Extracellular Polysaccharides Matrix — An Often Forgotten Virulence Factor in Oral Biofilm Research

http://www.ijos.org.cn Koo et al. Extracellular Polysaccharides Matrix doi: 10.4248/IJOS.09086 LETTER TO EDITOR Extracellular Polysaccharides Matrix — An Often Forgotten Virulence Factor in Oral Biofilm Research Hyun Koo*, Jin Xiao, Marlise I. Klein Eastman Department of Dentistry and Center for Oral Biology, University of Rochester Medical Center, USA Received Oct. 10, 2009; Revision accepted Nov. 15, 2009 Oral diseases related to dental biofilms continue to afflict the majority of the world’s population. Among them, dental caries continues to be the single most prevalent and costly oral infectious disease (Marsh, 2003; Dye et al., 2007). Dental caries results from the interaction of specific bacteria with constituents of the diet within a dental biofilm known as plaque (Bowen, 2002). Sucrose is considered to be the “arch criminal” from the dietary aspect because it serves as a substrate for synthesis of extracellular (EPS) and intracellular (IPS) polysaccharides in dental biofilm and is also fermentable (Bowen, 2002). However, it is important to emphasize that additional sugars and starch can certainly contribute to the pathogenesis (Bowen et al., 1980; Firestone et al., 1982; Thurnheer et al., 2008). Streptococcus mutans (S. mutans), a member of the oral microbial community, is generally regarded as the primary microbial culprit although additional microorganisms may be involved (Hamada and Slade, 1980; Loesche, 1986; Beighton, 2005). This bacterium (i) effectively utilizes dietary sucrose (and possibly starch) to synthesize large amounts of EPS through glucosyltransferases (Gtfs) and a fructosyltransferase (Ftfs), (ii) adheres tenaciously to glucan-coated surfaces, and (iii) is also acidogenic and acid-tolerant, which are critical virulence properties involved in the pathogenesis of dental caries. Biofilms and dental caries — the role of exoenzymes and EPS synthesis In nature, most of the biofilms develops from initial microbial attachment on a surface followed by formation of highly structured cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which are occurring in a complex extracellular matrix (Branda et al., 2005). The majority of biofilms matrices are rich in polysaccharides, and dental biofilms are no exception; up to 40% of the dry-weight of dental biofilm is composed of polysaccharides (as reviewed in Paes Leme et al., 2006). All the available evidence shows clearly that the primary sources of EPS in dental biofilms are products from the interaction of Gtfs and Ftfs with sucrose and starch (as reviewed in VaccaSmith et al., 1996; Kopec et al., 1997; Hayacibara et al., 2004; van Hijum et al., 2006; Klein et al., 2009). S. mutans is a key contributor to the formation of exopolysaccharide-matrix in dental biofilms. This bacterium produces three Gtfs, products of gtfB, gtfC and gtfD genes (Kuramitsu, 2003): GtfB, which synthesizes mostly insoluble glucans containing elevated amounts of α1,3-linked glucose; GtfC, which synthesizes a mixture of insoluble and soluble glucans [rich in α(1,6) linkages]; and GtfD which synthesizes predominantly soluble glucans. In addition, S. mutans produces a Ftfs, the Int J Oral Sci, 1(4): 229–234, 2009 - 229 - Extracellular Polysaccharides Matrix Koo et al. product of single ftf gene, which catalyzes the synthesis of fructans composed primarily of β(2,1) linkages. All of these exoenzymes and their polysaccharide products have been implicated in various roles in biofilm formation and dental caries process. For example, GtfB and GtfC are associated with bacterial adherence on tooth surface and structural stability/integrity of the extracellular matrix, and have been shown to be essential for the expression of virulence of S. mutans in rat caries model (Tanzer et al., 1985; Munro et al., 1991; Yamashita et al., 1993). Fructans are used as extracellular carbohydrate reservoir, which can be metabolized by bacteria during periods of nutrient deprivation (Burne et al., 1996). These observations show very clearly all these enzymes could be primary targets for therapeutic intervention to prevent biofilm formation and dental caries (Koo et al., 2006). The surface-adsorbed Gtfs and initial bacterial adherence on apatitic surfaces S. mutans cells can attach initially to saliva coated surfaces through sucrose-independent mechanisms mediated primarily by lectin-like interactions between specific pellicle proteins and bacterial adhesins (Gibbons, 1996). However, this bacterium binds to the glucan-coated surfaces, especially those synthesized by GtfB and GtfC, in larger numbers and with higher adhesion strength than to uncoated or saliva-coated apatitic surfaces (Kuramitsu, 1974; Schilling and Bowen, 1992; Cross et al., 2007). The Gtfs secreted by S. mutans, particularly GtfC, bind avidly to the pellicle formed on the tooth surface in an active form; a layer of polysaccharides is formed rapidly on the surfaces in the presence of sucrose (Rolla et al., 1983; Scheie et al., 1987; Schilling and Bowen, 1988; Vacca-Smith and Bowen, 1998; Hannig et al., 2008). In addition, starches can be digested by salivary α-amylases to maltose, maltodextrins and other oligosaccharides, some of which can be acceptors during glucan synthesis by Gtfs increasing the overall exopolysaccharide production (Fukui and Moriyama, 1983; Fu and Robyt, 1991; Vacca-Smith et al., 1996). The polysaccharides on the surface provide binding site for colonization by S. mutans (Schilling and Bowen, 1992) through several surface-proteins capable of binding glucans, - 230 - Int J Oral Sci, 1(4): 229–234, 2009 http://www.ijos.org.cn including the Gtfs and specific non-enzymatic glucan binding proteins (Banas and Vickerman, 2003). Moreover, it is apparent that glucan synthesized in situ by Gtf in pellicle provides enhanced binding for several oral microorganisms, including other oral streptococci, and Lactobacillus and Actynomyces species (Vacca-Smith et al., 1996; Bowen, 2002). The Gtfs, especially GtfB, also adhere to bacterial surfaces, and furthermore adhere to surfaces of bacteria that do not make enzyme, thereby converting them into de facto glucan producers (McCabe and Donkersloot, 1977; Hamada et al., 1978; Vacca-Smith and Bowen, 1998). Thus, glucans, and at a lesser extent fructans, formed in situ promote the accumulation of microorganisms on the tooth surface and to each other, and may explain the electron micrographs of even early dental biofilm-plaque, which display microorganisms enmeshed in and attached to polysaccharide on the surface of saliva-coated hydroxyapatite (VaccaSmith and Bowen, 2000). The attachment of bacterial cells on surfaces and formation of cellcluster (or microcolonies) within an extracellular matrix are critical steps for the initial formation and further development of pathogenic biofilms. The role of EPS in the development of cariogenic biofilms If the initial biofilm is allowed to remain on (...truncated)