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Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.

ABSTRACT: The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the -10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that the site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA-binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during GAS infection.

SUBMITTER: Churchward G

PROVIDER: S-EPMC4130213 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.

Churchward Gordon G Bates Christopher C Gusa Asiya A AA Stringer Virginia V Scott June R JR

Microbiology (Reading, England) 20090201 Pt 2

The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in ...[more]

PMID: 19202105

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Project description:The gram-positive human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes diseases ranging from mild and often self-limiting infections of the skin or throat to invasive and life-threatening illnesses. To cause such diverse types of disease, the GAS must be able to sense adverse environments and regulate its gene expression accordingly. The CovR/S two-component signal transduction regulatory system in GAS represses about 15% of the GAS genome, including many genes involved in virulence, in response to the environment. We report that CovR is still able to repress transcription from several promoters in the absence of the putative histidine kinase sensor for this system, CovS. We also show that a phosphorylation site mutant (D53A) of CovR is unable to repress gene expression. In addition, we report that a strain with a nonpolar mutation in CovS does not grow at a low pH, elevated temperature, or high osmolarity. The stress-related phenotypes of the CovS mutant were complemented by expression of covS from a plasmid. Selection for growth of a CovS mutant under stress conditions resulted in isolation of second-site mutations that inactivated covR, indicating that CovR and CovS act in the same pathway. Also, at 40 degrees C in the wild-type strain, CovR appeared to be less active on the promoter tested, which is consistent with the hypothesis that it was partially inactivated by CovS. We suggest that under mild stress conditions, CovS inactivates CovR, either directly or indirectly, and that this inactivation relieves repression of many GAS genes, including the genes needed for growth of GAS under stress conditions and some genes that are necessary for virulence. Growth of many gram-positive bacteria under multiple-stress conditions requires alteration of promoter recognition produced by RNA polymerase association with the general stress response sigma factor, sigma(B). We provide evidence that for GAS, which lacks a sigB ortholog, growth under stress conditions requires the CovR/S two-component regulatory system instead. This two-component system in GAS thus appears to perform a function for which other gram-positive bacteria utilize an alternative sigma factor.

Project description:BACKGROUND:The production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene. RESULTS:We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain. CONCLUSIONS:Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.

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Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.

Publications

Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.

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