Hydrogel-Encapsulated Biofilm Inhibitors Abrogate the Cariogenic Activity of Streptococcus mutans

Abstract

We designed and synthesized analogues of a previously identified biofilm inhibitor IIIC5 to improve solubility, retain inhibitory activities, and to facilitate encapsulation into pH-responsive hydrogel microparticles. The optimized lead compound HA5 showed improved solubility of 120.09 μg/mL, inhibited Streptococcus mutans biofilm with an IC₅₀ value of 6.42 μM, and did not affect the growth of oral commensal species up to a 15-fold higher concentration. The cocrystal structure of HA5 with GtfB catalytic domain determined at 2.35 Å resolution revealed its active site interactions. The ability of HA5 to inhibit S. mutans Gtfs and to reduce glucan production has been demonstrated. The hydrogel-encapsulated biofilm inhibitor (HEBI), generated by encapsulating HA5 in hydrogel, selectively inhibited S. mutans biofilms like HA5. Treatment of S. mutans-infected rats with HA5 or HEBI resulted in a significant reduction in buccal, sulcal, and proximal dental caries compared to untreated, infected rats.

Figure 1:

Chemical structures of G43 and IIIC5.

Figure 2:

Methoxy and hydroxy aurones

Figure 3:

Planktonic growth inhibitory activities of chalcones (3a-f), methoxyaurones (MA1–6), and hydroxyaurones (HA2–6). A) S. mutans UA159 were co-incubated with 50 μM of chalcones 3a-f and the planktonic growth was measured at OD₄₇₀. B) S. mutans UA159 were co-incubated with 50 μM of methoxy aurones, MA1–6 and the planktonic growth was measured at OD₄₇₀. C) S. mutans UA159 were co-incubated with 50 μM of hydroxyaurones, HA2–6 and the planktonic growth was measured at OD₄₇₀. Each experiment was repeated three times with triplicate microwells for each compound. Statistical significance was tested with one-way ANOVA. p<0.0001.

Figure 4:

Biofilm inhibitory activities of chalcones (3a-f), methoxyaurones (MA1–6) and hydroxyaurones (HA2–6). A) S. mutans UA159 were co-incubated with 50 μM of chalcones 3a-f and biofilm formation was measured at OD₅₆₂ using the crystal violet protocol. B) S. mutans UA159 were co-incubated with 50 μM of methoxyaurones, MA1–6 and biofilm formation was measured at OD₅₆₂ using the crystal violet protocol. C) S. mutans UA159 were co-incubated with 50 μM of hydroxyaurones HA2–6 and biofilm formation was measured at OD₅₆₂ using the crystal violet protocol. Each experiment was repeated three times with triplicate microwells for each compound. Statistical significance was tested with one-way ANOVA. p<0.0001.

Figure 5:

Inhibitory activities of hydroxyaurones (HA2–6) against commensal biofilms. A) S. gordonii DL1 were co-incubated with 50 μM of hydroxyaurones, HA2–6 or G43 and biofilm formation was measured at OD₅₆₂ using the crystal violet protocol. B) S. sanguinis SK36 were co-incubated with 50 μM of hydroxyaurones, HA2–6 or G43 and biofilm formation was measured at OD₅₆₂ using the crystal violet protocol. Each experiment was repeated three times with triplicate microwells for each compound. Statistical significance was tested with one-way ANOVA. p<0.0001.

Figure 6:

Biofilm inhibitory activities of compound HA5. A) S. mutans UA159 were co-incubated with HA5 at various concentrations and biofilm formation was measured at OD₅₆₂ using the crystal violet protocol. B) Gtfs precipitated from S. mutans culture were co-incubated with HA5 at various concentrations and the glucan production was quantified using cascade blue staining and subsequent image processing with ImageJ. C) Representative fluorescence microscopy images of UA159 biofilms after 16 h of treatment with various concentrations of HA5. Bacterial cells were stained with Syto-9 (green, panel I); glucans were stained with Cascade Blue–dextran conjugated dye (blue, panel II); eDNA was stained with propidium iodide (red, panel III) and a merged image of all three staining images (panel IV). D) S. mutans UA159, S. gordonii DL1 and S. sanguinis SK36 were co-incubated with HA5 at 50 μM and 100 μM and their growth were measured at OD₄₇₀. E) Chemical structure of HA5. Each of the biofilm, glucan and growth assays were conducted in triplicate and statistical significance was tested with one-way ANOVA. p<0.0001.

Figure 7:

A) High resolution X-ray co-crystal structure (PDB ID: 8FG8) of the inhibitor HA5 with the catalytic domain of GtfB. Inhibitor HA5 and BTB are displayed as green sticks. B) Expansion of the GtfB binding site showing the binding mode of HA5. C) Key active site interactions of HA5 with active site residues (grey sticks) along with its H-bong interactions with water (Wat) molecules and calcium (Ca²⁺) ion depicted as yellow dotted lines. Inhibitor HA5 is depicted as light blue sticks and BTB is depicted as lavender sticks.

Figure 8.

A) Optical images of empty (PMAA)₅ hydrogels microparticles. B) HA5-loaded hydrogel HEBI and HA5 in methanol (insert B). C) Atomic Force Microscopy (AFM) topography images of a tooth surface with height of 280 nm. D) AFM image after (PMAA)₅ hydrogel adsorption, cubical hydrogel particles are clearly seen sticking to the tooth surface. E) Amplitude error image of empty (PMAA)₅ hydrogels dried on the surface of a tooth. Scan size is 20 μm2 in both images, the height (z)-scale is 1.7 μm. F) S. mutans UA159 and two bacterial commensal species S. gordonii DL1 or S. sanguinis SK36 were co-incubated with HA5 or HEBI at 25 μM and their growth was measured at OD₄₇₀. G). S. mutans UA159, S. gordonii DL1 or S. sanguinis SK36 were co-incubated with 25 μM of HA5 or HEBI and biofilm formation was measured at OD₅₆₂ using the crystal violet protocol. Each of the biofilm and growth assays were conducted in triplicate and statistical significance was tested with one-way ANOVA. p<0.0001.

Scheme 1:

Synthesis of substituted aurones.