Virulence properties of cariogenic bacteria

The importance of Streptococcus mutans in the etiology of dental caries has been well documented. However, there is growing recognition that the cariogenic potential of dental plaque may be determined by the composite interactions of the total plaque bacteria rather than solely the virulence properties of a single organism. This study will examine how the interactions of S. mutans with other biofilm constituents may influence the cariogenicity of plaque samples. In order to begin to investigate the effects of nonmutans streptococci on the cariogenic potential of S. mutans, we have examined the effects of Streptococcus gordonii on the virulence properties of the former organisms. These studies have indicated that S.gordonii can attenuate several potential virulence properties of S. mutans including bacteriocin production, genetic transformation, and biofilm formation. Therefore, modulation of the interactions between plaque bacteria might be a novel approach for attenuating dental caries initiation.


Introduction
Although the incidence of dental caries has declined over the past few decades in highly industrialized countries, this disease is still a significant public health problem [1]. Despite the recognition of the importance of mutans streptococci, principally Streptococcus mutans, as primary etiological agents of caries and the role of environmental factors such as diet on this disease [2], no novel anticaries therapies based upon this information have been recently incorporated into routine dental practice. Intensive investigation into the virulence of S. mutans has identified a number of properties of these organisms which are likely to be important in cariogenesis including: sucrosedependent biofilm formation, relatively high aciduricity, and potent acidogenesis [2]. However, it is now recognized that other plaque constituents may also be relevant to caries initiation including nonmutans streptococci [3] and alkaline bacteria [4], as well as recently identified novel organisms [5].
As elegantly emphasized by Kleinberg [4], it is apparent that the interactions between cariogenic bacteria such as S. mutans with other nonmutans plaque constituents can modulate the cariogenic potential of a specific plaque sample. A more careful examination of this hypothesis would require a "systems biology" approach to dental plaque, i.e., determining how the individual plaque constituents interact to produce the resulting properties of a biofilm community. Therefore, in order to investigate how such interactions could affect the cariogenicity of a plaque sample, we have begun to investigate the effects of nonmutans streptococci on the cariogenic properties of S.
(page number not for citation purposes) mutans. Earlier studies have suggested an inverse relationship between the presence of S. mutans and strains classified as Streptococcus sanguis (including Streptococcus gordonii) in human dental plaque [6]. Therefore, an understanding of the molecular basis for such interactions could prove useful in designing new strategies to control human dental caries.

Bacterial strains
S. mutans strains GS5, LT11, NG8, and BM71 as well as S. gordonii Challis were routinely cultured in Todd-Hewitt broth (THB; Difco, Detroit, MI) and maintained on Tryticase soy agar plates. The construction of the Challis mutant BYW1 was recently described [7].

Transformation
Genetic transformation of S. mutans strains with Escherichia coli-streptococcal shuttle plasmid pPGS479 (Wang and Kuramitsu, unpub. results) in broth was carried out essentially as routinely carried out in this laboratory [8]. When mixed cultures of S. mutans and other oral streptococci were examined for transformation, the transformed cells were plated onto Mitis salivarius agar plates containing erythromycin (Erm, 10 µg/ml) to differentiate between S. mutans and nonmutans streptococci.
For transformation in biofilms, mixed biofilms were initially formed with S. mutans strains and S. gordonii Challis (10 5 cfu) in 0.10 ml one-fourth diluted THB-0.1% glucose containing 0.01% bovine mucin (Sigma, St. Louis, Mo.) in 96 well polystyrene microtiter plates. After 24 h, the plantonic cells were discarded and the biofilms washed with PBS. 0.10 ml of THB-10% horse serum was then added and the biofilms incubated for one h to induce maximum competence. Plasmid pPGS479 was then added and the biofilms incubated for an additional two hours prior to suspension and sonication of the biofilms followed by plating on Mitis salivarius-Erm agar plates.

Biofilm formation
Biofilm formation was evaluated in 48-well polystyrene plates essentially as recently described [7]. For mixed biofilms, S. gordonii Challis or the BYW1 mutant were initially incubated in THB-0.1% glucose for 24 h, the resultant biofilms washed with PBS and incubated with S. mutans GS5 cells in THG-0.5% sucrose for an additional 24 h. The biofilms were washed, sonicated to disperse the cells, and serially diluted for plating on Mitis-salivarius agar plates to differentiate the S. mutans and S. gordonii cells.

