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

Abstract

We used a murine model of postsurgical osteomyelitis (OM) to evaluate the relative virulence of the <i>Staphylococcus aureus</i> strain LAC and five isogenic variants that differ in the functional status of <i>saeRS</i> and <i>sarA</i> relative to each other. LAC and a variant in which <i>saeRS</i> activity is increased (<i>sae</i>^C) were comparably virulent to each other, while Δ<i>saeRS</i>, Δ<i>sarA</i>, Δ<i>saeRS</i>/Δ<i>sarA</i>, and <i>sae</i>^C/Δ<i>sarA</i> 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 <i>saeRS</i> attenuates virulence in our OM model owing primarily to the decreased production of <i>S. aureus</i> virulence factors, while mutation of <i>sarA</i> 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 <i>sarA</i> mutant than in the isogenic <i>saeRS</i> mutant. Irrespective of the mechanism involved, mutation of <i>saeRS</i> or <i>sarA</i> 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 (log₂ ≥ 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 <i>coa</i> 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 <i>sarA</i> mutant.