Elucidating SHP Pheromones, Rgg Receptors and Inhibitors of Gram-Positive Pathogen Quorum Sensing

Bacterial cell-to-cell signaling or quorum-sensing (QS) is the phenomenon used by bacteria to coordinate gene expression across their communities by production, secretion and recognition of chemical signals known as pheromones. This work focuses on studying QS in Streptococcus pyogenes, an obligate human colonizer and pathogen, which causes a variety of diseases including, but not limited to, pharyngitis, rheumatic fever and necrotizing fasciitis. It accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide pheromones, including those known as SHPs (short hydrophobic peptides). Upon transport into the bacterial cell, the pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. In S. pyogenes, four Rgg paralogs exist, each serving as transcriptional regulators of genes associated with pathogenesis (RopB), biofilm development (Rgg2 and Rgg3), or a cryptic competence regulon (ComR). The aims of this study were to : 1) identify the mature form of pheromones SHP2 and SHP3 from cell-free culture supernatants, 2) to discover antagonists of pheromone signaling by screening compound libraries for molecules that disrupt Rgg-pheromone interactions; and 3) characterize Rgg proteins biochemically. Bioluminescent reporters were developed to detect active pheromones in cell-free culture supernatants fractionated by reverse-phase chromatography. Mass spectrometry allowed for the identification of several peptide variants whose specific activities were subsequently tested by bioluminescence assay. Rgg-SHP interactions were investigated using a fluorescence-polarization (FP) assay, leading to the elucidation of the peptide-receptor affinities ranging between 500 nM and 5 µM. High-throughput screening (HTS) identified several compounds capable of disrupting Rgg:SHP complexes, the most potent being cyclosporin A, which was able to inhibit biofilm development. The x-ray crystal structure of Rgg without any ligand and in presence of cyclosporin A was solved in collaboration with another lab, and the contact residues of SHP with Rgg were identified by mutagenic analysis of Rgg.