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1993 4: 159CROBMHoward K. Kuramitsu

Virulence Factors of Mutans Streptococci: Role of Molecular Genetics

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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 mutansstreptococci (primarily Streptococcus mutans and 5. sobrinu). Traditional mutant analysis of these organismsfurther suggested the important role of several of these factors in cariogenicity. However, because these mutationswere not clearly defined, the utilization of cloned genes was necessary to verify their significance. The introductionof molecular genetic approaches for characterizing these factors has led not only to a clearer understanding of therole of these virulence factors in cariogenicity but has also suggested some novel approaches for reducing furtherthe incidence of dental caries.

KEY WORDS: Streptococcus mutans, S. sobrinus, molecular genetics, adhesins, sucrose-enhanced colonizationof teeth, glucosyltransferases.

I. INTRODUCTION

Despite the recent decline in dental cariesfrequency among children in technologically ad-vanced societies, tooth decay still remains a ma-jor health problem (Loesche, 1986; Tanzer, 1992).In addition, it is not clear yet whether or not thisdecline will continue into the next century. Thesignificant improvement in the oral health statusof Western children has been attributed primarilyto the widespread utilization of fluoride togetherwith improvements in oral care (Glass, 1982).Nevertheless, there still remains a sizeable pro-portion of the population that is at risk of devel-oping carious lesions (Krasse, 1985). Therefore, amore detailed understanding of the bacterial-hostinteractions that lead to dental caries may be ofvalue in developing additional anticaries strate-gies that may further decrease the frequency ofthis disease. In this respect, the recent introduc-tion of molecular genetic approaches for examin-ing the virulence of mutans streptococci (Loesche,1986) may yield innovative methods for both theidentification of children at high risk of develop-ing dental caries as well as the prevention ofdecay. Several recent reviews have addressedvarious aspects of this topic (Curtiss, 1985;

Macrina et ai, 1990; Russell, 1990), and thisarticle focuses primarily on the relationship be-tween the results of these newer approaches to theearlier investigations (Loesche, 1986; Tanzer,1992). Furthermore, since the publication of thesereviews, the results of animal studies utilizingdefined mutants constructed from cloned Strepto-coccus mutans genes are now available. In addi-tion, there is a discussion of several unresolvedissues relating to the cariogenicity of the mutansstreptococci.

II. VIRULENCE PROPERTIES OFMUTANS STREPTOCOCCI IDENTIFIEDFOLLOWING COMPARISON WITHOTHER ORAL BACTERIA

Despite the complexity of the human oralflora, pioneering animal model studies (Fitzgeraldand Keyes, 1960), as well as human epidemio-logical surveys (Loesche, 1986), have stronglyimplicated mutans streptococci (primarily S.mutans) as the principal etiological agents in hu-man dental caries. Therefore, for the past severaldecades there have been extensive efforts to iden-tify the virulence factors of these organisms as

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potential targets for anticaries prophylaxis. Ini-tially, potential virulence properties of the mutansstreptococci were identified following compari-son of these organisms with other oral strepto-cocci. One of the first putative virulence factorsof the mutans streptococci identified by this ap-proach was the ability of these organisms to colo-nize smooth surfaces in vitro in the presence ofsucrose (Gibbons etal, 1966). Subsequently, thisproperty was shown to be dependent on the syn-thesis of water-insoluble glucans from the disac-charide. Although several other oral streptococci(5. sanguis, S. salivarius) also have the ability tosynthesize these polysaccharides (Hamada andSlade, 1980), only the mutans streptococci gener-ally display sucrose enhanced colonization (colo-nization is defined in this review as the aggrega-tion of bacteria onto hard surfaces following ini-tial attachment and subsequent accumulation) ofsmooth surfaces (Loesche, 1986). One recentexception 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 estab-lished. Therefore, it was quite evident almost 30years ago that the mutans streptococci are capableof synthesizing colonization promoting glucansfrom sucrose. Because of the extensiveepidimeological evidence linking the incidenceof human dental caries with sucrose consumption(Newbrun, 1982), major emphasis was placed onexamining the mechanism of glucan synthesis.However, despite detailed investigations regard-ing the synthesis of these exopolysaccharides andthe role of glucosyltransferases (GTF) in this pro-cess (Loesche, 1986), the chemical nature of theglucans involved in colonization have not beenprecisely defined yet.

Because of this unique colonization propertyexhibited by the mutans streptococci, it was notsurprising that these organisms also exhibitedclumping (aggregation) when grown in the pres-ence of sucrose. This characteristic could be ra-tionalized as another example of sucrose-depen-dent attachment of cells to solid surfaces due toinsoluble glucan formation. However, in addi-tion, many of these organisms also aggregatedwhen cells were incubated in the presence of highmolecular weight glucans (especially water solublehigh molecular weight dextrans) (Gibbons andFitzgerald, 1969). This property was also uniqueto the mutans streptococci and was postulated to

play a role in colonization. More recently, it hasbeen suggested that such interactions may alsoaid in the initial attachment of the mutans strep-tococci to glucans formed within the pellicle ofteeth (Schilling and Bowen, 1992). Therefore,two of the earliest virulence properties associatedwith these organisms were their ability to synthe-size colonization, promoting insoluble glucans,and their interaction with these polysaccharides.

Despite the initial emphasis on the role ofinsoluble glucan formation in the colonization oftooth surfaces by the mutans streptococci, both invitro (Staat et al., 1980) as well as in vivo (VanHoute et aL, 1976) results have suggested thatthese organisms do not require sucrose for colo-nization. Subsequent approaches from severallaboratories (Douglas and Russell, 1984; Russell,1986, Lee et aL, 1989) have suggested that theseorganisms are capable of attaching to the pellicleof teeth by means of putative adhesin-like cellsurface molecules. As detailed below (SectionIV), molecular genetic approaches have suggestedseveral candidates for this phenomenon. Further-more, Gibbons et al. (1986) have demonstrateddifferences in tooth colonization mechanisms thatdepend on the group of mutans streptococci in-volved: S. mutans strains apparently attach byboth adhesin and glucan mediated mechanisms,whereas S. sobrinus strains utilize primarily thelatter process.

Because dental caries is ultimately related tothe acidogenicity of plaque bacteria, the fermen-tation patterns of the mutans streptococci havebeen examined extensively (Hamada and Slade,1980). These organisms were demonstrated toferment a wide variety of sugars and it was ofspecial interest that they appear to metabolizesucrose to lactic acid more rapidly than other oralbacteria (Minah and Loesche, 1977). This prop-erty of the mutans streptococci is undoubtedlyrelated to the multitude of enzyme systems ex-pressed by these organisms that are capable ofboth transporting and metabolizing sucrose(Loesche, 1986).

