The major role of <i>sarA</i> in limiting <i>Staphylococcus aureus</i> extracellular protease production <i>in vitro</i> is correlated with decreased virulence in diverse clinical isolates in osteomyelitis

We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in <i>Staphylococcus aureus</i> (Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of <i>sarA</i> results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on <i>in vitro</i> studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins <i>in vivo</i>. To this end, we compared the virulence of <i>mgrA</i>, <i>sarA</i>, <i>sarR</i>, <i>sarS</i>, <i>sarZ</i>, and <i>rot</i> mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of <i>mgrA</i> and <i>rot</i> limited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the <i>sarA</i> mutant exhibited reduced virulence in both strains. The reduced virulence of the <i>sarA</i> mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production <i>in vivo</i> in <i>S. aureus</i>.