AbstractInfections from opportunistic bacterial pathogens such as Staphylococcus aureus place a tremendous burdenon our healthcare system. The human skin and mucosal surfaces are coated with commensal bacteria thatmake up the healthy microbiome, and this natural protective layer is important for preventing infections. Thereis growing evidence that extensive interactions between commensal bacteria occur as they colonize a specificniche and compete for resources. One strategy the bacteria can employ to gain an advantage is to releasemolecules that negatively impact neighbors. There are growing reports that targeting quorum-sensing could bean effective strategy to reduce fitness and in turn slow growth of a competitor. S. aureus persistently colonizes20% of the healthy adult population, and this opportunistic pathogen uses quorum-sensing system to controlthe expression of enzymes, toxins, and immunomodulatory proteins that are essential to spread throughtissues and cause disease. This regulatory system is controlled by the production and sensing of a secretedcyclic peptide signal, also called an autoinducing peptide or AIP. In our preliminary studies, we discovered thata commensal staphylococcal strain, S. caprae, releases an AIP signal that competes with all S. aureus strains,including clinical methicillin-resistant S. aureus (MRSA) isolates. Using mass spectrometry, we identified theAIP structure, and this AIP prevents MRSA quorum-sensing activation and skin infection progression in amouse model. We hypothesize that commensal Staphylococci release AIP signals to compete with S. aureusto gain a competitive colonization advantage. This hypothesis is in part based on our previous findings that afunctional quorum-sensing system is necessary to colonize the host. Therefore, we propose that the purposeof the AIP cross-talk could be to gain a fitness advantage in the colonization environment. In Aim 1, we willinvestigate the mechanism of quorum-sensing inhibition by commensal Staphylococci. Besides S. caprae, wehave identified five additional staphylococcal strains that inhibit MRSA quorum-sensing, and we will employ ourmass spectrometric strategy to identify the corresponding AIP structures. We will confirm the AIP structuralassignment and test each of them in MRSA quorum-sensing reporter assays and toxin production tests. InAim 2, we will determine the ability of commensal AIPs to prevent MRSA infection using a mouse model. Wewill monitor MRSA agr activation using bioluminescence with real time mouse imaging in the presence ofinhibitory AIPs, and evaluate the ability of these AIPs to prevent development of a MRSA skin infection. Finally,in Aim 3, we will assess the impact of commensal Staphylococci on MRSA colonization. Using a developedmouse skin colonization model, we will assess MRSA colonization of living skin in the presence of varyinglevels of inhibitory AIPs, and perform competition assays between commensal Staphylococci and MRSA.Understanding how commensal bacteria can reduce the colonization and infection potential of problematicpathogens will improve our knowledge of the microbiome's contribution to human health.