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The novel peptide ScrA acts through the SaeRS two component system to regulate virulence gene expression in Staphylococcus aureus

ABSTRACT: Staphylococcus aureus is a Gram-positive opportunistic pathogen, which is carried by approximately 30 % of the population at any time. In addition to being a commensal S. aureus can cause an array of infections, ranging from mild skin and soft tissue infections to life threatening endocarditis and septicemia. S. aureus encodes a variety of virulence factors that facilitate its pathogenic lifestyle. Genes encoding these virulence factors are under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and protein-based systems, such as Two-Component Systems (TCS). Previous work in our lab demonstrated that overexpression of the sRNA tsr37 leads to an increase in cellular aggregation in the absence of host serum. We determined that the clumping phenotype was dependent on a previously unannotated 88 amino acid protein encoded within the tsr37 sRNA transcript (which we named ScrA for S. aureus clumping regulator A). In addition to increased clumping, overexpression of ScrA also leads to increased biofilm formation. To investigate the mechanism of action of ScrA we performed mass spectrometry proteomics and RNA-sequencing in the ScrA overexpressing strain. A variety of virulence factors, including several surface adhesins were upregulated while several proteases were downregulated. Moreover, results showed a significant upregulation of the SaeRS two component system, suggesting that ScrA is influencing SaeRS activity. We go on to demonstrate that overexpression of ScrA in a saeR mutant abrogates the clumping/biofilm phenotypes confirming that ScrA functions via the Sae system. Finally, we identified the ArlRS TCS as a potential positive regulator of scrA expression. Collectively our results show that ScrA is an activator of the SaeRS system and suggests that ScrA may act as an intermediary between the ArlRS and SaeRS systems.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Ronan Carroll

LAB HEAD: Ronan Carroll

PROVIDER: PXD028409 | Pride | 2022-08-11

REPOSITORIES: Pride

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The novel protein ScrA acts through the SaeRS two-component system to regulate virulence gene expression in Staphylococcus aureus.

Wittekind Marcus A MA Frey Andrew A Bonsall Abigail E AE Briaud Paul P Keogh Rebecca A RA Wiemels Richard E RE Shaw Lindsey N LN Carroll Ronan K RK

Molecular microbiology 20220415 5

Staphylococcus aureus is a Gram-positive commensal that can also cause a variety of infections in humans. S. aureus virulence factor gene expression is under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and two-component systems (TCS). Previous work in our laboratory demonstrated that overexpression of the sRNA tsr37 leads to an increase in bacterial aggregation. Here, we demonstrate that the clumping phenotype is dependent on a previously unannotated 88 a ...[more]

PMID: 35366366

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Project description:S. aureus possesses 16 two-component systems (TCS), two of which (GraRS & NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, conserved within the firmicutes. NsaRS has recently been documented as being important for nisin-resistance in S. aureus. In this study we present a detailed characterization of NsaRS and reveal that, as with other IM-HK TCS, it responds to disruptions in cell-wall stability. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-wall damaging antibiotics, including Phosphomycin, Ampicillin, Nisin and Penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall targetting antibiotic bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low affinity conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in both cell-wall and cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell-wall instability in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. We did four hybridizations for this experiment, including a biological replicate and a dye-swap experiment for each replicate to account for dye-bias. Spots flagged as empty or bad were excluded and the raw data from each slide was normalized using LOWESS method, with background correction. The data from the replicates were combined (using the median value) and a one-sample t-test was performed. The volcano plot was used with a fold change cut-off of >= 2 and a p-value of <0.05, to filter the genes that were differentially expressed.

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of gene regulon. The goal of this study is to investigate the genes regulated by Rsp in MRSA BD02-25 Methods: mRNA profiles of wild-type (WT) and Rsp knockout (â³Rsp) MRSA at mid-logarithmic growth phase (4h) were generated by deep sequencing, respectively in duplicate samples, using the Hiseq2000 (Illumina, CA) sequencer. The sequence reads that passed quality filters were aligned to S. aureus COL (RefSeq accession number NC_002951.2) using the Burrows-Wheeler Alignment tool (BWA) followed by ANOVA (ANOVA). Only the consistent data between the two WT or mutant samples were reserved for further analysis. qRTâPCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, RNA-seq analyses revealed that 328 genes were up-regulated and 176 genes were down-regulated in â³rsp compared with wild type. In order to explain the major relevant changes in â³rsp compare to wild-type, the differential expressed genes obtained in RNA-seq with three biological replicates were used to construct a regulation network. 108 up-regulated genes and 33 down-regulated genes in RNA-seq data had gathered in the protein-protein interaction network. 15 modules of biological processes were responsible, including ribosome, metabolism, biofilm formation, cell wall degradation, cytolysis, two-component system and so on. Biological processes related to biofilm formation, such as cell wall degradation and metabolism were up regulated, indicating that the defense systems were activated. However, the genes that responsible for the two-component system and cytolysis were down regulated, suggesting a possible pathway how Rsp controls the virulence. Conclusions: Our data provide new information to the virulence regulatory network in S. aureus. mRNA profiles of wild-type (WT) and Rsp knockout (â³Rsp) MRSA were generated by deep sequencing, in duplicate, using the Hiseq2000 (Illumina, CA) sequencer.

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