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RNAsequencing comparing WT S. aureus and an evolved mutant EVOL-A and EVOL-A ∆sarA

ABSTRACT: The goals of this experiment were to understand the basal changes in transcription between the EVOL-A strain that is an evolved strain in the NRS193 background with a mutation in Rel (Q672*) that has unknown impacts on transcription. The EVOL-A delSarA mutant was used to understand which changes in EVOL-A comapred to WT (NRS193) are dependent on the DNA-binding protein SarA.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE319106 | GEO | 2026/02/11

REPOSITORIES: GEO

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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.

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RNAsequencing comparing WT S. aureus and an evolved mutant EVOL-A and EVOL-A ∆sarA

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