Extracellular proteases inhibit protein-dependent biofilm formation in Staphylococcus aureus.

In Staphylococcus aureus, biofilm formation can be associated with the production of surface-anchored proteins, including Bap, SasG, FnBPs or Spa. By mutational analysis, and using a model strain in which biofilm formation was Bap-dependent, we found that sigma(B) was essential for protein-dependent biofilm development. Non-polar mutations of sigma(B) in genetically unrelated S. aureus strains lowered the Bap expression and completely impaired biofilm development. Although Northern blot analysis showed a slight reduction in bap transcription, we demonstrated that Aur and SspA, two proteases that are overexpressed in the sigB mutant strain and are capable of degrading Bap, inhibit biofilm formation. Interestingly, a double sigB-agr mutant, which showed a diminished capacity to express extracellular proteases, was able to restore biofilm formation. Since the vast majority of the S. aureus global regulators control the expression of the extracellular proteases, the results of this work demonstrate the existence of a new pathway controlling protein-mediated biofilm formation in which different global regulators modulate biofilm formation by controlling the expression of S. aureus extracellular proteases.

Role of mgrA and sarA in methicillin-resistant Staphylococcus aureus autolysis and resistance to cell wall-active antibiotics.

BACKGROUND: We have previously shown the importance of mgrA and sarA in controlling autolysis of Staphylococcus aureus, with MgrA and SarA both being negative regulators of murein hydrolases. METHODS: In this study, we analyzed the effects of mgrA and sarA on antibiotic-mediated lysis in vitro and on the responses to cell wall-active antibiotic therapy in an experimental endocarditis model by use of 2 representative MRSA strains: the laboratory strain COL and the community-acquired clinical strain MW2. RESULTS: We found that mgrA and sarA independently down-regulated sarV (a marker for autolysis), although the alteration in sarV expression did not correlate directly with the autolysis profiles of single mgrA and sarA mutants. Importantly, the mgrA/sarA double mutants of both strains were more autolytic than the single mutants in vitro. We demonstrated that, despite equivalent intrinsic virulences of the parent strains and their isogenic mgrA/sarA double mutants in the endocarditis model, oxacillin and vancomycin treatment of the mgrA/sarA double mutants yielded significant reductions in vegetation bacterial densities in vivo, compared with treatment of their respective parent strains. CONCLUSIONS: These results suggest that down-regulation of mgrA/sarA in combination with use of cell wall-active antibiotics may represent a novel approach to treat MRSA infections.

MgrA represses biofilm formation in Staphylococcus aureus.

MgrA is a pleiotropic regulator that controls autolysis, virulence, and efflux pump activity in Staphylococcus aureus. We recently found that mgrA mutants of strains RN6390, SH1000, and MW2 also displayed enhanced biofilm formation compared with their respective parents. The biofilms formed by mgrA mutants of RN6390 and MW2 are independent of sigB and ica loci, two genetic elements that have been previously associated with biofilm formation in S. aureus. Biofilms formed by mgrA mutants are dependent on the expression of surface proteins mediated by the sortase gene srtA. Extracellular DNA was also a crucial component of the early biofilm of mgrA mutants. Genetic analysis indicated that biofilm formation in mgrA mutants is mediated in part by agr RNAIII, a genetic locus regulated by mgrA. Additionally, SarA is important to biofilm formation in mgrA mutants since the double sarA mgrA mutants failed to form biofilms compared to single mgrA mutants of RN6390 and MW2. However, the SarA-mediated effect is independent of agr and proteases such as V8 protease and aureolysin. Collectively, our data showed MgrA to be a repressor of biofilm formation, and biofilms formed by mgrA mutants have features that are distinct from other reported biofilm types in S. aureus.

The SarA protein family of Staphylococcus aureus.

Staphylococcus aureus is widely appreciated as an opportunistic pathogen, primarily in hospital-related infections. However, recent reports indicate that S. aureus infections can now occur in other wise healthy individuals in the community setting. The success of this organism can be attributed to the large array of regulatory proteins, including the SarA protein family, used to respond to changing microenvironments. Sequence alignment and structural data reveal that the SarA protein family can be divided into three subfamilies: (1) single domain proteins; (2) double domain proteins; (3) MarR homologs. Structural studies have also demonstrated that SarA, SarR, SarS, MgrA and thus possibly all members of this protein family are winged helix proteins with minor variations. Mutagenesis studies of SarA disclose that the winged helix motifs are important for DNA binding and function. Recent progress concerning the functions and plausible mechanisms of regulation of SarA and its homologs are discussed.

SarA positively controls bap-dependent biofilm formation in Staphylococcus aureus.

The biofilm-associated protein Bap is a staphylococcal surface protein involved in biofilm formation. We investigated the influence of the global regulatory locus sarA on bap expression and Bap-dependent biofilm formation in three unrelated Staphylococcus aureus strains. The results showed that Bap-dependent biofilm formation was diminished in the sarA mutants by an agr-independent mechanism. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. As expected, the diminished capacity to form biofilms in the sarA mutants correlated with the decreased presence of Bap in the bacterial surface. Using transcriptional fusion and Northern analysis data, we demonstrated that the sarA gene product acts as an activator of bap expression. Finally, the bap promoter was characterized and the transcriptional start point was mapped by the rapid amplification of cDNA ends technique. As expected, we showed that purified SarA protein binds specifically to the bap promoter, as determined by gel shift and DNase I footprinting assays. Based on the previous studies of others as well as our work demonstrating the role for SarA in icaADBC and bap expression, we propose that SarA is an essential regulator controlling biofilm formation in S. aureus.