The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm

Abstract

Many infectious diseases in humans are caused or exacerbated by biofilms. Dental caries is a prime example of a biofilm-dependent disease, resulting from interactions of microorganisms, host factors, and diet (sugars), which modulate the dynamic formation of biofilms on tooth surfaces. All biofilms have a microbial-derived extracellular matrix as an essential constituent. The exopolysaccharides formed through interactions between sucrose- (and starch-) and Streptococcus mutans-derived exoenzymes present in the pellicle and on microbial surfaces (including non-mutans) provide binding sites for cariogenic and other organisms. The polymers formed in situ enmesh the microorganisms while forming a matrix facilitating the assembly of three-dimensional (3D) multicellular structures that encompass a series of microenvironments and are firmly attached to teeth. The metabolic activity of microbes embedded in this exopolysaccharide-rich and diffusion-limiting matrix leads to acidification of the milieu and, eventually, acid-dissolution of enamel. Here, we discuss recent advances concerning spatio-temporal development of the exopolysaccharide matrix and its essential role in the pathogenesis of dental caries. We focus on how the matrix serves as a 3D scaffold for biofilm assembly while creating spatial heterogeneities and low-pH microenvironments/niches. Further understanding on how the matrix modulates microbial activity and virulence expression could lead to new approaches to control cariogenic biofilms.

Keywords: Streptococcus mutans; dental caries; extracellular matrix; glucosyltransferases; heterogeneity; pH microenvironment.

Figure 1.

Assembly of an EPS-rich matrix and 3D biofilm architecture. This Fig. highlights the developmental process of EPS-matrix assembly and morphological and structural changes that occur to produce a mature biofilm structure. Panel (a) displays representative 3D renderings of the matrix in red and the bacterial microcolonies in green. These in vitro mixed-species (S. mutans, S. oralis, and A. naeslundii) biofilms were grown in culture medium with 1% sucrose on saliva-coated hydroxyapatite (sHA) surfaces for the times depicted (43, 67, 91, and 115 hrs). Panel (b) provides close-up cross-sectional images of the structural organization of bacterial cells, EPS, and overlay of both components. The arrow denotes an area where EPS connect 2 microcolonies. These images are adapted from Xiao et al. (2012).

Figure 2.

Three-dimensional in situ pH mapping of intact mixed-species biofilm. This Fig. provides representative 3D renderings of an in vitro mixed-species biofilm that has been subjected to 3D pH mapping. In panel (a), bacteria are highlighted in green and EPS in red, while the yellow box depicts an area of detailed pH mapping. Panel (b) is a set of orthogonal views, illustrating temporal changes of pH across the selected area following incubation in neutral sodium-phosphate-based buffer (pH 7.0). The dark areas indicate regions of low pH, while white or light areas indicate regions of pH that are more neutral, defined by the scale bar. In panel (c), a graph shows the distribution of pH values across the selected EPS-microcolony complex after 30 and 120 min of exposure to the buffer. The red boxes highlight the acidic pH regions. sHA: saliva-coated hydroxyapatite surface. This Fig. is adapted from Xiao et al. (2012).

Figure 3.

Acidic microenvironments across an EPS-microcolony complex and at the surface of biofilm attachment. This Fig. illustrates the heterogeneous pH distribution within the selected EPS-microcolony complex and at the biofilm/sHA interface. Panel (a) gives a detailed image of an EPS-microcolony complex attached to the sHA surface. EPS are depicted in red, bacteria are in green, and dark areas indicate acidic pH, with white areas indicating more neutral pH. The red arrows highlight acidic pH regions within the microcolony structure. Yellow arrows point out pH values close to neutral, which tend to occur at the microcolony/fluid phase interface. The red box denotes acidic pH at the interface between the microcolony complex and the surface of attachment (sHA). Panel (b-1) gives representative cross-sectional images at the sHA surface. White marks indicate the areas with surface-attached microcolonies, while red marks show the corresponding area in the pH channel. The table in Panel (b-2) lists the pH values at the biofilm/sHA interface in areas with and without surface-attached microcolonies following a 30-minute exposure to neutral buffer. The asterisk (*) indicates the values significantly different from each other (p < .05). This Fig. is adapted from Xiao et al. (2012).

Figure 4.

Exopolysaccharide synthesis in situ mediates assembly of matrix scaffold and cariogenic biofilm formation. This Fig. depicts the sequential assembly of the matrix scaffold of cariogenic biofilms. In panel (a), Gtf enzymes secreted by S. mutans become incorporated into the pellicle (particularly GtfC) and/or are adsorbed to bacterial surfaces (mainly GtfB). GtfB also absorbs to microorganisms that do not produce Gtfs (e.g., Actinomyces spp). In panel (b), GtfB and GtfC that are absorbed to surfaces within the oral cavity can rapidly utilize dietary sucrose (and starch hydrolysates). As a result, insoluble and soluble glucans are synthesized in situ. GtfD also produces soluble glucans that can serve as primers for GtfB to augment total insoluble EPS synthesis. In panel (c), the glucan molecules formed on surfaces provide avid binding sites for various resident microorganisms and especially S. mutans, which mediates bacterial clustering and adherence to the tooth enamel. This process occurs primarily through glucan-glucan and glucan-Gbp interactions (Bowen and Koo, 2011). Furthermore, bacteria coated by the Gtfs themselves become de facto glucan producers, so they could bind to tooth and microbial surfaces by mechanisms similar to those used by S. mutans. The surface-adsorbed Gtfs produce an insoluble matrix for dental plaque-biofilm in situ. Concomitantly, dietary carbohydrates are metabolized into acids by acidogenic/aciduric organisms, including S. mutans. In panel (d), once the EPS-rich matrix and the biofilm have been established, ecological pressures (e.g., pH, nutrient availability) determine which and how bacteria survive, facilitating the dominance of certain species within plaque, namely, cariogenic species (e.g., S. mutans) under frequent sucrose or other fermentable carbohydrate exposure. The presence of soluble polysaccharides in the matrix also provides additional sources of fermentable sugar. When the biofilm remains on tooth surfaces and the consumption of a carbohydrate-rich diet (especially sucrose) persists, the amount of EPS and extent of acidification of the matrix increase (e.g., as EPS-microlony complexes form). Such conditions elicit biochemical, ecological, and structural changes that favor the survival and dominance of highly acid-stress-tolerant organisms (Aas et al., 2008; Palmer et al., 2010; Gross et al., 2012) in these cohesive and firmly attached biofilms. The low-pH environment at the tooth-biofilm interface promotes demineralization of enamel. This model may explain the rapid accumulation of cariogenic plaque in the presence of sucrose (and starch hydrolysates), even if the initial S. mutans population is numerically low.