Biology of Streptococcus mutans- Derived Glucosyltransferases: Role in Extracellular Matrix Formation of Cariogenic Biofilms

The importance of Streptococcus mutans in the etiology and pathogenesis of dental caries is certainly controversial, in part because excessive attention is paid to the numbers of S. mutans and acid production while the matrix within dental plaque has been neglected. S. mutans does not always dominate within plaque; many organisms are equally acidogenic and aciduric. It is also recognized that glucosyltransferases from S. mutans (Gtfs) play critical roles in the development of virulent dental plaque. Gtfs adsorb to enamel synthesizing glucans in situ, providing sites for avid colonization by microorganisms and an insoluble matrix for plaque. Gtfs also adsorb to surfaces of other oral microorganisms converting them to glucan producers. S. mutans expresses 3 genetically distinct Gtfs; each appears to play a different but overlapping role in the formation of virulent plaque. GtfC is adsorbed to enamel within pellicle whereas GtfB binds avidly to bacteria promoting tight cell clustering, and enhancing cohesion of plaque. GtfD forms a soluble, readily metabolizable polysaccharide and acts as a primer for GtfB. The behavior of soluble Gtfs does not mirror that observed with surReceived: May 26, 2009 Accepted after revision: January 26, 2011 Published online: February 23, 2011 William H. Bowen, BDS, PhD University of Rochester, Center for Oral Biology 601 Elmwood Avenue, Box 611 Rochester, NY 14642 (USA) Tel. +1 585 275 0772, E-Mail William_Bowen @ urmc.rochester.edu © 2011 S. Karger AG, Basel Accessible online at: www.karger.com/cre D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 1/ 20 17 6 :5 7: 26 P M Bowen /Koo Caries Res 2011;45:69–86 70 time [Branda et al., 2005]. Clearly the structure of the polysaccharide matrix can play a critical role affecting the virulence of plaque by influencing the physical and biochemical properties of biofilm. It may enhance adherence of microorganisms, promote coherence, act as reserve source of energy, protect microorganisms from inimical influences, affect diffusion of substances into and out of biofilm, and help to concentrate metal ions and other physiological nutrients within a microenvironment [Tatevossian, 1990; Wilson and Ashley, 1990; Hayacibara et al., 2004; Paes Leme et al., 2006; Flemming and Wingender, 2010; Koo et al., 2010b]. Composition of Dental Plaque The broad composition of the biofilm known as dental plaque has been explored. Glucan (see below) comprises 10–20% dry weight of dental plaque and fructan approximately 1–2%; clearly these proportions will vary depending in part at least on the duration since the last intake of food [Critchley et al., 1967; Gibbons, 1968; Wood, 1969; Mayhall and Butler, 1976; Emilson et al., 1984]. Dental plaque also contains approximately 40% dry weight protein (mostly derived from bacteria and saliva), variable amounts of lipid, Ca, P, Mg and F compared with surrounding saliva [Hotz et al., 1972; Cole and Bowen, 1975; Paes Leme et al., 2006; Pessan et al., 2008]. Dental plaque in situ harbors approximately 80% water [Wilson and Ashley, 1990]. Electron histochemistry and transmission electron microscopy of dental plaque reveals microorganisms embedded in a matrix of polysaccharide [McDougall, 1963; Reese and Guggenheim, 2007], which appears to increase following exposure to sucrose [Saxton and Kolendo, 1967; Critchley et al., 1968]. All the available evidence shows clearly that the primary sources of EPS in dental plaque are products from the interaction of glucosyltransferases (Gtfs) and fructosyltransferases with sucrose and starch hydrolysates [Vacca-Smith et al., 1996a]. Role of Gtfs in Dental Plaque Formation Acquired Enamel Pellicle and Gtfs The earliest evidence of plaque formation is the appearance of a pellicle on the tooth surface [Leach and Saxton, 1966; Hay, 1967; Siqueira et al., 2007]. This material is repeatedly referred to as salivary pellicle even though it is clear that the pellicle harbors significant amounts of nonmammalian constituents [Eggen and Rölla, 1983; Rölla et al., 1983a; Vacca-Smith and Bowen, 1998; Hannig et al., 2008a] which influence the subsequent development of plaque. The saliva-derived constituents are selectively adsorbed to the surface, and include