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Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis.

The Hyp mouse is a murine homologue of human X-linked hypophosphatemia that displays hypo-mineralization in bone and dentin. In this study, we tested the hypothesis that the defect in Hyp mice leads to alterations in the expression of dentin matrix proteins that may be associated with the hypo-mineralization changes in the tissues. Quantitative RT-PCR analyses showed that expression of the osteocalcin gene in Hyp mice tooth germ samples was significantly higher than in wild-type mice, whereas the gene expressions of osteonectin, osteopontn, dentin matrix protein 1, and type I collagen in both types of mice were similar. Further, cultured Hyp mice tooth germ samples exhibited a higher expression of the osteocalcin gene than did those from wild-type mice, which was in accord with the results of our in vivo analysis. These findings suggest that osteocalcin mRNA is highly expressed in Hyp mice odontoblasts and may be associated with dentin hypo-mineralization.

Streptococcus mutans produces three glucosyltransferases, coded by gtfB, gtfC, and gtfD, whose cooperative action is essential for sucrose-dependent cellular adhesion. This cellular adhesion plays an important role in the formation of dental plaque and the initiation of dental caries. Since they bear genetic similarities and are large in size, differentiation of their gene expression has been difficult, and little is known about the dynamic process of gtf expression. Using a real-time reverse-transcription/polymerase chain-reaction, we determined the expression of each gtf. Under various conditions, the relative levels of transcription were gtfB > gtfD > gtfC. Sucrose enhanced gtfD expression, whereas it reduced that of gtfB and gtfC, suggesting the presence of independent promoters. Quantitative analyses demonstrated coincidence between the ratio of expression of each gtf and the ratio previously identified as optimal for sucrose-dependent adhesion in vitro, suggesting that S. mutans produces GTF at an optimal ratio to adhere to the tooth surface.

Streptococcus mutans produces 3 types of glucosyltransferase (GTF), whose cooperative action is considered to be essential for its cellular adherence to the tooth surface. However, the precise mechanisms for synthesizing adhesive glucans and the specific roles of each GTF in cellular adherence to smooth surfaces have not been elucidated. In the present study, seven types of isogenic mutants of S. mutans MT8148 lacking GTFB, GTFC, and/or GTFD activities were constructed by inactivation of the genes encoding GTFB, GTFC, and/or GTFD. Furthermore, recombinant GTFB, GTFC, and GTFD were prepared from Escherichia coli cells harboring recombinant plasmids containing each of the gtf genes. Using these GTF-deficient mutants and rGTFs, we reconstituted sucrose-dependent adherence of S. mutans resting cells and examined the role of each GTF in vitro. The highest level of sucrose-dependent adherence was found at the ratio of 20 rGTFB:l rGTFC:4 rGTFD in both the resting cells of GTF-deficient mutants and insoluble glucan synthesized by rGTFs. Moreover, when rGTFC and rGTFD were both present at concentrations of 1.5 mU and 6 mU, respectively, the insoluble glucan synthesized from sucrose by the rGTFs showed a high level of adhesiveness to smooth surfaces, even without rGTFB. These results suggest that the presence of all three GTFs at the optimum ratio is necessary for sucrose-dependent adherence of S. mutans, and that GTFC and GTFD may play significant roles in the synthesis of adhesive and insoluble glucan from sucrose.

The incisor dentin of hypophosphatemic (Hyp) mice was examined histopathologically to determine whether the multiple occurrences of interglobular dentin would be influenced by the serum phosphate level. Both normal and Hyp mice (12 weeks of age) were divided into two groups. The mice in one group were given a control diet (1.42% Ca, 1.16% P) and the other a high-calcium and high-phosphate diet (2.00% Ca, 3.00% P) for 30 days. Blood was collected from the mice every fifth day for measurement of the calcium and phosphate concentrations in serum. Both ground and decalcified cross-sections were prepared from incisors from the mandible and maxilla for microscopic examination. The levels of serum Ca and P were almost constant in normal mice, regardless of diet. On the other hand, serum P levels in Hyp mice fed the control diet were significantly lower than those in normal mice. The ten days' feeding of the high-Ca/-P diet significantly elevated the serum P level in Hyp mice, and it reached a level similar to that of the normal mice. However, histopathological examination showed no significant changes in incisor dentin of Hyp mice fed the high-Ca/-P diet, and interglobular dentin still occurred. These results suggest that the multiple formations of interglobular dentin, which is the most outstanding feature of X-linked hypophosphatemic vitamin-D-resistant rickets, are not influenced in Hyp mice by the short-time normalization of the serum phosphate level.

Hyp mice (murine homologue of human X-linked hypophosphatemia) have a disorder in phosphate homeostasis, and display hypomineralization in bones and teeth. We investigated whether a mutation of Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) has an effect on the expression level of type II sodium-dependent phosphate co-transporter (Npt2) in the developing teeth of the Hyp mouse. Quantitative RT-PCR analyses revealed that the amount of Npt2b mRNA, an isoform of Npt2, in Hyp mouse tooth germs was significantly lower than that in wild-type mice, in both in vivo and in vitro experiments. In addition, tooth germs from wild-type mice cultured in medium supplemented with antisense oligo-deoxynucleotide for Phex also showed a reduction of Npt2b mRNA expression. These findings suggest that the loss of Phex function is related to the defect of Npt2b expression in teeth, and Npt2b reduction is an intrinsic defect of Hyp murine teeth.

