Sugar metabolism by mutans streptococci

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1 Journal of Applied Microbiology Symposium Supplement 1997, 83, 80S 88S Sugar metabolism by mutans streptococci S.M. Colby and R.R.B. Russell Department of Oral Biology, Dental School, University of Newcastle, Newcastle upon Tyne, UK 1. Introduction, 80S 5.1 Glucosyltransferase, 83S 2. Oral streptococci, 80S 5.2 Dextranase, 84S 3. Carbohydrate metabolism in plaque, 81S 5.3 Dextranase inhibitor, 85S 4. Fructans in dental plaque 6. Glucan-binding proteins, 85S 4.1 Fructosyltransferase, 82S 7. Comparisons between species, 86S 4.2 Fructanase, 82S 8. Acknowledgements, 86S 5. Glucans in dental plaque 9. References, 86S 1. INTRODUCTION viridans group and advances in taxonomy in recent years (see Hardie and Whiley, pp. 1S 11S) have led to an appreci- Dental caries are arguably the most prevalent bacterial disease ation of their different properties and contribution to dental in developed countries and are responsible for enormous costs plaque formation. Plaque represents a highly complex ecoin terms of pain and discomfort, productivity losses due to system with many different types of bacteria present (Milnes toothache or dental appointments and provision of proet al. 1996) but streptococci generally comprise the majority fessional treatment. It is now just over a century since it was and so have attracted considerably more research interest first proposed that caries (dissolution of tooth enamel) is due than other plaque species. to the acids generated by bacterial fermentation of carbo- On the cleaned tooth surface, Streptococcus sanguis, Strep. hydrates and the link between dietary sugar and dental caries mitis and Strep. oralis predominate among the first bacteria to is well established (Moynihan 1996). While many carbocolonize and it is believed that these pioneer species help to hydrates may be utilized by plaque bacteria to generate acids, establish conditions for the subsequent development of the sucrose is recognized as being particularly important in the plaque biofilm by a combination of processes: growth and caries process because not only can it be fermented, but it division of attached bacteria to form microcolonies, estabalso serves as substrate for extracellular enzymes of plaque lishment of other species through coaggregation with the bacteria which synthesize sucrose-derived polymers. These pioneers (Kolenbrander 1993) and local changes in physiopolymers are of central importance in adhesive interactions logical conditions which encourage proliferation of other in plaque, where they mediate attachment of bacteria to the species. Of particular interest are the circumstances which tooth surface and to other bacteria thus stabilizing the plaque encourage colonization by Strep. mutans and Strep. sobrinus, biofilm, serve as energy stores aiding the survival of plaque which are the two species clearly implicated in the initiation bacteria and modulate the permeability of plaque and hence of caries. Studies from around the world have shown that the level of acid at the enamel surface. Streptococci are of 98% of adults carry Strep. mutans, while different studies central importance in the formation and metabolic activity of have found Strep. sobrinus in 7 35% of individuals. The plaque, and recent years have seen significant advances in majority of epidemiological studies exploring the association our understanding of the processes which can lead to the between streptococci and caries have determined the comdevelopment of caries. bined numbers of the two species so their relative importance is unclear, though in populations with a high caries prevalence 2. ORAL STREPTOCOCCI it is obvious that Strep. mutans must be the significant organism since it is mostly found in the absence of Strep. sobrinus. Some 19 distinct species of streptococci have the human oral There have, however, been suggestions that Strep. sobrinus is cavity as their natural habitat, all of them belonging to the more virulent when present (de Soet et al. 1993). Both species, which belong to the mutans group of streptococci, cause Correspondence to: Professor R. R. B. Russell, Department of Oral Biology, caries in experimental animals and both have been the subject Dental School, University of Newcastle, Newcastle upon Tyne NE2 4BW, UK. of extensive investigation at the molecular level (Russell 1997 The Society for Applied Bacteriology