but are not limited to proline-rich proteins, amylase, lysozyme, histatins, peroxidase, statherin and mucin 2 [Mayhall and Butler, 1976; Siqueira et al., 2007]. Some of these materials undergo conformational change when adsorbed to tooth surfaces and may then provide specific binding sites for some microorganisms [Hay et al., 1971; Gibbons et al., 1990]. The presence of bacterial cellular material in pellicle was reported by Armstrong [1967], but it remained for Rölla et al. [1983a] and Scheie et al. [1987] to identify the presence of soluble bacterial products in pellicle using immunological techniques. They identified fructosyltransferase, Gtf and lipoteichoic acid in pellicle formed in vitro and in vivo from whole saliva. These observations were subsequently confirmed and extended by VaccaSmith et al. [1996b] in pellicles formed on apatitic surface in situ. The Gtfs present in pellicle were shown by Schilling and Bowen [1988] to be active and capable of synthesizing glucan in situ from sucrose and provided binding sites for many oral microorganisms, including mutans streptococci. Gtfs present in whole saliva are incorporated into pellicle and form part of its structure; furthermore, Gtf added to ductal saliva (Gtf-free) is also adsorbed in active form onto hydroxyapatite (HA) surfaces [Schilling and Bowen, 1988]. Remarkably, Gtf incorporated into pellicle adsorbed to an HA surface displayed 3–4 times enhanced activity compared with similar amounts of enzyme in solution [Schilling et al., 1989; Venkitaraman et al., 1995; Vacca-Smith et al., 1996b]. Furthermore, it was observed that the enzymes when insolubilized remained highly active over a wide range of pH values [Schilling and Bowen, 1988; Venkitaraman et al., 1995; Vacca-Smith et al., 1996b]. It is clear that the presence of active Gtf within dental pellicle facilitates the formation of glucan in situ, thereby providing distinct binding sites for oral microorganisms. It can therefore be hypothesized that Gtfs from S. mutans influence the microbial colonization of tooth surfaces. Gtf Sources Gtf can be readily assayed in whole saliva from many persons, particularly those who are caries active [Scheie and Rölla, 1986; Scheie et al., 1987; Vacca-Smith et al., 2007]. Gtfs are remarkably stable even in whole saliva; measured amounts of purified Gtf enzymes added to D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 1/ 20 17 6 :5 7: 26 P M Biology of Streptococcus mutansDerived Glucosyltransferases Caries Res 2011;45:69–86 71 whole saliva remained completely active for up to 4 h at least [Vacca-Smith et al., 1996b]. Several groups of oral microorganisms produce Gtfs; these include Streptococcus sanguinis , Streptococcus mutans , Streptococcus sobrinus , Actinomyces spp., Streptococcus salivarius and Lactobacillus spp. [Newbrun, 1974]. The review presented here will focus on the Gtfs from S. mutans . S. mutans produces at least 3 genetically separate Gtfs, each of which synthesizes a structurally distinct glucan from sucrose. Details of the structural and functional organization of Gtfs are found elsewhere [for reviews, see Monchois et al., 1999; van Hijum et al., 2006]. GtfB (formally known as GtfI) synthesizes primarily insoluble glucan rich in -1,3-linkages, GtfC (GtfSI) produces a mixture of soluble (with mostly -1,6-linkages) and insoluble glucans, and GtfD (GtfS) forms predominantly soluble glucans [Aoki et al., 1986; Hanada and Kuramitsu, 1988, 1989]. Superficially, it may appear bizarre that microorganisms produce 3 distinct enzymes to act on the same substrate to form polysaccharides. We hypothesize with supporting data that each one plays a distinct role in the formation of dental plaque and, as a result, S. mutans has a far greater influence on the formation and composition of plaque than its population would appear to warrant. Gtfs in the Pellicle and on Bacterial Surfaces Gtf adsorbs to experimental pellicles formed on apatitic surfaces in situ with extraordinary rapidity; active Gtf is detected on HA disks within 1 min of placing them in the mouth. Prerinsing with sucrose enhances the amount of enzyme detected, possibly because Gtf will adhere to glucan formed in situ [Scheie et al., 1987; VaccaSmith and Bowen, 2000]. In vitro, Gtf binds poorly to uncoated HA and loses much of its activity [Schilling and Bowen, 