BackgroundThe accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci.Methodology/Principal FindingsWe generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to form a biofilm in the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard.Conclusions and SignificanceThe results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.

We demonstrate that mutation of the staphylococcal accessory regulator (sarA) limits the accumulation of alpha-toxin and phenol-soluble modulins (PSMs) in Staphylococcus aureus isolates of the USA300 clonal lineage. Degradation assays and experiments done with protease inhibitors suggested that this was due to the increased production of extracellular proteases rather than differences associated with the impact of sarA on transcription of the target gene (hla) or the accessory gene regulator (agr). This was confirmed by demonstrating that concomitant mutation of the gene encoding aureolysin (aur) reversed the alpha-toxin and PSM-deficient phenotypes of a USA300 sarA mutant. Mutation of sarA had little impact on the alpha-toxin or PSM phenotypes of the commonly studied strain Newman, which is known to have a mutation in saeS that results in constitutive activation of the saeRS regulatory system, and we also demonstrate that repair of this defect resulted in the increased production of extracellular proteases and reversed both the alpha-toxin and PSM-positive phenotypes of a Newman sarA mutant.

Mutation of the staphylococcal accessory regulator (sarA) limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L) or a Newman saeRS mutant (ΔsaeRS) resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level.

Recent studies suggest that extracellular DNA promotes biofilm formation in Staphylococcus aureus and, conversely, that extracellular nucleases limit the ability to form a biofilm. S. aureus produces at least two extracellular nucleases, and in the study described in this report, we examined the impact of each of these nucleases on biofilm formation under both in vitro and in vivo conditions. Our results demonstrate that both nucleases impact biofilm formation in the clinical isolate UAMS-1. Under certain in vitro conditions, this impact is negative, with mutation of either or both of the nuclease genes (nuc1 and nuc2) resulting in an enhanced capacity to form a biofilm. However, this effect was not apparent in vivo in a murine model of catheter-associated biofilm formation. Rather, mutation of either or both nuclease genes appeared to limit biofilm formation to a degree that could be correlated with increased susceptibility to daptomycin.A defining characteristic of many Staphylococcus aureus infections is formation of a biofilm. Because this compromises the efficacy of antimicrobial therapy, it is important to understand the mechanistic basis for biofilm formation. One factor recently shown to be relevant in this regard is extracellular DNA (eDNA). Under in vitro conditions, the two possible sources of eDNA are the growth medium and the bacteria themselves. In fact, recent data suggest that it is the latter that play a primary role (13). Current models also suggest that the S. aureus lytSR two-component regulatory system and CidR collectively control the release of eDNA by influencing expression of the cid and lrg operons, with the latter two operons serving opposing roles with respect to each other in modulating the production of murein hydrolases and, consequently, cell lysis (18). Specifically, mutation of cidA results in reduced production of murein hydrolases, reduced release of eDNA, and a reduced capacity to form a biofilm, while mutation of the lrgAB operon has the opposite effects (13,19). Additional results supporting this model include the fact that extracellular nuclease, whether applied exogenously or produced by S. aureus, limits biofilm formation, at least under certain in vitro conditions (3,13,22).The production of extracellular nuclease has also been associated with reduced susceptibility to phagocytosis owing to an enhanced capacity to escape from neutrophil extracellular traps (NETs) (4). Thus, from a pathogenesis point of view, the production of staphylococcal extracellular nuclease potentially plays the opposing roles of promoting escape from NETs but limiting the ability to form a biofilm. These opposing roles may be largely hypothetical owing to the ability of S. aureus to regulate nuclease production such that it is produced under conditions when avoiding phagocytosis is the primary concern (e.g., in the bloodstream) but repressed during the process of colonization and biofilm formation. However, these two conditions are not mutually exclusive, as evidenced by the observation that neut...

SUMMARY We investigated the in vivo relevance of the impact of sarA and saeRS on protease production using derivatives of the USA300 strain LAC. The results confirmed that mutation of saeRS or sarA reduces virulence in a bacteremia model to a comparable degree. However, while eliminating protease production restored virulence in the sarA mutant, it had little impact in the saeRS mutant. Additionally, constitutive activation of saeRS (saeRSC) enhanced the virulence of LAC and largely restored virulence in the isogenic sarA mutant. Based on these results, together with our analysis of the representative virulence factors alpha toxin, protein A (Spa), and extracellular nucleases, we propose a model in which the attenuation of saeRS mutants is defined primarily by decreased production of such factors, while constitutive activation of saeRS increases virulence, and reverses the attenuation of sarA mutants, because it results in both increased production and decreased protease-mediated degradation of these same factors. This regulatory balance was also apparent in a murine model of catheter-associated infection, with the results suggesting that the impact of saeRS on nuclease production plays an important role during the early stages of these infections that is partially offset by increased protease production in sarA mutants.