Virulence Factors of Mutans Streptococci Role of Molecular Genetics

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Virulence Factors of Mutans Streptococci: Role of Molecular Genetics

Howard K. Kuramitsu CROBM 1993 4: 159 DOI: 10.1177/10454411930040020201 The online version of this article can be found at: http://cro.sagepub.com/content/4/2/159
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Critical Reviews in Oral Biology and Medicine, 4(2):159-176 (1993)
Virulence Factors of Mutans Streptococci: Role of Molecular Genetics

Howard K. Kuramitsu
Departments of Pediatric Dentistry and Microbiology, University of Texas Health Science Center, San Antonio, TX ABSTRACT: Biochemical approaches were utilized initially to identify the virulence factors of the mutans streptococci (primarily Streptococcus mutans and 5. sobrinu). Traditional mutant analysis of these organisms further suggested the important role of several of these factors in cariogenicity. However, because these mutations were not clearly defined, the utilization of cloned genes was necessary to verify their significance. The introduction of molecular genetic approaches for characterizing these factors has led not only to a clearer understanding of the role of these virulence factors in cariogenicity but has also suggested some novel approaches for reducing further the incidence of dental caries. KEY WORDS: Streptococcus mutans, S. sobrinus, molecular genetics, adhesins, sucrose-enhanced colonization of teeth, glucosyltransferases.
I. INTRODUCTION Despite the recent decline in dental caries frequency among children in technologically advanced societies, tooth decay still remains a major health problem (Loesche, 1986; Tanzer, 1992). In addition, it is not clear yet whether or not this decline will continue into the next century. The significant improvement in the oral health status of Western children has been attributed primarily to the widespread utilization of fluoride together with improvements in oral care (Glass, 1982). Nevertheless, there still remains a sizeable proportion of the population that is at risk of developing carious lesions (Krasse, 1985). Therefore, a more detailed understanding of the bacterial-host interactions that lead to dental caries may be of value in developing additional anticaries strategies that may further decrease the frequency of this disease. In this respect, the recent introduction of molecular genetic approaches for examining the virulence of mutans streptococci (Loesche, 1986) may yield innovative methods for both the identification of children at high risk of developing dental caries as well as the prevention of decay. Several recent reviews have addressed various aspects of this topic (Curtiss, 1985;
Macrina et ai, 1990; Russell, 1990), and this article focuses primarily on the relationship between the results of these newer approaches to the earlier investigations (Loesche, 1986; Tanzer, 1992). Furthermore, since the publication of these reviews, the results of animal studies utilizing defined mutants constructed from cloned Streptococcus mutans genes are now available. In addition, there is a discussion of several unresolved issues relating to the cariogenicity of the mutans streptococci.
II. VIRULENCE PROPERTIES OF MUTANS STREPTOCOCCI IDENTIFIED FOLLOWING COMPARISON WITH OTHER ORAL BACTERIA Despite the complexity of the human oral flora, pioneering animal model studies (Fitzgerald and Keyes, 1960), as well as human epidemiological surveys (Loesche, 1986), have strongly implicated mutans streptococci (primarily S. mutans) as the principal etiological agents in human dental caries. Therefore, for the past several decades there have been extensive efforts to identify the virulence factors of these organisms as
1045-4411/93/S.50 1993 by CRC Press, Inc.