1988; Vacca-Smith and Bowen, 1998]; in contrast, Gtf adsorbs to saliva-coated HA (sHA) disks avidly with enhanced activity [Venkitaraman et al., 1995; Steinberg et al., 1996]. Results from early studies, before separate gene products were available, provided little information on which Gtf is present in pellicle [Schilling and Bowen, 1988]. As a result of cloning and gene deletion, the Gtf enzymes have been prepared separately and at a high level of purity which has led to rapid advances in the field [Hanada and Kuramitsu, 1988; Fukushima et al., 1992]. Although all 3 enzymes can bind to sHA, their affinity differs greatly [Vacca-Smith and Bowen, 1998]. GtfC has the greatest affinity for sHA, and in addition, based on Scatchard plots, GtfC displayed significantly more binding sites than did either GtfB or GtfD. Although GtfD binds to sHA, it displays relatively few binding sites. It is also noteworthy that the K m values for the 3 enzymes are lower by twoto eightfold following adsorption to sHA, an observation consistent with the reported enhanced activity of the insolubilized enzymes compared with the same enzymes in solution [Kuramitsu and Ingersoll, 1978; Venkitaraman et al., 1995; Steinberg et al., 1996]. The activity of GtfB is enhanced greatly by the inclusion of primer dextran in the test system [Koga et al., 1988; Venkitaraman et al., 1995; Vacca-Smith et al., 1996a; Kopec et al., 1997]. In contrast, Gtf detected in pellicle is not enhanced by primer dextran. Furthermore, the activity of GtfC is enhanced substantially when it is adsorbed to sHA; enhancement is not observed with GtfB adsorbed to sHA. The binding of GtfB to sHA in the presence of ductal parotid saliva supplemented with GtfC and GtfD was reduced when compared with its binding to sHA in the presence of parotid saliva alone. In contrast, the binding of GtfC or GtfD enzymes to sHA was unaffected by addition of other Gtfs [Vacca-Smith and Bowen, 2000]. We conclude therefore that the Gtf in pellicle has properties that are certainly consistent with it being GtfC [VaccaSmith et al., 1996b]. Data from in situ and in vitro studies suggest strongly that Gtf detected in pellicle is primarily GtfC from S. mutans [Venkitaraman et al., 1995; VaccaSmith and Bowen, 2000]. Gtfs also have the capacity to bind to many oral bacteria [McCabe and Donkersloot, 1977; Hamada et al., 1978; Vacca-Smith and Bowen, 1998], even those that do not synthesize Gtfs. We have observed that GtfB binds with greater avidity to oral microorganisms, e.g. Actinomyces viscosus , Lactobacillus casei and S. mutans , than do the other Gtfs from S. mutans and that the binding to bacteria occurs even in the presence of ductal saliva. Furthermore, the enzyme retains its activity when adsorbed to bacteria, thereby converting non-Gtf producers into de facto glucan formers [Vacca-Smith and Bowen, 1998]. The simultaneous synthesis of glucans by surface-adsorbed GtfB and GtfC is essential for the establishment of a matrix that enhances the coherence of bacterial cells and adherence to apatitic surfaces, allowing the formation of highly organized and dense cell clusters known as microcolonies [Tamesada et al., 2004; Koo et al., 2010b; Xiao and Koo, 2010]. These phenomena may well explain the observations on histochemical preparations of even early plaque that reveal microorganisms embedded in a polysaccharide matrix [Critchley et al., 1968; Venkitaraman et al., 1995; Reese and Guggenheim, 2007]. D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 1/ 20 17 6 :5 7: 26 P M Bowen /Koo Caries Res 2011;45:69–86 72 Adhesion of Gtfs to Surfaces The precise mechanisms involved in the binding of Gtfs to sHA and bacterial surfaces remain to be elucidated. The same type of basic structure is found in all Gtfs [Russell et al., 1988; Russell, 1994; Vacca-Smith and Bowen, 1998]. Each enzyme has at the amino terminus a signal peptide comprised of approximately 38 amino acids; adjacent to this peptide is a very variable domain constituted by about 200 amino acids characteristic for each of the enzymes [van Hijum et al., 2006]. The amino acid composition and structure of the Gtfs may clarify the distinct binding capacities of each enzyme. A series of repeat motifs of amino acids that varies for each enzyme is found at the C terminus. The C termini found in GtfB and GtfC differ in composition, which