It was also reasonable to assume that an im-portant virulence property of cariogenic bacteriashould be their ability to continue fermentation inthe absence of exogenous food supplies (condi-tions that are likely to be most conducive for toothdemineralization due to the reduction in salivaryflow during these periods) (Loesche, 1986). There-



fore, the observation that some strains of S. mutansthat were highly virulent in rats were capable ofintracellular polysaccharide storage (IPS) (Tanzeret a/., 1976), whereas others that were not capableof IPS were avirulent, suggested another potentialvirulence property of these organisms.

Because dental plaque pH becomes acidic inthe presence of a fermentable carbon source(Loesche, 1986), the relative aciduricity ofcariogenic bacteria may also serve as a virulencefactor. A comparison of the relative aciduricity oforal bacteria (Harper and Loesche, 1984) has in-deed demonstrated that strains of S. mutans aremore acid tolerant than all other bacteria exam-ined with the exception of the lactobacilli. Thisproperty appears to be related, in part, to therelative acid stability of the membrane-associatedH+-translocating ATPase of these organisms(Bender et al, 1986).

Another potential virulence factor of mutansstreptococci may be the bacteriocins produced bya large variety of these organisms (Hamada andOoshima, 1975). These antibacterial factors couldaid in the establishment of cariogenic bacteria byreducing the presence of potential competitors oftooth colonization. Several investigations (van derHoeven and Rogers, 1979; Hillman et al, 1987)have demonstrated that bacteriocin production canplay a role in enhancing the establishment ofmutans streptococci in rodents and in humans.However, both the large variety and the differen-tial spectrum of these inhibitors has made it dif-ficult to assess their role in the virulence of theseorganisms.

III. MUTANT CHARACTERIZATION ANDTHE IDENTIFICATION OF VIRULENCEFACTORS

The optimum approach for identifying a viru-lence factor of a pathogenic microorganism in-volves isolating a mutant of the organism defec-tive in a specific trait followed by comparison ofits pathogenicity with the parental organism in anappropriate animal model. Toward this end, anumber of mutants of mutans streptococci defec-tive in potential virulence traits have been iso-lated (Freedman et al, 1981). These, as well asthe other mutants described in this section, were

isolated as spontaneous or chemically inducedmutants. Therefore, the precise nature of thesemutations were not defined and the possibility ofmultiple defects in each cannot be discounted.

A. Colonization Defective Mutants

Among the first groups of mutants to be iso-lated were those defective in sucrose-enhancedcolonization of tooth surfaces because these couldbe readily detected on sucrose-containing agarplates. Subsequent examination revealed that eachof these was defective in insoluble glucan synthe-sis (Loesche, 1986). However, because the natureof these mutations was undefined and one of thesemutants, S. mutans C67-25, was shown to bedefective in several other potentially importantvirulence properties (Donoghue and Newman,1976), it was not possible to define precisely themutations at the molecular level. Nevertheless,the implantation of these mutants into rats yieldedresults that were compatible with the importantrole of insoluble glucan synthesis in dental caries.These results indicated that insoluble glucan-de-fective mutants colonized teeth less extensivelythan the parental organisms in the presence ofsucrose. In addition, these results revealed thatsucrose (and therefore glucan synthesis) was moresignificant for smooth surface caries when com-pared with fissure caries (Tanzer, 1979). Theseobservations were also consistent with earlier find-ings indicating that sucrose was not required forcolonization of rat teeth (van Houte et al, 1976).

As described previously, it was possible thatglucan-mediated aggregation of the mutans strep-tococci might also play an important role in toothcolonization. The isolation of mutants altered ineither dextran-mediated or sucrose-dependentaggregation (but not necessarily both properties)(Freedman and Tanzer, 1974; Murchison et al.1981) indicated that the former was not solelymediated by the glucan-binding properties of cell-associated GTFs. These results suggested thateither a specific glucan-binding molecule or pro-teolytic fragments of the GTFs associated withthe cell surface were involved. In addition, Russell(1979a) reported the isolation of a cell-surfaceglucan-binding protein from a S. mutans strainand similar proteins have been identified in other



mutans streptococci (McCabe etal., 1977; Drakeet al., 1988). Nevertheless, rigorous molecularcharacterization of these proteins is requiredfor identification of these molecules as nonen-zymic glucan-binding proteins, inasmuch as ithas been demonstrated that proteolytic frag-ments of GTFs lacking enzymatic activity havethe ability to bind glucans (Mooser and Wong,1988).

Several approaches (Staat et al., 1980; Curtiss,1985) have suggested that mutans streptococcalcolonization of teeth involves both a sucrose-dependent (glucan-mediated accumulation) andindependent (initial attachment) process. Subse-quent investigations have indicated that the latterphenomena could result from both specific (Leeet al., 1989) and nonspecific (Gibbons andEtherden, 1983) interactions of the organisms withthe pellicle of teeth. Furthermore, the identifica-tion of cell surface proteins in these organisms(Russell, 1979b) suggested the possibility thatadhesins might be involved in specific attachmentto teeth. Following the demonstration that a chemi-cally induced mutant of S. sobrinus 6715 alteredin a 210 kDa cell surface protein was defective inboth in vitro attachment to saliva-coated hydroxya-patite beads and in cariogencity in rats (Curtiss etal., 1986), it has been proposed that related pro-teins (antigen I/II, proteins B, PI, IF, and Pac)from other mutans streptococci may also be in-volved in sucrose-independent colonization oftooth surfaces (Ackermanns et al., 1985; Lee etal., 1989).

B. Mutants Defective in IntracellularMetabolism

Because tooth decay is primarily determinedby the acidogenicity of plaque bacteria, it wasnot surprising that mutants of some mutans strep-tococci defective in lactic dehydrogenase (LDH)activity were markedly less cariogenic in rodentmodel systems (Hillman, 1978; Fitzgerald et al.,1989). Likewise, mutants defective in the stor-age of intracellular polysaccharides displayedreduced cariogenicity at some tooth surfacesrelative to the parental organisms, especiallywhen fed at specified time intervals (Tanzer etal, 1976).

