Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation

Abstract

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 short hydrophobic peptides (SHPs), 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 supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants 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 although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization 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.

Keywords: Biofilm; Fluorescence Anisotropy; Gram-positive Bacteria; Pheromone; Quorum Sensing; Rgg; SHP; Streptococcus pyogenes (S. pyogenes).

FIGURE 1.

Identification of SHP pheromone variants from active fractions of culture supernatants. Culture supernatants from strains expressing shp2 (JCC177), shp3 (BNL187), or neither shp gene (BNL193) were fractionated with an acetonitrile (ACN) step gradient using C18 reverse-phase chromatography. A, maximum relative luciferase activity of elution fractions tested by reporter BNL178 (Δrgg3, shp3_GGGshp2_GGG::Pshp2-luxAB). B, extracted ion chromatograms of identified SHP variants. Peaks with gray shading demark integrated areas providing relative amounts of peptide ions detected.

FIGURE 2.

Synthetic SHP variant activities in bioluminescence reporter strains. Relative luminescence activity of the P_shp2-luxAB reporter in response to synthetic SHP variants in a strain (BNL177) incapable of producing endogenous SHPs (A) or in a wild-type genetic background strain (BNL148) (B). Plots indicate the means of at least three independent experiments, with EC₅₀ values calculated by applying linear regression analysis to dose-response curves using GraphPad Prism, version 6.01. A single concentration of SHP3-C8-reverse peptide was included as a control. The relative luminescence of BNL177 (C) and WT (D) is plotted versus time following addition of each peptide at its EC₅₀ concentration.

FIGURE 3.

Synthetic SHP variant interaction with purified Rgg proteins. A and B, direct FP of 10 nm FITC-labeled synthetic peptides titrated with purified MBP-Rgg2 (A) or Rgg3 (B) proteins. C and D, synthetic SHP variants were assessed for their ability to compete with FITC-labeled SHP2 for binding to Rgg proteins. Complexes of Rgg3:FITC-SHP2-C8 (formed under conditions containing 500 nm Rgg3, 10 nm FITC-SHP2-C8) were titrated with synthetic SHP2 variants (C) or SHP3 variants (D). E and F, synthetic SHP variants were assessed for their ability to compete with FITC-labeled SHP2-C8 for binding to Rggs. Complexes of MBP-Rgg2:FITC-SHP2-C8 (formed under conditions containing 1 μm MBP-Rgg2, 10 nm FITC-SHP2-C8) were titrated with synthetic SHP2 (E) and SHP3 (F) variants. Plots indicate the means of at least three independent experiments. K_d values were determined by applying linear regression on dose-response curves using GraphPad Prism (version 6.01).

FIGURE 4.

Full-length native SHP peptides do not bind Rgg proteins. Synthetic SHP variants were assessed for their ability to compete with FITC-labeled SHP2 for binding to Rgg proteins. Complexes of Rgg3:FITC-SHP2-C8 (pre-formed under conditions described in Fig. 3) were titrated with synthetic SHP3-C8 or full-length (FL) SHPs.

FIGURE 5.

SHP variants induce biofilm formation. NZ131 derivative strains were grown in 24-well plates and stimulated with varying concentrations of SHP variants. At 24 h, biofilms were assessed by a standard crystal violet staining method. Synthetic SHP variants were used to stimulate a strain (BNL170) unable to produce endogenous SHPs (top) or in a WT strain (bottom). Error bars indicate standard error from a minimum of three independent experiments.