Staphylococcus aureus is one of the most prominent of all bacterial pathogens. Its ability to cause disease is dependent on the production of a diverse array of virulence factors in a coordinated fashion. In many cases, this ultimately results in formation of a bacterial biofilm. This biofilm greatly compromises the efficacy of antimicrobial therapy. Current models of the regulatory circuits required to modulate these processes are based almost exclusively on experiments done with the laboratory strain RN6390. However, our preliminary results have demonstrated that there are important differences between RN6390 and clinical isolates. The central hypothesis behind this proposal is that defining global regulatory circuits in clinical isolates will facilitate our understanding of staphylococcal pathogenesis and biofilm formation and that this may lead to the identification of new therapeutic targets capable of limiting biofilm formation and thereby increasing the efficacy of conventional antimicrobial agents. Based on this, we propose to define regulatory circuits of S. aureus in the more relevant context of clinical isolates with a specific focus on biofilm formation and virulence in our animal models (Aim 1 ).These experiments will be done in a defined set of strains chosen based on the fact that they are representative of the most commonly encountered clinical isolates. We will also assess the impact of specific mutations that play a role in biofilm formation on the efficacy of antimicrobial therapy in vivo (Aim 2). Additionally, we have confirmed that mutation of one regulatory locus (sarA) in clinical isolates results in a reduced capacity to form a biofilm, reduced virulence in our animal models, and increases susceptibility to at least some anti-staphylococcal agents. Based on this, we have also included mechanistic studies of SarA function, the central issue being how SarA binds to cis DMA elements associated with its target genes (Aim 3). These studies will allow us to explore the possibility of using peptide-nucleic acids as specific inhibitors of SarA that can be used to limit the ability of clinical isolates to form a biofilm. Lay description: Staphylococcus aureus causes devastating infections, and our current understanding of how this happens is inaccurate because almost all studies have been done with a laboratory strain rather than the clinical isolates that most commonly cause disease. This proposal is aimed at correcting these inaccuracies and ultimately developing therapeutic agents that can inhibit the ability of S. aureus to cause disease.