C. Mutants Altered in Aciduricity

Until now, only one S. mutans mutant alteredin aciduricity and examined for cariogencity in ananimal model has been described in detail (deStoppelaar et al., 1971; Donoghue and Newman,1976). As might be anticipated, this mutant, S.mutans C67-25, was defective in cariogenicity onboth enamel and sulcal surfaces. However, as ispossible with any chemically induced mutant, itwas demonstrated that this mutant also displayedmultiple alterations relevant to cariogenicity (colo-nization, aggregation) (Loesche, 1986). In addi-tion, mutants of S. sobrinus altered in aciduricitydisplay reduced virulence in rats (Tanzer andFreedman, 1978). Therefore, the properties of thesemutants were consistent with the importance ofaciduricity as a virulence factor for the mutansstreptococci.

D. Mutants Altered in DextranaseActivity

An interesting class of mutants of S. mutansand S. sobrinus altered in exodextranase activityhas been described (Tanzer and Freedman, 1978;Tanzer, 1992). These chemically induced mu-tants displayed reduced cariogenicity when im-planted into Sprague-Dawley rats fed high su-crose diets on both smooth surfaces and fissuresof the teeth. However, the molecular basis forvirulence reduction in the mutants has not beenestablished.

IV. VIRULENCE FACTORSCHARACTERIZED UTILIZING CLONEDGENES

The introduction of recombinant DNAtechniques for investigating the virulence ofmutans streptococci has led to a more detailedunderstanding of the pathogenic factors ofthese organisms at the molecular level. Theisolation of defined mutants constructed fromcloned genes and their utilization in animalmodel systems has also provided a more un-equivocal basis for defining the virulence of



these organisms (Table 1). Such defined mu-tants can be utilized to obviate complicationsfrom multiple or pleiotrophic mutations thatcould confound some of the earlier interpreta-tions based upon the utilization of spontane-ously derived or chemically induced mutants(Loesche, 1986).

implanted into rats. However, because the spaAmutants were isolated following chemical mu-tagenesis and the possibility of pleiotrophic ef-fects in the mutants has not been evaluated, somecaution should be exercised in drawing the con-clusion that the spaA protein is a S. sobrinusadhesin required for tooth colonization. Unfor-

TABLE 1PUTATIVE S. mutatis

Gene

IdhATPasepacgtBgtiCgtio

gbpftf

gig

scrA

Role

AcidogenicltyAciduncityColonizationColonizationColonizationColonizationColonizationReservenutrient

Reservenutrient

Sucrosetransport

Virulence Genes

Mutants?

NoNoYesYesYesYesYesYes

Yes

Yes

In vivotesting?

NoNoYesYesYesYesNoYes

Yes

Yes

Cariesdecrease?

*

—NoYesYesNo—No

Yes

No

— = Not tested. See Section IV for references to investigationsoutlined in table.

A. Cloning of Genes Involved inSucrose-Independent Colonization ofTeeth

The initial identification of a molecule thatappeared to play a role in attachment of amutans streptococcus to the pellicle of teethresulted from the isolation of the spaA genefrom S. sobrinus 6715 (Holt et al., 1982). It wasdemonstrated that the product of this gene, a 210kDa protein, was primarily associated with thecell surface of the organism. Following chemi-cal mutagenesis and selection for mutants thatdid not react with anti-spaA antibody, mutantsdefective in this gene were isolated (Curtiss etal, 1983). Moreover, the spaA protein appearedto be a logical candidate for an adhesin involvedin sucrose-independent attachment to teeth be-cause a related protein from S. mutans appears tobe involved in attachment to saliva-coated hy-droxyapatite beads ((Russell, 1986) and the spaAmutants also produced fewer carious lesions when

tunately, a gene transfer system for S. sobrinusstrains has not been developed yet and it has notbeen possible to construct defined spaA mutantsfrom the isolated gene. In addition, the organiza-tion of the spaA gene on the chromosome ofstrain 6715 relative to other cell surface mol-ecules has yet to be determined and the possibil-ity of polar effects of the spaA mutation on thesemolecules needs to be evaluated. Nevertheless,the successful isolation and characterization ofthe spaA gene and its protein product providedthe initial impetus for the search for genes cod-ing for cell surface proteins that could play a rolein colonizing teeth.

Subsequent investigations have revealed thatproteins structurally related to the spaA proteinare also present on the cell surface of othermutans streptococci (Sommer et al., 1987; Leeet al, 1988; Takahashi et al, 1989) as well asin S. sanguis (Demuth et al, 1988). Becausesome strains of S. mutans are naturally trans-formable (Perry and Kuramitsu, 1981), one of



these was a logical candidate for the construc-tion of a defined mutant altered in the putativeadhesin. Bleweis and colleagues (Lee et al,1989) have constructed a mutant of S. mutansNG8 that is defective in the PI protein (theapparent functional equivalent of the spaA pro-tein of S. sobrinus) utilizing the cloned gene forinsertional inactivation following electro-poration. One of the resultant mutants, strain834, displayed reduced hydrophobicity relativeto the parental strain, and also did not bind to asalivary agglutinin as well as the parental strain.However, both the parental and mutant strainsdisplayed weak interactions with whole saliva-coated hydroxyapatite beads. Furthermore, bothstrains were equally cariogenic when implantedinto conventional pathogen-free rats fed a highsucrose (56%) diet (Bowen et al, 1991). Thislatter observation is consistent with the in vitroresults utilizing saliva-coated hydroxyapatitebeads and suggests that, although the PI pro-tein may play a role in tooth colonization,multiple interactions between S. mutans andthe pellicle of teeth are important for coloniza-tion. In addition, interactions between S. mutansand other plaque bacteria may also contributeto colonization by the former organisms. Like-wise, because previous investigations have sug-gested that S. mutans strains also interact withsalivary mucins and a proline-rich protein (Gib-bons, 1989), it is reasonable to assume thatmultiple adhesins may be present on the sur-face of these organisms. Moreover, becausethere is no information available presently re-garding the regulation of expression of theseputative adhesin molecules, the cellular levelsof these molecules in the environment of theoral cavity still need to be evaluated. It wouldalso be of great interest to compare the coloni-zation of these mutants with the parental organ-isms in the human oral cavity.

