PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a significant cause of morbidity and mortality due to a remarkable capacity tocolonize multiple host tissues. Consistent with this, S. aureus is the leading cause of skin and soft tissueinfections, bacteremia, osteomyelitis and endocarditis. Treating infections can be exceedingly challenging dueto the prevalence of antibiotic resistant isolates, which necessitate the development of new therapeuticstrategies. To proliferate within diverse tissues, S. aureus acquires essential nutrients by exploiting abundantnutrient reservoirs present in the host environment. S. aureus nutrient iron acquisition strategies have beenstudied for decades; however, the mechanisms employed by this pathogen to obtain the equally importantnutrient sulfur during infection are not known. Reduced and oxidized forms of the sulfur-containing molecules,glutathione and cysteine, are abundant in host tissues and support in vitro proliferation of S. aureus. Whetherthese molecules satisfy the sulfur requirement during pathogenesis is unresolved, because we do not understandhow S. aureus imports and catabolizes these molecules. To elucidate the mechanisms S. aureus employs toacquire host-derived glutathione, we completed a forward genetic screen and identified mutants that fail to growin medium supplemented with glutathione as the sole source of sulfur. A reverse genetic approach was pursuedto identify potential cysteine transporters. We constructed mutants inactivated for homologues of putativeoxidized cysteine transporters and show that the mutated strains are impaired for oxidized cysteine utilization invitro. Notably, one of the importers provides a competitive advantage in liver colonization in a murine model ofsystemic infection. These preliminary data represent identification of the first S. aureus sulfur acquisition systemsand support the hypothesis that during infection, S. aureus targets abundant host-derived sulfur-containingmolecules to satisfy the sulfur requirement. The proposed work will test this hypothesis by (i) establishing themechanisms that support S. aureus import and catabolism of host-derived GSH, (ii) identifying S. aureus reducedand oxidized cysteine acquisition strategies during infection, and (iii) determining sulfur source abundance anddistribution at the host-pathogen interface. Understanding how the host immune response to infection impactssulfur source availability in tissues is also a goal of this study. The completion of this work will reveal themechanisms S. aureus employs to acquire host-derived sulfur sources during colonization of distinct tissues.This work will provide novel therapeutic strategies to combat antibiotic resistant S. aureus by impeding nutrientsulfur acquisition. We predict that S. aureus sulfur source acquisition strategies are likely conserved in otherbacterial pathogens, broadening the scope and impact of the proposed work.