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potential targets for anticaries prophylaxis. Initially, potential virulence properties of the mutans streptococci were identified following comparison of these organisms with other oral streptococci. One of the first putative virulence factors of the mutans streptococci identified by this approach was the ability of these organisms to colonize smooth surfaces in vitro in the presence of sucrose (Gibbons etal, 1966). Subsequently, this property was shown to be dependent on the synthesis of water-insoluble glucans from the disaccharide. Although several other oral streptococci (5. sanguis, S. salivarius) also have the ability to synthesize these polysaccharides (Hamada and Slade, 1980), only the mutans streptococci generally display sucrose enhanced colonization (colonization is defined in this review as the aggregation of bacteria onto hard surfaces following initial attachment and subsequent accumulation) of smooth surfaces (Loesche, 1986). One recent exception to this rule has been reported for S. gordonii strains in vitro fVickerman et ai, 1991), but its significance in vivo has yet to be established. Therefore, it was quite evident almost 30 years ago that the mutans streptococci are capable of synthesizing colonization promoting glucans from sucrose. Because of the extensive epidimeological evidence linking the incidence of human dental caries with sucrose consumption (Newbrun, 1982), major emphasis was placed on examining the mechanism of glucan synthesis. However, despite detailed investigations regarding the synthesis of these exopolysaccharides and the role of glucosyltransferases (GTF) in this process (Loesche, 1986), the chemical nature of the glucans involved in colonization have not been precisely defined yet. Because of this unique colonization property exhibited by the mutans streptococci, it was not surprising that these organisms also exhibited clumping (aggregation) when grown in the presence of sucrose. This characteristic could be rationalized as another example of sucrose-dependent attachment of cells to solid surfaces due to insoluble glucan formation. However, in addition, many of these organisms also aggregated when cells were incubated in the presence of high molecular weight glucans (especially water soluble high molecular weight dextrans) (Gibbons and Fitzgerald, 1969). This property was also unique to the mutans streptococci and was postulated to
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play a role in colonization. More recently, it has been suggested that such interactions may also aid in the initial attachment of the mutans streptococci to glucans formed within the pellicle of teeth (Schilling and Bowen, 1992). Therefore, two of the earliest virulence properties associated with these organisms were their ability to synthesize colonization, promoting insoluble glucans, and their interaction with these polysaccharides. Despite the initial emphasis on the role of insoluble glucan formation in the colonization of tooth surfaces by the mutans streptococci, both in vitro (Staat et al., 1980) as well as in vivo (Van Houte et aL, 1976) results have suggested that these organisms do not require sucrose for colonization. Subsequent approaches from several laboratories (Douglas and Russell, 1984; Russell, 1986, Lee et aL, 1989) have suggested that these organisms are capable of attaching to the pellicle of teeth by means of putative adhesin-like cell surface molecules. As detailed below (Section IV), molecular genetic approaches have suggested several candidates for this phenomenon. Furthermore, Gibbons et al. (1986) have demonstrated differences in tooth colonization mechanisms that depend on the group of mutans streptococci involved: S. mutans strains apparently attach by both adhesin and glucan mediated mechanisms, whereas S. sobrinus strains utilize primarily the latter process. Because dental caries is ultimately related to the acidogenicity of plaque bacteria, the fermentation patterns of the mutans streptococci have been examined extensively (Hamada and Slade, 1980). These organisms were demonstrated to ferment a wide variety of sugars and it was of special interest that they appear to metabolize sucrose to lactic acid more rapidly than other oral bacteria (Minah and Loesche, 1977). This property of the mutans streptococci is undoubtedly related to the multitude of enzyme systems expressed by these organisms that are capable of both transporting and metabolizing sucrose (Loesche, 1986). It was also reasonable to assume that an important virulence property of cariogenic bacteria should be their ability to continue fermentation in the absence of exogenous food supplies (conditions that are likely to be most conducive for tooth demineralization due to the reduction in salivary flow during these periods) (Loesche, 1986). There-
fore, the observation that some strains of S. mutans that were highly virulent in rats were capable of intracellular polysaccharide storage (IPS) (Tanzer et a/., 1976), whereas others that were not capable of IPS were avirulent, suggested another potential virulence property of these organisms. Because dental plaque pH becomes acidic in the presence of a fermentable carbon source (Loesche, 1986), the relative aciduricity of cariogenic bacteria may also serve as a virulence factor. A comparison of the relative aciduricity of oral bacteria (Harper and Loesche, 1984) has indeed demonstrated that strains of S. mutans are more acid tolerant than all other bacteria examined with the exception of the lactobacilli. This property appears to be related, in part, to the relative acid stability of the membrane-associated H+-translocating ATPase of these organisms (Bender et al, 1986). Another potential virulence factor of mutans streptococci may be the bacteriocins produced by a large variety of these organisms (Hamada and Ooshima, 1975). These antibacterial factors could aid in the establishment of cariogenic bacteria by reducing the presence of potential competitors of tooth colonization. Several investigations (van der Hoeven and Rogers, 1979; Hillman et al, 1987) have demonstrated that bacteriocin production can play a role in enhancing the establishment of mutans streptococci in rodents and in humans. However, both the large variety and the differential spectrum of these inhibitors has made it difficult to assess their role in the virulence of these organisms.
isolated as spontaneous or chemically induced mutants. Therefore, the precise nature of these mutations were not defined and the possibility of multiple defects in each cannot be discounted.