B. Cloning of Genes Involved inSucrose-Enhanced Colonization ofTeeth

The efforts of many laboratories resulted inthe proposal that multiple GTFs were involved inthe synthesis of the glucans involved in sucrose-

enhanced colonization of teeth (Loesche, 1986).Verification of the expression of multiple GTFsin the mutans streptococci has been obtained re-cently by isolating individual gtf genes from sev-eral of these organisms (Gilpin et al, 1985; Aokietal, 1986; Hanada and Kuramitsu, 1988; Hanadaand Kuramitsu, 1989; Abo et al, 1990; Hanada etal, 1991). These results indicate that most strainsof S. mutans contain three distinct gtf genes: gtfBcoding for the GTF-I enzyme, gtfC expressing asimilar GTF-SI, and gtfD coding for the GTF-Senzyme. The first two enzymes synthesize prima-rily water-insoluble glucans, whereas the latterproduces water-soluble glucans. In addition, twogtf genes have been isolated from 5. downei(Ferretti et al, 1987; Gilmore et al, 1990), syn-thesizing insoluble and soluble glucans, respec-tively. Biochemical approaches have further sug-gested that one or two additional gtf genes mayreside on the chromosomes of this latter speciesand related S. sobrinus strains (Shimamura et al,1983; Yamashita et al, 1989). Therefore, it isclear that each strain of mutans streptococci ex-presses one or more GTF enzymes synthesizingsoluble glucans and at least one producing in-soluble glucans.

In order to examine the role of each of theGTFs in sucrose-enhanced colonization of teeth,S. mutans mutants defective in each of the gtfgenes have been isolated following insertionalinactivation of the genes (Munro et al, 1991;Yamashita et al, unpublished). These mutantshave been implanted into either specific pathogenfree or gnotobiotic rats fed sucrose diets for thepurpose of evaluating the relative contribution ofeach gene product to colonization andcariogenicity. Macrina and co-workers (Munro etal, 1991) have constructed mutants of S. mutansV403 defective in glucan synthesis, and implantedthese into gnotobiotic Fisher rats fed diet 305(UAB model). Mutants deleted for the two gtfgenes (B and C) coding for insoluble glucan syn-thesis exhibited reduced caries induction on thesmooth surfaces of the teeth, relative to the paren-tal strain. Furthermore, no additional reduction incariogencity was observed when either thefructosyltransferase (ftf) or gtfD genes were indi-vidually inactivated. These results were consis-tent with previous suggestions that water insolubleglucan synthesis was important, though not re-



quired, for cariogencity in rat models. However,the reductions in caries noted for the definedmutants were not as extensive as exhibited inearlier animal studies utilizing chemically inducedmutants of S. sobrinus 6715 defective in insolubleglucan synthesis (Tanzer et ai, 1974).

More recently, defined gtf mutants were con-structed in S. mutans UA130 for testing in spe-cific pathogen-free Sprague Dawley rats fed diet2000 (UR system) (Yamashita etaL, unpublished).These results also confirmed the role of insolubleglucan synthesis in cariogenicity on the smoothsurfaces of teeth but, in addition, revealed that thegtfB and C genes were both required for maximalcariogenicity. Furthermore, the reductions insmooth surface caries noted for the mutants inthis system were significantly greater than thoseexhibited in the UAB gnotobiotic system (Munroet a I., 1991) and were similar to that exhibited byS. sobrinus 6715 mutants defective in insolubleglucan synthesis fed a 56% sucrose diet (Tanzeret al., 1974). For example, smooth surface carieswas decreased by 80% for the gtfB'C mutant inthe pathogen-free UR rat model but only on theaverage of approximately 26% in the UAB gno-tobiotic rat system (Munro et aL, 1991). Like-wise, inactivation of either the gtfB or C genes ofstrain UA130 resulted in 76 and 85% reductions,respectively, in smooth surface caries. Thesecomparative results suggest that the UR conven-tional rat caries system appears to be much moresensitive to alterations in glucan synthesis by S.mutans than the UAB gnotobiotic rat model. Assuggested below, these differences cannot be as-cribed primarily to differences in the S. mutansstrains tested.

Additional support for the hypothesis that bothproducts of the gtfB and C genes are important forsucrose-enhanced colonization and cariogenicitywas obtained from an examination of the proper-ties of S. mutans UA101 (Yamashita etal., 1992).Unlike most strains of S. mutans examined (Chiaet aL. 1991), this strain contains only two gtfgenes on its chromosome: one expressing an en-zyme synthesizing insoluble glucan, gtfBC, andthe other corresponding to the gtfD gene (Hanadaand Kuramitsu, 1989). Following isolation of theformer gene, it was observed that this gene, gtfBC,was derived following homologous recombina-tion of the tandemly associated B and C genes in

a progenitor of strain UA101. This strain synthe-sizes approximately 30% of the water insolubleglucan relative to strains GS5 and UA130 anddisplays relatively weak in vitro sucrose-enhancedcolonization. Most importantly, when implantedinto the UR conventional rat model system, strainUA101 produced far fewer carious lesions on thesmooth surfaces of the rats, relative to strainUA130 on high (56%) sucrose diets (Table 2).Furthermore, introduction of the gtfC gene fromstrain GS5 into the chromosome of strain UA101following homologous recombination resulted ina derivative that restored cariogenicity on smoothsurfaces to a level similar to UA130. Likewise,incorporation of the gtfB gene into strain UA101restored the ability of the organism to colonizesmooth surfaces in vitro in the presence of su-crose. These results support the hypothesis that,in the presence of the gtfD gene, two copies of thegtf genes expressing enzymes synthesizing in-soluble glucan are required for maximum sucrose-enhanced colonization of smooth surfaces andcariogenicity by S. mutans strains. Because of theunavailability of data regarding the implantationof comparable g(f mutants for other mutans strep-tococci into animal models, it is not clear whichgtf genes are required for sucrose-enhanced colo-nization of teeth in these strains.

The utilization of S. mutans gtf mutants hassuggested that significant quantitative differencesare apparent in the UR pathogen-free (Bowen etaL, 1988) and UAB gnotobiotic (Munro et aL,1991) rat model caries systems. This is furthersupported by a comparison of the cariogenicity ofstrains UA101 and UA130 in the two systems(Table 2). In the UAB gnotobiotic system utiliz-ing diet 305 containing 5% sucrose, both strainsare equally cariogenic (Barletta et aL, 1988).However, in the UR conventional system UA101(containing two rather than three gtf genes) in-duced markedly reduced levels of smooth surfacecaries relative to strain UA130. As previouslysuggested from earlier mutant studies (Loesche,1986), these differences are minimized in regardto fissure caries. Therefore, smooth surface cariesin the UR conventional rat model system appearsto be more dependent upon insoluble glucan syn-thesis than in the UAB gnotobiotic system (Table2). This may result, in part, from the presence of"sticky" components of diet 305 (cellulose and



TABLE 2Comparison of UR and UAB Rat Models Relative to theRole of Glucan Formation in Smooth-Surface DentalCaries

UR ModelPathogen-free Sprague-Dawley rats

(56% sucrose diet)

UA101UA130

Yamashita et al., 1992.Barletta et al., 1988.

