Project description: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. To assess the relative impact of these two functions, we previously used a proteomics approach that measures protein abundance as a function of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assess the impact of this on the accumulation of S. aureus exoproteins1. While this approach confirmed that protease-mediated degradation has a significant impact on the S. aureus exoproteome, it was potentially limited in that it did not take into account the possibility that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, we present an expanded proteomics approach that utilizes a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels. Specifically, 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. Using methods that focus on total proteoforms vs. methods that focus specifically on full-length proteins, quantitative proteomic comparisons of sarA vs sarA/protease mutants identified proteins that were degraded in a protease dependent manner owing to mutation of sarA, while comparisons of a sarA/protease mutant vs 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 approach provided for a more comprehensive and robust analysis of the impact of mutating sarA and protease-mediated degradation on the S. aureus exoproteome.

Project description:Background The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in S. aureus wild type and ccpA-deleted mutant, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased enzymes of glycolytic pathway, indicating a higher glycolytic activity, while proteins required for the complete oxidation in the TCA cycle were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetylCoA to acetate with a concomitant substrate-level phosphorylation were neither regulated by glucose nor by CcpA. Most CcpA directly repressed genes were involved in utilization of amino acids as secondary carbon sources. More genes were found to be differentially expressed by CcpA in a glucose-independent manner than in the classical, glucose dependent way, suggesting that glucose-independent regulation by CcpA may be of particular importance in S. aureus. In the presence of glucose, CcpA was found to regulate expression of genes involved in metabolism, but that of genes coding for virulence determinants. Conclusions This study identified the CcpA regulon of exponentially growing S. aureus, for the first time. As in other bacteria, the CcpA-regulon of S. aureus comprised a large amount of metabolic genes but also some 50 genes associated with virulence. CcpA seemed to work in a glucose- as well as glucose-independent way. The transcriptomes of strain Newman and its isogenic ccpA-deleted mutant were determined in early exponential growth and 30 min after the addition of 10 mM glucose, under controlled pH conditions. In the absence of glucose, the wild type grew slightly faster than the mutant, reaching an OD600 of 1 approximately 20 min earlier than the mutant. Adding 10 mM glucose at OD600 1 increased the growth rate of the wild type but had only a minor effect on that of the mutant. 60 min after glucose addition, glucose was depleted down to 0.3 mM by the wild type, while still 3 mM glucose was left in the culture of the mutant. Despite increased glucose consumption rates in the wild type, acetate production was only slightly enhanced compared to the mutant. No lactate was excreted at any time point sampled. Acidification of the medium upon glucose metabolism was prevented by buffering, maintaining a pH of 7.5 for both strains and under both growth conditions for at least 2 h after glucose addition, allowing to rule out any pH effects.

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SarA regulon in S. aureus (ChIP-seq)

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