Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development

Abstract

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum.

Figure 1. Rgg regulators in the S. pyogenes NZ131 genome.

(A) The location of rgg2 and rgg3 and their neighboring genes is indicated by the gray box. Above the box, regions used to generate transcriptional reporters to luxAB are indicated by vertical dashed lines; these reporter constructs were inserted into the chromosome at the T12 bacteriophage attB site (tmRNA). Gene deletions are indicated below the box and correspond to genotypes listed in Table 1. (B) Alignment of pre-peptides SHP2 and SHP3 indicating identical amino acids in black font. The C-terminal eight residues are shaded.

Figure 2. Gene regulation mediated by Rgg proteins.

Overnight GAS cultures were diluted into CDM, and culture growth and reporter expression were measured over time. Data shown are representative of at least three independent experiments. Luciferase expression from (A) P_shp2 or (B) P_shp3 reporters integrated into wild-type (BNL148 and JCC157), Δrgg2 (BNL152 and JCC166), Δrgg3 (BNL149 and JCC158) and Δrgg2Δrgg3 (BNL153 and JCC167) strains indicate activator and repressor functions for Rgg2 and Rgg3, respectively. (C) Complementation of selected strains with rgg genes driven by their native promoters confirms the antagonistic effects of Rgg2 and Rgg3 on shp3 expression. Empty vector (pLZ12-Sp) was included as a control.

Figure 3. SHP stimulation of luciferase production.

For panels A and C, overnight GAS cultures were diluted into CDM and followed for growth and light production over time. For panels B and D-H, reporter strains were grown to log-phase (OD600 between 0.3 and 0.5) and then diluted 13-fold into conditioned (panel B) or fresh CDM containing 50 nM of the indicated peptide (panels D-H). For all synthetic peptide experiments, DMSO was included as a vehicle control. Data shown are representative of at least three independent experiments. (A) Expression of shp3 under its own promoter from a multi-copy plasmid (pSHP3) induces luciferase expression at both P_shp2 (triangles) and P_shp3 (squares); pLZ12-Sp is the vector-only control. (B) Luciferase-inducing activity is present in a cell-free culture supernatant prepared from mid-log phase Δrgg3 donor (JCC131) but not in wild-type (NZ131) or Δrgg3-shp3 (JCC132)-conditioned supernatants; Δrgg3-shp3 (JCC159) was used as the reporter strain, and fresh medium was included as a control. (C) Luciferase activity of a Δrgg3-shp3 strain (JCC168) expressing truncated versions of shp3 indicates the importance of the C-terminus. All truncation plasmids were derived from pSHP3; pSHP3_17-23 includes a methionine to initiate translation. (D) Synthetic full-length (sSHP3) and C-terminal eight amino acids (sSHP3-C8), but not reverse peptide (sSHP3-rev), induce luciferase activity in a Δrgg3-shp3 reporter strain (JCC168). (E) Amino acid substitutions within the C-terminus of SHP3 alter its reporter-inducing activity in a Δrgg3-shp3 reporter strain (JCC168). sSHP3-C8: DIIIIVGG; the sequences of synthetic peptides with amino acids substitutions are shown in the panel legend. (F) sSHP2-C8 and sSHP3-C8 stimulate induction and cross-induction of luciferase at P_shp2 (solid lines; BNL148) or P_shp3 (dotted lines; JCC174) in wild-type bacteria. Reverse peptide (sSHP2-C8-rev) was included as a control. (G) sSHP3-C8 induces a sustained response from the P_shp2 reporter in the wild-type strain (BNL148), while luciferase activity of a Δrgg3-shp3 strain (BNL150) wanes. (H) Synthetic C8 peptides stimulate modest luciferase activity in Δrgg2 strains with integrated P_shp2 (solid lines; BNL152) or P_shp3 (dotted lines; JCC175) reporters.

Figure 4. Rgg3 binds to shp3 and shp2 promoter regions but can be disrupted by cognate SHPs.

EMSA analysis was used to test the ability of recombinant Rgg3 to bind to target promoters and the effect of SHPs on this binding. (A) Rgg3 binds in a concentration-dependent manner to the promoters of both shp3 (upper panel) and shp2 (lower panel). (B) Binding to P_shp3 by Rgg3 is disrupted by the addition of pure (>95%) sSHP3-C8, and to a lesser extent by pure sSHP2-C8. Both peptides had a smaller effect on disruption of binding to P_shp2. Pure sSHP3-C8-rev was included as a control and did not affect binding to either probe. (C) Binding of P_shp3 and P_shp2 by Rgg3 is specific and can be disrupted by addition of 5-fold molar excess of unlabeled specific probe but not an equivalent amount of unlabeled rRNA probe (nonspecific). No binding of a negative control rRNA probe (P_rRNA) was detected. All reactions contained 10 nM probe, and 50 nM unlabeled competitor where indicated. Protein concentration is indicated above each lane in all panels.

Figure 5. Proteins previously identified in peptide signaling circuits are also important for Rgg-dependent gene regulation.

The oligopeptide permease and Eep protease are important for SHP signaling. (A) A ΔoppD mutant (JCC163) fails to respond to 50 nM sSHP3-C8 unless an oppD complementation vector, pOppD, is present. A wild-type reporter strain carrying empty vector (JCC157 (pLZ12-Sp)) was included as a control. (B) Expression of the C-terminus of SHP3 from a plasmid (pSHP3_15-23) bypasses the need for the permease in the ΔoppD mutant (JCC163). Wild-type (JCC157) and the Δrgg3 (JCC158) reporter strains carrying the vector (pLZ12-Sp) alone are included for reference. (C) The Eep metalloprotease contributes to signal production, as determined by measuring luciferase activity of the Δrgg3Δeep mutant (JCC161) expressing eep from a multi-copy plasmid (pEep) or carrying empty vector (pLZ12-Sp); Δrgg3 (JCC158) was included as a control. (D) Overexpression of shp3 (pSHP3) restores light induction in the Δeep mutant (JCC160) to near wild-type (JCC157) levels. Data shown are representative of experiments performed at least three times.

Figure 6. Influence of Rgg regulators on biofilm production.

SHPs can enhance biofilm biogenesis via the Rgg2/3 regulatory circuit and counter biofilm disassembly promoted by RopB. Crystal violet staining was performed to measure biofilm biomass of NZ131 derivatives grown 48 hours in 24-well polystyrene plates with (dark gray bars) or without (light gray bars) the addition of 50 nM sSHP3-C8. Levels of biofilm production were normalized to that of untreated NZ131 (wild type). Error bars indicate standard error from a minimum of three independent experiments.

Figure 7. Model for Rgg2/3-SHP2/3 regulation.

The pro-SHP peptide is subject to processing by Eep and is secreted by an unknown transporter. Once exported, a secondary processing step may produce active SHP (and, as seen in Figure 5D, process sSHP3). The oligopeptide permease (Opp) imports SHP to the cytoplasm where the pheromone directly engages Rgg proteins. In the absence of SHP, Rgg3 represses transcription of shp promoters. De-repression occurs upon Rgg3 binding of either peptide, whereas Rgg2-SHP interactions facilitate transcription activation.