PROJECT SUMMARY The human skin is the largest organ of the body. This physical barrier and the skin's innate immunity are an essential first defense against pathogens. Staphylococcus aureus is an opportunistic pathogen and the dominant microorganism of soft skin and tissue infections. These infections can quickly develop into systemic infections and worsen health outcomes. Increasingly, antibiotic-resistant epidemic S. aureus strains (e.g. methicillin-resistant S. aureus, MRSA) complicate treatment of these infections in the clinical setting. Although skin colonization increases risk of a MRSA infection, studying this aspect of host-pathogen interactions has been challenging. Current animal models lack the acidity, skin structure, and eccrine gland distribution of human skin and are not accessible to many research groups. Additionally, in vitro testing with media mimicking the human skin surface have not been published. To address these gaps, I developed an in vitro media that incorporates the metabolites, ions, and pH that MRSA would encounter on the human skin. I performed RNA-seq of MRSA grown in these conditions and compared these results to a previous RNA-seq experiment of MRSA inoculated on mouse skin. In both datasets, genes for metabolizing urea, derived from sweat glands; and urocanic acid, derived from the natural moisturizing factor, were significantly upregulated. I hypothesize that the metabolism of urea and urocanic acid contributes to S. aureus pH homeostasis and growth, respectively, on the skin. This hypothesis will be tested in the following aims: Aim 1: Ascertain the role of urease and related functions in MRSA growth and pH homeostasis in human skin-like in vitro models. Genetic and biochemical approaches will be used in an in vitro media and a differentiated keratinocyte model to investigate the role of urease, contribution of urea and nickel transport to urease function, and therapeutic approaches to inhibit urease activity Aim 2: Investigate the role of urocanic acid metabolism in MRSA skin colonization. Genetic and biochemical approaches will be used in the previously mentioned models to validate the predicted encoded functions of this pathway, investigate substrate specificity of the pathway, and better understand regulatory control. Epidemiology of pathway expression will be assessed by Western Blot analysis of clinical S. aureus skin isolates from atopic dermatitis patients and healthy controls. These studies will provide a foundation for future research of the skin microbiota, expand our understanding of MRSA physiology on the skin, and identify potential targets for future therapeutic development.