S. gordonii attenuates genetic transformation of S. mutans
Recent results in our laboratory have demonstrated that the presence of S. gordonii Challis, as well as other nonmutans oral streptococci, inhibited bacteriocin production by S. mutans GS5 and BM71 in broth cultures as well as in biofilms [7]. Such attenuation was mediated by the challisin protease secreted by strain Challis which degraded the competence stimulating peptide, CSP, a quorum sensing regulator in S. mutans which is required for bacteriocin production. In addition, the presence of strain Challis also markedly inhibited genetic transformation of S. mutans strains as exemplified for LT11 (Table 1). Such inhibition was also demonstrated for S. mutans strains GS5, NG8 and BM71 (data not shown). Despite the fact that CSP is also required for maximal transformation of S. mutans, mutants of strain Challis which do not express the challisin also inhibited transformation (data not shown). This suggested that in addition to attenuating the expression of the S. mutans CSP, S. gordonii also inhibited transformation of S. mutans via a CSP-independent mechanism. Furthermore, other oral streptococci such as S. sanguis, S. mitis, and S. oralis also were capable of inhibiting the transformation of S. mutans (data not shown).

S. gordonii attenuates biofilm formation by S. mutans
Since S. gordonii appears to be one of the earliest colonizers of human teeth [9], it was of interest to determine if the presence of these organisms could influence subsequent biofilm formation by S. mutans. Preformed biofilms of S. gordonii Challis were established in microtiter plates, washed, and S. mutans GS5 was allowed to colonize layers of strain Challis in the presence of sucrose ( Table 2). S. mutans GS5 did not colonize on S. gordonii as well as on the polystyrene plate surfaces (data not shown). Interestingly, strain GS5 colonized at higher rates on the challisin mutant, BYW1, than it did on the parental Chal- Thus, in addition to S. mutans GS5 bacteriocin production and transformation, S. gordonii also inhibits biofilm formation by the former strain.

Discussion
The present results suggest that S. gordonii, and perhaps additional nonmutans streptococci, can attenuate some of the virulence properties of S. mutans, in part, by altering the quorum sensitive dependent properties of the later organisms. The recent demonstration that S. gordonii Challis can inactivate the CSP of S. mutans via a proteasedependent mechanism [7] as well as the documented role of this signaling molecule in biofilm formation, genetic transformation, aciduricity [10] and bacteriocin production (Yonezawa and Kuramitsu, in press) by S. mutans supports such a model. In addition, quorum-sensing independent effects of strain Challis on S. mutans GS5 were also demonstrated.
Sucrose-dependent biofilm formation by S. mutans strains appears to be an important virulence property of these organisms [2,11]. Furthermore, the present investigation demonstrated that the presence of S. gordonii appears to inhibit sucrose-dependent biofilm formation. The observation that the Challis mutant BYW1 did not inhibit this process as much as did the parental Challis strain suggests that CSP production by strain GS5 plays a significant role in sucrose-dependent biofilm formation. Such a role has recently been demonstrated in several strains of S. mutans when glucose is the primary carbon source [10,12]. However, the molecular basis for the role of CSP in S. mutans biofilm formation has not yet been determined and is currently under investigation in this laboratory.
Genetic transformation of S. mutans may not only serve as a means for expanding the genetic capabilities of this organism but could also have evolved as a means of scavenging essential purine and pyrimidine nutrients. Therefore, it may be possible that the attenuation of this property could alter the survivability of these organisms under conditions of nutrient limitation. Thus, the inhibition of S. mutans transformation by S. gordonii may pro-vide the latter organism a competitive advantage in plaque where nucleotide precursors are limiting.
It is also possible that the presence of nonmutans streptococci such as S. gordonii could alter other quorum sensingdependent properties of S. mutans including aciduricity, but this has not yet been examined. In addition, recent results in this laboratory have suggested that the sensitivity of strain GS5 to antimicrobial agents is also dependent upon CSP expression (Nakano and Kuramitsu, unpub. results). Therefore, the presence of S. gordonii may also increase the sensitivity of S. mutans in dental plaque to endogenous or exogenously applied antimicrobial agents.

Conclusion
Taken together, the present results suggest that the presence of nonmutans streptococci in dental plaque could modulate the virulence properties of S. mutans. It will be of interest to subsequently determine if such interactions also occur in vivo and can modulate the cariogenicity of individual plaque samples. In addition, these results suggest potential novel strategies to attenuate the cariogenicity of S. mutans in plaque samples. These might include the development of antagonists of CSP activity or probiotic approaches involving commensal oral organisms which might attenuate S. mutans CSP activity.