Ursolic Acid Targets Glucosyltransferase and Inhibits Its Activity to Prevent Streptococcus mutans Biofilm Formation

Abstract

Streptococcus mutans (S. mutans), the prime pathogen of dental caries, can secrete glucosyltransferases (GTFs) to synthesize extracellular polysaccharides (EPSs), which are the virulence determinants of cariogenic biofilms. Ursolic acid, a type of pentacyclic triterpene natural compound, has shown potential antibiofilm effects on S. mutans. To investigate the mechanisms of ursolic acid-mediated inhibition of S. mutans biofilm formation, we first demonstrated that ursolic acid could decrease the viability and structural integrity of biofilms, as evidenced by XTT, crystal violet, and live/dead staining assays. Then, we further revealed that ursolic acid could compete with the inherent substrate to occupy the catalytic center of GTFs to inhibit EPS formation, and this was confirmed by GTF activity assays, computer simulations, site-directed mutagenesis, and capillary electrophoresis (CE). In conclusion, ursolic acid can decrease bacterial viability and prevent S. mutans biofilm formation by binding and inhibiting the activity of GTFs.

Keywords: biofilms; extracellular polysaccharides; glucosyltransferases; mechanism; ursolic acid.

Figure 1

Effects of ursolic acid on biofilms. (A): Percentage of bacterial viability within biofilms detected by XTT assay; (B): Inhibitory percentage of ursolic acid on biofilm formation detected by crystal violet staining. Data are presented as the mean±standard deviation. ^*p<0.05 and ^**p<0.01. (C): Effects of ursolic acid on biofilm structure. SYTO 9: green fluorescence, which stains both the dead and live bacterial cells; PI: red fluorescence, which stains dead bacterial cells. Control: ursolic acid untreated biofilms; CH: chlorhexidine (the positive control).

Figure 2

Effect of ursolic acid on glucosyltransferase (GTF) activities. GTFs were crudely extracted from bacterial culture medium and were then used to test the effect of ursolic acid on its activities by using an in vitro enzymatic reaction system. The final concentration of ursolic acid ranged from 0.04 to 0.12mg/ml. Extracellular polysaccharides (EPSs), including soluble and insoluble glucans, which are the catalytic products of GTFs, were measured as indicators of GTF activity. The data are presented as the mean ± standard deviation.

Figure 3

Interaction between ursolic acid and GTF-SI. (A-C) Binding model of ursolic acid with GTF-SI and its variants simulated by Gold software. (A) Wild-type GTF-SI; (B) GTF-SI variant A; (C) GTF-SI variant B. Ursolic acid, as the ligand, is shown in green. The protein is shown as a cartoon with pink color. The yellow dotted lines represent hydrogen bonds. (D-F): Interaction between ursolic acid and GTF-SI measured by CE assay. The black line represents the migration time of GTF-SI and its variants, whereas the red line is the migration time of GTF-SI or its variants together with ursolic acid. (D) Wild-type GTF-SI; (E) GTF-SI variant A; (F) GTF-SI variant B. Arrows point to the peak of the protein (black)/protein and ursolic acid complex (red).

Figure 4

Effect of ursolic acid on GTF catalyzed products. (A) Inhibitory percentage of ursolic acid on EPS production determined by phenol/H₂SO₄ analysis. After incubation with ursolic acid for 16h, the water-soluble glucans and water-insoluble glucans were prepared from bacterial culture using the method described above. Phenol/H₂SO₄ analysis was used to quantify the two types of EPSs. Data are presented as the mean ± standard deviation; (B) Effect of ursolic acid on fructose production by GTFs determined via CE assay. Data are presented as the mean ± standard deviation. ^*p<0.05 and ^**p<0.01.