Cariogenic virulence factors of Streptococcus mutans include acidogenicity, aciduricity, and extracellular polysaccharides (EPS) synthesis. The de novo designed antimicrobial peptide GH12 has shown bactericidal effects on S. mutans, but its interaction with virulence and regulatory systems of S. mutans remains to be elucidated. The objectives were to investigate the effects of GH12 on virulence factors of S. mutans, and further explore the function mechanisms at enzymatic and transcriptional levels. To avoid decrease in bacterial viability, we limited GH12 to subinhibitory levels. We evaluated effects of GH12 on acidogenicity of S. mutans by pH drop, lactic acid measurement and lactate dehydrogenase (LDH) assay, on aciduricity through survival rate at pH 5.0 and F₁F₀-ATPase assay, and on EPS synthesis using quantitative measurement, morphology observation, vertical distribution analyses and biomass calculation. Afterwards, we conducted quantitative real-time PCR to acquire the expression profile of related genes. GH12 at 1/2 MIC (4 mg/L) inhibited acid production, survival rate, EPS synthesis, and biofilm formation. The enzymatic activity of LDH and F₁F₀-ATPase was inhibited, and ldh, gtfBCD, vicR, liaR, and comDE genes were significantly downregulated. In conclusion, GH12 inhibited virulence factors of S. mutans, through reducing the activity of related enzymes, downregulating virulence genes, and inactivating specific regulatory systems.

Background:Targeting Streptococcus mutans, the principle cariogenic bacterium, could prove to be an effective means of preventing dental caries. A de novo designed antimicrobial peptide, GH12, has shown bactericidal effects on S. mutans and inhibitory effects on virulence and regulatory systems of S. mutans. However, the effects of GH12 on caries remain to be elucidated.Objectives: The objectives of this study were to evaluate the direct effects of GH12 on caries and numbers of S. mutansin vivo.Design: The Keyes score was evaluated in a rodent model and in vivo S. mutans was quantified by qPCR. To further interpret the in vivo anticaries efficacy, an in vitro biofilm model using short-term treatments with GH12 mimicked treatments in vivo was adopted. Results: In vivo data showed that GH12 at 8 mg/L reduced the incidence and severity of caries. Furthermore, GH12-treated animals had less S. mutans infection. In vitro data revealed that GH12 reduced the number of S. mutans within the biofilm, as well as reducing lactic acid and water-insoluble EPS synthesis of S. mutans biofilms. Afterwards, all rats showed no significant difference in weight gain, and no sign of harm to the mucosa, indicating that GH12 possessed good biocompatibility in vivo.Conclusion: Due to the combined inhibitory effects of GH12 on the biomass and cariogenic properties of S. mutans biofilms, the population of S. mutans in vivo was reduced, residual S. mutans biofilms were less acidic and compact, and thereby the onset and development of caries were arrested. Such findings collectively certified the clinical prospects for GH12.

Amelogenin and its various derived peptides play important roles in promoting biomimetic mineralization of enamel. Previously, an amelogenin-derived peptide named QP5 was proved to be able to repair demineralized enamel. The objective here was to interpret the mechanism of QP5 by elucidating the specific function of each domain for further sequence and efficacy improvement. Peptide QP5 was separated into domains (QPX)5 and C-tail. (QPX)3 was also synthesized to investigate how QPX repeats affect the mineralization process. Circular dichroism spectroscopy showed that two (QPX) repeats adopted a β-sheet structure, while C-tail exhibited a disordered structure. (QPX)5 showed more absorption in confocal laser scanning microscopy observation and a higher K value in Langmuir adsorption isotherms compared to C-tail, while (QPX)3 with better hydropathy had greater adsorption capability than (QPX)5. Meanwhile, calcium consumption kinetics, transmission electron microscopy and selected area electron diffraction indicated that (QPX)5, C-tail and (QPX)3 had similar inhibitory effects on the spontaneous calcium consumption and the morphology of their nucleation products were alike, while QP5 had a greater inhibitory effect than them and induced elongated plate-like crystals. X-Ray diffraction further showed that both C-tail and (QPX)3 had greater potential in improving the apatite crystal orientation degree. In conclusion, (QPX)5 was the major adsorption region, both (QPX)5 and C-tail inhibited the nucleation, and C-tail contributed more to improve the HAP orientation degree, so QP5 could exert a significant remineralization effect. By reducing two repeats, (QPX)3 showed higher hydropathicity than (QPX)5 and achieved higher binding affinity, and it was more potential in improving the HAP orientation degree with lower economic cost.

Sulforaphene (SFE) is a common nutritional supplement with antibacterial, anti-cancer, and anti-inflammatory effects. However, the effects of SFE on the cariogenicity of Streptococcus mutans and dental caries have not been reported. The objectives of this study were to investigate the caries-controlling potential of SFE. The effects of SFE on S. mutans were investigated using the broth microdilution method, crystal violet staining, SEM observation, acid tolerance assays, lactic acid quantification, and polysaccharide measurements. A rat caries model was established to evaluate the caries-controlling effects and biocompatibility of SFE in vivo. SFE inhibited S. mutans growth and biofilm formation. Furthermore, SFE restrained the cariogenic properties of S. mutans, including its acid production, acid tolerance, and extracellular polysaccharide production, without affecting the bacterial viability at sub-inhibitory levels. In the rat caries model, SFE significantly arrested the onset and development of dental caries. Moreover, no visible hemolytic phenomenon or cytotoxicity was detected in the SFE groups. After four weeks of SFE treatment, all rats remained in apparent good health with no significant differences in weight gain; their hemogram and biochemical parameters were normal; no pathological changes were observed in the oral mucosa, liver, or kidneys. In conclusion, SFE was safe and inhibited the development of caries effectively.

The acidogenicity of Streptococcus mutans is important for caries development. The antimicrobial peptide GH12 can affect the integrity of cellular membranes and the virulence factors of S. mutans. Combining GH12 and NaF (GF) efficiently controlled the development of caries, but its mechanisms remained unrevealed. This research intended to verify the effects of GF on the acidogenicity of S. mutans biofilms and to reveal the mechanisms. Lactic acid production assays and pH monitoring assays were conducted to investigate the regulatory effects of the GF treatment on the acidogenicity of S. mutans biofilms. RNA sequencing and bioinformatics analyses were conducted to screen the transcriptional profile affected by the GF treatment. The results demonstrated the GF group had significantly less lactic acid and maintained the broth’s pH values above 5.0 for longer times. Thereafter, GO/KEGG enrichment analyses and RT-qPCR validation revealed that the GF treatment mainly restrained the expression of genes related to the carbohydrates’ internalization and metabolism. Compared with NaF, the GF treatment further downregulated the carbohydrates transportation genes. Moreover, compared with GH12, the GF treatment affected the membrane’s integrity more significantly. Generally, GF treatment could arrest the acidogenicity of S. mutans biofilms, mainly through suppressing carbohydrates transportation and inhibiting overall metabolism.