Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus

Abstract

The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains.

New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates

“…Dual transcriptome sequencing (RNA-seq) (21) has afforded the ability to determine transcriptome changes using deep-sequencing technologies, and this technique has been exploited using wild-type strains of multiple GAS serotypes and mutant strains in which different virulence regulators have been inactivated (22)(23)(24)(25)(26)(27)(28). Recent development of dual RNA-seq analysis has assisted studies designed to measure transcripts of both pathogen and host during infection (29)(30)(31)(32).…”

Polymorphisms in Regulator of Cov Contribute to the Molecular Pathogenesis of Serotype M28 Group A Streptococcus

“…43 RocA is hypothesized to heterodimerize with CovS to affect CovR phosphorylation and downstream regulatory activity. 34,40,41,44 However, a physical interaction between RocA and CovS (or any other transcription regulator) has not been definitively shown. RocA inactivation alters CovR phosphorylation and transcription of CovRS regulated genes.…”

“…RocA inactivation alters CovR phosphorylation and transcription of CovRS regulated genes. 34,40,41 However, the molecular mechanism of the interaction remains unknown. Accessory proteins are often membrane proteins.…”

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group AStreptococcus

“…Group A Streptococcus (GAS) strains were routinely grown in Todd-Hewitt media supplemented with 0.2% yeast extract (THY) at 37°C with 5%CO2. A single amino acid change in ccpA was engineered into the wild type MGAS2221 strain by homologous recombination using the pBBL740 plasmid as described previously (Horstmann et al, 2018), to create the isoallelic strain 2221-CcpA-V301A.…”

“…Recombinant HPr protein was phosphorylated and purified as described previously (Shelburne et al, 2010). Cell lysates of GAS strains were prepared, separated on 12.5% SuperSep Phostag gels and detected using a polyclonal anti-HPr antibody (Horstmann et al, 2014) as described previously (Horstmann et al, 2018). Experiments were repeated at least twice using samples collected on separate days.…”