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 Macrina et ai, 1990; Russell, 1990), and this

Despite the recent decline in dental caries
frequency 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 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 pro-
portion of the population that is at risk of devel-
oping 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 strate-
gies that may further decrease the frequency of
this 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 the
identification of children at high risk of develop-
ing dental caries as well as the prevention of
decay. Several recent reviews have addressed
various aspects of this topic (Curtiss, 1985;
article focuses primarily on the relationship be-
tween 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 Strepto-
coccus mutans genes are now available. In addi-
tion, 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 epidemio-
logical surveys (Loesche, 1986), have strongly
implicated mutans streptococci (primarily S.
mutans) as the principal etiological agents in hu-
man dental caries. Therefore, for the past several
decades there have been extensive efforts to iden-
tify the virulence factors of these organisms as

1045-4411/93/S.50
© 1993 by CRC Press, Inc.
159
potential targets for anticaries prophylaxis. Ini-
tially, potential virulence properties of the mutans
streptococci were identified following compari-
son of these organisms with other oral strepto-
cocci. One of the first putative virulence factors
of the mutans streptococci identified by this ap-
proach was the ability of these organisms to colo-
nize smooth surfaces in vitro in the presence of
sucrose (Gibbons etal, 1966). Subsequently, this
property 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 to
synthesize these polysaccharides (Hamada and
Slade, 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) 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 estab-
lished. 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 regard-
ing the synthesis of these exopolysaccharides and
the role of glucosyltransferases (GTF) in this pro-
cess (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 pres-
ence of sucrose. This characteristic could be ra-
tionalized as another example of sucrose-depen-
dent attachment of cells to solid surfaces due to
insoluble glucan formation. However, in addi-
tion, 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
160
play a role in colonization. More recently, it has
been suggested that such interactions may also
aid in the initial attachment of the mutans strep-
tococci 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 synthe-
size 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 colo-
nization. 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. Further-
more, Gibbons et al. (1986) have demonstrated
differences in tooth colonization mechanisms that
depend on the group of mutans streptococci in-
volved: 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 fermen-
tation 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 prop-
erty of the mutans streptococci is undoubtedly
related to the multitude of enzyme systems ex-
pressed by these organisms that are capable of
both transporting and metabolizing sucrose
(Loesche, 1986).
It was also reasonable to assume that an im-
portant virulence property of cariogenic bacteria
should be their ability to continue fermentation in
the absence of exogenous food supplies (condi-
tions 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 in-
deed demonstrated that strains of S. mutans are
more acid tolerant than all other bacteria exam-
ined 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 differen-
tial spectrum of these inhibitors has made it dif-
ficult to assess their role in the virulence of these
organisms.
III. MUTANT CHARACTERIZATION AND
THE IDENTIFICATION OF VIRULENCE
FACTORS
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 of
its pathogenicity with the parental organism in an
appropriate animal model. Toward this end, a
number of mutants of mutans streptococci defec-
tive in potential virulence traits have been iso-
lated (Freedman et al, 1981). These, as well as
the other mutants described in this section, were
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 iso-
lated 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 synthe-
sis (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-de-
fective 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 com-
pared with fissure caries (Tanzer, 1979). These
observations were also consistent with earlier find-
ings 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 strep-
tococci 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 cell-
associated GTFs. These results suggested that
either a specific glucan-binding molecule or pro-
teolytic 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
161
mutans streptococci (McCabe etal., 1977; Drake
et al., 1988). Nevertheless, rigorous molecular
characterization of these proteins is required
for identification of these molecules as nonen-
zymic glucan-binding proteins, inasmuch as it
has been demonstrated that proteolytic frag-
ments 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 sucrose-
dependent (glucan-mediated accumulation) and
independent (initial attachment) process. Subse-
quent 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 identifica-
tion 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 chemi-
cally induced mutant of S. sobrinus 6715 altered
in a 210 kDa cell surface protein was defective in
both in vitro attachment to saliva-coated hydroxya-
patite beads and in cariogencity in rats (Curtiss et
al., 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 of
tooth surfaces (Ackermanns et al., 1985; Lee et
al., 1989).
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 strep-
tococci 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 stor-
age 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).
162
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 (colo-
nization, aggregation) (Loesche, 1986). In addi-
tion, 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 mu-
tants displayed reduced cariogenicity when im-
planted into Sprague-Dawley rats fed high su-
crose diets on both smooth surfaces and fissures
of the teeth. However, the molecular basis for
virulence reduction in the mutants has not been
established.
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 un-
equivocal basis for defining the virulence of
these organisms (Table 1). Such defined mu-
tants can be utilized to obviate complications
from multiple or pleiotrophic mutations that
could 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 spaA
mutants were isolated following chemical mu-
tagenesis and the possibility of pleiotrophic ef-
fects in the mutants has not been evaluated, some
caution should be exercised in drawing the con-
clusion that the spaA protein is a S. sobrinus
adhesin required for tooth colonization. Unfor-

TABLE 1
PUTATIVE S. mutatis Virulence Genes

In vivo Caries
Gene Role Mutants? testing? decrease?