A. Colonization Defective Mutants Among the first groups of mutants to be isolated were those defective in sucrose-enhanced colonization of tooth surfaces because these could be readily detected on sucrose-containing agar plates. Subsequent examination revealed that each of these was defective in insoluble glucan synthesis (Loesche, 1986). However, because the nature of these mutations was undefined and one of these mutants, S. mutans C67-25, was shown to be defective in several other potentially important virulence properties (Donoghue and Newman, 1976), it was not possible to define precisely the mutations at the molecular level. Nevertheless, the implantation of these mutants into rats yielded results that were compatible with the important role of insoluble glucan synthesis in dental caries. These results indicated that insoluble glucan-defective mutants colonized teeth less extensively than the parental organisms in the presence of sucrose. In addition, these results revealed that sucrose (and therefore glucan synthesis) was more significant for smooth surface caries when compared with fissure caries (Tanzer, 1979). These observations were also consistent with earlier findings indicating that sucrose was not required for colonization of rat teeth (van Houte et al, 1976). As described previously, it was possible that glucan-mediated aggregation of the mutans streptococci might also play an important role in tooth colonization. The isolation of mutants altered in either dextran-mediated or sucrose-dependent aggregation (but not necessarily both properties) (Freedman and Tanzer, 1974; Murchison et al. 1981) indicated that the former was not solely mediated by the glucan-binding properties of cellassociated GTFs. These results suggested that either a specific glucan-binding molecule or proteolytic fragments of the GTFs associated with the cell surface were involved. In addition, Russell (1979a) reported the isolation of a cell-surface glucan-binding protein from a S. mutans strain and similar proteins have been identified in other
III. MUTANT CHARACTERIZATION AND THE IDENTIFICATION OF VIRULENCE FACTORS The optimum approach for identifying a virulence factor of a pathogenic microorganism involves isolating a mutant of the organism defective in a specific trait followed by comparison of its pathogenicity with the parental organism in an appropriate animal model. Toward this end, a number of mutants of mutans streptococci defective in potential virulence traits have been isolated (Freedman et al, 1981). These, as well as the other mutants described in this section, were
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mutans streptococci (McCabe etal., 1977; Drake et al., 1988). Nevertheless, rigorous molecular characterization of these proteins is required for identification of these molecules as nonenzymic glucan-binding proteins, inasmuch as it has been demonstrated that proteolytic fragments of GTFs lacking enzymatic activity have the ability to bind glucans (Mooser and Wong, 1988). Several approaches (Staat et al., 1980; Curtiss, 1985) have suggested that mutans streptococcal colonization of teeth involves both a sucrosedependent (glucan-mediated accumulation) and independent (initial attachment) process. Subsequent investigations have indicated that the latter phenomena could result from both specific (Lee et al., 1989) and nonspecific (Gibbons and Etherden, 1983) interactions of the organisms with the pellicle of teeth. Furthermore, the identification of cell surface proteins in these organisms (Russell, 1979b) suggested the possibility that adhesins might be involved in specific attachment to teeth. Following the demonstration that a chemically induced mutant of S. sobrinus 6715 altered in a 210 kDa cell surface protein was defective in both in vitro attachment to saliva-coated hydroxyapatite beads and in cariogencity in rats (Curtiss et al., 1986), it has been proposed that related proteins (antigen I/II, proteins B, PI, IF, and Pac) from other mutans streptococci may also be involved in sucrose-independent colonization of tooth surfaces (Ackermanns et al., 1985; Lee et al., 1989).
C. Mutants Altered in Aciduricity Until now, only one S. mutans mutant altered in aciduricity and examined for cariogencity in an animal model has been described in detail (de Stoppelaar et al., 1971; Donoghue and Newman, 1976). As might be anticipated, this mutant, S. mutans C67-25, was defective in cariogenicity on both enamel and sulcal surfaces. However, as is possible with any chemically induced mutant, it was demonstrated that this mutant also displayed multiple alterations relevant to cariogenicity (colonization, aggregation) (Loesche, 1986). In addition, mutants of S. sobrinus altered in aciduricity display reduced virulence in rats (Tanzer and Freedman, 1978). Therefore, the properties of these mutants were consistent with the importance of aciduricity as a virulence factor for the mutans streptococci.
D. Mutants Altered in Dextranase Activity An interesting class of mutants of S. mutans and S. sobrinus altered in exodextranase activity has been described (Tanzer and Freedman, 1978; Tanzer, 1992). These chemically induced mutants displayed reduced cariogenicity when implanted into Sprague-Dawley rats fed high sucrose diets on both smooth surfaces and fissures of the teeth. However, the molecular basis for virulence reduction in the mutants has not been established.
B. Mutants Defective in Intracellular Metabolism Because tooth decay is primarily determined by the acidogenicity of plaque bacteria, it was not surprising that mutants of some mutans streptococci defective in lactic dehydrogenase (LDH) activity were markedly less cariogenic in rodent model systems (Hillman, 1978; Fitzgerald et al., 1989). Likewise, mutants defective in the storage of intracellular polysaccharides displayed reduced cariogenicity at some tooth surfaces relative to the parental organisms, especially when fed at specified time intervals (Tanzer et al, 1976). IV. VIRULENCE FACTORS CHARACTERIZED UTILIZING CLONED GENES
The introduction of recombinant DNA techniques for investigating the virulence of mutans streptococci has led to a more detailed understanding of the pathogenic factors of these organisms at the molecular level. The isolation of defined mutants constructed from cloned genes and their utilization in animal model systems has also provided a more unequivocal basis for defining the virulence of
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these organisms (Table 1). Such defined mutants can be utilized to obviate complications from multiple or pleiotrophic mutations that could confound some of the earlier interpretations based upon the utilization of spontaneously derived or chemically induced mutants (Loesche, 1986).
implanted into rats. However, because the spaA mutants were isolated following chemical mutagenesis and the possibility of pleiotrophic effects in the mutants has not been evaluated, some caution should be exercised in drawing the conclusion that the spaA protein is a S. sobrinus adhesin required for tooth colonization. Unfor-
TABLE 1 PUTATIVE S. mutatis Virulence Genes