UAB ModelGnotobiotic Fisher rats

(5% sucrose diet)

Smooth surface lesions

4.08a

34.811.0b

11.6

corn starch) not found in diet 2000 which couldobviate the requirement for adhesive insolubleglucan synthesis for smooth surface caries initia-tion (Yamashita et al, 1992).

Earlier biochemical analysis utilizing puri-fied GTFs (Fukushima et al, 1981; Kuramitsuand Wondrack, 1983) suggested that the glucaninvolved in sucrose-enhanced colonization re-quired both the GTF-I and GTF-S enzymes. How-ever, the results utilizing S. mutans mutants de-fective in the gtfD gene coding for GTF-S activitysuggests that this enzyme is not required for su-crose-enhanced colonization in vivo (Munro etal, 1991; Yamashita et al, 1992). Nevertheless,because the GTF-I and GTF-SI enzymes are ca-pable of synthesizing some water soluble glucans(Aoki etal, 1986; Hanada and Kuramitsu, 1988),these results do not necessarily obviate a role forsoluble glucan synthesis in the sucrose-enhancedcolonization process. In addition, because thenumber and nature of the GTFs produced by theother mutans streptococci are apparently distinctfrom S. mutans (Shimamura et al, 1983;Yamashita etal, 1989), the GTF-S enzymes couldbe required in these other strains.

C. Role of Glucan Binding inColonization

Because the interaction of the mutans strepto-cocci with glucans synthesized by the GTFs couldplay a role in sucrose-enhanced colonization oftooth surfaces, it is important to identify the mol-ecules involved in such binding. The isolation of

the gbp gene (Russell et al, 1985) indicated thata glucan binding protein distinct from a proteolyticfragment of a GTF was expressed by strains of S.mutans. Although the isolation of comparablegenes from other mutans streptococci has not beendocumented yet, it is likely that such proteins arepresent on the cell surfaces of these organisms(McCabe et al, 1977; Drake et al, 1988). How-ever, if glucan binding is a significant factor incolonization, it is likely that the glucan bindingprotein is not required for such interactions. Thisis suggested by a recent demonstration that a S.mutans mutant defective in the gbp gene colo-nizes smooth surfaces in vitro in the presence ofsucrose as well as the parental organism, althoughthe resultant plaques appear to be qualitativelydistinct (Banas and Gilmore, 1991). Neverthe-less, because this mutant has not yet been testedin vivo, it is premature to exclude this gene as apotential virulence factor. However, based on thesein vitro results, it is likely that more than oneprotein is involved in glucan binding by theseorganisms. Likely candidates are the GTFs ortheir proteolytic fragments (Mooser and Wong,1988). Because the GTFs apparently have differ-ent affinities for various glucans (Koga et al,1983), additional investigations are required toassess their relative roles in colonization that re-sults from glucan binding, in addition to glucansynthesis. Whether such interactions are impor-tant in cariogenesis remains to be determinedbecause previous results (Tanzer and Freedman,1978) with chemically induced nonaggregatingmutants of S. sobrinus indicate that such interac-tions may be obviated in the oral cavity.



D. Isolation of Genes Involved inIntracellular Metabolism

Because of the potential for utilizing LDH"mutants of S. mutans in replacement therapy(Hillman et al, 1987), it was of interest to isolatethe Idh gene from these organisms as an initialstep in constructing isogenic mutants. The genefrom S. mutans JH1000 has been cloned recentlyand characterized (Hillman et al, 1990). How-ever, the construction of an isogenic mutant fromthe cloned gene has yet to be reported althoughthe gene has been insertionally inactivated inEscherichia coll (Duncan and Hillman, 1991).

Because sucrose metabolism is clearly rel-evant to the cariogenicity of the mutans strepto-cocci, an examination of mutants altered in su-crose metabolism in animal systems is of interest.Because the majority of the sucrose metabolizedby these strains is transported into the cells andmetabolized intracellularly (Tanzer et al, 1972),the enzymes involved in this process could beconsidered potential virulence factors. Evidencefor multiple uptake systems for sucrose has beenobtained previously (Slee and Tanzer, 1982) andthe 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 genecoding for the Enz IISUC in the gnotobiotic ratindicated that this mutant was as cariogenic as theparental organism (Macrina et al, 1991). Thiswas not unexpected because additional pathwaysfor metabolizing sucrose are present in these or-ganisms (Chassy, 1983) and sucrose may be trans-ported into the cells via the trehalose PTS (Poyand Jacobson, 1990) or putative non-PTS sucroseuptake system (Slee and Tanzer, 1982).

E. Isolation and Characterization ofGenes Involved in StoragePolysaccharide Metabolism

Because the early utilization of chemically-induced mutants defective in the storage of intra-cellular polysaccharides suggested that this prop-erty may be an important virulence factor (Tanzeret al, 1976), it was of interest to construct definedmutants with this phenotype. Recently, Harris andCurtiss (1991) have isolated genes involved in

intracellular glycogen storage from S. mutans andhave constructed isogenic mutants defective inthis property. Implantation of these mutants ofstrain UA130 into gnotobiotic rats indicated thatthe mutants are significantly less cariogenic thanthe parental organism when the animals are fedeither ad libitum or following programmed feed-ing (Harris etal, personal communication). Theseresults clearly indicate that the storage of intra-cellular polysaccharides by S. mutans is an im-portant virulence property, as previously suggested(Tanzer et al, 1976).

Other potential plaque storage polysaccha-rides are the extracellular fructans synthesized byS. mutans as well as by other plaque bacteria(Carlsson, 1970). Several recent investigationsutilizing rat model systems (Schroeder et al, 1989;Yamashita et al., unpublished) have suggestedthat S. mutans mutants defective in fructan syn-thesis are normally cariogenic. More recently, thegene coding for the fructanase of S. mutans hasbeen isolated (Burne et al., 1987), an isogenicfruA mutant constructed in strain UA159, andimplanted into specific pathogen-free rats. Theseresults also indicated that the mutant is ascariogenic as the parental organism (Burne, per-sonal communication). However, because theseexperiments were carried out with rats fed adlibitum, it will be useful to examine these mutantsunder programmed feeding conditions in order tofurther assess their relative roles in cariogenicity.Nevertheless, it is important to note that strains ofS. sobrinus that synthesize little or no detectablefructan are highly virulent in rodent caries models(Tanzer, 1992).

