The ability of <i>sarA</i> to limit protease production plays a key role in the pathogenesis of <i>Staphylococcus aureus</i> osteomyelitis irrespective of the functional status of <i>agr</i>

We demonstrate that mutation of the staphylococcal accessory regulator A (<i>sarA</i>) in the USA300 strain LAC limits virulence in a murine osteomyelitis model to a greater extent than mutation of the accessory gene regulator (<i>agr</i>) and that it does so irrespective of the functional status of <i>agr</i>. Protease production was decreased in the <i>agr</i> mutant but increased in <i>sarA</i> and <i>sarA/agr</i> mutants to a degree that limited biofilm formation. Extracellular protein A (eSpa) and full-length extracellular nuclease (Nuc1) were absent in the conditioned medium (CM) from <i>sarA</i> and <i>sarA</i>/<i>agr</i> mutants, and their abundance was restored in both mutants by eliminating protease production. Cytotoxicity of CM for osteoblasts and osteoclasts was also reduced in both mutants. Cytotoxicity was restored in a protease-deficient <i>sarA</i> mutant but not in the protease-deficient <i>sarA</i>/<i>agr</i> mutant. Reduced cytotoxicity was correlated with the reduced abundance of full-length α-toxin, LukF, and LukS in <i>sarA</i> and <i>sarA</i>/<i>agr</i> mutants. The abundance of these toxins in their full-length form was increased in the protease-deficient <i>sarA</i> mutant by comparison to LAC, demonstrating that mutation of <i>sarA</i> increases the production of these toxins but increased protease production limits their abundance in full-length and presumably functional forms. Most importantly, eliminating protease production enhanced the virulence of <i>sarA</i> and <i>sarA/agr</i> mutants, but had no impact in the <i>agr</i> mutant. We conclude that a key factor in the attenuation of LAC <i>sarA</i> and <i>sarA</i>/<i>agr</i> mutants in osteomyelitis is the increased production of extracellular proteases and its impact on virulence factors that contribute to biofilm formation and cytotoxicity.<b>IMPORTANCE</b>The persistent emergence of antibiotic-resistant strains has rekindled interest in anti-virulence strategies to combat <i>S. aureus</i> infections. Numerous reports describe anti-virulence strategies focusing on key regulatory elements that globally influence virulence factor production, the two most commonly targeted being the accessory gene regulator (<i>agr</i>) and the staphylococcal accessory regulator A (<i>sarA</i>). We demonstrate that mutation of <i>sarA</i> limits virulence to a greater extent than mutation of <i>agr</i> and that this can be attributed to increased protease production in both <i>sarA</i> and <i>sarA</i>/<i>agr</i> mutants. This illustrates the critical role of <i>sarA</i> in protease-mediated post-translational regulation in <i>S. aureus</i>. It also suggests that an inhibitor of <i>sarA</i> would be more effective than an inhibitor of <i>agr</i> in overcoming the therapeutic recalcitrance of osteomyelitis and that such an inhibitor would remain effective even in the context of <i>agr</i> mutants known to arise <i>in vivo</i> during the transition from acute to chronic infection.