Idh Acidogeniclty No *
No
ATPase Aciduncity No No —
pac Colonization Yes Yes No
gtB Colonization Yes Yes Yes
gtiC Colonization Yes Yes Yes
gtio Colonization Yes Yes No
gbp Colonization Yes No —
ftf Reserve Yes Yes No
nutrient
gig Reserve Yes Yes Yes
nutrient
scrA Sucrose Yes Yes No
transport

— = Not tested. See Section IV for references to investigations

outlined in table.

A. Cloning of Genes Involved in tunately, a gene transfer system for S. sobrinus

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 chemi-
cal 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 be-
cause a related protein from S. mutans appears to
be involved in attachment to saliva-coated hy-
droxyapatite beads ((Russell, 1986) and the spaA
mutants also produced fewer carious lesions when
strains has not been developed yet and it has not
been possible to construct defined spaA mutants
from the isolated gene. In addition, the organiza-
tion of the spaA gene on the chromosome of
strain 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 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 cod-
ing 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 trans-
formable (Perry and Kuramitsu, 1981), one of

163
these was a logical candidate for the construc-
tion 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 pro-
tein of S. sobrinus) utilizing the cloned gene for
insertional inactivation following electro-
poration. 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 saliva-
coated 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 pro-
tein may play a role in tooth colonization,
multiple interactions between S. mutans and
the pellicle of teeth are important for coloniza-
tion. In addition, interactions between S. mutans
and other plaque bacteria may also contribute
to colonization by the former organisms. Like-
wise, because previous investigations have sug-
gested that S. mutans strains also interact with
salivary mucins and a proline-rich protein (Gib-
bons, 1989), it is reasonable to assume that
multiple adhesins may be present on the sur-
face of these organisms. Moreover, because
there is no information available presently re-
garding 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 coloni-
zation of these mutants with the parental organ-
isms 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-
164
enhanced colonization of teeth (Loesche, 1986).
Verification of the expression of multiple GTFs
in the mutans streptococci has been obtained re-
cently by isolating individual gtf genes from sev-
eral 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 prima-
rily 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), syn-
thesizing insoluble and soluble glucans, respec-
tively. Biochemical approaches have further sug-
gested 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 ex-
presses one or more GTF enzymes synthesizing
soluble glucans and at least one producing in-
soluble 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 syn-
thesis exhibited reduced caries induction on the
smooth surfaces of the teeth, relative to the paren-
tal strain. Furthermore, no additional reduction in
cariogencity was observed when either the
fructosyltransferase (ftf) or gtfD genes were indi-
vidually inactivated. These results were consis-
tent with previous suggestions that water insoluble
glucan synthesis was important, though not re-
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 con-
structed in S. mutans UA130 for testing in spe-
cific 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 gno-
tobiotic rat system (Munro et aL, 1991). Like-
wise, 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 conven-
tional 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 as-
cribed 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 proper-
ties 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 en-
zyme 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 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 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 su-
crose. These results support the hypothesis that,
in the presence of the gtfD gene, two copies of the
gtf genes expressing enzymes synthesizing in-
soluble glucan are required for maximum sucrose-
enhanced 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 strep-
tococci into animal models, it is not clear which
gtf genes are required for sucrose-enhanced colo-
nization 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 utiliz-
ing 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) in-
duced 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 syn-
thesis than in the UAB gnotobiotic system (Table
2). This may result, in part, from the presence of
"sticky" components of diet 305 (cellulose and
165
 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)
UA101
UA130
Yamashita et al., 1992.
Barletta et al., 1988.
corn starch) not found in diet 2000 which could
obviate the requirement for adhesive insoluble
glucan synthesis for smooth surface caries initia-
tion (Yamashita et al, 1992).
Earlier biochemical analysis utilizing puri-
fied GTFs (Fukushima et al, 1981; Kuramitsu
and Wondrack, 1983) suggested that the glucan
involved 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 activity
suggests that this enzyme is not required for su-
crose-enhanced colonization in vivo (Munro et
al, 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 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 strepto-
cocci with glucans synthesized by the GTFs could
play a role in sucrose-enhanced colonization of
tooth surfaces, it is important to identify the mol-
ecules involved in such binding. The isolation of
166
UAB Model
Gnotobiotic Fisher rats
(5% sucrose diet)
Smooth surface lesions
4.08a 11.0 b
34.8 11.6
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). How-
ever, 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 colo-
nizes 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). Neverthe-
less, 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 differ-
ent affinities for various glucans (Koga et al,
1983), additional investigations are required to
assess their relative roles in colonization that re-
sults from glucan binding, in addition to glucan
synthesis. Whether such interactions are impor-
tant in cariogenesis remains to be determined
because previous results (Tanzer and Freedman,
1978) with chemically induced nonaggregating
mutants of S. sobrinus indicate that such interac-
tions may be obviated in the oral cavity.
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). How-
ever, 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 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 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 or-
ganisms (Chassy, 1983) and sucrose may be trans-
ported 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 chemically-
induced mutants defective in the storage of intra-
cellular polysaccharides suggested that this prop-
erty 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 feed-
ing (Harris etal, personal communication). These
results 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 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 syn-
thesis 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, per-
sonal 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) repre-
sents an exodextranase and not the extracellular
endodextranase that can be detected in culture
fluids of these strains (Schachtele et al., 1975). It
167
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 synthe-
