Identification of rocA, a positive regulator of covR expression in the group A streptococcus.
ABSTRACT: In the group A streptococcus (GAS; Streptococcus pyogenes), a two-component system known as CovRS (or CsrRS) regulates about 15% of the genes, including several important virulence factors like the hyaluronic acid capsule. Most of these genes, including covR itself, are negatively regulated by CovR. We have isolated two independent ISS1 insertions in an open reading frame (ORF) that increases CovR expression as measured by a Pcov-gusA reporter fusion in single copy in the GAS chromosome. This ORF, named rocA for "regulator of Cov," activates covR transcription about threefold. As expected, a rocA mutant is mucoid and produces more transcript from the has promoter since this promoter is repressed by CovR. This effect is dependent on the presence of a wild-type covR gene. In contrast to its activation of Pcov, RocA negatively regulates its own expression. This autoregulation is not dependent on the presence of the covR gene. All the phenotypes of the rocA mutant were complemented by the presence of the rocA gene on a plasmid. The rocA gene is present in strains of all nine M serotypes of GAS tested and is absent from strains representing 11 other groups of streptococci and related bacteria, including strains of the closely related group C and G streptococci. It seems likely that rocA plays an important role in the pathogenesis of GAS since it affects expression of the global regulator CovR.
Project description:The control of the virulence response regulator and sensor (CovR-CovS) two-component regulatory system in group A Streptococcus (GAS) strains regulates more than 15% of gene expression and has critical roles in invasive GAS infection. The membrane-embedded CovS has kinase and phosphatase activities, and both are required for modulating the phosphorylation level of CovR. Regulator of Cov (RocA) is a positive regulator of covR and also been shown to be a pseudokinase that interacts with CovS to enhance the phosphorylation level of CovR; however, how RocA modulates the activity of CovS has not been determined conclusively. Although the phosphorylation level of CovR was decreased in the rocA mutant in the exponential phase, the present study shows that phosphorylated CovR in the rocA mutant increased to levels similar to those in the wild-type strain in the stationary phase of growth. In addition, acidic stress, which is generally present in the stationary phase, enhanced the phosphorylation level of CovR in the rocA mutant. The phosphorylation levels of CovR in the CovS phosphatase-inactivated mutant and its rocA mutant were similar under acidic stress and Mg2+ (the signal that inhibits CovS phosphatase activity) treatments, suggesting that the phosphatase activity, but not the kinase activity, of CovS is required for RocA to modulate CovR phosphorylation. The phosphorylation level of CovR is crucial for GAS strains to regulate virulence factor expression; therefore, the growth phase- and pH-dependent RocA activity would contribute significantly to GAS pathogenesis.IMPORTANCE The emergence of invasive group A streptococcal infections has been reported worldwide. Clinical isolates that have spontaneous mutations or a truncated allele of the rocA gene (e.g., emm3-type isolates) are considered to be more virulent than isolates with the intact rocA gene (e.g., emm1-type isolates). RocA is a positive regulator of covR and has been shown to enhance the phosphorylation level of intracellular CovR regulator through the functional CovS protein. CovS is the membrane-embedded sensor and modulates the phosphorylation level of CovR by its kinase and phosphatase activities. The present study shows that the enhancement of CovR phosphorylation is mediated via the repression of CovS's phosphatase activity by RocA. In addition, we found that RocA acts dominantly on modulating CovR phosphorylation under neutral pH conditions and in the exponential phase of growth. The phosphorylation level of CovR is crucial for group A Streptococcus species to regulate virulence factor expression and is highly related to bacterial invasiveness; therefore, growth phase- and pH-dependent RocA activity and the sequence polymorphisms of rocA gene would contribute significantly to bacterial phenotype variations and pathogenesis.
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: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.
Project description:Variable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes The nature of the polymorphism is a one-unit deletion in a three-tandem-repeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes.
Project description:Group A streptococcal isolates of serotype M18 are historically associated with epidemic waves of pharyngitis and the non-suppurative immune sequela rheumatic fever. The serotype is defined by a unique, highly encapsulated phenotype, yet the molecular basis for this unusual colony morphology is unknown. Here we identify a truncation in the regulatory protein RocA, unique to and conserved within our serotype M18 GAS collection, and demonstrate that it underlies the characteristic M18 capsule phenotype. Reciprocal allelic exchange mutagenesis of rocA between M18 GAS and M89 GAS demonstrated that truncation of RocA was both necessary and sufficient for hyper-encapsulation via up-regulation of both precursors required for hyaluronic acid synthesis. Although RocA was shown to positively enhance covR transcription, quantitative proteomics revealed RocA to be a metabolic regulator with activity beyond the CovR/S regulon. M18 GAS demonstrated a uniquely protuberant chain formation following culture on agar that was dependent on excess capsule and the RocA mutation. Correction of the M18 rocA mutation reduced GAS survival in human blood, and in vivo naso-pharyngeal carriage longevity in a murine model, with an associated drop in bacterial airborne transmission during infection. In summary, a naturally occurring truncation in a regulator explains the encapsulation phenotype, carriage longevity and transmissibility of M18 GAS, highlighting the close interrelation of metabolism, capsule and virulence.
