Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation [Gene Regulation]

June 23rd, 2014 by Aggarwal, C., Jimenez, J. C., Nanavati, D., Federle, M. J.

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as SHPs (short hydrophobic peptides), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernates that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernates fractionated by reverse-phase chromatography and mass-spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and though activities differed widely among the group, peptides comprising C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg-SHP interactions were determined using a fluorescence-polarization (FP) assay that utilized FITC-labeled peptide ligands. Peptide-receptor affinities were seen to be as low as 500 nM and their binding affinities directly correlated with observed bioactivity. Revelation of naturally-produced pheromones, along with determination of their affinity for cognate receptors, are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication.
  • Posted in Journal of Biological Chemistry, Publications
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