Additional potential storage polysaccharidesmay be the glucans that are synthesized in dentalplaque 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 areknown to be elaborated by S. mutans strains(Schachtele et al., 1975) could be important inthis respect. However, although a dextranase genehas been cloned from S. sobrinus 6715 (Barrett etal., 1987), the comparable gene has not yet beenisolated from a S. mutans strain. The previouslyisolated dextranase gene that maps close to thegtfA gene of S. mutans (Burne et al., 1986) repre-sents an exodextranase and not the extracellularendodextranase that can be detected in culturefluids of these strains (Schachtele et al., 1975). It



is likely that a combination of both dextranases isnecessary for strains of mutans streptococci tometabolize dextran molecules. In addition, theextracellular endodextranase could play a role inmodifying the structure of the glucans synthe-sized by the GTFs of these organisms (SchachteleetaU 1975).

F. Genes Involved in the Aciduricity ofMutans Streptococci

Biochemical characterization of S. mutansstrains has established a primary role for themembrane-associated ATPases in the aciduricityof these organisms (Bender et ai, 1986). How-ever, the genes that code for the multiple subunitsof this enzyme complex have yet to be isolatedand characterized. An initial step in this approachhas been reported recently (Quivey, personal com-munication), whereby a DNA fragment codingfor one of these subunits has been isolated follow-ing PCR amplification. Therefore, because thegenes that code for the comparable complex in E.coli constitute a single operon (Walker et al.,1984), it is likely that gene walking from thisinitial fragment could ultimately lead to the isola-tion of the entire operon. Subsequently, mutantsdefective in this activity could be constructed andexamined for cariogenicity in animal model sys-tems.

Tn916 mutagenesis of S. mutans UA96 hasled to the preliminary isolation of mutants thatexhibit altered aciduricity (Marchman et al.,1990). Although none of these mutants has beencharacterized extensively (Caufield, personalcommunication), Southern blot analysis has re-vealed that the transposon was inserted outsideof the ATPase locus into different positions inthe mutant chromosomes. Therefore, it is likelythat multiple genes are involved in maintainingthe aciduricity of these organisms. More re-cently, a similar strategy has been utilized toisolate a mutant of S. mutans GS5 that exhibitsmarkedly reduced aciduricity and also displaystemperature-sensitive growth (Yamashita andKuramitsu, unpublished). Molecular character-ization of these mutants will be required toidentify the genes that are involved in this viru-lence property of S. mutans.

G. Isolation of Bacteriocin Genes

Because there have been suggestions that theelaboration of bacteriocins (mutacins) by themutans streptococci may play a role in coloniza-tion (van der Hoeven and Rogers, 1979), it wouldbe of interest to construct defined bacteriocin"mutants of these organisms for testing in animalmodels. Utilizing a Tn916 mutagenesis strategy,a mutant defective in mutacin activty in S. mutansUA96 has been isolated recently (Caufield et al,1990). Therefore, it should be possible to test thebacteriocin" mutant in appropriate animal modelsystems.

V. SOME UNRESOLVED QUESTIONSREGARDING THE VIRULENCE OFMUTANS STREPTOCOCCI

Both biochemical (Loesche, 1986) and ge-netic (Macrina et al., 1990) approaches have de-fined the important roles of the following viru-lence factors of mutans streptococci in dentalcaries: colonization of teeth both in the presenceand absence of sucrose, GTF-mediated synthesisof water insoluble glucans, strong fermentation ofa variety of sugars, metabolism of storgagepolysaccharides, and significant aciduricity. Assuggested above, there are still a number of ques-tions regarding each of these as well as otherpotential virulence factors that still remained un-answered. In many cases, each of these can beapproached utilizing the techniques of moleculargenetics.

A. Colonization of Teeth

As noted previously, the identity of theadhesins involved in the initial sucrose-indepen-dent attachment of S. mutans strains to teethhave yet to be convincingly established. Becauseit has been suggested that more than one adhesinmay be involved in this process (Gibbons, 1989;Bowen et al., 1991), it will be of interest toidentify both biochemically and genetically thecell surface proteins of these organisms that spe-cifically bind to human salivary mucins or pro-



line-rich proteins (Gibbons, 1989) in addition tosalivary agglutinin (Lee etal, 1989). Mutants of5. mutans could then be constructed containingalterations in individual and multiple putativeadhesins. Subsequently, these mutants could beexamined in vitro, as well as following implan-tation into rats and humans under different di-etary conditions (glucose vs. sucrose). In addi-tion, the question concerning the role of S. mutansbinding to pellicle containing GTF and glucansin tooth colonization needs to be resolved (Gib-bons et ai, 1986; Schilling and Bowen, 1992).Such interactions appear to be significant in thecolonization of S. sobrinus strains to teeth (Gib-bons et al, 1986).

Another question that has not been an-swered satisfactorily yet is the role of sucrose(glucan synthesis) in the development of hu-man fissure and approximal caries. The resultsfrom animal model experiments have consis-tently demonstrated that the presence of su-crose in the diet is more critical for the devel-opment of smooth surface relative to sulcaland proximal lesions (Loesche, 1986; Tanzer,1992). In addition, the utilization of definedS. mutans mutants defective in insoluble glucansynthesis implanted into rats also has suggestedthat glucan synthesis is not a major factor incaries development in fissures (Munro et al.,1991; Yamashita et al, 1992). However, oneearlier investigation (Tanzer, 1979) hasdemonstrated significant enhancement of5. mutans-induced fissure caries in rats in thepresence of sucrose. Furthermore, human epi-demiological studies (Newbrun, 1982) have sug-gested that sucrose is a major factor in thedevelopment of human carious lesions (whichoccur primarily on the occlusal surfaces andsecondarily in the proximal regions of teeth).Therefore, a question can be raised as towhether the pathogen-free conventional orgnotobiotic rat systems as presently employedare suitable model systems for quantitativelyassessing the role of sucrose (and glucans) inthe development of S. mutans-induced cariouslesions in the sulcal areas. Differences in themorphology of human vs. rat fissures couldobscure or minimize the role of sucrose incaries induction in these regions of humanteeth.