may explain in part at least the preferential adsorption of GtfC enzyme to sHA and of GtfB enzyme to bacterial surfaces. The GtfB and GtfD are comparable in structure and both are hydrophilic [Shiroza et al., 1987; Honda et al., 1990]. GtfC is also hydrophilic; however, in contrast to the GtfB and GtfD enzymes, it harbors a hydrophobic domain within the direct repeat units in the C terminus [Ueda et al., 1988]. The presence of small hydrophobic domains in GtfC may be associated with pellicle-binding activity of this enzyme, possibly through interactions with specific salivary macromolecules found in the pellicle, such as lysozyme and -amylase [Vacca-Smith et al., 1996a; Vacca-Smith and Bowen, 1998]. Conversely, the presence of specific carboxyl-terminal repeat units of GtfB has a major role in the binding of the enzyme to the cell surface of oral streptococci [Kato and Kuramitsu, 1991]. It is noteworthy [Vickerman et al., 1996, 2003] that the ability of Gtf enzymes from S. gordonii to bind to biotindextran was dramatically reduced following deletion of the C-terminal hydrophilic residue. Using a series of deletion derivatives for the C terminus, it has been shown that direct repeat units constitute part of the minimum domain required for the lectin type glucan binding by S. mutans GtfD [Mooser and Wong, 1988; Lis et al., 1995]. The C-terminal glucan-binding domain of GtfD contains five 65-amino-acid direct repeat units. The removal of the single direct repeat unit significantly reduced the ability of the protein to bind glucans [Lis et al., 1995] and may be necessary for enzyme catalytic activity [van Hijum et al., 2006]. However, the precise role of the glucanbinding domain on the conformation-function relationship of Gtf enzymes, including the presumed ( / )8 barrel structure in the catalytic domain, remains largely unknown [van Hijum et al., 2006]. Nevertheless, the tertiary structure of the glucan-binding domain (presumably containing a high percentage of  -sheet) may be essential for the lectin type glucan binding by GtfD [Mooser and Wong, 1988; Banas and Vickerman, 2003; van Hijum et al., 2006]. In addition, Gtfs also bind to other components of the salivary pellicle such as salivary peroxidase, amylase and lysozyme [Korpela et al., 2002; Hannig et al., 2005; Kho et al., 2005]. Available data shows clearly that Gtfs interact with salivary amylase in pellicle [Vacca-Smith et al., 1996a] inhibiting activity and blocking adsorption of salivary amylase to HA [Hannig et al., 2005]. Lactoperoxidase in the absence of substrates, which is structurally and enzymatically similar to salivary peroxidase, inhibits all 3 Gtf enzymes in solution or adsorbed to sHA beads [Korpela et al., 2002]. The presence of lysozyme reduced the activity of GtfB in solution and on a surface but was without apparent effect on the structure of glucan formed [Kho et al., 2005]. The interactions of Gtf with peroxidase and lysozyme in salivary pellicle could result in decreased colonization of tooth surfaces as a consequence of depressed glucan production. It is also noteworthy that many oral microorganisms, including S. mutans , adsorb lysozyme, amylase and other salivary proteins [Douglas, 1983; Douglas and Russell, 1984; Ambatipudi et al., 2010], which may influence the binding of GtfB on the bacterial cell surface. The simultaneous binding of GtfB and salivary amylase and their interactions create the opportunity to synthesize complex insoluble glucans directly on the bacterial surface [Vacca-Smith et al., 1996a; Chaudhuri et al., 2007]. Gtf Activity and Glucan Structure The attention to oral biofilms has been enhanced greatly in recent years largely because it is recognized that microorganisms behave differently in biofilms compared with that in the planktonic state [Marsh and Bradshaw, 1995; Hall-Stoodley et al., 2004]. Enzymes too may behave differently when adsorbed to a surface compared with solubilized enzymes [Fears and Latour, 2009]. The adsorption of Gtf to sHA results in substantial enhancement of activity compared with activity of the same enzyme in solutions [Schilling and Bowen, 1988]. In contrast to what occurs on sHA, Gtf adsorbs comparatively poorly to uncoated HA and loses some activity. Even more remarkable is the difference in structure of glucans formed by GtfB and GtfC when adsorbed to sHA