sized 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). How-
ever, 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 com-
munication), whereby a DNA fragment coding
for one of these subunits has been isolated follow-
ing 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 isola-
tion of the entire operon. Subsequently, mutants
defective in this activity could be constructed and
examined for cariogenicity in animal model sys-
tems.
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 re-
vealed 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 re-
cently, 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 character-
ization of these mutants will be required to
identify the genes that are involved in this viru-
lence property of S. mutans.
168
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 coloniza-
tion (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 ge-
netic (Macrina et al., 1990) approaches have de-
fined the important roles of the following viru-
lence 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 ques-
tions regarding each of these as well as other
potential virulence factors that still remained un-
answered. 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-indepen-
dent 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 spe-
cifically bind to human salivary mucins or pro-
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 implan-
tation into rats and humans under different di-
etary conditions (glucose vs. sucrose). In addi-
tion, the question concerning the role of S. mutans
binding to pellicle containing GTF and glucans
in tooth colonization needs to be resolved (Gib-
bons et ai, 1986; Schilling and Bowen, 1992).
Such interactions appear to be significant in the
colonization 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 results
from 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 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 epi-
demiological studies (Newbrun, 1982) have sug-
gested 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 ap-
proaches 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 "adhe-
sive" glucan that is involved in this process. Nei-
ther 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 in-
volved in adhesive glucan synthesis may be dis-
tinct, 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 addi-
tional 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 viru-
lence of pathogenic microorganisms can be influ-
enced by the environmental conditions within the
host (DiRita and Mekalanos, 1989). Therefore,
one aspect of the cariogenicity of mutans strepto-
cocci 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 poten-
tial virulence factors in the mutans streptococci
have been isolated and characterized (Macrina et
al, 1990), little information is currently available
169
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 utili-
zation of chemostat-grown cells of mutans strep-
tococci (Elwood, 1976) has suggested that the
growth rate can influence the differential expres-
sion 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 organ-
isms.
A very recent study (Caufield, personal com-
munication) 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 neces-
sary for the critical colonization of newly erupted
teeth by S. mutans strains, which are transmitted
primarily from the child's mother. It will be im-
portant 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 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. 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 hu-
170
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 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 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 inter-
est to determine if these purified antigens are also
protective in non-human primates, because ear-
lier results evaluating these enzymes as potential
vaccines have suggested that intact GTF mol-
ecules 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 strep-
tococci has been proposed previously by Curtiss
and co-workers utilizing a different strategy that
involves ingestion of genetically engineered Sal-
monella 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 im-
mune response.
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. Vari-
ous strategies to continuously produce such in-
hibitors 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-glu-
cose 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-
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 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 sig-
nificant for human occlusal and interproximal
caries.
C. Inhibitors of S. Mutans Growth
Sandham and colleagues (1988) have demon-
strated that the application of chlorhexidine onto
tooth surfaces can quite effectively eliminate S.
mutans from such surfaces. Therefore, this com-
pound incorporated into a number of oral vehicles
is currently under evaluation as an effective
anticaries strategy. A similar strategy may be fea-
sible 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).
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
171
oral cavity (Loesche, 1986), the further develop-
ment 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 in-
sertional inactivation of the cloned gene and by
gene transfer into appropriate transformable
strains. To further enhance the colonizing poten-
tial 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 sug-
gested recently whereby the genes for the argin-
ine deiminase system (Burne et ai, 1989) may be
inserted into an S. mutans strain to produce a less
cariogenic strain. The resultant organism, pro-
ducing elevated levels of NH3, would be much
less acidogenic and could be used as a replace-
ment therapy vehicle. Alternatively, a LDH" argi-
nine 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 unal-
tered 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 re-
duction in cariogenicity. A practical point of con-
sideration in this respect must ultimately be the
political and social obstacles to utilizing geneti-
cally 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. Neverthe-
less, because of uncertainties regarding the future
decline in caries with current strategies, these
approaches should be evaluated carefully.
VII. SUMMARY
The utilization of biochemical approaches in
conjunction with animal models has led to the
172
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 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 of
these mutants into animal models were clearly
important in defining some of the cariogenic prop-
erties of the mutans streptococci. Those individu-
als 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 in-
vestigations 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 de-
tailed understanding of the molecular basis for
these properties. Specifically, these approaches
can 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 were
not recognized by strictly biochemical ap-
proaches. 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 resolu-
tion of these issues should provide an even bet-
ter undertstanding of the molecular basis for
cariogenicity, and it could provide novel strate-
gies for identification of individuals at high risk
of 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. In
addition, the critical comments of Dr. J. M. Tanzer
were very helpful and are much appreciated.
 The work described from the author's labora-
tory is supported in part by National Institutes of
Health grants DE03258 and DE09864.
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