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

Abstract

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.

The Impacts of msaABCR on sarA -Associated Phenotypes Are Different in Divergent Clinical Isolates of Staphylococcus aureus

“…Genes within the msa operon encode a putative protein (MsaA) with no known function, a DNA binding protein (MsaB) shown to act as a transcription factor that regulates expression of numerous genes, and a regulatory RNA (msaC) and an antisense RNA (msaR) complementary to msaB (27). As would be expected based on the phenotypes of sarA mutants (3,4,13,15,16,28) and the role of msaABCR in enhancing expression of sarA, mutation of msaABCR (hereinafter referred to as msa) has been correlated with increased protease production and a decreased capacity to form a biofilm (25,27,29).…”

“…Based on this, we examined the impact of mutating msa on nuclease production with a specific focus on the Nuc1 extracellular nuclease. This was facilitated by the availability of an anti-Nuc1 antibody (16), which allowed us to investigate this issue using Western blots of CM harvested from overnight cultures of each strain. It is important to recognize that Nuc1 is produced in two forms, the smaller of which (NucA) is proteolytically derived from the larger (NucB), and both of which are enzymatically active (41).…”

“…We have attempted to address this by directly comparing different regulatory mutants generated in divergent clinical isolates of S. aureus using both in vitro and in vivo assays (3,4,44). The results of these studies have led us to focus on sarA and to hypothesize that a primary factor contributing to the impact of mutating sarA on virulence and virulenceassociated phenotypes is the increased production of extracellular proteases and the limitation this imposes on the accumulation of both surface-associated and extracellular virulence factors (1,16). To date, we have not included the msaABCR operon in these studies, and it is important to do so given that msa has been shown to function upstream of sarA and to impact sarA-associated phenotypes, including biofilm formation and protease production (25)(26)(27)29).…”

“…Western blotting. SarA Western blotting was performed with an anti-SarA antibody and appropriate secondary antibodies, as previously described (1,15,16). Western blots included at least two biological replicates.…”

Assessing the Contribution of an HtrA Family Serine Protease During Borrelia turicatae Mammalian Infection

“…Proteins were identified and quantified by LC-MS/MS based on spectral counts of tryptic peptides for the proteins of interest. Sample preparation, protein identification, and analysis was carried out by the UAMS Proteomics Core as previously described (Zielinska et al, 2012; Byrum et al, 2018). Proteins were then identified using Mascot version 2.5.1 (Matrix Science, Boston, MA) and the B. turicatae strain 91E315 database (GenBank assembly accession: GCA_000012085.2).…”