Inhibition of Streptococcus mutans adhesion and biofilm formation with small-molecule inhibitors of sortase A from Juniperus chinensis

Abstract

Background: Streptococcus mutans, an important Gram-positive pathogen in dental caries, uses sortase A (SrtA) to anchor surface proteins to the bacterial cell wall, thereby promoting biofilm formation and attachment to the tooth surface.

Design: Based on activity-guided separation, inhibitors of S. mutans SrtA were isolated from Juniperus chinensis and identified through combined spectroscopic analysis. Further effects of isolated SrtA inhibitor on S. mutans were evaluated on bacterial aggregation, adherence and biofilm formation.

Results: Six compounds (1-6) were isolated from the dried heartwood of J. chinensis. A novel compound designated 3',3"-dihydroxy-(-)-matairesinol (1) was identified, which exhibited potent inhibitory activity toward S. mutans SrtA (IC₅₀ = 16.1 μM) without affecting microbial viability (minimum inhibitory concentration > 300 μM). The results of subsequent bioassays using compound 1 indicated that this compound inhibits S. mutans aggregation, adhesion and biofilm formation on solid surfaces by inhibiting SrtA activity. The onset and magnitude of inhibition of adherence and biofilm formation in S. mutans treated with compound 1 at 4× the SrtA IC₅₀ are comparable to the behaviors of the untreated srtA-deletion mutant.

Conclusion: Our findings suggest that small-molecule inhibitors of S. mutans SrtA may be useful for the prevention of dental plaque and treatment of dental microbial diseases.

Keywords: Adhesion; Juniperus chinensis; Streptococcus mutans; biofilm formation; sortase A inhibitor.

Figure 1.

Structures of compounds 1–6 from Juniperus chinensis: 3’,3”-dihydroxy-(−)-matairesinol (1), (−)-matairesinol (2), quercetin (3), 4,6-dihydroxy-2-methoxyacetophenone (4), 5-hydroxyhinokitiol (5), and juniperone A (6).

Figure 2.

Key correlations found in the ¹H correlated spectroscopy (bold) and heteronuclear multiple bond correlation (arrows) experiments for compound 1.

Figure 3.

Inhibitory effects of compound 1 on saliva-induced aggregation of Streptococcus mutans NG8. Cells with approximate optical density of 1.0 at 700 nm were incubated aerobically at 37°C with human saliva for 2 h. (A) Saliva-induced aggregation of S. mutans NG8 (wild type), srtA-deletion mutant (ΔsrtA), and srtA-complemented mutant (ΔsrtA + srtA). NG8-buffer refers to the aggregation assay performed with S. mutans NG8 in the absence of saliva. (B) S. mutans NG8 treated with compound 1. The aggregation assay was performed with S. mutans NG8 in the presence of 16.1 μM (1× IC₅₀), 32.2 μM (2× IC₅₀) and 64.4 μM (4× IC₅₀) compound 1. Each point indicates the mean ± standard deviation of three independent experiments. Results were compared using two-way analysis of variance with the post-hoc Dunnett’s test. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus controls.

Figure 4.

Inhibitory effects of compound 1 on Streptococcus mutans adherence to saliva-coated hydroxyapatite beads (S-HAs). Attachment of S. mutans NG8, srtA-deletion mutant (ΔsrtA), and srtA-complemented mutant (ΔsrtA+srtA) was induced for 90 min at 37°C under aerobic condition, followed by dispersal via sonication (50 W, 30s) after three washes, and then colony-forming unit counting on Mitis-Salivarius agar (3.2 mg/mL bacitracin) after incubation for 48 h at 37°C. The concentration of compound 1 ranged from 1× the IC₅₀ (16.1 μM) to 4× the IC₅₀ (64.4 μM). Data are presented as the mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001 based on Student’s t-test).

Figure 5.

Inhibition of Streptococcus mutans biofilm formation by compound 1. Biofilm formation assays were performed using a polystyrene 96-well plate (A) and artificial resin teeth (B) at 37°C for 24 h under aerobic condition. The concentration of compound 1 ranged from 1× the IC₅₀ (16.1 μM) to 4× the IC₅₀ (64.4 μM) and biomass of the biofilm was measured via 0.1% safranin staining. 0.1% NaF was used as a positive control. Data are presented as the mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001 based on Student’s t-test).