Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms

Abstract

Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.

FIG. 1.

(a) Water-insoluble glucans synthesized by S. mutans glucosyltransferase B (labeled with Alexa Fluor 647; maximum absorbance wavelength, 647 nm; maximum fluorescence emission wavelength, 668 nm). (a-1) Phase-contrast image of glucans before excitation with a laser at 633 nm (×20 oil objective; numerical aperture, 0.70). (a-2) Fluorescence image of glucans (adapted from reference 22). (b) Saliva-coated hydroxyapatite (sHA) biofilm model: simultaneous visualization of EPS (red) and bacteria and microcolonies (green) in a three-dimensional image of an S. mutans biofilm formed on the surface of an sHA disk.

FIG. 2.

(a) Representative three-dimensional images of biofilms formed by S. mutans UA159 and gtf mutant strains in the presence of 1% (wt/vol) sucrose. (b) COMSTAT analysis of the distribution of bacteria and EPS from the disk surface to the fluid phase interface.

FIG. 3.

Representative three-dimensional images of multispecies biofilms grown in the presence of sucrose (a) or glucose (b) and a two-species biofilm grown in the presence of sucrose (c). The images are three-dimensional images of bacteria (green) and EPS (red) and selected enlarged areas for visualization of the structural relationship between bacterial cells and glucans.

FIG. 4.

Biomasses of EPS and bacterial cells in multispecies biofilms formed in the presence of 1% (wt/vol) sucrose or 1% (wt/vol) glucose and in sucrose-grown two-species (A. naeslundii and S. oralis) biofilms, as determined by COMSTAT analysis. The data are means ± standard deviations (n = 30) from three independent experiments. The values for the EPS and bacterial biomasses for the sucrose-grown multispecies biofilm at 67 h, 91 h, and 115 h are significantly different from the values for the glucose-grown multispecies and sucrose-grown two-species biofilms (P < 0.05, as determined by ANOVA for all pairs using the Tukey-Kramer honestly significant difference test).

FIG. 5.

Biochemical analyses of multispecies biofilms formed in the presence of 1% (wt/vol) sucrose or 1% (wt/vol) glucose. (a) Average number of CFU recovered per biofilm. (b) Dry weight and biochemical composition of the biofilms. The data are means ± standard deviations (n = 9) from three independent experiments. The dry weight and the total amounts of protein and insoluble (INS) and soluble (SOL) polysaccharides in the sucrose-grown multispecies biofilm at 115 h are significantly different from the values for the glucose-grown multispecies biofilm (P < 0.05, as determined by ANOVA for all pairs using the Tukey-Kramer honestly significant difference test).