Dataset Information

RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus.

ABSTRACT: Regulating gene expression during infection is critical to the ability of pathogens to circumvent the immune response and cause disease. This is true for the group A Streptococcus (GAS), a pathogen that causes both invasive (e.g., necrotizing fasciitis) and noninvasive (e.g., pharyngitis) diseases. The control of virulence (CovRS) two-component system has a major role in regulating GAS virulence factor expression. The regulator of cov (RocA) protein, which is a predicted kinase, functions in an undetermined manner through CovRS to alter gene expression and reduce invasive disease virulence. Here, we show that the ectopic expression of a truncated RocA derivative, harboring the membrane-spanning domains but not the dimerization or HATPase domain, is sufficient to complement a rocA mutant strain. Coupled with a previous bioinformatic study, the data are consistent with RocA being a pseudokinase. RocA reduces the ability of serotype M1 GAS isolates to express capsule and to evade killing in human blood, phenotypes that are not observed for M3 or M18 GAS due to isolates of these serotypes naturally harboring mutant rocA alleles. In addition, we found that varying the RocA concentration attenuates the regulatory activity of Mg2+ and the antimicrobial peptide LL-37, which positively and negatively regulate CovS function, respectively. Thus, we propose that RocA is an accessory protein to the CovRS system that influences the ability of GAS to modulate gene expression in response to host factors. A model of how RocA interacts with CovRS, and of the regulatory consequences of such activity, is presented.

SUBMITTER: Jain I

PROVIDER: S-EPMC5649027 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus.

Jain Ira I Miller Eric W EW Danger Jessica L JL Pflughoeft Kathryn J KJ Sumby Paul P

Infection and immunity 20171018 11

Regulating gene expression during infection is critical to the ability of pathogens to circumvent the immune response and cause disease. This is true for the group A Streptococcus (GAS), a pathogen that causes both invasive (e.g., necrotizing fasciitis) and noninvasive (e.g., pharyngitis) diseases. The control of virulence (CovRS) two-component system has a major role in regulating GAS virulence factor expression. The regulator of cov (RocA) prote ...[more]

PMID: 28808155

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Project description:Group B Streptococcus (GBS; Streptococcus agalactiae) is a leading cause of sepsis in neonates and pregnant mothers worldwide. Whereas the hyper-virulent serogroup III clonal cluster 17 has been associated with neonatal disease and meningitis, serogroup III ST283 was recently implicated in invasive disease among non-pregnant adults in Asia. Here, through comparative genome analyses of invasive and non-invasive ST283 strains, we identified a truncated DNA-binding regulator of a two-component system in a non-invasive strain that was homologous to Bacillus subtilis bceR, encoding the bceRSAB response regulator, which was conserved among GBS strains. Using isogenic knockout and complementation mutants of the ST283 strain, we demonstrated that resistance to bacitracin and the human antimicrobial peptide cathelicidin LL-37 was reduced in the ΔbceR strain with MICs changing from 64 and 256 μg/ml to 0.25 and 64 μg/ml, respectively. Further, the ATP-binding cassette transporter was upregulated by sub-inhibitory concentrations of bacitracin in the wild-type strain. Upregulation of dltA in the wild-type strain was also observed and thought to explain the increased resistance to antimicrobial peptides. DltA, an enzyme involved in D-alanylation during the synthesis of wall teichoic acids, which mediates reduced antimicrobial susceptibility, was previously shown to be regulated by the bceR-type regulator in Staphylococcus aureus. In a murine infection model, we found that the ΔbceR mutation significantly reduced the mortality rate compared to that with the wild-type strain (p < 0.01). Moreover, this mutant was more susceptible to oxidative stress compared to the wild-type strain (p < 0.001) and was associated with reduced biofilm formation (p < 0.0001). Based on 2-DGE and mass spectrometry, we showed that downregulation of alkyl hydroperoxide reductase (AhpC), a Gls24 family stress protein, and alcohol dehydrogenase (Adh) in the ΔbceR strain might explain the attenuated virulence and compromised stress response. Together, we showed for the first time that the bceR regulator in GBS plays an important role in bacitracin and antimicrobial peptide resistance, virulence, survival under oxidative stress, and biofilm formation.

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.

Dataset Information

RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus.

Publications

RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus.

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