Increased production of aureolysin and staphopain A is a primary determinant of the reduced virulence of mutants in osteomyelitis.

We previously demonstrated that mutation of in limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 mutants by subsequent mutation of and , while mutation of had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of was also required to mimic the phenotype of mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of and had the greatest impact on restoring the virulence of LAC and UAMS-1 mutants, with concurrent mutation of and the operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that -mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of virulence factors in diverse clinical isolates and diverse forms of infection.