2 STREPTOCOCCAL SUGAR METABOLISM 81S 1994). Other members of the mutans group of streptococci, mation on certain species will give clues as to the capabilities Streptococcus cricetus, Streptococcus rattus and Streptococcus of others. downei, were originally isolated from animals and are rarely, Streptococcus mutans is remarkably versatile in the range of if ever, found in humans. These species can, however, provide carbohydrates which it can utilize and this may be one of the useful model organisms for the study of various biological features which enables it to outgrow other species when the properties which they share with the species found in diet is rich in carbohydrate, regardless of the particular sugars humans. present. Versatility of Strep. mutans is also manifested with In healthy adults with a low level of caries experience and regard to sugar uptake and aciduricity. Although the oral an exemplary diet, i.e. one with a low sucrose consumption cavity might be considered to be an ideal place for bacteria and no snacking between meals, the mutans streptococci may to grow, being warm, moist and well supplied with nutrients comprise less than 0 01% of the plaque bacteria. A switch to (host salivary glycoproteins supplemented with three or more a sugar-rich diet leads to an increase in the mutans species meals a day), local conditions in dental plaque are subject and mutans streptococci may comprise more than half of the to considerable fluctuation. The main continuous source of viable bacteria in plaque at tooth sites where caries are active. sugars comes from the breakdown of saliva but during meals Longitudinal studies have demonstrated that a high level of it has been estimated that the available sugar may rise mutans streptococci in plaque at a caries-prone tooth site fold from 1 mmol l 1 to 100 mmol l 1 while the ph may vary (particularly in fissures or between teeth) gives a high risk of from greater than 6 7 (the average ph of saliva) to less than subsequently developing caries at that site (Loesche 1986). ph 4 0. In order to flourish in such challenging conditions We are therefore faced with two questions: what conditions Strep. mutans has a multiplicity of transport mechanisms. For encourage the mutans streptococci, and what properties make example, sucrose is taken up by three distinct systems with them so cariogenic once they are established? This article will K m values of mol l 1, mol l 1 and not concern itself with the surface components of streptococci mol l 1 so that there is efficient uptake over a which may be involved in adhesive interactions with other broad range of external sucrose concentrations (Slee and bacteria or with host macromolecules but will focus on the Tanzer 1982). The uptake systems have been identified as physiological processes involving carbohydrates. a sucrose-specific phosphotransferase (PTS), the trehalose- PTS and a third transport system which may correspond to the multiple sugar metabolism system (Tao et al. 1993). Once inside the cell, sugars are fermented by the glycolytic pathway and Strep. mutans can maintain glycolysis at ph values as low 3. CARBOHYDRATE METABOLISM IN as 3 8. Streptococcus sobrinus is even more aciduric, though it PLAQUE is capable of utilizing a narrower range of sugars than Strep. Pioneer species of streptococci are not in general strong acid mutans. The molecular basis of aciduricity of the mutans producers but, in the presence of fermentable carbohydrate streptococci is not yet understood but is the subject of investigation in the diet, they will lower the ph so that acid-tolerant in a number of laboratories. bacteria will be favoured. A sugar-rich diet can thus induce Clearly, any of the sugars which can be fermented may an ecological shift in the plaque microflora and a resultant contribute to acid production but of particular interest is the increase in aciduric species, especially mutans streptococci metabolism of the sugar which has been termed the archcriminal and lactobacilli (Marsh 1994). There is little detailed information in dental caries, sucrose. The levels of mutans strepand available on sugar transport and metabolic pathways tococci in the mouth respond rapidly to changes in the in the pioneer species of streptococci, though it is clear that amount of sucrose in the diet (Wennerholm et al. 1995) and even within individual species there is variation in acid- a number of properties may explain this. Direct uptake of producing capacity. It is thus apparent that some strains of sucrose has already been alluded to above, and it has been species which have not shown an overall association with estimated that 90% of the sucrose encountered by Strep. caries may still be important in the development of caries, mutans is taken up and enters glycolysis, leading to acid and account for the group of non-mutans streptococci generation. However, since this pathway is common to all detected in caries lesions where no mutans streptococci are sugars, the intracellular metabolism of sucrose may be con- detectable (Sansone et al. 1993). Simple classification to the sidered to offer no insights into its unique propensity to species level therefore seems to be inadequate in delineating contribute to caries. On the other hand, the metabolism of the properties which may make a bacterium cariogenic and it sucrose by extracellular enzymes does offer some distinctive is unwise to consider the mutans streptococci to have a mono- features and its different fates are summarized in Fig. 1. Extracellular enzymes synthesizing fructans or glucans, enzymes degrading these polymers and glucan-binding pro- teins have all been identified. poly of properties which may contribute to caries. Nevertheless, it seems probable that the virulence properties of all cariogenic streptococci are comparable, and detailed infor-

