Two Group A Streptococcal Peptide Pheromones Act through Opposing Rgg Regulators to Control Biofilm Development

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum

Peroxide Stimulon and Role of PerR in Group A Streptococcus▿†

We have characterized group A Streptococcus (GAS) genome-wide responses to hydrogen peroxide and assessed the role of the peroxide response regulator (PerR) in GAS under oxidative stress. Comparison of transcriptome changes elicited by peroxide in wild-type bacteria with those in a perR deletion mutant showed that 76 out of 237 peroxide-regulated genes are PerR dependent. Unlike the PerR-mediated upregulation of peroxidases and other peroxide stress defense mechanisms previously reported in Gram-positive species, PerR-dependent genes in GAS were almost exclusively downregulated and encoded proteins involved in purine and deoxyribonucleotide biosynthesis, heme uptake, and amino acid/peptide transport, but they also included a strongly activated putative transcriptional regulator (SPy1198). Of the 161 PerR-independent loci, repressed genes (86 of 161) encoded proteins with functions similar to those coordinated by PerR, in contrast to upregulated loci that encoded proteins that function in DNA damage repair, cofactor metabolism, reactive oxygen species detoxification, pilus biosynthesis, and hypothetical proteins. Complementation of the perR deletion mutant with wild-type PerR restored PerR-dependent regulation, whereas complementation with either one of two PerR variants carrying single mutations in two predicted metal-binding sites did not rescue the mutant phenotype. Metal content analyses of the recombinant wild type and respective PerR mutants, in addition to regulation studies in metal-supplemented and iron-depleted media, showed binding of zinc and iron by PerR and an iron requirement for optimal responses to peroxide. Our findings reveal a novel physiological contribution of PerR in coordinating DNA and protein metabolic functions in peroxide and identify GAS adaptive responses that may serve to enhance oxidative stress resistance and virulence in the host