Characterization of the Effect of the Histidine Kinase CovS on Response Regulator Phosphorylation in Group A Streptococcus

Abstract

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.

Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

“…However, the model cannot explain the difference in the effects of RocA and CovS mutations on the expression of speB and sagA at the stationary phase in vitro and in vivo. RocA and CovS mutations cause similar levels of downregulation of RopB expression (24,53), and thus, RopB appears not to play a role in the differential effects of RocA and CovS mutations on speB expression. There are also other unique features of speB and sagA expression described in the literature.…”

“…In vitro CovR phosphorylation induces dimerization and enhances CovR binding to the promoter regions of hasA, sagA, covRS, speMF, and speB (49)(50)(51)(52). In bacteria, CovS senses Mg 2ϩ to phosphorylate CovR (53). Mutation or deletion of covS reduces the level of CovR phosphorylation by about 50% in M3 GAS strain MGAS1870 with a natural rocA-null mutation and about 70% in M1 strain MGAS2221 with wild-type rocA.…”

Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes

“…The sequences of the TaqMan primers and probes for nga, slo, and speB are listed in Table S2 in the supplemental material. The sequences of TaqMan primers and probes for measuring grab and prtS have been previously published (22). Each experiment was performed in triplicate with mean values (Ϯ standard deviations) shown.…”

Use of a Phosphorylation Site Mutant To Identify Distinct Modes of Gene Repression by the Control of Virulence Regulator (CovR) in Streptococcus pyogenes

“…Despite being studied for greater than 15 years, new information about the critical role of the CovRS system and its mechanistic principles continues to accumulate at a high rate (23)(24)(25)(26). It has recently been discovered that several regulatory pathways play a part in fine-tuning CovR phosphorylation status in response to different environmental cues (22-24, 27, 28).…”