In vivo testing? No No Yes Yes Yes Yes No Yes Yes Yes Caries decrease? * No Yes Yes No No Yes No
Gene Idh ATPase pac gtB

gtiC gtio
Role Acidogeniclty Aciduncity Colonization Colonization Colonization Colonization Colonization Reserve nutrient Reserve nutrient Sucrose transport
Mutants? No No Yes Yes Yes Yes Yes Yes Yes Yes
gbp ftf gig

scrA
= Not tested. See Section IV for references to investigations outlined in table.
A. Cloning of Genes Involved in Sucrose-Independent Colonization of Teeth The initial identification of a molecule that appeared to play a role in attachment of a mutans streptococcus to the pellicle of teeth resulted from the isolation of the spaA gene from S. sobrinus 6715 (Holt et al., 1982). It was demonstrated that the product of this gene, a 210 kDa protein, was primarily associated with the cell surface of the organism. Following chemical mutagenesis and selection for mutants that did not react with anti-spaA antibody, mutants defective in this gene were isolated (Curtiss et al, 1983). Moreover, the spaA protein appeared to be a logical candidate for an adhesin involved in sucrose-independent attachment to teeth because a related protein from S. mutans appears to be involved in attachment to saliva-coated hydroxyapatite beads ((Russell, 1986) and the spaA mutants also produced fewer carious lesions when
tunately, a gene transfer system for S. sobrinus strains has not been developed yet and it has not been possible to construct defined spaA mutants from the isolated gene. In addition, the organization of the spaA gene on the chromosome of strain 6715 relative to other cell surface molecules has yet to be determined and the possibility of polar effects of the spaA mutation on these molecules needs to be evaluated. Nevertheless, the successful isolation and characterization of the spaA gene and its protein product provided the initial impetus for the search for genes coding for cell surface proteins that could play a role in colonizing teeth. Subsequent investigations have revealed that proteins structurally related to the spaA protein are also present on the cell surface of other mutans streptococci (Sommer et al., 1987; Lee et al, 1988; Takahashi et al, 1989) as well as in S. sanguis (Demuth et al, 1988). Because some strains of S. mutans are naturally transformable (Perry and Kuramitsu, 1981), one of
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these was a logical candidate for the construction of a defined mutant altered in the putative adhesin. Bleweis and colleagues (Lee et al, 1989) have constructed a mutant of S. mutans NG8 that is defective in the PI protein (the apparent functional equivalent of the spaA protein of S. sobrinus) utilizing the cloned gene for insertional inactivation following electroporation. One of the resultant mutants, strain 834, displayed reduced hydrophobicity relative to the parental strain, and also did not bind to a salivary agglutinin as well as the parental strain. However, both the parental and mutant strains displayed weak interactions with whole salivacoated hydroxyapatite beads. Furthermore, both strains were equally cariogenic when implanted into conventional pathogen-free rats fed a high sucrose (56%) diet (Bowen et al, 1991). This latter observation is consistent with the in vitro results utilizing saliva-coated hydroxyapatite beads and suggests that, although the PI protein may play a role in tooth colonization, multiple interactions between S. mutans and the pellicle of teeth are important for colonization. In addition, interactions between S. mutans and other plaque bacteria may also contribute to colonization by the former organisms. Likewise, because previous investigations have suggested that S. mutans strains also interact with salivary mucins and a proline-rich protein (Gibbons, 1989), it is reasonable to assume that multiple adhesins may be present on the surface of these organisms. Moreover, because there is no information available presently regarding the regulation of expression of these putative adhesin molecules, the cellular levels of these molecules in the environment of the oral cavity still need to be evaluated. It would also be of great interest to compare the colonization of these mutants with the parental organisms in the human oral cavity.
B. Cloning of Genes Involved in Sucrose-Enhanced Colonization of Teeth The efforts of many laboratories resulted in the proposal that multiple GTFs were involved in the synthesis of the glucans involved in sucrose-
enhanced colonization of teeth (Loesche, 1986). Verification of the expression of multiple GTFs in the mutans streptococci has been obtained recently by isolating individual gtf genes from several of these organisms (Gilpin et al, 1985; Aoki etal, 1986; Hanada and Kuramitsu, 1988; Hanada and Kuramitsu, 1989; Abo et al, 1990; Hanada et al, 1991). These results indicate that most strains of S. mutans contain three distinct gtf genes: gtfB coding for the GTF-I enzyme, gtfC expressing a similar GTF-SI, and gtfD coding for the GTF-S enzyme. The first two enzymes synthesize primarily water-insoluble glucans, whereas the latter produces water-soluble glucans. In addition, two gtf genes have been isolated from 5. downei (Ferretti et al, 1987; Gilmore et al, 1990), synthesizing insoluble and soluble glucans, respectively. Biochemical approaches have further suggested that one or two additional gtf genes may reside on the chromosomes of this latter species and related S. sobrinus strains (Shimamura et al, 1983; Yamashita et al, 1989). Therefore, it is clear that each strain of mutans streptococci expresses one or more GTF enzymes synthesizing soluble glucans and at least one producing insoluble glucans. In order to examine the role of each of the GTFs in sucrose-enhanced colonization of teeth, S. mutans mutants defective in each of the gtf genes have been isolated following insertional inactivation of the genes (Munro et al, 1991; Yamashita et al, unpublished). These mutants have been implanted into either specific pathogen free or gnotobiotic rats fed sucrose diets for the purpose of evaluating the relative contribution of each gene product to colonization and cariogenicity. Macrina and co-workers (Munro et al, 1991) have constructed mutants of S. mutans V403 defective in glucan synthesis, and implanted these into gnotobiotic Fisher rats fed diet 305 (UAB model). Mutants deleted for the two gtf genes (B and C) coding for insoluble glucan synthesis exhibited reduced caries induction on the smooth surfaces of the teeth, relative to the parental strain. Furthermore, no additional reduction in cariogencity was observed when either the fructosyltransferase (ftf) or gtfD genes were individually inactivated. These results were consistent with previous suggestions that water insoluble glucan synthesis was important, though not re-
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quired, for cariogencity in rat models. However, the reductions in caries noted for the defined mutants were not as extensive as exhibited in earlier animal studies utilizing chemically induced mutants of S. sobrinus 6715 defective in insoluble glucan synthesis (Tanzer et ai, 1974). More recently, defined gtf mutants were constructed in S. mutans UA130 for testing in specific pathogen-free Sprague Dawley rats fed diet 2000 (UR system) (Yamashita etaL, unpublished). These results also confirmed the role of insoluble glucan synthesis in cariogenicity on the smooth surfaces of teeth but, in addition, revealed that the gtfB and C genes were both required for maximal cariogenicity. Furthermore, the reductions in smooth surface caries noted for the mutants in this system were significantly greater than those exhibited in the UAB gnotobiotic system (Munro et a I., 1991) and were similar to that exhibited by S. sobrinus 6715 mutants defective in insoluble glucan synthesis fed a 56% sucrose diet (Tanzer et al., 1974). For example, smooth surface caries was decreased by 80% for the gtfB'C mutant in the pathogen-free UR rat model but only on the average of approximately 26% in the UAB gnotobiotic rat system (Munro et aL, 1991). Likewise, inactivation of either the gtfB or C genes of strain UA130 resulted in 76 and 85% reductions, respectively, in smooth surface caries. These comparative results suggest that the UR conventional rat caries system appears to be much more sensitive to alterations in glucan synthesis by S. mutans than the UAB gnotobiotic rat model. As suggested below, these differences cannot be ascribed primarily to differences in the S. mutans strains tested. Additional support for the hypothesis that both products of the gtfB and C genes are important for sucrose-enhanced colonization and cariogenicity was obtained from an examination of the properties of S. mutans UA101 (Yamashita etal., 1992). Unlike most strains of S. mutans examined (Chia et aL. 1991), this strain contains only two gtf genes on its chromosome: one expressing an enzyme synthesizing insoluble glucan, gtfBC, and the other corresponding to the gtfD gene (Hanada and Kuramitsu, 1989). Following isolation of the former gene, it was observed that this gene, gtfBC, was derived following homologous recombination of the tandemly associated B and C genes in
