A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes.
ABSTRACT: A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/?covR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.
Project description:To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneously during invasive infections and, in in vivo models of infection, rapidly become dominant. Here, we compared wild-type GAS with covR, covS, and covRS isogenic mutant strains to investigate the heterogeneity in the types of natural mutations that occur in covR and covS and the phenotypic consequences of covR or covS mutation. We found that the response regulator CovR retains some regulatory function in the absence of CovS and that CovS modulates CovR to significantly enhance repression of one group of genes (e.g., the speA, hasA, and ska genes) while it reduces repression of a second group of genes (e.g., the speB, grab, and spd3 genes). We also found that different in vivo-induced covR mutations can lead to strikingly different transcriptomes. While covS mutant strains show increased virulence in several invasive models of infection, we determined that these mutants are significantly outcompeted by wild-type GAS during growth in human saliva, an ex vivo model of upper respiratory tract infection. We propose that CovS-mediated regulation of CovR activity plays an important role in the ability of GAS to cycle between pharyngeal and invasive infections.
Project description:The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains.
Project description:CovR/CovS is a two-component regulatory system in group A Streptococcus and primarily acts as a transcriptional repressor. The D53 residue of CovR (CovRD53) is phosphorylated by the sensor kinase CovS, and the phosphorylated CovRD53 protein binds to the intergenic region of rgg-speB to inhibit speB transcription. Nonetheless, the transcription of rgg and speB is suppressed in covS mutants. The T65 residue of CovR is phosphorylated in a CovS-independent manner, and phosphorylation at the D53 and T65 residues of CovR is mutually exclusive. Therefore, how phosphorylation at the D53 and T65 residues of CovR contributes to the regulation of rgg and speB expression was elucidated. The transcription of rgg and speB was suppressed in the strain that cannot phosphorylate the D53 residue of CovR (CovRD53A mutant) but restored to levels similar to those of the wild-type strain in the CovRT65A mutant. Nonetheless, inactivation of the T65 residue phosphorylation in the CovRD53A mutant cannot derepress the rgg and speB transcription, indicating that phosphorylation at the T65 residue of CovR is not required for repressing rgg and speB transcription. Furthermore, trans complementation of the CovRD53A protein in the strain that expresses the phosphorylated CovRD53 resulted in the repression of rgg and speB transcription. Unlike the direct binding of the phosphorylated CovRD53 protein and its inhibition of speB transcription demonstrated previously, the present study showed that inactivation of phosphorylation at the D53 residue of CovR contributes dominantly in suppressing rgg and speB transcription.IMPORTANCE CovR/CovS is a two-component regulatory system in group A Streptococcus (GAS). The D53 residue of CovR is phosphorylated by CovS, and the phosphorylated CovRD53 binds to the rgg-speB intergenic region and acts as the transcriptional repressor. Nonetheless, the transcription of rgg and Rgg-controlled speB is upregulated in the covR mutant but inhibited in the covS mutant. The present study showed that nonphosphorylated CovRD53 protein inhibits rgg and speB transcription in the presence of the phosphorylated CovRD53 in vivo, indicating that nonphosphorylated CovRD53 has a dominant role in suppressing rgg transcription. These results reveal the roles of nonphosphorylated CovRD53 in regulating rgg transcription, which could contribute significantly to invasive phenotypes of covS mutants.
Project description:CovR/CovS is an important two-component regulatory system in human pathogen group A Streptococcus (GAS). Epidemiological studies have shown that inactivation of the sensor kinase CovS is correlated with invasive clinical manifestations. The phosphorylation level of response regulator CovR decreases dramatically in the absence of CovS, resulting in the derepression of virulence factor expression and an increase in bacterial invasiveness. Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease and is negatively regulated by CovR; however, the expression of SpeB is almost completely repressed in the covS mutant. The present study found that in the emm1-type A20 strain, non-phosphorylated CovR acts as a transcriptional repressor for SpeB-positive regulator Rgg. In addition, the expression of Rgg-negative regulator LacD.1 is upregulated in the covS mutant. These results suggest that inactivation of Rgg in the covS mutant would directly mediate speB repression. The current study showed that overexpression of rgg but not inactivation of lacD.1 in the covS mutant partially restores speB expression, indicating that only rgg repression, but not lacD.1 upregulation, contributes to the speB repression in the covS mutant.
