Label-Free Proteomic Approach to Characterize Protease-Dependent and -Independent Effects of sarA Inactivation on the Staphylococcus aureus Exoproteome

The staphylococcal accessory regulator A ( sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. We previously used a proteomics approach that measures protein abundance of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assesses the impact of this on the accumulation of S. aureus exoproteins. Our previous approach was limited as it did not take into account that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, our goal was to use an expanded proteomics approach utilizing a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels to alleviate these false negatives and thereby provide for characterization of protease-dependent and -independent effects of sarA mutation on the S. aureus exoproteome. Proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases ( sarA/protease) were resolved using one-dimensional gel electrophoresis. Quantitative proteomic comparisons of sarA versus sarA/protease mutants identified proteins that were cleaved in a protease-dependent manner owing to mutation of sarA, and comparisons of sarA/protease mutant versus the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This expanded approach provided for a more comprehensive analysis of the impact of mutating sarA on the S. aureus exoproteome.