RocA Regulates Phosphatase Activity of Virulence Sensor CovS of Group A Streptococcus in Growth Phase- and pH-Dependent Manners.

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.

Effect of Phosphatase Activity of the Control of Virulence Sensor (CovS) on Clindamycin-Mediated Streptolysin O Production in Group A Streptococcus.

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.

Acidic stress enhances CovR/S-dependent gene repression through activation of the covR/S promoter in emm1-type group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus) is a clinically important gram-positive bacterium that causes severe diseases with high mortality. Spontaneous mutations in genes encoding the CovR/CovS two-component regulatory system have been shown to derepress expression of virulence factors and are significantly associated with invasiveness of infections. Sensor kinase CovS senses environmental signals and then regulates the levels of phosphorylated CovR. In addition, CovS is responsible for survival of group A Streptococcus under acidic stress. How this system regulates the expression of CovR-controlled genes under acidic stress is not clear. This study shows that the expression of CovR-controlled genes, including hasA, ska, and slo, is repressed under acidic conditions by a CovS-dependent mechanism. Inactivation of CovS kinase activity or CovR protein phosphorylation derepresses the transcription of these genes under acidic conditions, suggesting that the phosphorylation of CovR is required for the repression of the CovR-controlled genes. Furthermore, the promoter activity of the covR/covS operon (pcov) was upregulated after 15min of incubation under acidic conditions. Replacement of pcov with a constitutively activated promoter abrogated the acidic-stress-dependent repression of the genes, indicating that the pH-dependent pcov activity is directly involved in the repression of CovR-controlled genes. In summary, the present study shows that inactivation of CovS not only derepresses CovR-controlled genes but also abrogates the acidic-stress-dependent repression of the genes; these phenomena may significantly increase bacterial virulence during infection.