Project Summary Staphylococcus aureus infects every niche of the human host and is the leading cause of Gram-positive sepsis. There are over 900,000 severe S. aureus infections in the United States annually, emphasizing the need for new antibiotics to treat these infections. Understanding the molecular mechanisms used by the host to kill S. aureus and by S. aureus to defend against host killing will identify and validate novel targets for antimicrobial design. When the host encounters S. aureus, immune cells excrete various bactericidal small molecules. One class of bactericidal small molecules are polyunsaturated fatty acids, which are toxic to many bacterial species, but until recently the mechanism of toxicity was undefined. We discovered that arachidonic acid (AA), an abundant host polyunsaturated fatty acid, is bactericidal against S. aureus through a lipid peroxidation mechanism. AA is oxidized in S. aureus to a,b-unsaturated carbonyls and g-ketoaldehydes that are electrophilic, reacting with nucleophilic amino acids of the S. aureus proteome. Scavenging either oxidants that initiate lipid peroxidation or electrophiles generated through lipid peroxidation protects S. aureus from AA killing, confirming lipid peroxidation generated electrophiles as the bactericidal effectors of AA. Discovering the mechanism of AA toxicity against S. aureus is just the first step in identifying and validating lipid peroxidation as an antimicrobial strategy. We do not know the lipid electrophile species generated in S. aureus and what S. aureus proteins are targeted by electrophiles. We also do not know the S. aureus processes that produce the oxidants responsible for initiating lipid peroxidation or the identity of the oxidants produced in S. aureus. Finally, we do not know the specific host niches where lipid peroxidation is bactericidal or which host niches are most promising to test lipid peroxidation as an antimicrobial therapy. This proposal will expand the understanding of the bactericidal mechanisms of AA both in vitro and in vivo by testing three main hypotheses. In Aim 1, we will determine the lipid electrophile species and pathways of formation in S. aureus. This aim will test the hypothesis that oxidants derived from S. aureus respiration initiate lipid peroxidation resulting in a diverse array of bactericidal lipid electrophiles. In Aim 2, we will discover the protein targets of AA-derived lipid electrophiles in S. aureus. This aim will test the hypothesis that lipid electrophiles exert toxicity by disrupting enzymes in essential S. aureus processes through post-translational modification. In Aim 3, we will define the role of AA release in S. aureus pathogenesis. This aim will test the hypothesis that inhibiting host AA release results in increased S. aureus pathogenesis in a murine model of systemic infection and that the bactericidal role of AA will be different across the multiple host tissues tested. The insights gained from this proposal will ...