3 82S S.M. COLBY AND R.R.B. RUSSELL (a) Strep. mutans sucrose ftf frua gtf B gtf C gtf D fructan + fructose (b) Strep. sobrinus gtf I glucans + glucose fructose dexa + modified glucans isomaltosaccharides dei msm glucose dexb modified glucans cleaves sucrose to release free glucose (which is available for uptake into the cell) while linking the fructose parts of the molecule together to yield an inulin-like fructan. Until the introduction of gene cloning techniques, it was not clear how many FTF Strep. mutans were produced because multiple bands of FTF activity ranging in size from to Da could be resolved by SDS-PAGE (Russell and Gilpin 1987). A single ftf gene has now been cloned and nucleotide sequencing has shown it to have a deduced molecular weight of (Shiroza and Kuramitsu 1988). The reason for the appearance of multiple electrophoretic forms is not clear, but post-translational modification by proteolysis is a common feature of the extracellular enzymes of Strep. mutans (Russell et al. 1986). Streptococcus sobrinus does not have a fructosyltransferase. glucans sucrose gtf S 4.2 Fructanase dexa gtf T + It is likely that a variety of different plaque bacteria can gtf U exploit fructan as a substrate and Strep. mutans itself has a + isomaltosaccharides fructanase. This enzyme is an exofructosidase which releases fructose single fructose units which can be taken up and metabolized. Fructanase is found in the supernatant fluid of liquid cultures so an unexpected finding from the sequencing of the frua gene was the presence of an LPXTG motif close to the C- terminus (Burne and Penders 1992). Such a motif, together Fig. 1 The metabolism of sucrose by extracellular enzymes in with a C-terminal hydrophobic tail, is characteristic of pro- (a) Streptococcus mutans and (b) Strep. sobrinus. Gene symbols: teins which are covalently anchored to peptidoglycan. Two gtf, glucosyltransferase; dexa, dextranase; ftf, wall-associated proteins of Strep. mutans (Ferretti et al. 1989; fructosyltransferase; frua, fructanase; msm, multiple sugar Okahashi et al. 1989) and the dextranase described below also metabolism operon; dexb, dextran glucosidase; dei, dextranase have these wall-anchor motifs and a region rich in proline or inhibitor. Both species also have mechanisms for the direct uptake of sucrose serine threonine which is thought to correspond to a wall- spanning segment. Although LPXTG proteins may be covalently linked to peptidoglycan (Schneewind et al. 1995) all the 4. FRUCTANS IN DENTAL PLAQUE Strep. mutans proteins with this motif are released, the extent of release depending upon growth conditions (Lee 1992; Fructans are linear polymers consisting solely of fructose Burne and Penders 1994). The surface protein WapA has a units, synthesized from sucrose. The fructose units may be site of cleavage outside the wall-spanning layer (Ferretti et joined by b(2 6)-linkages, in which case they are referred to al. 1989) and fructanase also is subject to proteolysis (Burne as levan, or b(2 1)-linkages as are found in inulin from and Penders 1994) but the sites of cleavage have not been dahlia tubers. Despite evidence for a high rate of synthesis of defined. There may be more than one release mechanism fructan, only low levels are found in plaque and this is and the biological significance of the phenomenon remains believed to be due to a high rate of turnover, with fructan unclear. It has been suggested that the release of surface being rapidly degraded. Fructan thus serves as a short-term components by a surface protein releasing enzyme (SPRE) extracellular energy storage molecule. There is conflicting can modulate the balance between immobilized cells and evidence as to whether the ability to make fructan contributes planktonic ones which would be free to migrate and colonize to the virulence of Strep. mutans (Kuramitsu 1993). other sites in the mouth (Lee et al. 1996). An alternative viewpoint is to envisage varying conditions in plaque when 4.1 Fructosyltransferase there may be different advantages to Strep. mutans of having In Strep. mutans, the most active extracellular enzyme acting on sucrose is fructosyltransferase (FTF), an enzyme which enzymes such as fructanase surface-associated (benefiting the single cell) or a roving fructanase which would benefit the entire local microbial community.