B. Sucrose-Enhanced Colonization ofTooth Surfaces

Although both biochemical and genetic ap-proaches have outlined the role of glucans intooth colonization (Loesche, 1986), a number ofsignificant questions still have not been resolved.Foremost among these is the nature of the "adhe-sive" glucan that is involved in this process. Nei-ther the precise chemical nature of the theseglucans (molecular size, degree of branching ofalpha-1,6- and -1,3-glucose linkages ) nor thespecific roles of each GTF in synthesizing thesemolecules has yet been defined precisely for themutans streptococci. Because the properties ofeach specific S. mutans GTF appears to differsomewhat from the comparable enzymes fromother 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 in-volved in adhesive glucan synthesis may be dis-tinct, depending upon the species investigated. Asadditional gtf genes are isolated from differentstrains, expressed in other streptococci (S. millerlS. lactis ), and defined mutants constructed, thesequestions should be answered more adequately.Moreover, the insoluble glucans may play addi-tional roles in cariogenesis besides colonization,as recently proposed (van Houte et al, 1989), andthese could also be evaluated utilizing specificmutants and appropriate animal model systems.

C. Environmental Influences on theCariogenicity of the MutantsStreptococci

It is becoming increasingly clear that the viru-lence of pathogenic microorganisms can be influ-enced by the environmental conditions within thehost (DiRita and Mekalanos, 1989). Therefore,one aspect of the cariogenicity of mutans strepto-cocci that has not yet been addressed adequatelyis the influence of the environment in the oralcavity on the cariogenicity of these organisms.Although a number of genes that express poten-tial virulence factors in the mutans streptococcihave been isolated and characterized (Macrina etal, 1990), little information is currently available



regarding the regulation of their expression. It islikely that the localized environment within plaque(acidic pH, lower O2 tension, limiting nutrients)could affect the expression of these genes. Forexample, it has been proposed recently (Hudsonand Curtiss, 1990) that the expression of the gtfgenes of S. mutans is influenced by attachment ofthe organisms to tooth surfaces. However, themolecular basis for such apparent regulation hasnot been determined yet. In this respect, the utili-zation of chemostat-grown cells of mutans strep-tococci (Elwood, 1976) has suggested that thegrowth rate can influence the differential expres-sion of the GTF-I and GTF-S enzymes of some ofthese organisms. Moreover, the observation thatS. mutans cells embedded in an insoluble glucanmatrix ferment sugars at a more rapid rate relativeto cells colonized in the absence of glucan (vanHoute et a/., 1989) suggests that physical factorscan also influence the physiology of these organ-isms.

A very recent study (Caufield, personal com-munication) has suggested that the future cariesexperience of a child may well be determined ata crucial "window" stage within the first 2 yearsof life. It is reasonable to assume that during thisperiod an environment is developed that is neces-sary for the critical colonization of newly eruptedteeth by S. mutans strains, which are transmittedprimarily from the child's mother. It will be im-portant to determine what these factors are andtheir influence on the pathogenic properties ofthese organisms. The presence or absence of otheroral microorganisms may also play a critical rolein this process. Little information is currently avail-able regarding the influence of other early colo-nizers of teeth, such as S. sanguis, on the viru-lence properties of S. mutans. Coculture ap-proaches involving the utilization of S. mutansstrains genetically engineered to express reportergenes fused to genes involved in cariogenicitymay be important in this respect. This approachmay help to define relevant environmental factorsthat may alter the expression of virulence factorsin these organisms.

D. Cariogenicity of Different Strains ofS. Mutans

Kohler and Krasse (1988) have described thedifferential cariogenicity in rats of two fresh hu-

man oral isolates of S. mutans. Therefore, it islikely that a range of cariogenic potentials may beexpressed in different strains of these organismsharbored by humans. However, no information iscurrently available regarding the correlation be-tween the incidence of caries in a study popula-tion relative to the specific S. mutans strains har-bored by each individual. One reason for the ab-sence of such studies has been the lack of a simple,sensitive test that can distinguish between differ-ent strains of 5. mutans. The introduction of re-striction fragment length polymorphism (RFLP)analysis of these organisms (Caufield and Walker,1989) has suggested a highly sensitive techniqueto distinguish between strains of these organisms.Such a survey, together with a comparison of thebiochemical properties of the strains identified,may reveal previously unrecognized virulencefactors that may be important in cariogenicity.

VI. STRATEGIES TO NEUTRALIZE THEVIRULENCE FACTORS OF MUTANSSTREPTOCOCCI

A. Anticaries Vaccines

Several excellent reviews have discussed theprospects for developing an anticaries vaccine(Curtiss etal, 1986; Michalek and Childers, 1990)and the reader is urged to consult these for adetailed analysis of this subject. Extensive workis currently in progress to identify and isolatepurified antigens that can be utilized in producingan effective vaccine. More recently, Smith andTaubman (1991) have utilized small antigenicpeptides corresponding to the GTF-Is from mutansstreptococci that appear to protect rodents againstchallenge by S. mutans strains. It will be of inter-est to determine if these purified antigens are alsoprotective in non-human primates, because ear-lier results evaluating these enzymes as potentialvaccines have suggested that intact GTF mol-ecules were not protective in these animals (RussellandColman, 1981).

Because the presentation of an antigen to thehost is an important factor in the elaboration of animmune response (Michalek and Childers, 1990),several recent investigations in the oral cavityhave proposed novel approaches toward this end.



Macrina and colleagues (Dertzbaugh et ai, 1990)have constructed genetic fusions containing partof the cholera toxin B subunit together with asmall peptide of the S. mutans GS5 gtfB geneproduct as a potential oral immunogen. However,such fusions have not been examined yet as ananticaries vaccine. Another enhancement strategyfor the oral immune response against mutans strep-tococci has been proposed previously by Curtissand co-workers utilizing a different strategy thatinvolves ingestion of genetically engineered Sal-monella strains expressing S. mutans antigens(Curtiss et ai, 1988). In addition, the continuedexpression of S. mutans antigens by geneticallyengineered noncariogenic oral microorganisms(S. sanguis) implanted into the oral cavity mayprove beneficial in inducing an anticaries im-mune response.

