Although the CDC's 2019 report on Antibiotic Resistance Threats in U.S. shows that methicillin-resistant Staphylococcus aureus (MRSA) cases have declined, nearly 85% of all deaths attributed to antibiotic resistance between 2005 and 2016 were the result of MRSA infections. Bloodstream infections from community-acquired MRSA have declined less sharply than hospital-acquired MRSA during this time (-17% and -6.9%, respectively), whereas cases of community-associated methicillin-susceptible S. aureus (MSSA) have risen nearly 4%. Nearly half of all health-care-associated infections are now attributed to MSSA rather than MRSA. At the same time, MSSA and MRSA are being identified as the cause of infective endocarditis with greater frequency. The changing dynamics of S. aureus infections demonstrate the critical need to better our understanding of its pathogenesis in order to develop new approaches for prevention and treatment. One of the competitive advantages S. aureus has within the infection site is its ability to scavenge host-derived fatty acids to satisfy its own lipid biosynthesis needs. The mechanisms of the fatty acid kinase pathway are well-documented for monounsaturated fatty acids like oleic acid. However, the fatty acid content of the human body varies dramatically between different organs and tissues. Despite their prevalence in the human body, we have overlooked the role of host-derived saturated, straight-chain fatty acids (SCFAs) in investigations of the fatty acid kinase pathway. Our long-term goal is to increase the efficacy of existing antibacterial therapies by purposefully and selectively remodeling the staphylococcal membrane during infection. The overall objective for this application is to define the lipid composition of S. aureus within the complex environment of the mammalian host and identify the impacts of host-derived FAs on the antimicrobial susceptibilities of S. aureus. Our central hypothesis is that the S. aureus membrane mimics the fatty acyl composition of the infection site to a greater extent than previously thought and that the resulting impacts on membrane physiology modify the tolerance of S. aureus to antimicrobials. The rationale for this project is that more effective antimicrobial treatment regimens will be developed by accounting for or exploiting the lipid composition of S. aureus when residing within the mammalian host. In Aim 1, we will determine the prevalence and fate of mammalian-derived saturated and unsaturated fatty acids using advanced liquid chromatography-mass spectrometry-based lipidomics. In Aim 2, we will identify the impact of S. aureus' use of exogenous fatty acids on its membrane physiology and cell morphology. In Aim 3, we will test the hypothesis that host-derived fatty acids can influence antibiotic tolerances of S. aureus in vitro and in vivo. We expect that the results of our research will prompt greater appreciation and consideration of host-derived fatty acids as modulators of antibi...