compared with the product formed when the same enzyme is in solution. Based on susceptibility to dextranase and mutanase, products of digestion and structural analyses, D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 1/ 20 17 6 :5 7: 26 P M Biology of Streptococcus mutansDerived Glucosyltransferases Caries Res 2011;45:69–86 73 there is a dramatic increase in the number of -1,3-linkages and a higher percentage of 3-linked branch points (e.g. 2,3-, 3,4-, 3,6and 3,4,6-linked glucose) in the surface-formed glucan compared with that formed by the same enzymes in solution. The presence of starch hydrolysates, associated with gradual digestion of starch by salivary -amylase (free in solution, adsorbed onto apatite and/or microbial surfaces), also influences the formation and structure of glucan by GtfB [Vacca-Smith et al., 1996a]. The hydrolysates, which include a range of high-molecular-weight oligomers ( 1 1 kDa), maltotriose and maltose/isomaltose [Klein et al., 2010], can serve as acceptor sites for polymer formation, constituting an integral part of the polymer molecule [Fukui and Moriyama, 1983; Koga et al., 1988; Fu and Robyt, 1991]. Furthermore, it appears that these carbohydrates can serve as substrate for the addition of branches to glucan even in the absence of sucrose [McCabe and Hamelik, 1983]. The combination of starch hydrolysates and sucrose with surface-adsorbed GtfB results as expected in enhanced formation of predominantly insoluble glucan and an increase in the number of -1,4and -1,3linkages with 3,4-linked glucose as the major branch point [Kopec et al., 1997]. Inclusion of starch hydrolysates with sucrose is without effect on the structure of product formed by GtfC and GtfD. It is noteworthy that parts of Gtf molecules have substantial sequence similarity with members of the -amylase family [van Hijum et al., 2006]. The significance of Gtf similarity is underlined by Gtf/ -amylase residues conserved in all but one -amylase invariant residue [Devulappalle et al ., 1997]. Moreover, the presence of amylase increased both the sucrase and transferase component activities of GtfB [Chaudhuri et al., 2007]. The product from the starch-sucrose substrate resembles in some measure the structure reported for plaque polysaccharide [Hotz et al., 1972; Birked and Rosell, 1975]. Furthermore it provides distinct binding sites for some oral microorganisms. For example, Actinomyces spp. and S. mutans bind in larger numbers to glucan formed on sHA surface in the presence of starch hydrolysates as compared to glucan formed in the absence of starch [Vacca-Smith et al., 1996a]. Sucrose is frequently consumed with starch, and this combination could clearly influence the composition, structure and physical properties of plaque matrix (more details later). Clearly, the glucans formed on surfaces are highly insoluble, and furthermore provide distinct binding sites for microorganisms [Kopec et al., 1997] and affect the 3-dimensional structure of the matrix [Koo et al., 2010b]. Whether similar phenomena occur with glucans formed on bacterial surfaces remains to be determined. Gtf-Glucan-Mediated Bacterial Adherence The adherence of microorganisms to the tooth surface is critical in the formation of dental plaque, and understandably much attention has been focused on microbial binding to uncoated tooth surfaces, HA, polystyrene (plastic) or glass [Morris and McBride, 1984; Gibbons and Hay, 1989; Schilling and Bowen, 1992]. Non-pellicle-covered teeth rarely if ever occur in vivo. Salivary glycoproteins and bacterial products can be detected on HA disks within 30 s of being placed in the mouth [Hay et al., 1971; Vacca-Smith and Bowen, 2000; Hannig et al., 2008a]. Saliva-coated disks prepared in situ assayed directly for enzyme activity revealed that fructosyltransferase activity increased up to 1 min of exposure and decreased when kept in the mouth for longer periods. Gtf activity on the disks in contrast increased the longer the disks were kept in the mouth [Scheie et al., 1987; Vacca-Smith and Bowen, 2000]. Many oral microorganisms do not adhere in large numbers to uncovered HA [Schilling and Bowen, 1992] and even fewer adhere to sHA [Schilling and Bowen, 1988; Schilling et al., 1989; Venkitaraman et al., 1995; Vacca-Smith and Bowen, 1998] even though considerable selectivity is apparent, and novel