Colonization of the Murine Oropharynx by Streptococcus pyogenes Is Governed by the Rgg2/3 Quorum Sensing System.

Streptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors are known to contribute to persistent colonization of this niche, and many are important in mucosal immunity and vaccine development. In this work, mice were infected intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system-a peptide-based signaling system conserved in sequenced isolates of S. pyogenes Deletion of the QS system's transcriptional activator (Δrgg2) dramatically diminished the percentage of colonized mice, while deletion of the transcriptional repressor (Δrgg3) increased the percentage of colonized mice compared to that of the wild type (WT). Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that QS-dependent colonization is responsive to the intrinsic conditions within the host upper respiratory tract. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative reverse transcription-PCR (qRT-PCR) subsequently confirmed QS upregulation within 1 h of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the Δrgg2 bacteria suggests that their rapid elimination failed to elicit the previously characterized response to intranasal inoculation of GAS. This work identifies a new transcriptional regulatory system governing the ability of S. pyogenes to colonize the nasopharynx and provides knowledge that could help lead to decolonization therapeutics.

A novel chemical inducer of Streptococcus quorum sensing acts by inhibiting the pheromone-degrading endopeptidase PepO.

Bacteria produce chemical signals (pheromones) to coordinate behaviors across a population in a process termed quorum sensing (QS). QS systems comprising peptide pheromones and their corresponding Rgg receptors are widespread among Firmicutes and may be useful targets for manipulating microbial behaviors, like suppressing virulence. The Rgg2/3 QS circuit of the human pathogen Streptococcus pyogenes controls genes affecting resistance to host lysozyme in response to short hydrophobic pheromones (SHPs). Considering that artificial activation of a QS pathway may be as useful in the objective of manipulating bacteria as inhibiting it, we sought to identify small-molecule inducers of the Rgg2/3 QS system. We report the identification of a small molecule, P516-0475, that specifically induced expression of Rgg2/3-regulated genes in the presence of SHP pheromones at concentrations lower than typically required for QS induction. In searching for the mode of action of P516-0475, we discovered that an S. pyogenes mutant deficient in pepO, a neprilysin-like metalloendopeptidase that degrades SHP pheromones, was unresponsive to the compound. P516-0475 directly inhibited recombinant PepO in vitro as an uncompetitive inhibitor. We conclude that this compound induces QS by stabilizing SHP pheromones in culture. Our study indicates the usefulness of cell-based screens that modulate pathway activities to identify unanticipated therapeutic targets contributing to QS signaling.

A Quorum Sensing-Regulated Protein Binds Cell Wall Components and Enhances Lysozyme Resistance in Streptococcus pyogenes.

The Rgg2/3 quorum sensing (QS) system is conserved among all sequenced isolates of group A Streptococcus (GAS; Streptococcus pyogenes). The molecular architecture of the system consists of a transcriptional activator (Rgg2) and a transcriptional repressor (Rgg3) under the control of autoinducing peptide pheromones (SHP2 and SHP3). Activation of the Rgg2/3 pathway leads to increases in biofilm formation and resistance to the bactericidal effects of the host factor lysozyme. In this work, we show that deletion of a small gene, spy49_0414c, abolished both phenotypes in response to pheromone signaling. The gene encodes a small, positively charged, secreted protein, referred to as StcA. Analysis of recombinant StcA showed that it can directly interact with GAS cell wall preparations containing phosphodiester-linked carbohydrate polymers but not with preparations devoid of them. Immunofluorescence microscopy detected antibody against StcA bound to the surface of paraformaldehyde-fixed wild-type cells. Expression of StcA in bacterial culture induced a shift in the electrostatic potential of the bacterial cell surface, which became more positively charged. These results suggest that StcA promotes phenotypes by way of ionic interactions with the GAS cell wall, most likely with negatively charged cell wall-associated polysaccharides.IMPORTANCE This study focuses on a small protein, StcA, that is expressed and secreted under induction of Rgg2/3 QS, ionically associating with negatively charged domains on the cell surface. These data present a novel mechanism of resistance to the host factor lysozyme by GAS and have implications in the relevance of this circuit in the interaction between the bacterium and the human host that is mediated by the bacterial cell surface.

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides.

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

Modeling Streptococcus pyogenes Pharyngeal Colonization in the Mouse.

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a human-restricted pathogen most commonly found in the posterior oropharynx of the human host. The bacterium is responsible for 600 million annual cases of pharyngitis globally and has been found to asymptomatically colonize the pharynxes of 4-30% of the population. As such, many studies have utilized animals as models in order to decipher bacterial and host elements that contribute to the bacterial-pharyngeal interaction and determine differences between acute infection and asymptomatic colonization. The aim of this review is to first describe both bacterial and host factors that are important for the pharyngeal persistence of GAS in humans, then to detail the bacterial and host factors that are important for colonization in murine model, and finally to compare the two in order to evaluate the strength of murine pharyngeal colonization as a model for the human-GAS pharyngeal interaction.