4 STREPTOCOCCAL SUGAR METABOLISM 83S 5. GLUCANS IN DENTAL PLAQUE (Dibdin and Shellis 1988; van Houte et al. 1989). Exposure to sucrose thus has a number of effects, aiding colonization In contrast to the rapid turnover of fructan, glucans are a by adhesion to the tooth and increasing the total plaque bulk constant feature of plaque (Hotz et al. 1972) and consist of while at the same time enhancing the overall rate of acid glucose units joined by a(1 6)- and/or a(1 3)-linkages. The production relative proportions of these two linkages and the degree of branching determine the ultimate properties of the glucan. A glucan which is essentially a linear a(1 6)-linked chain is referred to as a dextran and is soluble (the trivial name 5.1 Glucosyltransferase dextransucrase is often applied to the enzyme which produces Because of the importance of glucans in plaque, the glucosyltransferases dextran); in contrast mutan is a water-insoluble glucan with (GTF) which make glucans have attracted a high proportion of a(1 3)-linkages. In practice, the glucan considerable interest. Streptococcus mutans produces three distinct in plaque will be a highly complex macromolecular network GTF while Strep. sobrinus produces four. Each GTF is resulting from the action of synthetic and degradative encoded by a separate gene and each enzyme has distinctive enzymes described below. However, experiments have shown properties, varying in its requirement for a primer molecule that both soluble and insoluble glucans are important in cell to start the polymerization reaction, the proportion of a(1 cell and cell surface adhesive interactions in dental plaque, 6)- and a(1 3)-linkages and degree of branching it introduces with dextran-mediating bacterial aggregation while a(1 3)- into the glucan, and the total length of the glucan chain rich, branched insoluble glucans have been shown to be the produced. The glucans made by the individual GTF of Strep. major contributor to adherence to hard surfaces (Ebisu et al. mutans have not been well characterized, but the GTF of 1974). Strep. sobrinus all have individual characteristics (Table 1). Besides their adhesive properties, a range of other func- Within dental plaque, the overall properties of the glucan tions for glucans has been suggested, including acting as an present will depend on the relative activity of the different extracellular energy store (see below) and protecting strep- GTF present and also on their interactions, since one GTF tococci from attack by host defences, bacteriophage or bacteriocins. may modify the product of another. With regard to the caries process, it has been The gene sequences of 12 streptococcal GTF are now proposed that their major significance may be in establishing available, as well as two from Leuconostoc mesenteroides. Mul- an intercellular matrix which determines the density of bac- tiple alignment of the deduced amino acid sequences has terial cells in plaque: in the presence of sucrose large amounts revealed common features of GTF. All are of high molecular of glucan are produced and effectively reduce the number of weight ( kda) and composed of distinct domains Strep. mutans in a unit volume. However, the looser plaque (Fig. 2). The challenge facing those working in the field of structure results in more efficient diffusion of nutrients GTF is to identify those features of the protein structure through the matrix and this leads to an overall greater metabolic which determine function. We need to understand what it is activity and hence higher rate of acid production that determines why a particular GTF makes a particular Table 1 Glucosyltransferases of Streptococcus sobrinus Glucosyltransferase Glucan Enzyme Water Molecular Gene names Primer Structure solubility weight gtf I I* P3 Dependent Branched, Insoluble High mostly, a(1 3)-linkages (insoluble) gtf U S1 P4 Dependent Branched, Soluble a(1 6)- and a(1 3)-linkages gtf S S3 P2 Independent Linear, Soluble ³5300 a(1 6)-linkages gtf T S4 P1 Independent Branched, Soluble a(1 6)- and a(1 3)-linkages Nomenclature used by Cheetham et al. (1991)* and Hanada et al. (1993). Data on primer dependence and glucan properties are also taken from these authors.