a progenitor of strain UA101. This strain synthesizes approximately 30% of the water insoluble glucan relative to strains GS5 and UA130 and displays relatively weak in vitro sucrose-enhanced colonization. Most importantly, when implanted into the UR conventional rat model system, strain UA101 produced far fewer carious lesions on the smooth surfaces of the rats, relative to strain UA130 on high (56%) sucrose diets (Table 2). Furthermore, introduction of the gtfC gene from strain GS5 into the chromosome of strain UA101 following homologous recombination resulted in a derivative that restored cariogenicity on smooth surfaces to a level similar to UA130. Likewise, incorporation of the gtfB gene into strain UA101 restored the ability of the organism to colonize smooth surfaces in vitro in the presence of sucrose. These results support the hypothesis that, in the presence of the gtfD gene, two copies of the gtf genes expressing enzymes synthesizing insoluble glucan are required for maximum sucroseenhanced colonization of smooth surfaces and cariogenicity by S. mutans strains. Because of the unavailability of data regarding the implantation of comparable g(f mutants for other mutans streptococci into animal models, it is not clear which gtf genes are required for sucrose-enhanced colonization of teeth in these strains. The utilization of S. mutans gtf mutants has suggested that significant quantitative differences are apparent in the UR pathogen-free (Bowen et aL, 1988) and UAB gnotobiotic (Munro et aL, 1991) rat model caries systems. This is further supported by a comparison of the cariogenicity of strains UA101 and UA130 in the two systems (Table 2). In the UAB gnotobiotic system utilizing diet 305 containing 5% sucrose, both strains are equally cariogenic (Barletta et aL, 1988). However, in the UR conventional system UA101 (containing two rather than three gtf genes) induced markedly reduced levels of smooth surface caries relative to strain UA130. As previously suggested from earlier mutant studies (Loesche, 1986), these differences are minimized in regard to fissure caries. Therefore, smooth surface caries in the UR conventional rat model system appears to be more dependent upon insoluble glucan synthesis than in the UAB gnotobiotic system (Table 2). This may result, in part, from the presence of "sticky" components of diet 305 (cellulose and
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TABLE 2 Comparison of UR and UAB Rat Models Relative to the Role of Glucan Formation in Smooth-Surface Dental Caries
UR Model Pathogen-free Sprague-Dawley rats (56% sucrose diet) UAB Model Gnotobiotic Fisher rats (5% sucrose diet)
Smooth surface lesions UA101 UA130 Yamashita et al., 1992. Barletta et al., 1988. 4.08a 34.8 11.0 b 11.6
corn starch) not found in diet 2000 which could obviate the requirement for adhesive insoluble glucan synthesis for smooth surface caries initiation (Yamashita et al, 1992). Earlier biochemical analysis utilizing purified GTFs (Fukushima et al, 1981; Kuramitsu and Wondrack, 1983) suggested that the glucan involved in sucrose-enhanced colonization required both the GTF-I and GTF-S enzymes. However, the results utilizing S. mutans mutants defective in the gtfD gene coding for GTF-S activity suggests that this enzyme is not required for sucrose-enhanced colonization in vivo (Munro et al, 1991; Yamashita et al, 1992). Nevertheless, because the GTF-I and GTF-SI enzymes are capable of synthesizing some water soluble glucans (Aoki etal, 1986; Hanada and Kuramitsu, 1988), these results do not necessarily obviate a role for soluble glucan synthesis in the sucrose-enhanced colonization process. In addition, because the number and nature of the GTFs produced by the other mutans streptococci are apparently distinct from S. mutans (Shimamura et al, 1983; Yamashita etal, 1989), the GTF-S enzymes could be required in these other strains. C. Role of Glucan Binding in Colonization Because the interaction of the mutans streptococci with glucans synthesized by the GTFs could play a role in sucrose-enhanced colonization of tooth surfaces, it is important to identify the molecules involved in such binding. The isolation of
the gbp gene (Russell et al, 1985) indicated that a glucan binding protein distinct from a proteolytic fragment of a GTF was expressed by strains of S. mutans. Although the isolation of comparable genes from other mutans streptococci has not been documented yet, it is likely that such proteins are present on the cell surfaces of these organisms (McCabe et al, 1977; Drake et al, 1988). However, if glucan binding is a significant factor in colonization, it is likely that the glucan binding protein is not required for such interactions. This is suggested by a recent demonstration that a S. mutans mutant defective in the gbp gene colonizes smooth surfaces in vitro in the presence of sucrose as well as the parental organism, although the resultant plaques appear to be qualitatively distinct (Banas and Gilmore, 1991). Nevertheless, because this mutant has not yet been tested in vivo, it is premature to exclude this gene as a potential virulence factor. However, based on these in vitro results, it is likely that more than one protein is involved in glucan binding by these organisms. Likely candidates are the GTFs or their proteolytic fragments (Mooser and Wong, 1988). Because the GTFs apparently have different affinities for various glucans (Koga et al, 1983), additional investigations are required to assess their relative roles in colonization that results from glucan binding, in addition to glucan synthesis. Whether such interactions are important in cariogenesis remains to be determined because previous results (Tanzer and Freedman, 1978) with chemically induced nonaggregating mutants of S. sobrinus indicate that such interactions may be obviated in the oral cavity.
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D. Isolation of Genes Involved in Intracellular Metabolism Because of the potential for utilizing LDH" mutants of S. mutans in replacement therapy (Hillman et al, 1987), it was of interest to isolate the Idh gene from these organisms as an initial step in constructing isogenic mutants. The gene from S. mutans JH1000 has been cloned recently and characterized (Hillman et al, 1990). However, the construction of an isogenic mutant from the cloned gene has yet to be reported although the gene has been insertionally inactivated in Escherichia coll (Duncan and Hillman, 1991). Because sucrose metabolism is clearly relevant to the cariogenicity of the mutans streptococci, an examination of mutants altered in sucrose metabolism in animal systems is of interest. Because the majority of the sucrose metabolized by these strains is transported into the cells and metabolized intracellularly (Tanzer et al, 1972), the enzymes involved in this process could be considered potential virulence factors. Evidence for multiple uptake systems for sucrose has been obtained previously (Slee and Tanzer, 1982) and the genes involved in the sucrose PTS isolated (Lunsford and Macrina, 1986; Hayakawa et al, 1986; Sato et al., 1989). An examination of a S. mutans V403 mutant defective in the scrA gene coding for the Enz IISUC in the gnotobiotic rat indicated that this mutant was as cariogenic as the parental organism (Macrina et al, 1991). This was not unexpected because additional pathways for metabolizing sucrose are present in these organisms (Chassy, 1983) and sucrose may be transported into the cells via the trehalose PTS (Poy and Jacobson, 1990) or putative non-PTS sucrose uptake system (Slee and Tanzer, 1982).
