RocA truncation underpins hyper-encapsulation, carriage longevity and transmissibility of serotype M18 group A streptococci.
ABSTRACT: 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: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: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: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 important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.
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: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:Two-component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine-tune their activity. In group A Streptococcus (GAS), regulator of Cov (RocA) is an accessory protein that functions with the control of virulence regulator/sensor TCS, which regulates approximately 15% of the GAS transcriptome. Whole-genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid-altering) and nonsense (protein-truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally occurring clinical isolates with rocA polymorphisms (n = 48), an isogenic rocA deletion mutant strain, and five isogenic rocA polymorphism mutant strains to perform genome-wide transcript analysis (RNA sequencing), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change, causing a wild-type-like virulence phenotype, or a substantial transcriptome change, leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Taken together, our data show that naturally occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.
Project description:Background:Streptococcus agalactiae can cause urinary tract infection (UTI). The role of the S. agalactiae global virulence regulator, CovR, in UTI pathogenesis is unknown. Methods:We used murine and human bladder uroepithelial cell models of UTI and S. agalactiae mutants in covR and related factors, including ?-hemolysin/cytolysin (?-h/c), surface-anchored adhesin HvgA, and capsule to study the role of CovR in UTI. Results:We found that covR-deficient serotype III S. agalactiae 874391 was significantly attenuated for colonization in mice and adhesion to uroepithelial cells. Mice infected with covR-deficient S. agalactiae produced less proinflammatory cytokines than those infected with wild-type 874391. Acute cytotoxicity in uroepithelial cells triggered by covR-deficient but not wild-type 874391 was associated with significant caspase 3 activation. Mechanistically, covR mutation significantly altered the expression of several genes in S. agalactiae 874391 that encode key virulence factors, including ?-h/c and HvgA, but not capsule. Subsequent mutational analyses revealed that HvgA and capsule, but not the ?-h/c, exerted significant effects on colonization of the murine urinary tract in vivo. Conclusions:S. agalactiae CovR promotes bladder infection and inflammation, as well as adhesion to and viability of uroepithelial cells. The pathogenesis of S. agalactiae UTI is complex, multifactorial, and influenced by virulence effects of CovR, HvgA, and capsule.
Project description:A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.