5 84S S.M. COLBY AND R.R.B. RUSSELL type of glycosyl linkage, what determines the length of glucan chain which it produces and what influences the kinetics of the enzyme reaction, such as the need for a primer glucan. A B C D Detailed knowledge of the individual GTF properties will Fig. 2 Representation of the domain structure common to all help in elucidating the way in which they interact with each glucosyltransferases (GTFs). A, Signal peptide (42% homology other. between GTFs); B, variable region of unknown function (ca 200 Once the deduced amino acid sequence of GTF became residues) which is unique to each GTF; C, conserved region (43% available, a search of the databanks indicated a degree of homology between GTFs) of ca 800 residues. This is thought to relatedness to a-amylase (Ferretti et al. 1987) and suggested be the catalytic domain. The solid bar indicates the region that comparison with other enzymes acting on carbohydrate homologous to the (b/a) 8 barrel; D, repeat region essential for glucan binding. This domain consists of a series of tandem substrates would be informative. GTF can be regarded as repeats (ca 30 amino acids each) which are also found in proteins glycosyl hydrolases, which have the ability to hydrolyse of other organisms and may represent a common type of sugar sucrose and also to transfer the released glucose to a growing binding domain acceptor chain. Information on other glycosidic enzymes should thus aid our understanding of GTF. For example, it is known that the catalytic mechanism of glycosidases involves glutamic acid or aspartic acid residues, providing a nucleo- helices and the central domain of GTF sequences can also be philic carboxylate and a general acid catalyst (Sinnot 1990). aligned in this way (Fig. 2). The striking and distinctive While multiple alignment of related sequences can often help feature of the GTF alignment is that the first helix detected identify conserved residues essential for catalysis, alignment in the GTF sequence aligns with a helix H3 of the other of GTF sequences has failed to locate the critical residues proteins, while the b and a segments normally found at the (there are 23 points where glutamate is conserved in all GTF!) start of proteins such as amylase (E1 H1 E2 H2 E3) come in so progress depends on advances in structural determination the latter part of the GTF sequence. In other words, in the or comparison with heterologous sequences. Mooser et al. GTF there is a totally novel construction in which the usual (1991) were able to trap a glucosyl-enzyme intermediate for- arrangement of alternating sheets and helices has undergone med during the reaction of GTF with sucrose and isolated cyclic permutation (MacGregor et al. 1996). Nevertheless, all the relevant active site peptide from GTF, demonstrating the elements required to form the predicted standard (b/a) 8 that it was homologous to a common glycosidase motif, with barrel are present in GTF and this model has the potential an aspartic acid implicated as the core residue. Kato et al. of a major advance in understanding GTF structure/function (1992) have confirmed the importance of this residue by relationships. There is now a considerable body of knowledge site-directed mutagenesis. From our knowledge of enzyme on the amino acid residues involved in the active site of mechanisms, we can anticipate that a number of amino acid amylase and some of the other glycosidases, so super- residues well separated on the primary sequence but juxta- imposition of the amylase and GTF sequences allows prediction posed when the protein is folded into its tertiary structure of which residues are important in GTF. Although will play critical roles in GTF function. The large size of amylases show very great sequence diversity, four well conserved GTF makes them extremely challenging molecules for crystallization regions have been identified and the corresponding and X-ray crystallography and no 3-D structural regions of GTF can be recognized. Site-directed mutagenesis model is yet available. However, there has been a recent of specific amino acids can now proceed on a rational basis exciting advance in determining the structure of GTF by and yield new insights into GTF mechanisms. MacGregor et al. (1996), who applied a combination of sequence alignment, hydrophobic cluster analysis and structure prediction strategies to GTF. 5.2 Dextranase Glycosyl hydrolases have been grouped