SUMMARY Staphylococcus aureus is a Gram-positive pathogen that can infect every niche of the vertebrate host. S. aureus infections range from minor skin infections to fatal bacteremia. In the US alone, there are nearly 900,000 cases of severe S. aureus skin and soft tissue infections diagnosed each year. The prevalence of this pathogen and the frequent use of antibiotics selected for antibiotic resistant strains, including methicillin resistant S. aureus (MRSA) with resistance to the -lactam class of antibiotics. Once primarily a nosocomial infection, MRSA is now often acquired in community settings, causing about 10% of severe S. aureus infections. These strains also exhibit increased virulence, presenting the desperate need to develop new therapies. Cystic fibrosis (CF) is a genetic disease resulting in thicker than normal lung mucus, predisposing patients to pneumonia, especially from S. aureus, which is present in 70% of CF patients. The immune system of healthy patients kills pathogens through mechanisms involving the release of reactive oxidants like HOCl that oxidize and inactivate proteins necessary for bacterial survival. HOCl kills cells through non-specific chemistry so both host and pathogen are damaged. CF patients have an increased number of activated neutrophils in their lungs from a young age, and the HOCl generated by those neutrophils contributes to deceased pulmonary function and shortened life expectancy. This proposal will define the role of HOCl-induced oxidation of methionine in S. aureus physiology and pathogenesis. The S. aureus methionine sulfoxide reductases (Msr) revert methionine sulfoxide to methionine, repairing oxidatively damaged proteins. This process prevents the organism from needing to synthesize oxidized proteins de novo, a significant energy savings for the cell. Staphylococci are unique among bacterial species in having four Msr enzymes, and the role of these enzymes in pathogenesis is not understood. The Beavers lab recently demonstrated that Msr enzymes protect S. aureus from killing by neutrophils as well as HOCl, the primary oxidant produced by neutrophils, but not other oxidants present at the host-pathogen interface. The CF lung is a particularly relevant setting to explore the role of Msr enzymes in S. aureus pathogenesis because S. aureus persists in the CF lung despite high concentrations of neutrophils and a harsh oxidizing environment. This proposal will test the hypothesis that Msr enzymes protect S. aureus during CF pneumonia by defining the mechanisms by which S. aureus uses Msr enzymes to avoid killing by the host immune system and determining the role of each methionine sulfoxide reductase during S. aureus pneumonia in the CF lung. This will provide a foundation for future research focused on understanding bacterial physiology and responses to host-pathogen oxidants. Additionally, the identified and validated pathways represent therapeutic weaknesses in S. aureus and thus targets for the de...