Ectopic expression of the sensor CovS cytosolic domain confers phosphatase activity and full virulence to the M1T1 CovSY39H attenuated variant of group A Streptococcus

Group A Streptococcus (GAS) causes a wide variety of diseases ranging from mild, noninvasive, such as pharyngitis and impetigo, to life-threatening infections, such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The two-component CovR/S system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence. An attenuated pharyngeal colonizing variant (S126) possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H) was recovered in France from a member of a family in which another individual developed NF and STSS caused by the M1T1 WT strain (S119). We employed transcriptome analyses (RNA-seq), quantitative determinations of CovR phosphorylation, measurements of virulence factor activity, and a murine model of human NF to demonstrate that CovS of strain S126 almost lost its entire phosphatase activity but retained its kinase and phosphotransfer activities. Moreover, we reversed its attenuated phenotype by ectopically expressing the cytosolic domain of wild-type CovS. Culturing the corresponding strain S126covS-3 in a chemically defined medium (CDM) supplemented with asparagine (Asn), conditions that produce an excess of cytosolic ADP over ATP, stimulated the ectopically expressed phosphatase activity. Consequently, this led to dephosphorylation of CovR[~]P and increased the expression of virulence factors. Most importantly, S126covS-3 reverted to the wild-type phenotype of S119 in the mouse model of human GAS NF. Our study provides a new mechanistic tool that enables the manipulation of CovS phosphatase activity both in vitro and in vivo. IMPORTANCEThe shift from high to low virulence and back in GAS is critical for understanding its pathogenesis and developing new treatments to control GAS infections. The two-component CovR/S system, comprising the sensor kinase/phosphatase CovS and the transcription regulator CovR, regulates the degree of GAS virulence. In the M1T1 serotype, covR/S mutations are typically associated with a loss of CovR/S function, leading to hypervirulent phenotypes. However, an attenuated pharyngeal-colonizing variant possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H strain S126) was isolated in France. Here, we demonstrate that S126 is attenuated because the mutation in CovS inhibits its phosphatase activity but preserves its kinase and phosphotransfer activities. By expressing the cytosolic domain of WT CovS in the S126 background, we endowed the resulting strain, S126covS-3, with phosphatase activity, which was further stimulated by ADP when formed in chemically defined medium (CDM) supplemented with asparagine (Asn). Most importantly, S126covS-3 switched to full virulence, similar to that of S119 in a mouse model of human GAS NF. These findings underscore the importance of comprehensive analyses of disease-related covR/S mutants in understanding the virulence and persistence of GAS.