The major role ofsarAin limitingStaphylococcus aureusextracellular protease production is correlated with decreased virulence in diverse clinical isolates in osteomyelitis

Abstract

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 inStaphylococcus aureus. We also showed that mutation ofsarAresults 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 onin vitrostudies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteinsin vivo. To this end, we compared the virulence ofmgrA, sarA, sarR, sarS, sarZ, androtmutants 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 ofmgrAandrotlimited virulence to a statistically significant extent in UAMS-1, but not in LAC. In contrast, thesarAmutant 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 thesarAmutant 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 thesein vitrophenotypes, either alone or in combination with each other, may be useful in prioritizing additional mutants forin vivoevaluation. Most importantly, they illustrate the significance of limiting protease productionin vivoin S.aureus, and confirm that SarA plays the primary role in this regard.Author SummaryStaphylococcus aureuscauses 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 theS. aureusvirulence 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 soin 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 ofsarAattenuated virulence in both strains. This illustrates the importance of limiting protease production as a means of post-translational regulatory control inS. aureusand confirms thatsarAplays a predominant role in this regard.