and classified into The nature and amount of glucan in plaque is influenced not a series of families on the basis of amino acid sequence similarities only by the activity of the GTFs but also by that of extra- and, as structures become available, larger groupings cellular dextranase. A wide range of bacterial species associ- of structurally related families are being formed (Davies and ated with dental plaque have been shown to produce Henrissat 1995). One major grouping consists of enzymes dextranase (Johnson 1990) and those of Strep. mutans and with a (b/a) 8 barrel structure also referred to as a TIM Strep. sobrinus have been studied in some detail (Wanda and barrel in which eight b strands (E1 E8) alternate with eight Curtiss 1994; Igarashi et al. 1995). a helices (H1 H8). Included in this group are a-amylase, Dextranase (Dex) is able to break down glucans to iso- cycloglucanotransferase, isoamylase and the DexB dextran glucosidase from Strep. mutans. The sequences of all these proteins can be aligned on the basis of their b sheets and a maltosaccharides 3 4 glucose units long by cleaving a(1 6)- linkages within the dextran chain (Pulkownik and Walker 1977; Walker et al. 1981). This activity may modify glucans

6 STREPTOCOCCAL SUGAR METABOLISM 85S by altering the ratio of a(1 6)- to a(1 3)-linked chains, hence 5.3 Dextranase inhibitor influencing solubility and adhesive properties. Dex activity Despite the similarity in amino acid sequence, there appear therefore influences sucrose-dependent adherence by reducto be differences in the regulation of dextranase in the oral ing the number of a(1 6)-linkages in the glucan and this has streptococci. A heat-stable protein which inhibits dextranase been demonstrated in Strep. mutans where Dex mutants activity, known as Dei, has been detected in Strep. sobrinus accumulate onto a smooth surface to a markedly greater extent and Strep. downei but not in Strep. mutans (Hamelik and than the wild type (Colby et al. 1995). A further consequence McCabe 1982; Sun et al. 1994). The activity of this protein of Dex activity may be to provide primer or branch points is specific since Dei from Strep. sobrinus will only inhibit for GTF thus contributing to the complexity of the glucan dextranase from Strep. sobrinus and the closely related Strep. structure (Germaine et al. 1977). downei and Strep. cricetus but not from Strep. mutans (Pearce Apart from its effect on the adhesiveness of glucans, the et al. 1991; Sun et al. 1994). Under conditions of carbohydrate dextranase of Strep. mutans breaks down glucans to isolimitation of Strep. sobrinus, Dei levels are high and so little maltosaccharides which may then be transported into the cell active dextranase can be detected. When growth rates via the multiple sugar metabolism (msm) operon (Russell et increase, however, as would occur in plaque at meal times, al. 1992; Tao et al. 1993) where they are further degraded to the relative proportions and binding of Dex and Dei alter glucose by a dextran glucosidase, DexB (Colby et al. 1995). so that under these conditions free Dex becomes available Streptococcus sobrinus does not have such an uptake mech- (Wellington et al. 1994). This contrasts with Strep. mutans anism and is unable to utilize dextrans or isomaltosaccharides where available Dex is released from early exponential phase as sole carbon sources (Ellis and Miller 1977). (Walker et al. 1981). There is significant homology between the deduced amino acid sequences of Strep. mutans and Strep. sobrinus dextranases. Both have N-terminal signal peptide sequences as would be expected of extracellular enzymes and, near the C-terminal 6. GLUCAN-BINDING PROTEINS end, the LPXTG motif followed by a hydrophobic region The glucan-binding domain of GTF may have dual functions: and a charged tail similar to the proposed wall-anchor region at least one or two of the repeat units are needed for in fructanase (Fig. 3). Dextranase differs from other LPXTG glucan synthesis (Ferretti et al. 1987) but the binding capacity proteins in not having a clearly defined region rich in the will also lead to glucan-mediated binding of bacterial cells hydrophilic amino acids serine, threonine or proline but it is through surface-associated GTF. Free GTF is also found in remarkable in having a large portion of the molecule apparently saliva and on the tooth surface where it remains active so that not needed for dextranase activity. The size