Quorum quenching of Streptococcus mutans via the nano-quercetin-based antimicrobial photodynamic therapy as a potential target for cariogenic biofilm

Abstract

Background: Quorum sensing (QS) system can regulate the expression of virulence factors and biofilm formation in Streptococcus mutans. Antimicrobial photodynamic therapy (aPDT) inhibits quorum quenching (QQ), and can be used to prevent microbial biofilm. We thereby aimed to evaluate the anti-biofilm potency and anti-metabolic activity of nano-quercetin (N-QCT)-mediated aPDT against S. mutans. Also, in silico evaluation of the inhibitory effect of N-QCT on the competence-stimulating peptide (CSP) of S. mutans was performed to elucidate the impact of aPDT on various QS-regulated genes.

Methods: Cytotoxicity and intracellular reactive oxygen species (ROS) generation were assessed following synthesis and confirmation of N-QCT. Subsequently, the minimum biofilm inhibitory concentration (MBIC) of N-QCT against S. mutans and anti-biofilm effects of aPDT were assessed using colorimetric assay and plate counting. Molecular modeling and docking analysis were performed to confirm the connection of QCT to CSP. The metabolic activity of S. mutans and the expression level of various genes involved in QS were evaluated by flow cytometry and reverse transcription quantitative real-time PCR, respectively.

Results: Successful synthesis of non-toxic N-QCT was confirmed through several characterization tests. The MBIC value of N-QCT against S. mutans was 128 μg/mL. Similar to the crystal violet staining, the results log₁₀ CFU/mL showed a significant degradation of preformed biofilms in the group treated with aPDT compared to the control group (P < 0.05). Following aPDT, metabolic activity of S. mutans also decreased by 85.7% (1/2 × MBIC of N-QCT) and 77.3% (1/4 × MBIC of N-QCT), as compared to the control values (P < 0.05). In silico analysis showed that the QCT molecule was located in the site formed by polypeptide helices of CSP. The relative expression levels of the virulence genes were significantly decreased in the presence of N-QCT-mediated aPDT (P < 0.05).

Conclusions: The combination of N-QCT with blue laser as a QQ-strategy leads to maximum ROS generation, disrupts the microbial biofilm of S. mutans, reduces metabolic activity, and downregulates the expression of genes involved in the QS pathway by targeting genes of the QS signaling system of S. mutans.

Keywords: Antimicrobial photodynamic therapy; Competence-stimulating peptide; Dental caries; Quorum sensing; Streptococcus mutans.

Fig. 1

Characterization of synthesized nano-QCT (N-QCT): a Field emission scanning electron microscope (FESEM) image (Scale bar = 2 μm), b The size distribution profile of N-QCT, c Elemental mapping of N-QCT

Fig. 2

Ultraviolet–visible spectroscopy of N-QCT

Fig. 3

Effects N-QCT on cell viability of human gingival fibroblast cells: A Inverted light microscope images of treated cells with varying concentrations of N-QCT (magnification × 40); a Control (Untreated cells), b 128 μg/mL, c 256 μg/mL, and d 512 μg/mL, B. The mean percentage viability versus concentrations of N-QCT using MTT assay

Fig. 4

Anti-biofilm effect of N-QCT at the different concentrations against S. mutans. *Significantly different from the control group (no treatment), P < 0.05

Fig. 5

Effects of different treatment groups on the production of intracellular reactive oxygen species (ROS) in S. mutans. *Significantly different from the control group (no treatment), P < 0.05

Fig. 6

Biofilm disruption effects of different treatment groups against S. mutans. *Significantly different from the control group (no treatment), P < 0.05

Fig. 7

Effect of different treatment groups on cell viability of S. mutans. *Significantly different from the control group (no treatment), P < 0.05

Fig. 8

Effect of different treatment groups on biofilm metabolic activity in S. mutans; A measured by flow cytometry: a Untreated biofilm cells, b 0. 2% CHX, c N-QCT at 1/2 × MBIC, d N-QCT at 1/4 × MBIC, e Blue laser light, f aPDT using 1/2 × MBIC of N-QCT, and g aPDT using 1/4 × MBIC of N-QCT. B Percentage of biofilm living cells. *Significantly different from the control group (no treatment), P < 0.05

Fig. 9

The PPI network of CSP protein identified in S. mutans

Fig. 10

Molecular modeling of QCT-bound CSP. The residues of CSP and QCT are represented using a ball-and-tinctorial stick physical model: a Binding site of QCT in CSP, b Conformation of QCT in the binding site of CSP. The bonds between the ligand and the protein are presented by blue dashed lines

Fig. 11

Effect of different treatment groups on the expression of various genes involved in quorum sensing of S. mutans: a comA, b comB, c comDE, and d gtfB. *Significantly different from the control group (no treatment), P < 0.05