E. Isolation and Characterization of Genes Involved in Storage Polysaccharide Metabolism Because the early utilization of chemicallyinduced mutants defective in the storage of intracellular polysaccharides suggested that this property may be an important virulence factor (Tanzer et al, 1976), it was of interest to construct defined mutants with this phenotype. Recently, Harris and Curtiss (1991) have isolated genes involved in
intracellular glycogen storage from S. mutans and have constructed isogenic mutants defective in this property. Implantation of these mutants of strain UA130 into gnotobiotic rats indicated that the mutants are significantly less cariogenic than the parental organism when the animals are fed either ad libitum or following programmed feeding (Harris etal, personal communication). These results clearly indicate that the storage of intracellular polysaccharides by S. mutans is an important virulence property, as previously suggested (Tanzer et al, 1976). Other potential plaque storage polysaccharides are the extracellular fructans synthesized by S. mutans as well as by other plaque bacteria (Carlsson, 1970). Several recent investigations utilizing rat model systems (Schroeder et al, 1989; Yamashita et al., unpublished) have suggested that S. mutans mutants defective in fructan synthesis are normally cariogenic. More recently, the gene coding for the fructanase of S. mutans has been isolated (Burne et al., 1987), an isogenic fruA mutant constructed in strain UA159, and implanted into specific pathogen-free rats. These results also indicated that the mutant is as cariogenic as the parental organism (Burne, personal communication). However, because these experiments were carried out with rats fed ad libitum, it will be useful to examine these mutants under programmed feeding conditions in order to further assess their relative roles in cariogenicity. Nevertheless, it is important to note that strains of S. sobrinus that synthesize little or no detectable fructan are highly virulent in rodent caries models (Tanzer, 1992). Additional potential storage polysaccharides may be the glucans that are synthesized in dental plaque not only by S. mutans but also by S. sanguis (and S. gordonii) strains (Hamada and Slade. 1980). It is possible that the dextranases that are known to be elaborated by S. mutans strains (Schachtele et al., 1975) could be important in this respect. However, although a dextranase gene has been cloned from S. sobrinus 6715 (Barrett et al., 1987), the comparable gene has not yet been isolated from a S. mutans strain. The previously isolated dextranase gene that maps close to the gtfA gene of S. mutans (Burne et al., 1986) represents an exodextranase and not the extracellular endodextranase that can be detected in culture fluids of these strains (Schachtele et al., 1975). It
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is likely that a combination of both dextranases is necessary for strains of mutans streptococci to metabolize dextran molecules. In addition, the extracellular endodextranase could play a role in modifying the structure of the glucans synthesized by the GTFs of these organisms (Schachtele etaU 1975). F. Genes Involved in the Aciduricity of Mutans Streptococci Biochemical characterization of S. mutans strains has established a primary role for the membrane-associated ATPases in the aciduricity of these organisms (Bender et ai, 1986). However, the genes that code for the multiple subunits of this enzyme complex have yet to be isolated and characterized. An initial step in this approach has been reported recently (Quivey, personal communication), whereby a DNA fragment coding for one of these subunits has been isolated following PCR amplification. Therefore, because the genes that code for the comparable complex in E. coli constitute a single operon (Walker et al., 1984), it is likely that gene walking from this initial fragment could ultimately lead to the isolation of the entire operon. Subsequently, mutants defective in this activity could be constructed and examined for cariogenicity in animal model systems. Tn916 mutagenesis of S. mutans UA96 has led to the preliminary isolation of mutants that exhibit altered aciduricity (Marchman et al., 1990). Although none of these mutants has been characterized extensively (Caufield, personal communication), Southern blot analysis has revealed that the transposon was inserted outside of the ATPase locus into different positions in the mutant chromosomes. Therefore, it is likely that multiple genes are involved in maintaining the aciduricity of these organisms. More recently, a similar strategy has been utilized to isolate a mutant of S. mutans GS5 that exhibits markedly reduced aciduricity and also displays temperature-sensitive growth (Yamashita and Kuramitsu, unpublished). Molecular characterization of these mutants will be required to identify the genes that are involved in this virulence property of S. mutans.
G. Isolation of Bacteriocin Genes Because there have been suggestions that the elaboration of bacteriocins (mutacins) by the mutans streptococci may play a role in colonization (van der Hoeven and Rogers, 1979), it would be of interest to construct defined bacteriocin" mutants of these organisms for testing in animal models. Utilizing a Tn916 mutagenesis strategy, a mutant defective in mutacin activty in S. mutans UA96 has been isolated recently (Caufield et al, 1990). Therefore, it should be possible to test the bacteriocin" mutant in appropriate animal model systems.
V. SOME UNRESOLVED QUESTIONS REGARDING THE VIRULENCE OF MUTANS STREPTOCOCCI Both biochemical (Loesche, 1986) and genetic (Macrina et al., 1990) approaches have defined the important roles of the following virulence factors of mutans streptococci in dental caries: colonization of teeth both in the presence and absence of sucrose, GTF-mediated synthesis of water insoluble glucans, strong fermentation of a variety of sugars, metabolism of storgage polysaccharides, and significant aciduricity. As suggested above, there are still a number of questions regarding each of these as well as other potential virulence factors that still remained unanswered. In many cases, each of these can be approached utilizing the techniques of molecular genetics.
A. Colonization of Teeth As noted previously, the identity of the adhesins involved in the initial sucrose-independent attachment of S. mutans strains to teeth have yet to be convincingly established. Because it has been suggested that more than one adhesin may be involved in this process (Gibbons, 1989; Bowen et al., 1991), it will be of interest to identify both biochemically and genetically the cell surface proteins of these organisms that specifically bind to human salivary mucins or pro-
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line-rich proteins (Gibbons, 1989) in addition to salivary agglutinin (Lee etal, 1989). Mutants of 5. mutans could then be constructed containing alterations in individual and multiple putative adhesins. Subsequently, these mutants could be examined in vitro, as well as following implantation into rats and humans under different dietary conditions (glucose vs. sucrose). In addition, the question concerning the role of S. mutans binding to pellicle containing GTF and glucans in tooth colonization needs to be resolved (Gibbons et ai, 1986; Schilling and Bowen, 1992). Such interactions appear to be significant in the colonization of S. sobrinus strains to teeth (Gibbons et al, 1986). Another question that has not been answered satisfactorily yet is the role of sucrose (glucan synthesis) in the development of human fissure and approximal caries. The results from animal model experiments have consistently demonstrated that the presence of sucrose in the diet is more critical for the development of smooth surface relative to sulcal and proximal lesions (Loesche, 1986; Tanzer, 1992). In addition, the utilization of defined S. mutans mutants defective in insoluble glucan synthesis implanted into rats also has suggested that glucan synthesis is not a major factor in caries development in fissures (Munro et al., 1991; Yamashita et al, 1992). However, one earlier investigation (Tanzer, 1979) has demonstrated significant enhancement of 5. mutans-induced fissure caries in rats in the presence of sucrose. Furthermore, human epidemiological studies (Newbrun, 1982) have suggested that sucrose is a major factor in the development of human carious lesions (which occur primarily on the occlusal surfaces and secondarily in the proximal regions of teeth). Therefore, a question can be raised as to whether the pathogen-free conventional or gnotobiotic rat systems as presently employed are suitable model systems for quantitatively assessing the role of sucrose (and glucans) in the development of S. mutans-induced carious lesions in the sulcal areas. Differences in the morphology of human vs. rat fissures could obscure or minimize the role of sucrose in caries induction in these regions of human teeth.