Project description:The two-component control of virulence (Cov) regulator (R)-sensor (S) (CovRS) regulates the virulence of Streptococcus pyogenes (group A Streptococcus [GAS]). Inactivation of CovS during infection switches the pathogenicity of GAS to a more invasive form by regulating transcription of diverse virulence genes via CovR. However, the manner in which CovRS controls virulence through expression of extended gene families has not been fully determined. In the current study, the CovS-regulated gene expression profiles of a hypervirulent emm23 GAS strain (M23ND/CovS negative [M23ND/CovS(-)]) and a noninvasive isogenic strain (M23ND/CovS(+)), under different growth conditions, were investigated. RNA sequencing identified altered expression of ∼ 349 genes (18% of the chromosome). The data demonstrated that M23ND/CovS(-) achieved hypervirulence by allowing enhanced expression of genes responsible for antiphagocytosis (e.g., hasABC), by abrogating expression of toxin genes (e.g., speB), and by compromising gene products with dispensable functions (e.g., sfb1). Among these genes, several (e.g., parE and parC) were not previously reported to be regulated by CovRS. Furthermore, the study revealed that CovS also modulated the expression of a broad spectrum of metabolic genes that maximized nutrient utilization and energy metabolism during growth and dissemination, where the bacteria encounter large variations in available nutrients, thus restructuring metabolism of GAS for adaption to diverse growth environments. From constructing a genome-scale metabolic model, we identified 16 nonredundant metabolic gene modules that constitute unique nutrient sources. These genes were proposed to be essential for pathogen growth and are likely associated with GAS virulence. The genome-wide prediction of genes associated with virulence identifies new candidate genes that potentially contribute to GAS virulence.The CovRS system modulates transcription of ∼ 18% of the genes in the Streptococcus pyogenes genome. Mutations that inactivate CovR or CovS enhance the virulence of this bacterium. We determined complete transcriptomes of a naturally CovS-inactivated invasive deep tissue isolate of an emm23 strain of S. pyogenes (M23ND) and its complemented avirulent variant (CovS(+)). We identified diverse virulence genes whose altered expression revealed a genetic switching of a nonvirulent form of M23ND to a highly virulent strain. Furthermore, we also systematically uncovered for the first time the comparative levels of expression of a broad spectrum of metabolic genes, which reflected different metabolic needs of the bacterium as it invaded deeper tissue of the human host.
Project description:Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections.
Project description:We sought to determine how CovRS mutations varying CovR phosphorylation levels affect the gene expression profile of group A streptococcus Overall design: There were 8 strains analyzed, each in quadruplicate replicates: 1) wild-type GAS serotype M1; 2) covS-E281A GAS serotype M1; 3) covS-T284A GAS serotype M1 4) covR-D53A GAS serotypep M1; 5) wild-type GAS serotype M3; 6) covS-E281A GAS serotype M3; 7.) covS-T284A GAS serotype M1; 8.) covR-D53A GAS serotypep M3
Project description:Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [SseA+] and normal SpeB activity [SpeBA+]). SseA+ SpeBA+ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an SseA+ SpeBA+ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other SseA+ SpeBA+ isolates also had nonsense mutations or small indels in rocA RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.
Project description:The group A Streptococcus (GAS) causes diseases that range from mild (e.g. pharyngitis) to severely invasive (e.g. necrotizing fasciitis). Strain- and serotype-specific differences influence the ability of isolates to cause individual diseases. At the center of this variability is the CovR/S two-component system and the accessory protein RocA. Through incompletely defined mechanisms, CovR/S and RocA repress the expression of more than a dozen immunomodulatory virulence factors. Alleviation of this repression is selected for during invasive infections, leading to the recovery of covR, covS or rocA mutant strains. Here, we investigated how RocA promotes CovR/S activity, identifying that RocA is a pseudokinase that interacts with CovS. Disruption of CovS kinase or phosphatase activities abolishes RocA function, consistent with RocA acting through the modulation of CovS activity. We also identified, in conflict with a previous study, that the RocA regulon includes the secreted protease-encoding gene speB. Finally, we discovered an inverse correlation between the virulence of wild-type, rocA mutant, covS mutant and covR mutant strains during invasive infection and their fitness in an ex vivo upper respiratory tract model. Our data inform on mechanisms that control GAS disease potential and provide an explanation for observed strain- and serotype-specific variability in RocA function.
Project description:Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated-a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.