B. Inhibitors of S. Mutans Colonization

Because one or more adhesins on the cellsurface of S. mutans may be important in theinitial colonization of these organisms to toothsurfaces (see Section IV), one potential strategyfor reducing the colonization of these organismsmay be to bathe the oral cavity with an active-sitepeptide of the adhesins. Such peptides could actas competitive inhibitors of the colonization of S.mutans. Both biochemical and genetic analysis ofthe potential adhesin molecules should identifythe functional domains of these proteins and couldlead to the synthesis of inhibiting peptides. Vari-ous strategies to continuously produce such in-hibitors in the oral cavity could be developed andexamined in animal models and ultimately inhuman implantation experiments.

Because the inhibition of glucan synthesisfrom sucrose by S. mutans should result in adecrease of colonization and cariogencity(Loesche, 1986), it has been proposed that thepresence of glucanases in the oral cavity may bebeneficial (Guggenheim et a/., 1972). A fungalglucanase that can hydrolyze the alpha- 1-3-glu-cose linkages present in insoluble glucans hasbeen purified recently and characterized (Krigerand Quivey, 1990). If the gene for this enzymecould be engineered into a noncariogenic oralmicroorganism such as S. sanguis, the coloniza-

tion of teeth by these altered organisms may re-duce the subsequent colonization of S. mutans.Whether such enzymatic approaches will be ef-fective in vivo remains to be determined becauseit is not clear whether or not such enzymes will beeffective under these conditions.

Strategies designed to interfere with adhesinor glucan-mediated attachment of S. mutans toteeth may be limited primarily to affecting smoothsurface caries. It is possible that other nonspecificfactors (bacterial entrapment) could be more sig-nificant for human occlusal and interproximalcaries.

C. Inhibitors of S. Mutans Growth

Sandham and colleagues (1988) have demon-strated that the application of chlorhexidine ontotooth surfaces can quite effectively eliminate S.mutans from such surfaces. Therefore, this com-pound incorporated into a number of oral vehiclesis currently under evaluation as an effectiveanticaries strategy. A similar strategy may be fea-sible based upon the elaboration of anti-S. mutansbacteriocins in the oral cavity. Such inhibitors areproduced by several microorganisms (Hamadaand Ooshima, 1975), and it may be possible toisolate the genes coding for one of these andexpress the gene in an oral plaque bacterium. Thiscould result in the continuous production of thebacteriocin in the oral cavity. However, a numberof important questions must be addressed beforeconsidering this approach in human therapy (thestability of the bacteriocins in the oral cavity,specificity of the bacteriocin against the majorstrains of S. mutans present in the oral cavity, andpotential development of resistance to suchagents).

D. Replacement Therapy

Several years ago, Hillman (1978) proposedthat the colonization of the oral cavity by S. mutansstrains defective in LDH could be used as a basisfor replacement therapy to reduce the incidenceof dental caries. This proposal was based on theproperties of a chemically induced LDH" mutantof S. rattus. However, because these organismsapparently are not good colonizers of the human

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oral cavity (Loesche, 1986), the further develop-ment of this strategy was dependent upon theisolation of specific LDH" mutants of a S. mutansstrain. In view of the recent isolation of the LDHgene from 5. mutans JH1000 (Hillman et ai,1990), it should now be possible to constructdefined LDH mutants of S. mutans following in-sertional inactivation of the cloned gene and bygene transfer into appropriate transformablestrains. To further enhance the colonizing poten-tial of the LDH' mutant, it has been proposed thatan elevated bacteriocin producer of this strainwould make the best candidate for a replacementtherapy vehicle (Hillman et a/., 1987).

A variation of this strategy has been sug-gested recently whereby the genes for the argin-ine deiminase system (Burne et ai, 1989) may beinserted into an S. mutans strain to produce a lesscariogenic strain. The resultant organism, pro-ducing elevated levels of NH3, would be muchless acidogenic and could be used as a replace-ment therapy vehicle. Alternatively, a LDH" argi-nine deiminase+ construct of S. mutans could beutilized for this purpose. Moreover, it may bepossible to reduce dental caries by competitivedisplacement of mutans streptococci with unal-tered oral microorganisms such as S. salivariusTOVE-R (Tanzer et ai, 1985).

It will be of interest to determine if novelstrategies based upon genetic manipulations canbe utilized ultimately in children for further re-duction in cariogenicity. A practical point of con-sideration in this respect must ultimately be thepolitical and social obstacles to utilizing geneti-cally engineered organisms in humans, especiallychildren. Because of the questions being raisedregarding the utilization of genetically engineeredorganisms, it may be difficult to convince thegeneral public to use these therapies in view ofthe continuing decline in the incidence of dentalcaries without such novel approaches. Neverthe-less, because of uncertainties regarding the futuredecline in caries with current strategies, theseapproaches should be evaluated carefully.

VII. SUMMARY

The utilization of biochemical approaches inconjunction with animal models has led to the

identification of a number of important virulenceproperties of the mutans streptococci. Prior to theutilization of cloned genes to construct definedmutants in S. mutans, mutants of mutans strepto-cocci were isolated following traditional mutagen-esis protocols, which could not preclude the gen-eration of multiple alterations in the organisms.Nevertheless, the results from the implantation ofthese mutants into animal models were clearlyimportant in defining some of the cariogenic prop-erties of the mutans streptococci. Those individu-als now utilizing the modern powerful tools ofmolecular genetics to further define thecariogenicity of these organisms (including thisauthor) should be reminded that none of the majorvirulence factors proposed from these earlier in-vestigations has yet to be revised based on morerecent molecular biological approaches.

The more recent introduction of moleculargenetic approaches, besides substantiating mostof these earlier findings, has led to a more de-tailed understanding of the molecular basis forthese properties. Specifically, these approachescan be utilized to define more precisely the ac-tive virulence factors (adhesin binding sites,enzyme active sites, etc.). In addition, the appli-cation of these strategies has led to the identifi-cation of potential virulence factors that werenot recognized by strictly biochemical ap-proaches. Despite the fact that the general basisfor the cariogenicity of mutans streptococci isnow well recognized, a number of importantquestions still remain unresolved. The resolu-tion of these issues should provide an even bet-ter undertstanding of the molecular basis forcariogenicity, and it could provide novel strate-gies for identification of individuals at high riskof developing carious lesions as well as suggest-ing additional preventive therapies.

ACKNOWLEDGMENTS

The author gratefully acknowledges the com-munication 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. Inaddition, the critical comments of Dr. J. M. Tanzerwere very helpful and are much appreciated.



The work described from the author's labora-tory is supported in part by National Institutes ofHealth grants DE03258 and DE09864.

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