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
further validate lipid peroxidation as an antimicrobial therapy, identify S. aureus proteins to target for
antimicrobial development, and expand the understanding of the bactericidal role of AA in vivo.