Dental Biofilm and Streptococcus mutans

Dental caries and periodontal disease are typical oral diseases that are prevalent worldwide, and still represent the primary cause of tooth loss, so effective management measures are required [1,2]. Oral bacteria form dental plaque, which is a kind of biofilm formed on the supragingival and the subgingival surfaces of tooth and the root surface of tooth [3]. Microorganisms in the dental plaque metabolize carbohydrates to organic acids, thereby destroying the hard tissues of teeth and causing dental caries. Periodontitis results from an inflammatory response of the periodontal tissues to oral bacteria [4-7].

S. mutans is an oral bacterium representing mutans streptococci. It is one of important bacteria for formation of supragingival dental plaque which is a dental biofilm formed on the supragingival tooth surface, and of dental caries [2,8].

Acquired Pellicle Formation and Bacterial Capture

Formation of the dental plaque occurs via complex multistage processes. The first step is the acquired pellicle forma-tion (Fig. 1A). The enamel surface surrounded by the hydration layer has negative charge because it has a high concentration of phosphate group [2]. Cations such as calcium ions bind to the negative charge of the enamel surface, eventually changing the enamel surface to positive charge. In saliva, there are acidic proteins such as phosphoproteins and sulfate glycoproteins, which are negatively charged. Acidic proteins bind to the enamel surface via calcium ions to form acquired pellicle [2]. Calcium ion acts as a bridge between the negative charge on the enamel surface and the negative charge of acid proteins. Oral bacteria are captured on the acquired pellicle of tooth surface (Fig. 1B). Bacterial capture in the early stage is reversible attachment which is caused by ionic bond or van der Waals interaction [2].

Bacterial Adhesion

The captured oral bacteria form irreversible adhesions to the tooth surface covered by the acquired pellicle (Fig. 1B). Calcium bridge, hydrophobic interaction, polymer bridge, or covalent bond between the tooth surface covered with the acquired pellicle and bacterial surface are the main mechanisms of irreversible adhesion [2], and also cell surface adhesins of S. mutans, such as glucan-binding proteins (GBPs) and spaP, are important for bacterial adhesion to tooth surface. S. mutans carry gbps genes (gbpA, gbpB, and gbpC) related with the adhesion. The gbpA, gbpB, and gbpC genes encode GBP A, B, and C, which are known to play an important role in the adhesion of S. mutans to glucan molecules, a kind of extracellular polysaccharide of plaque matrix (Fig. 2). In particular, the GBP C is involved in dextran (glucan)-dependent aggregation (DDAG) of oral bacteria [9,10]. S. mutans also carry the spaP gene, which encodes the cell surface antigen, spaP. The spaP is also known as Ag I/II, PAc, AgB, Pl, Sr, SpaA, PAg, SspA, SspB and SoaA, which adheres to salivary agglutinin glycoprotein (SAG) and proline-rich protein of the acquired pellicle on the tooth surface as a kind of surface fibrillar adhesin (Fig. 2) [11-14]. The irreversible adhesion is followed by oral bacterial growth, division and then colonization.

Extracellular Polysaccharide Formation

Matrix of dental plaque is formed after colonization of the oral microorganisms (Fig. 1C). The plaque matrix can be formed in the absence of food. The plaque matrix is classified into protein matrix and extracellular polysaccharide matrix. S. mutans has gtfB, gtfC, gtfD, and ftf genes each of which encode glycosyltransferases (GTFs) B, C, D, and fructosyltransferase (ftf), respectively [2,12,13]. After the protein matrix of the dental plaque is formed, S. mutans decomposes sucrose in the oral cavity into glucose and fructose using bacterial invertase, and then synthesizes glucan by polymerizing glucose using GTF. ftf synthesizes polysaccharides such as fructan by polymerizing fructose. The extracellular polysaccharides thicken and harden the dental plaque and lower the oxygen permeability, and play a decisive role in dental plaque maturation (Figs. 1D, 3).