of the glucans made on the tooth form another site of attachment dextranases differs and it seems reasonable to assume that for bacteria (Schilling and Bowen 1992). Besides GTF, a the common region is responsible for dextranase activity. number of other glucan-binding proteins (GBP) without Multiple active forms of Dex have been reported for Strep. known enzyme activity can be found. Streptococcus mutans sobrinus, ranging in size from to (Barrett et al. produces two GBPs, one of which consists of a series of 1987) and a similar phenomenon is seen with Strep. mutans tandem repeat units homologous to those found in the car- (Russell and Ferretti 1990; Igarashi et al. 1992; Colby et al. boxyterminal one-third of GTF (Banas et al. 1990). The 1995) and also Strep. salivarius (Lawman and Bleiweis 1991). occurrence of this independent GBP is consistent with the The points of cleavage have not been determined but it seems idea that GTF have a modular structure and this is supported likely that the multiple forms result from sequential removal by the fact that the Dei dextranase inhibitor also contains of C-terminal stretches of amino acids. However, the role of homologous repeat units (Sun et al. 1995). Furthermore, the non-enzymatic C-terminal regions of these large molecules homologous sequences are found in proteins from other remains an enigma. organisms which also have a modular structure: autolysin of Strep. pneumoniae, fibronectin-binding protein of Strep. pyogenes and toxin A of Clostridium difficile (Wren 1991; Talay Strep. mutans Dex et al. 1994; Yother and White 1994). In all these cases the repeat-containing module is thought to be involved in binding to a macromolecular carbohydrate substrate, and subtle differences Strep. sobrinus Dex in protein sequence must determine the specificity A B C D of binding. Fig. 3 Representation of the structure of the extracellular A second GBP which induces a strong immune response dextranase enzymes of Streptococcus mutans and Strep. sobrinus. Both in children has been described in Strep. mutans (Smith et al. proteins possess a signal peptide (A), a conserved domain with 1994) and Strep. sobrinus also produces a number of GBP (Ma 57% homology (C), and two variable regions (B and D) et al. 1996; Wu-Yuan and Gill 1996). Ma et al. (1996) have

7 86S S.M. COLBY AND R.R.B. RUSSELL produced evidence that only certain of these, which they call 9. REFERENCES glucan-binding lectins (GBL), can mediate aggregation by Banas, J.A., Russell, R.R.B. and Ferretti, J.J. (1990) Sequence glucan. analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt. Infection and Immunity 58, Barrett, J.F., Barrett, T.A. and Curtiss, R. (1987) Purification and partial characterization of the multicomponent dextranase com- 7. COMPARISONS BETWEEN SPECIES plex of Streptococcus sobrinus and cloning of the dextranase gene. Infection and Immunity 55, Streptococcus mutans and Strep. sobrinus occupy a similar eco- Burne, R.A. and Penders, J.E.C. (1992) Characterization of the logical niche and both share the capacity to produce extra- Streptococcus mutans GS-5 frua gene encoding exo-b-d-fruccellular polymers from sucrose and to adhere to hard surfaces tosidase. Infection and Immunity 60, by means of these polymers. Nevertheless, as Fig. 1 illus- Burne, R.A. and Penders, J.E.C. (1994) Differential localization of trates, the details of these processes are distinct in each spec- the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA. ies. Streptococcus mutans has three GTF and an FTF as FEMS Microbiology Letters 121, compared to four GTF (and no FTF) in Strep. sobrinus. While Cheetham, N.W.H., Walker, G.J., Pearce, B.J., Fiala-Beer, E. and both species produce dextranase, the controlling inhibitor Dei Taylor, C. (1991) Structures of water-soluble a-d-glucans syn- thesized from sucrose by glucosyltransferases isolated from Strepis found only in Strep. sobrinus while Strep. mutans can not tococcus sobrinus culture filtrates. Carbohydrate Polymers 14, only degrade dextran but also utilize the resultant oligo- Colby, S.M., Whiting, G.C., Tao, L. and Russell, R.R.B. (1995) saccharides for intracellular metabolism. The number and Insertional inactivation of the Streptococcus mutans dexa (dextransize of GBP also differs between the two species but the way in ase) gene results in altered adherence and dextran catabolism. which these are involved in adhesion is still unclear. Further Microbiology 