Identification and Mechanistic Elucidation of the Rgg2/3 Quorum Sensing Circuit of Streptococcus pyogenes

Bacterial cell to cell communication, known as quorum sensing (QS), exists in a wide range of bacterial species and in many cases the responses elicited by QS signals contribute directly to pathogenesis. Recently, a new quorum sensing system was discovered in multiple streptococcal species in which a peptide pheromone modulates control of target genes through transcriptional regulators belonging to the Rgg-family. The Gram-positive bacterium Streptococcus pyogenes, also known as Group A streptococcus (GAS), is a strictly human commensal that poses large health and economic burdens worldwide deriving from its ability to cause self-limiting acute infections, life-threatening invasive disease, and post-infection sequelae. GAS has no known conserved quorum sensing systems, but has four rgg paralogs encoded in all 13 sequenced genomes. Two of these Rgg proteins (Rgg2 and Rgg3) are located in the genome adjacent to small open reading frames encoding putative peptides (SHP2, SHP3) with similar properties to pheromones of other QS systems. Using a combination of genetic and biochemical approaches, we have identified a novel QS system in GAS which utilizes both Rgg2 and Rgg3 in conjunction with SHP2 and SHP3 to control target gene expression. We demonstrate that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes. The C-terminal eight amino acids of the SHP peptides are identical at seven of eight positions and comprise the active signaling molecules, and both mature peptides function intracellularly to induce system activation through de-repression of Rgg3 and activation of Rgg2. Intriguingly, Rgg2 and Rgg3 share a highly conserved binding site within target promoters, and direct competition between the two regulators results in concentration-dependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP peptide. Studies presented herein also suggest some degree of Rgg-SHP specificity and demonstrate that both shp gene dosage and identity contribute to system activation. This is a unique regulatory circuit among known peptide-responsive QS systems, and further insight into Rgg function is anticipated to be of large importance to the understanding of both regulatory-network architecture and intercellular communication in Rgg-containing species.