Exploiting Correlations between Protein Abundance and the Functional Status of saeRS and sarA To Identify Virulence Factors of Potential Importance in the Pathogenesis of Staphylococcus aureus Osteomyelitis

Abstract

We used a murine model of postsurgical osteomyelitis (OM) to evaluate the relative virulence of the Staphylococcus aureus strain LAC and five isogenic variants that differ in the functional status of saeRS and sarA relative to each other. LAC and a variant in which saeRS activity is increased (sae C ) were comparably virulent to each other, while ΔsaeRS, ΔsarA, ΔsaeRS/ΔsarA, and sae C /ΔsarA mutants were all attenuated to a comparable degree. Phenotypic comparisons including a mass-based proteomics approach that allowed us to assess the number and abundance of full-length proteins suggested that mutation of saeRS attenuates virulence in our OM model owing primarily to the decreased production of S. aureus virulence factors, while mutation of sarA does so owing to protease-mediated degradation of these same virulence factors. This was confirmed by demonstrating that eliminating protease production restored virulence to a greater extent in a LAC sarA mutant than in the isogenic saeRS mutant. Irrespective of the mechanism involved, mutation of saeRS or sarA was shown to result in reduced accumulation of virulence factors of potential importance. Thus, using our proteomics approach we correlated the abundance of specific proteins with virulence in these six strains and identified 14 proteins that were present in a significantly increased amount (log 2 ≥ 5.0) in both virulent strains by comparison to all four attenuated strains. We examined biofilm formation and virulence in our OM model using a LAC mutant unable to produce one of these 14 proteins, specifically staphylocoagulase. The results confirmed that mutation of coa limits biofilm formation and, to a lesser extent, virulence in our OM model, although in both cases the limitation was reduced by comparison to the isogenic sarA mutant.

SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates ofStaphylococcus aureusLAC and UAMS-1

“…This was also true of a sarA mutant with the exception of the spl operon, with expression levels of this operon being essentially unchanged in a sarA mutant [ 34 ]. As noted above, this is consistent with other reports examining the impact of sarA on spl transcription [ 6 , 14 ], but contradicted by our proteomics studies demonstrating that these proteases are present in increased amounts in CM from a LAC sarA mutant [ 27–29 ]. In contrast, mutation of mgrA was associated with decreased expression of all protease genes/operons other than that encoding scpA , which was largely unaffected in an mgrA mutant [ 34 ].…”

“…SarA has been shown to bind to the aur and sspABC promoter regions [ 49 , 52 ] but to our knowledge this is the first report demonstrating it also binds the scpA and splA-F promoters. The fact that SarA was captured using the spl bait is particularly significant in light of the discrepancy between reports suggesting that mutation of sarA does not impact expression of the spl operon [ 34 ] and our demonstration that these proteases, as well as aureolysin, ScpA, SspA, and SspB are present in increased amounts in conditioned medium (CM) from stationary phase cultures of a LAC sarA mutant by comparison to CM from LAC itself [ 27–29 ]. In addition, we previously used pCM11 [ 55 ] to generate transcriptional reporters in which DNA fragments upstream of each protease-encoding gene/operon were used to drive expression of the superfolder green fluorescent protein (sGFP), and when these were introduced into the parent strains and their isogenic sarA mutants, fluorescence was significantly higher in the sarA mutants with all four reporters including the spl::gfp reporter, and this was true in both LAC and UAMS-1 [ 56 ].…”

“…However, there is some contradictory evidence in this regard. For instance, several reports concluded that mutation of sarA has no impact on transcription of the spl operon [ 6 , 14 , 34 ] but we have demonstrated that all six Spl proteases are present in increased amounts in conditioned media (CM) from stationary phase cultures of a LAC sarA mutant by comparison to CM from LAC itself [ 27–29 ]. Irrespective of this discrepancy, it is clear that mutation of agr results in a decrease in overall protease activity to a degree that can be correlated with an increased capacity to form a biofilm, while mutation of sarA results in an increase in overall protease activity to a degree that can be correlated with a decreased capacity to form a biofilm [ 32 ].…”

“…Based on such observations, it would be anticipated that eliminating the ability of S. aureus to produce these proteases would result in decreased virulence, but we and others have demonstrated that doing so in the USA300 strain LAC increases rather than decrease virulence in diverse forms of infection including sepsis and osteomyelitis [ 26–28 ]. Similarly, it would be anticipated that increased production of these proteases would increase virulence, but we have also demonstrated that mutation of the staphylococcal accessory regulator ( sarA ) results in the increased production of all of these proteases but reduced virulence in these same animal models [ 27–29 ]. These paradoxical observations can be explained by the fact that eliminating protease production results in the increased abundance of S. aureus surface-associated and extracellular virulence factors [ 26 ], while the increased production of proteases in sarA mutants results in the decreased accumulation of these virulence factors [ 27–29 ].…”

Virulence factors and clonal diversity of Staphylococcus aureus in colonization and wound infection with emphasis on diabetic foot infection

“…aureus strains collected longitudinally from a chronic osteomyelitis patient showed agrC frameshift mutations over time resulting in reduced virulence and less tissue damage [ 29 ]. Mass-based proteomics approach in a murine osteomyelitis model demonstrated that mutations in exoprotein regulatory protein saeRS and staphylococcal accessory regulator sarA attenuates virulence by downregulating virulence factor production and degradation of virulence factors respectively [ 30 ]. Víquez-Molina et al [ 31 ] compared the prevalence of virulence genes encoding for pvl, etA, etB, and tsst in S .…”