Berberine chloride hydrate impairs Streptococcus mutans biofilm formation via inhibiting sortase A activity

Abstract

Dental caries is a biofilm-associated chronic progressive disease, results from the dissolution of mineralized tooth tissue by acidic generated from bacterial sugar fermentation. S. mutans, a prominent pathogen of dental caries, is acknowledged for its role in cariogenic biofilm formation, utilizing Sortase A (SrtA) to catalyse surface proteins, thus promoting biofilm formation. In our previous studies, the inhibitory effect of the berberine chloride hydrate (BH) on S. mutans biofilms was confirmed. Here, we further investigate the influence of BH on S. mutans biofilm-induced bovine enamel caries model and explore the effect of BH on S. mutans SrtA activity. We found that BH inhibited S. mutans biofilm formation in bovine enamel model, leading to a reduction in demineralization. Furthermore, we identified and characterized SrtA, which might catalyze SpaP of S. mutans to form fibrillar amyloid aggregates. Our findings showed that BH inhibited SrtA activity by binding to essential amino acid residues LEU-111, MET-123, and ARG-213. BH inhibited amyloid fibers formation by downregulating the expression of srtA gene, thus disrupting S. mutans biofilm formation. Taken together, our study provides new insight into the mechanism of antibiofilm activity of BH and reveals great potential for anticaries clinical applications.

Fig. 1. BH shows good antimicrobial effect against S. mutans biofilms.

A Effect of BH on S. mutans free-floating bacterial cells by OD 600 measurement. B Effects of BH on living S. mutans free-floating bacterial cells by CFU counts. C Effects of BH on S. mutans biofilm formation on culture plates detected by crystal violet staining. D Effects of BH on metabolic activity of pre-established S. mutans biofilm on culture plates. E Effects of BH on living cells within S. mutans pre-established biofilm on culture plates by CFU counts. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 2. BH shows anti-biofilm formation effects on bovine enamel blocks.

A Representative SEM images of S. mutans biofilms on the bovine enamel. B Representative CLSM images of S. mutans biofilms on the bovine enamel. C The proportion of dead bacteria with live bacteria within the biofilms (n = 9). D Quantitative measurement of total biomass in the biofilms (n = 9). * p < 0.05.

Fig. 3. The anti-demineralization effect of BH against cariogenic model induced by S. mutans biofilms.

A Representative images from micro-CT- of bovine enamel undergoing 7-day biofilm-induced demineralization. Regions with high density represent sound enamel, whereas low-density shadows indicate lesions similar to caries. B The calculated demineralization depths (n = 9). C The calculated mineral densities (n = 9). * p < 0.05, ** p < 0.01.

Fig. 4. BH directly targeted SrtA to inhibit the activity of SrtA.

A The inhibitory effect of BH on SrtA activity. B Predicted interaction between BH and SrtA based on molecular docking. C The RMSD analysis of BH with SrtA. D The RMSF analysis of BH with SrtA. E SASA analysis of BH with SrtA. F The Rg analysis of BH with SrtA. G The energy decomposition analysis of the interactions between BH and SrtA.

Fig. 5. BH reduces amyloid fibrils formation by inhibiting the srtA gene.

A TEM images of amyloid fibrils in △srtA biofilms and S. mutans biofilms with or without BH treatment. Red arrows indicate amyloid fibrils. B Quantification of srtA and spaP mRNA expression levels in bacterial cells within biofilms treated with BH using qRT-PCR. C Fluorescence intensity of biofilms was used to record the influence of BH on amyloid fibrils. D Representative images of △srtA biofilms and S. mutans biofilms with or without BH treatment imaged by CLSM. E The total biomass within the biofilms. F The thickness of the biofilms. * p < 0.05, ** p < 0.01, *** p < 0.001.