141, investigations of the function of all these different comglycosyl hydrolases. Current Opinion in Structural Biology 3, 853 Davies, G. and Henrissat, B. (1995) Structures and mechanisms of ponents should throw light on their relative contributions to the complex process of growth and adherence in the oral 859. cavity. In particular, now that many of the genes have been de Soet, J.J., Holbrook, W.P., Magnusdottir, M.O. and De Graaff, J. (1993) Streptococcus sobrinus and Streptococcus mutans in a longicloned, targeted gene inactivation allows comparison of tudinal study of dental caries. Microbial Ecology in Health and mutants with wild type in in vitro models of adhesion (Colby Disease 6, et al. 1995) and in vivo, where the capacity to cause caries Dibdin, G.H. and Shellis, R.P. (1988) Physical and biochemical in experimental animals can be explored (Kuramitsu 1993). studies of Streptococcus mutans sediments suggest new factors Improved knowledge of the structure of critical molecules linking the cariogenicity of plaque with its extracellular polysaccharide content. Journal of Dental Research 67, also sets the scene for rational design of specific inhibitors, or identification of targets for vaccine development (Taubman Ebisu, S., Misaki, A., Kato, K. and Kotani, S. (1974) The structure et al. 1995). of water-insoluble glucans of cariogenic Streptococcus mutans, for- This review has presented a number of instances which med in the absence and presence of dextranase. Carbohydrate Research 38, illustrate instructive parallels between research on oral strep- Ellis, D.W. and Miller, C.H. (1977) Extracellular dextran hydrolase tococci and subjects in quite different areas. Examples are from Streptococcus mutans strain Journal of Dental Research the structural similarities between GTF of streptococci and 56, Leuconostoc mesenteroides (the commercial source of dextran), Ferretti, J.J., Gilpin, M.L. and Russell, R.R.B. (1987) Nucleotide between GTF and amylase, the finding of homologous carbo- sequence of a glucosyltransferase gene from Streptococcus sobrinus hydrate binding domains in bacteria from different habitats, MFe28. Journal of Bacteriology 169, and differing mechanisms of cell wall anchorage in proteins Ferretti, J.J., Russell, R.R.B. and Dao, M.L. (1989) Sequence analy- which initially appear to have common features. Studies of sis of the wall-associated protein precursor of Streptococcus mutans the oral streptococci thus have much to contribute to, as well antigen A. Molecular Microbiology 3, Germaine, G.R., Harlander, S.K., Leung, W.-L.S. and Schachtele, as to learn from, a wide range of microbiological systems. C.F. (1977) Streptococcus mutans dextransucrase: functioning of primer dextran and endogenous dextranase in water-soluble and water-insoluble glucan synthesis. Infection and Immunity 16, ACKNOWLEDGEMENTS Hamelik, R.M. and McCabe, M.M. (1982) An endodextranase inhibitor from batch cultures of Streptococcus mutans. Biochemical Research in the authors laboratory has been supported by and Biophysical Research Communications 106, the Medical Research Council (Grant Nos and Hanada, N., Katayama, T., Kunimori, A., Yamashita, Y. and Take- G CB) and Public Health Service grant DE08191 hara, T. (1993) Four different types of glucans synthesized by from the US National Institutes of Health, in collaboration glucosyltransferases from Streptococcus sobrinus. Microbios 73, 23 with the University of Oklahoma. 35.

8 STREPTOCOCCAL SUGAR METABOLISM 87S Hotz, P., Guggenheim, B. and Schmid, R. (1972) Carbohydrates in Pulkownik, A. and Walker, G.J. (1977) Purification and substrate pooled dental plaque. Caries Research 6, specificity of an endo-dextranase of Streptococcus mutans K1-R. Igarashi, T., Yamamoto, A. and Goto, N. (1992) Characterization Carbohydrate Research 54, of the dextranase purified from Streptococcus mutans Ingbritt. Russell, R.R.B. (1994) The application of molecular genetics to the Microbiology and Immunology 36, microbiology of dental caries. Caries Research 28, Igarashi, T., Yamamoto, A. and Goto, N. (1995) Sequence analysis Russell, R.R.B. and Ferretti, J.J. (1990) Nucleotide sequence of the of the Streptococcus mutans Ingbritt dexa gene encoding extra- dextran glucosidase (dexb) gene of Streptococcus mutans. Journal cellular dextranase. Microbiology and Immunology 39, of General Microbiology 136, Johnson, I.H. 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