Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance is a growing challenge and methicillin-resistant S. aureus (MRSA) infections are more difficult to treat, resulting in increased burden for both patients and healthcare systems. S. aureus causes the majority of skin infections in active-duty military and veterans, and skin colonization is a known precursor of infection. How this pathogen colonizes the skin is unknown. In recent microscopy studies, S. aureus developed biofilm communities during skin colonization and the quorum-sensing pathway was found to be activated. To investigate this further, we developed a new mouse skin colonization model and determined the MRSA global transcriptional response. Based on these RNAseq studies, our central hypothesis is that MRSA has a unique expression program that supports adherence and survival in the skin environment. We will address the innovative idea that MRSA specifically induces a select group of virulence factors to survive in the harsh skin environment and evade innate defenses. In Specific Aim 1, we will investigate the MRSA skin adherence and regulatory profile. We hypothesize that MRSA adapts to the skin environment by expressing a specific subset of adhesins, toxins and exo-enzymes. We will evaluate the contributions of specific virulence factors using in vitro adherence to human keratinocytes, skin explant colonization, wound healing assays and mouse models of skin colonization. In Specific Aim 2, we will determine the contribution of quorum-sensing and skin upregulated factors to MRSA immune evasion. We hypothesize that MRSA evades skin immunity using quorum-sensing regulated factors. Toward this end, we will define the role of urease in pH homeostasis and biofilm development, and the function of SasF surface protein in skin fatty acid resistance. We will use our developed human skin-like media to aid functional and regulatory studies on MRSA immune evasion factors. We will also determine the contribution of quorum-sensing factors in resistance mechanisms to antimicrobial peptides and skin fatty acids. In Specific Aim 3, we will assess the function of surface proteins in MRSA dissemination from colonization. In preliminary studies, we discovered an important role for SasF in skin infection and immune evasion. We hypothesize that MRSA strains with SasF have enhanced capacity to cause systemic disease. We will determine the function of SasF in response to neutrophil challenge, and we will evaluate the contribution of this protein to systemic dissemination in mouse models. Finally we will characterize civilian and military MRSA strain collections for presence and function of SasF to determine whether this protein is a biomarker of invasive disease. An improved understanding of how MRSA colonizes the skin and transitions to infection could open avenues to developing therapeutic strategies for minimizing the skin in...