The ability of to limit protease production plays a key role in the pathogenesis of osteomyelitis irrespective of the functional status of .

We demonstrate that mutation of the staphylococcal accessory regulator A () in the USA300 strain LAC limits virulence in a murine osteomyelitis model to a greater extent than mutation of the accessory gene regulator () and that it does so irrespective of the functional status of . Protease production was decreased in the mutant but increased in and 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 and / 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 mutant but not in the protease-deficient / mutant. Reduced cytotoxicity was correlated with the reduced abundance of full-length α-toxin, LukF, and LukS in and / mutants. The abundance of these toxins in their full-length form was increased in the protease-deficient mutant by comparison to LAC, demonstrating that mutation of 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 and mutants, but had no impact in the mutant. We conclude that a key factor in the attenuation of LAC and / mutants in osteomyelitis is the increased production of extracellular proteases and its impact on virulence factors that contribute to biofilm formation and cytotoxicity.The persistent emergence of antibiotic-resistant strains has rekindled interest in anti-virulence strategies to combat 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 () and the staphylococcal accessory regulator A (). We demonstrate that mutation of limits virulence to a greater extent than mutation of and that this can be attributed to increased protease production in both and / mutants. This illustrates the critical role of in protease-mediated post-translational regulation in . It also suggests that an inhibitor of would be more effective than an inhibitor of in overcoming the therapeutic recalcitrance of osteomyelitis and that such an inhibitor would remain effective even in the context of mutants known to arise during the transition from acute to chronic infection.