Mechanistic and Physiological Regulation of Rgg2/3 Quorum Sensing in Streptococcus pyogenes

All isolates of the Gram-positive, human-restricted pathogen Streptococcus pyogenes (Group A Streptococcus, GAS) possess the Rgg2/3 quorum sensing (QS) pathway, predicted to be involved in asymptomatic carriage of GAS. The Rgg2/3 QS pathways consists of the positive transcriptional regulator Rgg2 and the negative regulator Rgg3, both of which engage with a pair of short hydrophobic peptide (SHP) pheromones. Rgg2 and Rgg3 are both members of the Rgg-Rap-Npr-PlcR-PrgX (RRNPP) family of regulators. SHP production is stimulated in response to cell density and environmental condition (e.g. growth on mannose, low iron), and interact with both Rgg2 and Rgg3 to induce additional SHP production, biofilm formation and lysozyme resistance. The mechanisms by which the Rgg2/3 QS pathway functions, both at the whole-cell physiological level and at the protein mechanistic level are active research questions in the Federle Lab. Here, we present work demonstrating that activation of the Rgg2/3 QS pathway may be abrogated in response to environmental stressors (such as anti-microbial peptide exposure) via directed SHP degradation by the CovRS-regulated endopeptidase PepO. These data are the first reported example of attenuation of an RRNPP-type QS pathway via enzymatic activity and support our global hypothesis that Rgg2/3 QS may be active during asymptomatic carriage. Furthermore, we present additional studies assessing the mechanism by which Rgg2 serves as a positive regulator of transcription. Building on data derived from the recently solved structure of Rgg2 of Streptococcus dysgalactiae, we used a selection to identify eight mutants with constitutive (e.g. peptide-independent) activity. These mutants were characterized in vivo and in vitro, and these mutations increased Rgg2 sensitivity for both active SHP and variants of the SHP pheromones, as well decreasing Rgg2 sensitivity to the Rgg2-SHP competitive inhibitor cyclosporine A. Finally, we have shown that Rgg2 R153 is critical for peptide interaction in vivo, and that this residue is predicted to interacts with the N-terminal amino acid of the SHP. Collectively, these findings improve our understanding of the Rgg2/3 QS pathway, and will inform ongoing studies seeking to ascertain the role of the QS pathway in shaping host-bacteria interaction.