Development of simple and high sensitive method for caries risk diagnosis by molecular genetics

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First, we cloned the genes responsible for the serotype c-specific glucose side-chain formation of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. There were five ORFs in this region, and two novel genes (rgpH and rgp) were required for glucose side-chain formation. RGP produced in E.coli containing genes required for rhamnan synthesis and rgpH reacted with serotype c-specific rabbit antiserum, whereas rgpl did not produce glucose side chains. The addition of rgpl was drastically decreased the reactivity of RGP with rhamnan-specific antiserum. These results suggest that rgpH may be a unique glucosyltransferase for glucose side chain formation of RGP, whereas rgpl may be a regulatory gene which involve in the up-regulation of glucosylation. Next, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The rgpA-F homologs were well conserved among the three serotypes. By contrast, the regions downstream from the rgpF homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotype-specific glucose side-chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes.S.mutans was isolated from 198 of 432 preschool children (3〜4 years old). The serotypes of S.mutans in all but one were identified with serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occurred rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection of multiple serotypes of S.mutans was significantly higher than that in the group with a mono infection of a single serotype.