变异链球菌srtA基因多态性与S-ECC易感性关系的分子流行病学研究

The cell surface protein antigen c (Pac) of streptococcus mutans (S. mutans) plays a vital role in bacterial initial adherence. It was reported that the polymorphisms of srtA gene of standard strain of S. mutans could influnce the adhension ability via Pac. In our recent study with a small sample, we found that srtA genes of S. mutans from the dental plaque of children showed a number of mutation sites. However, the distribution of the mutation in children with different caries susceptibility is still unknown. Therefore, we plan to conduct a large sample molecular epidemiology study to reveal the relationship betweeen the polymorphisms of srtA gene and caries susceptibility, and explore the inflence of mutation of srtA gene on the cariogenicity of S. mutans. We will detect the srtA gene of S. mutans isolates from severe early childhood caries (S-ECC) and caries-free children by DNA sequencing at both directions and compare the distribution of different mutation types in the two groups. Furthermore, Pac of S. mutans in different types of mutation will be detected by western blotting method. Adhension to saliva-coated hydroxylapatite will be evaluated by 3H-TDR radioiodination, and the charateristic of biofilm be observed by confocal laser scanning microscope. In sum, mutation types of srtA gene possibly related to S-ECC susceptibility will be screened, and they could become future targets for the prediction and prevention of dental caries.

变异链球菌（变链菌）表面蛋白Pac对其与牙面的初始粘附起关键作用。以往对变链菌标准株研究提示srtA 基因存在多态性并可影响表面蛋白Pac介导的粘附力。我们前期小样本研究发现儿童牙菌斑变链菌srtA基因存在多个突变位点，但其在不同龋易感人群的分布尚不明了。为进一步探讨变链菌srtA基因多态性与龋易感性的关系，并揭示其对变链菌致龋性的影响，本课题拟进行大样本分子流行病学研究，对重症婴幼儿龋(S-ECC)和无龋儿童牙菌斑变链菌进行srtA基因全长双向测序，分析各突变类型在两组儿童的分布差异，并采用western blotting、放射性同位素标记及激光共聚焦技术分别检测变链菌表面蛋白Pac、对唾液包被羟基磷灰石颗粒的粘附力及所形成生物膜的特点，比较不同突变类型的srtA基因对这些指标的影响。筛选出与S-ECC易感相关的srtA基因突变类型，为寻找龋病预测及预防新靶标提供实验依据。

本课题首先进行了大样本分子流行病学研究，对121株来自重症婴幼儿龋(S-ECC)和121株来自无龋儿童牙菌斑的变链菌进行了srtA基因测序，分析各突变类型在两组间的分布差异。单因素结果显示，T168G和G470A突变在两组间频率分布有统计学差异。采用logistic回归控制乳牙龋相关的危险因素，结果显示，除了社会、行为学因素，srtA突变位点中只有168位点与乳牙高龋相关。发生该突变菌株多见于无龋组。. 接着，我们评估了srtA基因T168G和G470A突变对变链菌转肽酶活性的影响。采用pET32a(+)表达质粒，构建srtA基因重组表达载体，获得以上两种突变类型菌株和变链菌UA159的SrtA酶。通过酶促反应动力学实验测定酶促反应常数。结果显示，与UA159相比，T168G 突变可显著降低 SrtA 酶的活性。. 除了单个碱基突变，我们也从基因型角度分析了srtA的突变情况。单因素结果显示，3A基因型（176、470、671碱基同时突变）和3E基因型（168、176、671碱基同时突变）的频率分布在两组间有统计学差异。多因素结果显示，除了社会、行为学因素，3A基因型与乳牙高龋相关。3A基因型的菌株多见于无龋组。.我们接着进一步研究了变链菌 srtA 基因多态性对其生物学表型的影响。以pFW5 自杀质粒为载体构建重组质粒，通过同源重组技术构建变链菌srtA 基因T168G点突变和3A基因型突变株。比较UA159及其 srtA 基因突变株的生长曲线、表面蛋白 Pac 的胞壁锚定、粘附和生物膜形成能力。结果显示，变链菌T168G点突变和3A基因型不影响其生长周期，但可影响 SrtA 酶对表面蛋白 Pac 的胞壁锚定作用，降低变链菌的粘附能力和生物膜形成能力。 .综上，本研究筛选出了与S-ECC易感相关的srtA基因突变类型，并为寻找龋病预测及预防新靶标提供了实验依据。