PROJECT SUMMARY Staphylococcus aureus is a leading cause of a wide range of bacterial infections globally and the most common bacterial cause of mortality in the United States. These poor outcomes are driven by the high rates of antibiotic treatment failure (15-40%) seen with many types of S. aureus infections including osteomyelitis, prosthetic joint infections, and endocarditis. Antibiotic tolerance, which is defined as the ability of bacteria to survive in the face of antibiotics through phenotypic changes without the acquisition of antibiotic resistance, is the major driver of antibiotic treatment failure in S. aureus infections. Even with appropriate antibiotic therapy, mortality rates can exceed 20% for some of these infections. Given the high incidence and the frequent treatment failure, new and improved treatment options are needed to combat S. aureus infections. Despite the clinical significance of antibiotic tolerance, the mechanisms by which it occurs are poorly understood. To gain new understanding of treatment failure in S. aureus infections, my fellowship research focused on understanding antibiotic tolerance by conducting comprehensive, unbiased genetic and proteomic screens of S. aureus during antibiotic exposure. With this approach, I identified a previously unknown relationship between antibiotic tolerance and arginine metabolism where antibiotic tolerance in mature S. aureus communities increases when arginine is depleted. In addition to being an essential amino acid for S. aureus growth, arginine is also required for the production of nitric oxide by host immune cells such as neutrophils and macrophages. Nitric oxide production is important for the host response to S. aureus infections, which establishes arginine as an important contributor to the ability of the immune system to combat S. aureus. Collectively, these results support the hypothesis that S. aureus influences arginine levels during infection by carefully regulating arginine metabolism as a means to survive both in the face of antibiotics and the innate immune response. Through this proposal, I plan to test this hypothesis by (1) elucidating the mechanism(s) by which arginine metabolism influences antibiotic tolerance in S. aureus, (2) determining the role of S. aureus arginine metabolism in persistence in the presence of innate immune effector cells, and (3) identifying the contribution of S. aureus arginine metabolism to persistence and antibiotic treatment failure during infection. Together, these experiments will better define the role of an essential amino acid at the host-pathogen interface. The work in this proposal has the potential to uncover new therapeutics targets for the treatment of recalcitrant infections that fail conventional therapies. In addition, through this award, I will receive important mentorship while gaining specific research skills in animal models, eukaryotic cell culture, and imaging mass spectrometry. This NIH K08 Mentored Clinical ...