B. Sucrose-Enhanced Colonization of Tooth Surfaces Although both biochemical and genetic approaches have outlined the role of glucans in tooth colonization (Loesche, 1986), a number of significant questions still have not been resolved. Foremost among these is the nature of the "adhesive" glucan that is involved in this process. Neither the precise chemical nature of the these glucans (molecular size, degree of branching of alpha-1,6- and -1,3-glucose linkages ) nor the specific roles of each GTF in synthesizing these molecules has yet been defined precisely for the mutans streptococci. Because the properties of each specific S. mutans GTF appears to differ somewhat from the comparable enzymes from other mutans streptococci (Shimamura et al, 1983; Aoki et al, 1986; Hanada and Kuramitsu, 1988; Hanada and Kuramitsu, 1989; Yamashita et al, 1989), the respective roles of the enzymes involved in adhesive glucan synthesis may be distinct, depending upon the species investigated. As additional gtf genes are isolated from different strains, expressed in other streptococci (S. millerl S. lactis ), and defined mutants constructed, these questions should be answered more adequately. Moreover, the insoluble glucans may play additional roles in cariogenesis besides colonization, as recently proposed (van Houte et al, 1989), and these could also be evaluated utilizing specific mutants and appropriate animal model systems.
C. Environmental Influences on the Cariogenicity of the Mutants Streptococci It is becoming increasingly clear that the virulence of pathogenic microorganisms can be influenced by the environmental conditions within the host (DiRita and Mekalanos, 1989). Therefore, one aspect of the cariogenicity of mutans streptococci that has not yet been addressed adequately is the influence of the environment in the oral cavity on the cariogenicity of these organisms. Although a number of genes that express potential virulence factors in the mutans streptococci have been isolated and characterized (Macrina et al, 1990), little information is currently available
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regarding the regulation of their expression. It is likely that the localized environment within plaque (acidic pH, lower O2 tension, limiting nutrients) could affect the expression of these genes. For example, it has been proposed recently (Hudson and Curtiss, 1990) that the expression of the gtf genes of S. mutans is influenced by attachment of the organisms to tooth surfaces. However, the molecular basis for such apparent regulation has not been determined yet. In this respect, the utilization of chemostat-grown cells of mutans streptococci (Elwood, 1976) has suggested that the growth rate can influence the differential expression of the GTF-I and GTF-S enzymes of some of these organisms. Moreover, the observation that S. mutans cells embedded in an insoluble glucan matrix ferment sugars at a more rapid rate relative to cells colonized in the absence of glucan (van Houte et a/., 1989) suggests that physical factors can also influence the physiology of these organisms. A very recent study (Caufield, personal communication) has suggested that the future caries experience of a child may well be determined at a crucial "window" stage within the first 2 years of life. It is reasonable to assume that during this period an environment is developed that is necessary for the critical colonization of newly erupted teeth by S. mutans strains, which are transmitted primarily from the child's mother. It will be important to determine what these factors are and their influence on the pathogenic properties of these organisms. The presence or absence of other oral microorganisms may also play a critical role in this process. Little information is currently available regarding the influence of other early colonizers of teeth, such as S. sanguis, on the virulence properties of S. mutans. Coculture approaches involving the utilization of S. mutans strains genetically engineered to express reporter genes fused to genes involved in cariogenicity may be important in this respect. This approach may help to define relevant environmental factors that may alter the expression of virulence factors in these organisms. D. Cariogenicity of Different Strains of S. Mutans Kohler and Krasse (1988) have described the differential cariogenicity in rats of two fresh hu170
man oral isolates of S. mutans. Therefore, it is likely that a range of cariogenic potentials may be expressed in different strains of these organisms harbored by humans. However, no information is currently available regarding the correlation between the incidence of caries in a study population relative to the specific S. mutans strains harbored by each individual. One reason for the absence of such studies has been the lack of a simple, sensitive test that can distinguish between different strains of 5. mutans. The introduction of restriction fragment length polymorphism (RFLP) analysis of these organisms (Caufield and Walker, 1989) has suggested a highly sensitive technique to distinguish between strains of these organisms. Such a survey, together with a comparison of the biochemical properties of the strains identified, may reveal previously unrecognized virulence factors that may be important in cariogenicity.
VI. STRATEGIES TO NEUTRALIZE THE VIRULENCE FACTORS OF MUTANS STREPTOCOCCI A. Anticaries Vaccines Several excellent reviews have discussed the prospects for developing an anticaries vaccine (Curtiss etal, 1986; Michalek and Childers, 1990) and the reader is urged to consult these for a detailed analysis of this subject. Extensive work is currently in progress to identify and isolate purified antigens that can be utilized in producing an effective vaccine. More recently, Smith and Taubman (1991) have utilized small antigenic peptides corresponding to the GTF-Is from mutans streptococci that appear to protect rodents against challenge by S. mutans strains. It will be of interest to determine if these purified antigens are also protective in non-human primates, because earlier results evaluating these enzymes as potential vaccines have suggested that intact GTF molecules were not protective in these animals (Russell andColman, 1981). Because the presentation of an antigen to the host is an important factor in the elaboration of an immune response (Michalek and Childers, 1990), several recent investigations in the oral cavity have proposed novel approaches toward this end.
Macrina and colleagues (Dertzbaugh et ai, 1990) have constructed genetic fusions containing part of the cholera toxin B subunit together with a small peptide of the S. mutans GS5 gtfB gene product as a potential oral immunogen. However, such fusions have not been examined yet as an anticaries vaccine. Another enhancement strategy for the oral immune response against mutans streptococci has been proposed previously by Curtiss and co-workers utilizing a different strategy that involves ingestion of genetically engineered Salmonella strains expressing S. mutans antigens (Curtiss et ai, 1988). In addition, the continued expression of S. mutans antigens by genetically engineered noncariogenic oral microorganisms (S. sanguis) implanted into the oral cavity may prove beneficial in inducing an anticaries immune response.
