The major role of sarA in limiting Staphylococcus aureus extracellular protease production 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 Staphylococcus aureus. We also showed that mutation of sarA 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 in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo. To this end, we compared the virulence of mgrA, sarA, sarR, sarS, sarZ, and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1. As assessed based on an overall osteomyelitis pathology score derived from the incidence of bone fracture, bacterial burdens in the bone, cortical bone destruction, and reactive bone formation, mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC. In contrast, the sarA mutant exhibited reduced virulence in both strains. This illustrates the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence. The reduced virulence of the sarA 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. This suggests that these in vitro phenotypes, either alone or in combination with each other, may be useful in prioritizing additional mutants for in vivo evaluation. Most importantly, they illustrate the significance of limiting protease production in vivo in S. aureus, and confirm that SarA plays the primary role in this regard. Author Summary Staphylococcus aureus causes a diverse array of infections due to its ability to produce an arsenal of virulence factors. Among these are extracellular proteases, which serve several purposes on behalf of the bacterium. However, it has become increasingly apparent that it is also critical to limit the production of these proteases to prevent them from compromising the S. aureus virulence factor repertoire. Many regulatory loci have been implicated in this respect, but it is difficult to draw relative conclusions because few reports have made direct comparisons, and fewer still have done so in vivo. We addressed this by assessing the impact on virulence of six regulatory loci previously implicated in protease production. We did this in the clinical context of osteomyelitis using mutants generated in two divergent clinical isolates. Our results confirm significant strain-dependent differences, reinforcing the importance of considering such diverse clinical isolates when evaluating targets for potential therapeutic intervention. In this respect, only mutation of sarA attenuated virulence in both strains. This illustrates the importance of limiting protease production as a means of post-translational regulatory control in S. aureus and confirms that sarA plays a predominant role in this regard.