binding sites in salivary glycoproteins are revealed (cryptotopes) as a result of adsorption to HA surface [Gibbons et al., 1990]. These observations on adherence are in marked contrast to what occurs when glucan is formed in situ on the surface of HA. With glucan synthesized from surface Gtfs (particularly GtfB and GtfC but not GtfD), there is a threefold increase in the number of S. mutans adhering to the glucan-coated surface compared with the number adhering to an uncoated HA or sHA surface [Kuramitsu, 1974; Schilling and Bowen, 1992; Venkitaraman et al., 1995]. The binding of S. mutans to glucans formed in situ is mediated by the presence of cell-associated Gtf enzymes and non-Gtf glucan-binding proteins (Gbps). At least 4 Gbps have been identified in S. mutans, GbpA, GbpB, GbpC and GbpD [Guggenheim, 1970; Russell, 1979; Banas and Vickerman, 2003]. GbpA and GbpD contain carboxyl-terminal repeats similar to those found in the glucan-binding domain of Gtf enzymes. The glucanbinding domain of GbpC has not been identified although it shares homology with the AgI/II family of proteins; GbpB is similar to peptidoglycan hydrolase [Banas and Vickerman, 2003]. Among them, GbpC (and possibly GbpB) is cell wall bound and may function as cell surface glucan receptors in S. mutans [Banas and Vickerman, D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 1/ 20 17 6 :5 7: 26 P M Bowen /Koo Caries Res 2011;45:69–86 74 2003]. Each of the Gbps appears to have a role in sucrosedependent adhesion and biofilm formation by S. mutans , although loss of cell-surface-anchored GbpC was the most disruptive [Lynch et al., 2007]. The inclusion of dextran ( -1,6-linked glucan; molecular weight 9,000) in the model system effectively blocks or reduces adherence of microorganisms to glucan-coated apatitic surface. Furthermore, treating the in situ formed glucan with dextranase [Schilling et al., 1989; Venkitaraman et al., 1995] prevented or reduced bacterial adherence whereas mutanase treatment ( -1–3) was without effect. These observations suggest strongly that presence of -1,6-linkages in GtfBand GtfC-derived glucans is conferring a specific structure or conformation that provides at least 1 binding site. Results from using atomic force microscopy demonstrate that attachment of S. mutans to tooth surfaces was largely mediated by glucan production and that attachment strengthens with time [Cross et al., 2007]. Evidence is lacking that ionic interactions play a role in the binding of S. mutans to glucan [Cross et al., 2007], especially considering that glucan formed in vitro is uncharged. Furthermore, it is apparent that glucan synthesized in situ by Gtf in pellicle provides enhanced binding for several oral microorganisms. Thus, it appears that a glucan surface on HA (mostly produced by GtfC) provided distinct binding sites for oral microorganisms that do not bind readily to HA or sHA. Concomitantly, glucan formed by Gtf adsorbed to the surface of cells (primarily GtfB) provides an opportunity for aggregation and accumulation of cells and structural support for development of microcolonies ( fig. 1 ) at the same time increasing the bulk of plaque. It is apparent that the formation of cell clusters and microcolonies tightly adherent on the surface of HA is a result of interplay between GtfB and GtfC enzymes and their glucan products at different loci [Koo et al., 2010b]. The presence of highly adherent and insoluble glucans in situ increases mechanical stability by binding the bacterial cells together and to the apatite surface ( fig. 1 ) allowing them to persist on tooth enamel in high levels for a prolonged period. These polymers, in addition to interactions with specific Gbps expressed on S. mutans (and other oral microorganisms) are critical in maintaining the 3-dimensional structure of microcolonies over time ( fig. 1 ), thereby modulating the development of cariogenic biofilms [Lynch et al., 2007; Koo et al., 2010b; Xiao and Koo, 2010]. Based on these observations, we have revised and updated the glucan-mediated bacterial adherence model initially proposed by Rölla et al. [1983b] as shown in figure 2 . Exopolysaccharide Matrix and Cariogenic Plaque Development The precise role(s) of EPS in the pathogenesis of dental caries is in some measure controversial. Nevertheless, there is considerable agreement on