CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus.

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

Contribution of AmyA, an extracellular alpha-glucan degrading enzyme, to group A streptococcal host-pathogen interaction.

alpha-Glucans such as starch and glycogen are abundant in the human oropharynx, the main site of group A Streptococcus (GAS) infection. However, the role in pathogenesis of GAS extracellular alpha-glucan binding and degrading enzymes is unknown. The serotype M1 GAS genome encodes two extracellular proteins putatively involved in alpha-glucan binding and degradation; pulA encodes a cell wall anchored pullulanase and amyA encodes a freely secreted putative cyclomaltodextrin alpha-glucanotransferase. Genetic inactivation of amyA, but not pulA, abolished GAS alpha-glucan degradation. The DeltaamyA strain had a slower rate of translocation across human pharyngeal epithelial cells. Consistent with this finding, the DeltaamyA strain was less virulent following mouse mucosal challenge. Recombinant AmyA degraded alpha-glucans into beta-cyclomaltodextrins that reduced pharyngeal cell transepithelial resistance, providing a physiologic explanation for the observed transepithelial migration phenotype. Higher amyA transcript levels were present in serotype M1 GAS strains causing invasive infection compared with strains causing pharyngitis. GAS proliferation in a defined alpha-glucan-containing medium was dependent on the presence of human salivary alpha-amylase. These data delineate the molecular mechanisms by which alpha-glucan degradation contributes to GAS host-pathogen interaction, including how GAS uses human salivary alpha-amylase for its own metabolic benefit.