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

首先，我们克隆了变形链球菌C型特异性鼠李糖-葡萄糖多糖（RGP）葡萄糖侧链形成的基因。该区域有5个开放阅读框，其中2个新基因（rgpH和rgp）是葡萄糖侧链形成所必需的。在大肠杆菌中产生的RGP含有鼠李糖合成和rgpH所需的基因，与血清型c特异性兔抗血清反应，而rgpl不产生葡萄糖侧链。rgpl的加入大大降低了RGP与鼠李糖特异性抗血清的反应性。这些结果表明，rgpH可能是RGP葡萄糖侧链形成的一个独特的葡萄糖基转移酶，而rgpl可能是一个参与葡萄糖基化上调的调控基因。接下来，从血清型e和f菌株中分离相应的染色体区域并进行表征。rgpA-F同源物在三种血清型之间具有很好的保守性。相比之下，从rgpF同源物的下游区域在三种血清型之间差异很大。在不同血清型菌株中替换这些区域可转化其血清型表型，表明这些区域参与了每种血清型菌株中的特定型葡萄糖侧链形成。根据这些区域DNA序列的差异，建立了一种PCR方法来确定血清型，从432名学龄前儿童（3 ~ 4岁）中分离出198株变形链球菌。除1株外，其余菌株均用血清分型PCR进行了血清型鉴定。在日本学龄前儿童中，C型占优势（84.8%），其次是E型（13.3%），F型很少发生（1.9%）。多种血清型混合感染组患龋率明显高于单一血清型感染组。

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.