A blood isolate of Streptococcus mutans strain TW871 shows relatively low homology with MT8148, a reference oral isolate strain, and lacks the serotype-specific polysaccharide antigen, suggesting that other cell-surface structures correlate with cariogenicity. We compared cariogenicity of TW871 with MT8148 (serotype c) and blood isolate TW964 (serotype f) in rats. Strain TW871 showed significantly lower cariogenicity than MT8148 or TW964 and expressed significantly lower sucrose-independent cellular adhesion to saliva-coated hydroxyapatite and dextran-binding activity than strain MT8148. Strains TW871 and TW964 showed a defect in the gbpA gene by Southern hybridization analysis, while sequencing analysis revealed gbpC variation in TW871. These results suggest that variation in GbpC may alter cellular adherence properties and can be correlated with the cariogenicity of S. mutans in this strain.

The cacao bean husk has been shown to possess two types of cariostatic substances, one showing anti-glucosyltransferase (GTF) activity and the other antibacterial activity, and to inhibit experimental dental caries in rats infected with mutans streptococci. In the present study, chromatographic purification revealed high-molecular-weight polyphenolic compounds and unsaturated fatty acids as active components. The former, which showed strong anti-GTF activity, were polymeric epicatechins with C-4β and C-8 intermolecular bonds estimated to be 4636 in molecular weight in an acetylated form. The latter, which showed bactericidal activity against Streptococcus mutans, were determined to be oleic and linoleic acids, and demonstrated a high level of activity at a concentration of 30 μg/mL. The cariostatic activity of the cacao bean husk is likely caused by these biologically active constituents.

Xerostomia is a serious side-effect of radiotherapy for head and neck cancer and often enhances caries activity. However, the relationship between caries induction and the IgA level in saliva in rats subjected to irradiation of the salivary glands is unclear. The effect of salivary gland irradiation on salivary function was examined in specific pathogen-free Sprague-Dawley rats infected with or without Streptococcus mutans MT8148R (serotype c). The flow rate of saliva and the protein concentration in saliva were significantly reduced in irradiated rats, regardless of infection of S. mutans. The caries activity was enhanced in these rats, and an irradiation level of 50 Gy significantly increased the caries score. However, longitudinal study indicated that the salivary concentration of IgA reactive with S. mutans whole cells maintained similar or significantly higher levels in irradiated rats, when compared with those of nonirradiated rats. In addition, there was no negative correlation between the caries score and the salivary concentration of IgA reactive with S. mutans. These findings suggest that the secreted IgA against S. mutans may not play a significant role in the caries induction of hyposalivated rats.

SPF Sprague-Dawley rats and ICR mice were inoculated with either Streptococcus mutans MT8148R (serotype c) or 6715 (g), and the influence of inoculum size, inoculum frequency, and sucrose on the establishment of S. mutans in the oral cavity was examined. Successful colonization of S. mutans in the experimental animals was absolutely dependent on the number of the cells introduced orally. Furthermore, inoculum frequency and sucrose seemed to act as secondary factors to modify the establishment of S. mutans, and it is suggested that high inoculum frequency may decrease the inoculum size necessary for the colonization of S. mutans in the oral cavity.

Streptococcus mutans possesses the abilities to adhere to pellicle-coated tooth surfaces and to form acids - two characteristics associated with the cariogenicity of this micro-organism. De novo synthesis of insoluble glucan by S. mutans glucosyltransferase from sucrose is essential in the adherence process. Therefore, agents which interfere with the adherence ability of S. mutans would be useful for controlling dental caries. In the present report, we have summarized our recent findings concerning virulence factors of S. mutans and means for prevention of S. mutans- induced dental caries.

Susceptibility of rats, hamsters, and mice to carious infection by S. mutans serotypes c and d was compared. S. mutans serotype c induced a similar level of carious lesions at experimental periods of 68, 82, and 98 d in rats, hamsters, and mice, respectively. On the other hand, S. mutans serotype d developed a high level of caries at those experimental periods in rats and hamsters, whereas in mice it showed weak caries activity.

Teeth from the C57BLl6J-Hyp mouse (Hyp mouse) were examined histologically, radiographically, and crystallographically. Microscopic examinations of the ground sections, the decalcified and H-E-stained sections, and the contact microradiograms of molars and incisors from the Hyp mouse showed several abnormalities-such as large pulp chamber, wide predentin, thin dentin at the pulp floor, and multiple occurrences of interglobular dentin. Powder and microbeam x-ray diffraction analyses showed that the crystallinity of hydroxyapatite in incisor globular dentin from the Hyp mouse was higher than that in incisor dentin from the normal mouse. On the other hand, the findings of transmission electron microscopy demonstrated that the sizes of hydroxyapatite crystals of globular dentin in the Hyp incisor were larger than those in normal incisor dentin. These results demonstrated that the findings in Hyp mouse teeth were in accord with those of human XLH teeth.

Streptococcus mutans strains were surveyed to determine their lysogenicity. Growth inhibition of the indicator strains around the inoculum of stab culture of some S mutans strains was demonstrated. Many gram-positive bacteria, including mycobacteria and streptomycetes, were sensitive to the antibacterial activity.