen during S. aureus infection.
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