tion of teeth by these altered organisms may reduce the subsequent colonization of S. mutans. Whether such enzymatic approaches will be effective in vivo remains to be determined because it is not clear whether or not such enzymes will be effective under these conditions. Strategies designed to interfere with adhesin or glucan-mediated attachment of S. mutans to teeth may be limited primarily to affecting smooth surface caries. It is possible that other nonspecific factors (bacterial entrapment) could be more significant for human occlusal and interproximal caries. C. Inhibitors of S. Mutans Growth Sandham and colleagues (1988) have demonstrated that the application of chlorhexidine onto tooth surfaces can quite effectively eliminate S. mutans from such surfaces. Therefore, this compound incorporated into a number of oral vehicles is currently under evaluation as an effective anticaries strategy. A similar strategy may be feasible based upon the elaboration of anti-S. mutans bacteriocins in the oral cavity. Such inhibitors are produced by several microorganisms (Hamada and Ooshima, 1975), and it may be possible to isolate the genes coding for one of these and express the gene in an oral plaque bacterium. This could result in the continuous production of the bacteriocin in the oral cavity. However, a number of important questions must be addressed before considering this approach in human therapy (the stability of the bacteriocins in the oral cavity, specificity of the bacteriocin against the major strains of S. mutans present in the oral cavity, and potential development of resistance to such agents).
B. Inhibitors of S. Mutans Colonization Because one or more adhesins on the cell surface of S. mutans may be important in the initial colonization of these organisms to tooth surfaces (see Section IV), one potential strategy for reducing the colonization of these organisms may be to bathe the oral cavity with an active-site peptide of the adhesins. Such peptides could act as competitive inhibitors of the colonization of S. mutans. Both biochemical and genetic analysis of the potential adhesin molecules should identify the functional domains of these proteins and could lead to the synthesis of inhibiting peptides. Various strategies to continuously produce such inhibitors in the oral cavity could be developed and examined in animal models and ultimately in human implantation experiments. Because the inhibition of glucan synthesis from sucrose by S. mutans should result in a decrease of colonization and cariogencity (Loesche, 1986), it has been proposed that the presence of glucanases in the oral cavity may be beneficial (Guggenheim et a/., 1972). A fungal glucanase that can hydrolyze the alpha- 1-3-glucose linkages present in insoluble glucans has been purified recently and characterized (Kriger and Quivey, 1990). If the gene for this enzyme could be engineered into a noncariogenic oral microorganism such as S. sanguis, the coloniza-
D. Replacement Therapy Several years ago, Hillman (1978) proposed that the colonization of the oral cavity by S. mutans strains defective in LDH could be used as a basis for replacement therapy to reduce the incidence of dental caries. This proposal was based on the properties of a chemically induced LDH" mutant of S. rattus. However, because these organisms apparently are not good colonizers of the human
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oral cavity (Loesche, 1986), the further development of this strategy was dependent upon the isolation of specific LDH" mutants of a S. mutans strain. In view of the recent isolation of the LDH gene from 5. mutans JH1000 (Hillman et ai, 1990), it should now be possible to construct defined LDH mutants of S. mutans following insertional inactivation of the cloned gene and by gene transfer into appropriate transformable strains. To further enhance the colonizing potential of the LDH' mutant, it has been proposed that an elevated bacteriocin producer of this strain would make the best candidate for a replacement therapy vehicle (Hillman et a/., 1987). A variation of this strategy has been suggested recently whereby the genes for the arginine deiminase system (Burne et ai, 1989) may be inserted into an S. mutans strain to produce a less cariogenic strain. The resultant organism, producing elevated levels of NH3, would be much less acidogenic and could be used as a replacement therapy vehicle. Alternatively, a LDH" arginine deiminase+ construct of S. mutans could be utilized for this purpose. Moreover, it may be possible to reduce dental caries by competitive displacement of mutans streptococci with unaltered oral microorganisms such as S. salivarius TOVE-R (Tanzer et ai, 1985). It will be of interest to determine if novel strategies based upon genetic manipulations can be utilized ultimately in children for further reduction in cariogenicity. A practical point of consideration in this respect must ultimately be the political and social obstacles to utilizing genetically engineered organisms in humans, especially children. Because of the questions being raised regarding the utilization of genetically engineered organisms, it may be difficult to convince the general public to use these therapies in view of the continuing decline in the incidence of dental caries without such novel approaches. Nevertheless, because of uncertainties regarding the future decline in caries with current strategies, these approaches should be evaluated carefully.
identification of a number of important virulence properties of the mutans streptococci. Prior to the utilization of cloned genes to construct defined mutants in S. mutans, mutants of mutans streptococci were isolated following traditional mutagenesis protocols, which could not preclude the generation of multiple alterations in the organisms. Nevertheless, the results from the implantation of these mutants into animal models were clearly important in defining some of the cariogenic properties of the mutans streptococci. Those individuals now utilizing the modern powerful tools of molecular genetics to further define the cariogenicity of these organisms (including this author) should be reminded that none of the major virulence factors proposed from these earlier investigations has yet to be revised based on more recent molecular biological approaches. The more recent introduction of molecular genetic approaches, besides substantiating most of these earlier findings, has led to a more detailed understanding of the molecular basis for these properties. Specifically, these approaches can be utilized to define more precisely the active virulence factors (adhesin binding sites, enzyme active sites, etc.). In addition, the application of these strategies has led to the identification of potential virulence factors that were not recognized by strictly biochemical approaches. Despite the fact that the general basis for the cariogenicity of mutans streptococci is now well recognized, a number of important questions still remain unresolved. The resolution of these issues should provide an even better undertstanding of the molecular basis for cariogenicity, and it could provide novel strategies for identification of individuals at high risk of developing carious lesions as well as suggesting additional preventive therapies.
ACKNOWLEDGMENTS The author gratefully acknowledges the communication of relevant information from Drs. A. Bleiweis, R. Burne, G. Harris, J. Ferretti, J. P. Klein, F. L. Macrina, R. Marquis, S. Michalek, R. Quivey, R. R. B. Russell, and J. M. Tanzer. In addition, the critical comments of Dr. J. M. Tanzer were very helpful and are much appreciated.
VII. SUMMARY The utilization of biochemical approaches in conjunction with animal models has led to the
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The work described from the author's laboratory is supported in part by National Institutes of Health grants DE03258 and DE09864.
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Virulence Factors of Mutans Streptococci Role of Molecular Genetics

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Critical Reviews http://cro.sagepub.

com/ in Oral Biology & Medicine

Virulence Factors of Mutans Streptococci: Role of Molecular Genetics

Critical Reviews in Oral Biology and Medicine, 4(2):159-176 (1993)

Virulence Factors of Mutans Streptococci: Role of Molecular Genetics

1045-4411/93/S.50 1993 by CRC Press, Inc.

TABLE 1 PUTATIVE S. mutatis Virulence Genes

Gene Idh ATPase pac gtB

Mutants? No No Yes Yes Yes Yes Yes Yes Yes Yes

gbp ftf gig

= Not tested. See Section IV for references to investigations outlined in table.

Streptococcus sobrinus gtf Gene that Encodes a

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