many aspects [Paes Leme et al., 2006]. Glucan formed on the tooth surface synthesized by Gtf incorporated into the pellicle from sucrose can provide enhanced binding sites for a number of oral microorganisms, especially S. mutans . In turn, microorganisms that synthesize glucans, or adsorb Gtf and become de facto glucan producers, form highly stable and persistent microcolonies. The exopolymers produced in situ provide a coherent, adherent and mechanically stable matrix, and offer a continuum of a diverse range of binding sites; the structure of glucan is altered over time by the presence of mutanases and dextranases [Guggenheim and Burckhardt, 1974; Walker et al., 1981; Hayacibara et al., 2004]. In addition, Gtfs may bind to the glucan already formed within dental plaque [Tamasada et al., 2004]. Glucan adds to the bulk of plaque thereby leading to a dense concentration of microorganisms on cloistered sites of tooth surfaces protected from inimical influences [Thurnheer et al., 2003; Kreth et al., 2008; Xiao and Koo, 2010]. In addition, soluble glucans may be readily digested and used as a reserve source of energy and contribute in part at least to the low pH values observed in cariogenic plaque [as reviewed in Paes Leme et al., 2006]. It is also noteworthy that elevated amounts of insoluble glucans in dental plaque significantly reduced the inorganic concentration in the matrix, particularly Ca, P and F [Cury et al., 2000; Paes Leme et al., 2006, 2008]. Plaque should not be viewed as a homogeneous accumulation of microorganisms within a matrix but as a collection of highly organized microenvironments which can display distinct structures, compositions and diverse pH values (ranging from as low as pH 4 at the tooth-biofilm interface to as high as pH 6.5 at the biofilm-fluid phase) [Xiao et al., unpubl. data]. The physical role that glucan in plaque plays in cariogenesis is subject to diverse opinions. It is postulated that glucan limits diffusion of charged ions into and out of plaque whereas uncharged substances such as sucrose may diffuse readily [Melvaer et al., 1972, 1974; Melsen et al., 1979]. Available evidence suggests that the EPS from S. mutans and possibly additional oral microorganisms may be charged possibly by the incorporation of lipoteichoic acid [Melvaer et al., 1972, 1974; Melsen et al., 1979; Kuramitsu et al., 1980; Rölla et al., 1980; Vickerman and Jones, 1992]. In addition, Rölla et al. [1980] have observed elevated levels of lipoteichoic acid in sucrose-induced dental plaque in vivo. Based on extensive in vitro modelD ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 1/ 20 17 6 :5 7: 26 P M Biology of Streptococcus mutansDerived Glucosyltransferases Caries Res 2011;45:69–86 75 ing, Dibdin and Shellis [1988] suggest that polysaccharides within plaque have little or no influence on diffusion and that spaces within the matrix of plaque provide ‘storage’ of acids and microbial products thereby providing effective maintenance of an elevated concentration of acid in contact with the tooth surface; they further suggest that this property alone may account for the virulence of the acid attack from dental plaque without invoking diffusion limitations. These observations are supported in part by Hata and Mayanagi [2003], who noted that EPS had little effect on acid diffusion in artificial plaque. In contrast, Tatevossian [1990] and others [Wilson and Ashley, 1990] note that concentrations of solutes in plaque fluid differ markedly from those observed in saliva, an observation which suggests that there is some restriction between the interior of dental plaque and its external environment. Furthermore, it appears that diffusion rates measured by different methods are widely diverse. Tatevossian [1990] further notes: ‘We have much to learn about the fluid environment of the teeth and about dynamic changes in plaque fluid composition and properties during perturbations.’ Polysaccharide within dental plaque is not evenly distributed, and its density is enhanced at the tooth interface [Saxton and Kolendo, 1967; Reese and Guggenheim, 2007]. The presence of EPS-rich matrix and microcolonies appears to create a myriad of microenvironments displaying a distinct range of in situ pH values [Xiao et al., unpubl. data]. 24 h Cross-sectional image of selected area 3D reconstruction of biofilm