Project description:Serotype M28 group A streptococcus (GAS) is a common cause of infections such as pharyngitis ("strep throat") and necrotizing fasciitis ("flesh-eating" disease). Relatively little is known about the molecular mechanisms underpinning M28 GAS pathogenesis. Whole-genome sequencing studies of M28 GAS strains recovered from patients with invasive infections found an unexpectedly high number of missense (amino acid-changing) and nonsense (protein-truncating) polymorphisms in rocA (regulator of Cov), leading us to hypothesize that altered RocA activity contributes to M28 GAS molecular pathogenesis. To test this hypothesis, an isogenic rocA deletion mutant strain was created. Transcriptome sequencing (RNA-seq) analysis revealed that RocA inactivation significantly alters the level of transcripts for 427 and 323 genes at mid-exponential and early stationary growth phases, respectively, including genes for 41 transcription regulators and 21 virulence factors. In contrast, RocA transcriptomes from other GAS M protein serotypes are much smaller and include fewer transcription regulators. The rocA mutant strain had significantly increased secreted activity of multiple virulence factors and grew to significantly higher colony counts under acid stress in vitro RocA inactivation also significantly increased GAS virulence in a mouse model of necrotizing myositis. Our results demonstrate that RocA is an important regulator of transcription regulators and virulence factors in M28 GAS and raise the possibility that naturally occurring polymorphisms in rocA in some fashion contribute to human invasive infections caused by M28 GAS strains.
Project description:Phenotypic heterogeneity is commonly observed between isolates of a given pathogen. Epidemiological analyses have identified that some serotypes of the group A Streptococcus (GAS) are non-randomly associated with particular disease manifestations. Here, we present evidence that a contributing factor to the association of serotype M3 GAS isolates with severe invasive infections is the presence of a null mutant allele for the orphan kinase RocA. Through use of RNAseq analysis, we identified that the natural rocA mutation present within M3 isolates leads to the enhanced expression of more than a dozen immunomodulatory virulence factors, enhancing phenotypes such as hemolysis and NAD(+) hydrolysis. Consequently, an M3 GAS isolate survived human phagocytic killing at a level 13-fold higher than a rocA complemented derivative, and was significantly more virulent in a murine bacteremia model of infection. Finally, we identified that RocA functions through the CovR/S two-component system as levels of phosphorylated CovR increase in the presence of functional RocA, and RocA has no regulatory activity following covR or covS mutation. Our data are consistent with RocA interfacing with the CovR/S two-component system, and that the absence of this activity in M3 GAS potentiates the severity of invasive infections caused by isolates of this serotype.
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:Streptococcus mutans, the primary aetiological agent of dental caries, is one of the major bacteria of the human oral cavity. The pathogenicity of this bacterium is attributed not only to the expression of virulence factors, but also to its ability to respond and adapt rapidly to the ever-changing conditions of the oral cavity. The two-component signal transduction system (TCS) CovR/S plays a crucial role in virulence and stress response in many streptococci. Surprisingly, in S. mutans the response regulator CovR appears to be an orphan, as the cognate sensor kinase, CovS, is absent in all the strains. We found that acetyl phosphate, an intracellular phosphodonor molecule known to act in signalling, might play a role in CovR phosphorylation in vivo. We also found that in vitro, upon phosphorylation by potassium phosphoramide (a high-energy phophodonor) CovR formed a dimer and showed altered electrophoretic mobility. As expected, we found that the conserved aspartic acid residue at position 53 (D53) was the site of phosphorylation, since neither phosphorylation nor dimerization was seen when an alanine-substituted CovR mutant (D53A) was used. Surprisingly, we found that the ability of CovR to act as a transcriptional regulator does not depend upon its phosphorylation status, since the D53A mutant behaved similarly to the wild-type protein in both in vivo and in vitro DNA-binding assays. This unique phosphorylation-mediated inhibition of CovR function in S. mutans sheds light on an unconventional mechanism of the signal transduction pathway.
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