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

Abstract

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.

Keywords: CHAP domain; S-layers; biofilms; cationic peptides; murein hydrolases; pheromone; surface protein; wall polysaccharides.

FIG 1

Biofilm formation experiments carried out following systematic mutation of genes regulated by the Rgg2/3 QS system. (A) Targets of Rgg2/3 regulation revealed by previous transcriptional analysis. Rgg2 and Rgg3 antagonistically compete to regulate two target promoters, P_shp3 and P_shp2, which promote transcription of their corresponding pheromone and downstream genes (17, 18). (B) Representative image showing biofilm formation with either the SHP2-C8 or SHP3-C8 pheromone. (C) Biofilm formation was examined with and without pheromone in strains with mutations in the operons controlled by the QS system, and the spy49_0412 to spy49_0414c region was complemented on a p7INT plasmid under the control of its native, pheromone-responsive promoter. (D) Genes in the spy49_0412 to spy49_0414c region were systematically knocked out, and spy49_0414c was reintroduced on a p7INT plasmid under the control of its native, pheromone-responsive promoter. (E) Expression of the spy49_0414c gene from a constitutive P_recA promoter generates an increase in biofilm formation in the absence of QS signaling. Quantification of biofilm formation was performed in both a wild-type background and a Δrgg2 mutant background; the latter was unable to activate the QS system. The error bars represent the standard deviations from three independent experiments, each carried out in duplicate.

FIG 2

Pheromone-dependent lysozyme resistance is attributable to StcA across GAS serotypes. (A) Pheromone-dependent challenge in 2 mg ml⁻¹ lysozyme after pretreatment with 100 nM SHP-C8 or rev-SHP, which is the control peptide, in WT and ΔstcA strains. (B) Pheromone-dependent challenge in 2 mg ml⁻¹ lysozyme in a strain containing the stcA gene on plasmid pFED761 under the control of the constitutive promoter P_recA and a strain containing the empty pFED761 vector. Error bars indicate standard deviations. (C) Lysozyme challenge of isolate MGAS315 of the M3 serotype after pretreatment with 100 nM SHP-C8 or rev-SHP. The concentration of lysozyme used for MGAS315 challenge was 50 mg ml⁻¹. The mean and standard deviation represent biological duplicates done in technical duplicate. (D) Lysozyme challenge of isolate MGAS10394 of the M6 serotype after pretreatment with 100 nM SHP-C8 or Rev-SHP peptide. The concentration of lysozyme used for MGAS10394 challenge was 20 mg ml⁻¹. The bars represent the standard deviations of biological duplicates done in technical duplicate.

FIG 3

(A) The 89-amino-acid sequence of StcA, showing the predicted 20-amino-acid secretion signal in red, the 15 positively charged amino acids in blue, and the highlighted polyproline region, with the flanking basic lysines underlined. (B) Alignment of the Rgg2 regulons of GAS and S. canis. In S. canis FSL-Z3, a putative transglutaminase is encoded downstream of the SHP2 pheromone. Alignment of the GAS NZ131 IGR shows high homology to the transglutaminase. (C) Biofilm formation of the wild type and nine cysteine proteinase deletion mutants. Quantification of biofilm formation in three experiments in the presence or absence of the SHP3-C8 pheromone, with the bars displaying standard deviations.

FIG 4

Investigation of Isp2-StcA interaction. (A) Pheromone-dependent challenge in 2 mg ml⁻¹ lysozyme after pretreatment with 100 nM SHP-C8 or rev-SHP in WT and Δisp2 strains. Bars display standard deviations for biological duplicates done in technical duplicate. (B) FITC-labeled cell wall was incubated with recombinant Isp2, and cleavage of the cell wall was observed over the course of 1 h. Addition of 10 nM recombinant StcA resulted in no significant change to the hydrolytic capability of Isp2. (C) Cell wall was incubated with lysozyme in the same manner, with or without the addition of 10 nM StcA. AU, arbitrary units.

FIG 5

In vitro aggregation of purified GAS cell wall upon addition of recombinant StcA. Remazol brilliant blue-labeled GAS cell wall with the addition of buffer or 10 nM recombinant StcA after shaking at room temperature for 30 min. (B) Aggregation of Remazol brilliant blue-labeled GAS cell wall with StcA was disrupted with 250 mM NaCl. (C) Cell wall treated with hydrofluoric acid (HF) to remove lipoteichoic acid was incubated with 10 nM StcA before and after treatment. Each experiment was performed in triplicate, with bars representing standard deviations.

FIG 6

Immunofluorescence microscopy. WT and ΔstcA cells were induced with 100 nM SHP-C8 pheromone for 1 h before being fixed with paraformaldehyde. The cells were then stained with a rabbit polyclonal anti-StcA antibody and an Αlexa Fluor 488-coupled anti-rabbit goat immunoglobulin antibody. WT and ΔstcA cell imaging showing anti-StcA binding in the green fluorescent protein channel and DAPI (cytoplasm) in the blue channel is shown. Magnifications, ×126.

FIG 7

Cytochrome c binding assay. NZ131 and mutant cells were grown to an OD₆₀₀ of 0.3 with 50 nM SHP-C8 pheromone or 50 nM rev-SHP pheromone before incubation with 1 mg ml⁻¹ cytochrome c. The percentage of the total bound cytochrome c was quantified spectrophotometrically at 535 nm. Asterisks indicate conditions that were significant at a P value of <0.01. Experiments were performed in biological triplicate, and bars represent standard deviations.