Exploiting Conserved Quorum Sensing Signals in Streptococcus mutans and Streptococcus pneumoniae

Abstract

Bacterial species of the Streptococcus genera are considered either commensal bacteria or potential pathogens, according to their metabolic evolution and production of quorum sensing (QS)-controlled virulence factors. S. mutans, in particular, has become one of the best-studied examples of bacteria that are able to get along or cheat commensal species, even of the same genera. S. mutans and S. pneumoniae share homolog QS pathways and a competence stimulating peptide (CSP) for regulating bacteriocin production. Intriguingly, the abundance of S. pneumoniae and S. mutans alternates in complex microbial communities, thus opening the role for the fratricide communication of homolog QS systems. Since the inhibition of the QS has been proposed in treating bacterial infections, in this study, we designed and synthesized analogs of S. pneumoniae CSP with precise residual modifications. We reported that S. pneumoniae CSP analogs reduced the expression of genes involved in the QS of S. mutans and biofilm formation without affecting bacterial growth. The CSP analogs inhibited bacteriocin production in S. mutans, as reported by co-cultures with commensal bacteria of the oral cavity. The peptide CSP1AA, bearing substitutions in the residues involved in QS receptor recognition and activation, reported the most significant quorum-quenching activities. Our findings provide new insights into specific chemical drivers in the CSP sequences controlling the interconnection between S. mutans and S. pneumoniae. We think that the results reported in this study open the way for new therapeutic interventions in controlling the virulence factors in complex microbial communities such as the oral microbiota.

Keywords: bacteriocin; competence-stimulating peptide; microbiota; peptide synthesis; quorum quenching.

Figure 1

Effects of peptides on prokaryotic and eukaryotic cell growth and viability. (a) Cultures of S. mutans (1 × 10⁶ CFU/mL) were incubated with 20 µM of peptides or the vehicle. Bacterial growth was monitored for 36 h by measuring the absorbance at 620 nm (A620 nm). Experiments were performed three times. Data are reported as the mean ± SEM. (b) Human primary gingival fibroblasts (HGFs) were cultured with 20 μM peptides for 24 h. Cell viability was assessed by an MTT assay. Experiments were performed two times. Data are reported as the percentage of cell viability calculated over the control (vehicle).

Figure 2

Peptides interfere in S. mutans biofilm formation. (a) Cultures of S. mutans were seeded in 96-well plates and incubated in 1:10 BHI:AS medium at 37 °C. Adhering cells were determined at the specified time points after incubation for 20 min with resazurin 0.01%. Biofilm formation was evaluated by measuring the relative fluorescence units (RFU) using a fluorimeter (Ex = 530–570 nm, Em = 590–620 nm). Experiments were performed three times. Data are reported as the mean ± SEM. (b) Percentage of residual biofilm. S. mutans was incubated with peptides (20 μM) or chlorhexidine (CHX, 0.63 μg/mL) for 72 h. Stimuli were renewed every 24 h. Biofilms were evaluated by crystal violet staining. Cultures incubated with the vehicle were assigned as 100% biofilm. Data are reported as the mean ± SEM of three independent experiments. (c) To evaluate the effects on mature biofilm, S. mutans was incubated for 48 h. CHX (0.63 μg/mL) and peptides (20 μM) were added, and biofilms were evaluated 24 h later by crystal violet staining. Cultures incubated with the vehicle were assigned as 100% biofilm. Data are reported as the mean ± SEM of three independent experiments. * denotes p < 0.05 vs. the vehicle; ° denotes p < 0.05 vs. CSP1.

Figure 3

Bactericidal activity of synthesized peptides. (a) Cultures of S. mutans were incubated with the vehicle or CSP1 (20 μM) and spotted on 0.7% agar media on the surface of agar plates previously inoculated with S. mitis, S. oralis, or S. sanguinis. Plates were incubated under microaerophilic conditions at 37 °C for 16 h. Cultures of S. mutans were incubated with CSP1 and analogs (20 µM) and plated as described above. Cultures were spotted on agar plates inoculated with S. mitis (b), S. oralis (c), or S. sanguinis (d). Plates were incubated under microaerophilic conditions at 37 °C for 16 h. S. mutans cultures were spotted on agar plates inoculated with F. nucleatum and incubated under anaerobic conditions at 37 °C for 16 h (e). At the end of the incubations, the diameters of clear areas indicating bactericidal effects were measured. Data are reported as the mean ± SEM of at least five independent experiments, counting ten spots for each experimental group. * denotes p < 0.05 vs. CSP1.

Figure 4

Effects of synthesized peptides on QS-related gene expression. (a) Cultures of S. mutans were incubated with the vehicle or CSP1 (20 μM) for 2–24 h. Bacteria were collected and subjected to RNA extraction. Expression of the bip gene was investigated by qPCR. (b) Cultures of S. mutans were incubated with CSP1 with or without the synthesized peptides (each one at 20 μM). The expression of the bip gene was investigated 8 h later by qPCR. (c) S. mutans were diluted in BHI containing 0.2% w/v sucrose and AS (1:10) media to obtain biofilm. Cultures were added with the vehicle or CSP1 (20 μM) and incubated for 2–24 h. Adherent bacteria were collected and subjected to RNA extraction. Expression of the vicK gene was investigated by qPCR. (d) S. mutans was incubated with CSP1 with or without synthesized peptides (each one at 20 μM) and cultured to obtain biofilm. The expression of the vicK gene was investigated 16 h later by qPCR. Planktonic cultures of S. mutans were incubated for 8 h with CSP1 with or without synthesized peptides (each one at 20 μM). Bacteria were collected and subjected to RNA extraction. Expressions of comD (e) or comE (f) genes were investigated by qPCR. The results are reported as the log₂ of relative gene expression. The data are expressed as the mean ± SEM of three independent experiments. * denotes p < 0.05 vs. CSP1.

Figure 5

Schematic representation of the main experimental approaches and results obtained in the study. As indicated, S. mutans in planktonic or biofilm cultures was incubated with the natural CSP1 of S. pneumoniae or CSP1 analogs at different times. Cultures were then subjected to the measurement of cell viability by spectrophotometry, bacteriocin production by co-culture experiments, and the measurement of the zone of inhibition, biofilm evaluation, and qPCR to evaluate the expression of genes involved in the QS system.