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

Abstract

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.

Keywords: CovR-CovS; RocA; group A Streptococcus; pH.

FIG 1

Phosphorylation levels of CovR in the wild-type strain (A20), the CovS kinase-inactivated mutant (CovS_H280A), the CovS phosphatase-inactivated mutant (CovS_T284A), and their rocA mutants (ΔrocA). (A) Phosphorylation levels of CovR in the A20, CovS_H280A mutant, CovS_T284A mutant, and their rocA mutants. Bacteria were cultured to the exponential phase of growth, and total proteins were extracted for Phostag Western blot analysis. (B) Growth curves of A20, the CovS_T284A mutant, and their rocA mutants. OD₆₀₀, optical density at 600 nm. (C) Phosphorylation levels of CovR in A20, the CovS_T284A mutant, and their rocA mutants. Total proteins were extracted from bacteria cultured for 3 to 7 h and analyzed by Phostag Western blotting. CovR∼P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein was used as the loading control. *, P < 0.05.

FIG 2

The phosphorylation level of CovR in the exponential-phase and stationary-phase wild-type strain (A20), the CovS phosphatase-inactivated mutant (CovS_T284A), and their rocA mutants (ΔrocA) after Mg²⁺ stimulus. Bacteria were cultured to (A) the exponential phase (4 h of incubation) or (B) the stationary phase (6 h) and treated or not treated with 20 mM Mg²⁺ for an additional 1 h. After treatment, the total proteins were extracted and analyzed by Phostag Western blotting. CovR∼P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein was used as the loading control. *, P < 0.05.

FIG 3

The phosphorylation level of CovR and the expression of CovR-controlled SLO in the wild-type strain (A20), CovS phosphatase-inactivated mutant (CovS_T284A), and their rocA mutants (ΔrocA) under different pH and Mg²⁺-treatment conditions. (A) The phosphorylation level of CovR in A20, CovS_T284A mutant, and their rocA mutants after neutral and acidic broths treatments. Bacteria were cultured for 2 h in TSBY broth and treated with neutral and acidic broths (with or without 20 mM Mg²⁺) for an additional 1 h before the Phostag Western blot analysis. Total protein was used as the loading control. (B) Transcription and (C) expression of streptolysin O (SLO) in A20, its rocA mutant, and the CovS_T284A rocA mutant after neutral and acidic broth treatments. Bacteria were cultured for 2 h in TSBY broth and treated with neutral and acidic broth for an additional 1 h. RNA was extracted for reverse transcription-PCR (RT-qPCR) analysis, and bacterial culture supernatants were collected for detecting SLO by Western blotting. Biological replicate experiments were performed using three independent preparations. The expression of slo was normalized to that of gyrA. *, P < 0.05.

FIG 4

The phosphorylation level of CovR and the expression of CovR-controlled slo, ska, and hasA expression in the wild-type A20 strain, its rocA mutant (ΔrocA), and the vector control (Vec), and rocA trans-complementary strains (Comp). (A) The phosphorylation level of CovR in A20, its rocA mutant, and the vector control and rocA trans-complementary strains. Bacteria were cultured to the exponential phase of growth, and total proteins were extracted and analyzed by Phostag Western blotting. CovR∼P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein was used as the loading control. (B) The expression of SLO and (C) the transcription of CovR-controlled slo, ska, and hasA in A20, its rocA mutant, and the vector-control and rocA trans-complementary strains after neutral and acidic broth treatments. Bacteria were cultured for 2 h in TSBY broth and treated with neutral and acidic broths for an additional 1 h. Bacterial culture supernatants were collected for detecting SLO by Western blotting, and RNAs were extracted for reverse transcription-quantitative PCR (RT-qPCR) analysis. Biological replicate experiments were performed using three independent preparations. The expression of slo, ska, and hasA was normalized to that of gyrA. *, P < 0.05.

FIG 5

The phosphorylation level of CovR and the expression of CovR-controlled slo, ska, and hasA expression in the wild-type A20 strain, the rocA-deletion mutant (ΔrocA), the mutant with the truncated rocA allele from the emm3-type isolate (rocA_emm3), and its rocA trans-complementary strain (Comp). (A) The phosphorylation level of CovR in A20, its rocA mutants, and the rocA trans-complementary strains. Bacteria were cultured to the exponential phase of growth, and total proteins were extracted and analyzed by Phostag Western blotting. CovR∼P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein was used as the loading control. (B) The transcription of CovR-controlled slo, ska, and hasA in A20, its rocA mutants, and the rocA trans-complementary strain after neutral and acidic broth treatments. Bacteria were cultured for 2 h in TSBY broth and treated with neutral and acidic broths for an additional 1 h. Bacterial RNAs were extracted for RT-qPCR analysis. Biological replicate experiments were performed using three independent preparations. The expression of slo, ska, and hasA was normalized to that of gyrA. *, P < 0.05. The lower panels show the colony morphology of A20 and its rocA mutants on the blood agar plate after 16 h of incubation at 37°C and under 5% CO₂ conditions.

FIG 6

Model showing the difference in modulating CovR phosphorylation in the wild-type and rocA-mutated strains under Mg²⁺ or acidic pH stimuli. The diagram shows that RocA modulates the phosphorylation level of CovR via dominantly repressing CovS’s phosphatase activity under neutral pH and in the exponential phase of growth. In the rocA-mutated strains (the rocA isogenic mutant or RocA-truncated emm3-type isolates), the phosphatase activity of CovS is derepressed and resulted in low levels of phosphorylated CovR compared to those in the wild-type strain. Also, under acidic pH or Mg²⁺ stimuli (signals to inactivate the phosphatase activity of CovS), the increase of phosphorylated CovR is more pronounced in the rocA-mutated strains than that the wild-type strain, suggesting that RocA inactivation would result in increased susceptibility of GAS strains in response to changes of environmental conditions (e.g., pH, Mg²⁺, and growth phases).