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

Abstract

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