Biofilm Formation

The dental plaque formed in oral cavity is a mixed population of biofilms made by various bacteria combining themselves. Oral streptococci species such as Streptococcus sanguinis, S. mutans, Streptococcus gordonii, Streptococcus mitis, and Streptococcus oralis play the important role in the formation of supragingival plaque and dental caries. Actinomyces species such as Actinomyces viscosus are closely related to the development of dental plaque and dental caries on the root surface [2,15].

These bacteria combine with the protein matrix formed by the oral bacteria and the extracellular polysaccharides such as glucan and fructan to form the dental plaque on the tooth surface. The gene relA carried by S. mutans encodes RelA, which is known to regulate the formation of biofilm and to contribute to quorum-sensing [16]. Among the genes carried by the S. mutans, smu630 is reported to be important for sucrosedependent and sucrose-independent dental biofilm formation [12]. The gene brpA in S. mutans is also known to regulate biofilm formation [17]. The gene comD encodes a histidine kinase receptor and the gene comE encodes a cognate response regulator of the competence-stimulating peptide, which are part of the quorum-sensing cascade of S. mutans [17].

Sugar Uptake and Metabolism

Oral bacteria uptake sugars that are consumed in food and metabolize them with glucose, then use glucose to get the necessary energy via glycolysis and fermentation, and produce organic acids as metabolic products (Fig. 4) [2,7]. The relA gene of S. mutans encodes RelA, which is known to contribute to the regulation of phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), the glucose uptake system [16]. The gene eno of S. mutans encodes the bacterial enolase, which is a major component of the PTS in the bacteria, and is known to contribute to bacterial sugar uptake (Fig. 4) [18]. In addition, the gene ldh of S. mutans encodes lactic acid dehygbpA drogenase, which contributes to lactic acid formation.

Acid Tolerance

Oral bacteria metabolize the carbohydrates in the food to produce organic acids. Then the surrounding environment becomes acidic because the proton concentration is increased. S. mutans can survive and grow in the acidic environment because of its several genes enabling to overcome acidic environment. The gene atpD in S. mutans encodes the F₁F₀- ATPase. F₁F₀-ATPase is a proton pump that discharges H+ from within the bacteria to the outside, to overcome acid stress and maintain acid tolerance (Fig. 5) [18,19]. Moreover, F₁F₀-ATPase has another function as an ATP synthase [19]. The inside of bacterial cells maintains neutral pH, but when the pH is low outside the cell, proton gradient is formed on the boundary of the cell membrane. Proton gradient causes a proton motive force in which H+ tries to enter from outside the cell. F₁F₀- ATPase uses proton motive force to synthesize ATP required by bacteria. F-ATPase can play a dual role in obtaining acid tolerance by releasing protons from inside of cells, and also producing ATP for the growth and survival of bacteria. In addition, aguD encodes the agmatine deiminase system (AgDS), which produces alkali, enabling to overcome acid stress and maintain acid tolerance [18]. The gene brpA and relA also contribute to acid tolerance.

Regulation

The gene vicR encodes putative histidine kinase, which regulates expression of gbpB, gtfB, gtfC, gtfD, and ftf [18]. The gene brpA encoding regulatory protein BrpA, which regulates biofilm formation [20]. The gene relA encoding regulatory protein RelA, which is guanosine tetra (penta)-phosphate synthetase, and regulates biofilm formation and glucose uptake system [16].

Conclusions

Virulence genes in S. mutans can be classified into genes involved in bacterial adhesion, extracellular polysaccharide formation, biofilm formation, sugar uptake and metabolism, acid tolerance, and regulation (Table 1). The genes related with bacterial adhesion are gbps (gbpA, gbpB, and gbpC) and spaP. The genes related with extracellular polysaccharide formation are gtfs (gtfB, gtfC, and gtfD) and ftf. The genes involved in biofilm formation are smu630, relA and comDE. The genes involved in sugar uptake and metabolism are eno, ldh, and relA. The genes related with acid tolerance are atpD, aguD, brpA, and relA. The genes involved in the regulation are vicR, brpA, and relA.