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.
Highlights
The ability of bacteria to modify gene expression levels in adaptation to external influences is key to many aspects of bacterial pathogenesis [1]
Tight control of production of virulence factors is critical to group A streptococcus (GAS) pathogenesis, and the control of virulence two-component signaling system (CovRS) is central to this process
We discovered that GAS strains lacking CovS phosphatase activity resulting in high CovR phosphorylation level (CovR~P) levels had markedly impaired infectivity
Summary
The ability of bacteria to modify gene expression levels in adaptation to external influences is key to many aspects of bacterial pathogenesis [1]. TCS are absent in humans but abundant in a wide range of bacteria. They usually consist of a membrane-embedded histidine kinase that determines the regulatory activity of its cognate response regulator by altering its phosphorylation status [2, 4]. The control of virulence regulator sensor (CovRS, called CsrRS for capsule synthesis regulator) system of group A streptococcus (GAS) is one of the best-studied TCS in connection with bacterial pathogenesis [5, 6]. In tandem with the histidine kinase CovS, CovR is the central regulator of GAS virulence factor production [5, 10]. Similar to other OmpR/PhoB family members, CovR is phosphorylated at a conserved aspartic acid residue (D53) to create CovR~P, which is considered to be the active regulatory form of the protein [11,12,13]
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