2-AI/T对变形链球菌生物膜的作用及其机制初探

Streptococcus mutans is a major cariogenic bacterium. It has adapted to the biofilm lifestyle, which is essential for pathogenesis of dental caries. In our previous study, with a series of small molecules library from marine natural product, we identified a derivate 2-aminoimidazole/ tridazole(2-AI/T) which has the most activity to selectively inhibit cariogenic S. mutans biofilm formation. Therefore, in this study, we focus on this compound 2-AI/T for further effect and mechanism. In vitro experiment, the formed biofilm of S.mutans is treated by 2-AI/T to observe whether it can disperse biofilm. S.mutans is cocultured with 2-AI/T at the 50% inhibitory concentration(MIC50) and then inoculated onto plate to investigate its effects on the viability of cells. In its safety assay, mucosal epithelial cell and gingival fibroblast are treated with 2-AI/T and its cytotoxicity are detected by MTT assay. In vivo experiment, BALB/c mice are orally infected with S.mutans, and then treated with 2-AI/T. At 1day,3day,7day and 14day,these mice are sacrificed and the biofilms on the tooth surface are observed by scanning electron microscope. The caries level is determinated by microCT. According to our previous results of microarray, from these detected genes, we selected genes which have down-regulated significantly, they are gbpA, gtfB, gtfC and ptsG. To confirm these results, real-time PCR and northern blot are used to detect these genes' expression in mRNA level. To determine the level of these selected protein after treated with 2-AI/T, the whole protein and cell wall protein are extracted and detected with specific antibody by western blot respectively.The GTF activity and glucose uptake are detected by radioactivity assay. The compound 2-AI/T we identified may function as a decoy signaling molecule to disrupt normal expression of biofilm-associated genes and proteins in S.mutans so it can disturb cariogenic biofilm. Although the exact mechanism should be further investigated,this study will provide a novel and effective method to prevent and treat dental plaque.

在项目组前期研究中, 以海洋天然产物的衍生物氨基唑类化合物为研究对象，从这些化合物的小分子库中筛选出能选择性抑制变形链球菌致龋生物膜形成的小分子2-氨基咪唑/三唑2-AI/T。 本项目就是在此基础上,进一步研究其对已形成的变形链球菌生物膜的作用；通过监测在含有2-AI/T的培养基中，变形链球菌的生长情况，来确定该分子对细菌活力的影响；通过激光共聚焦显微镜对比观察经2-AI/T处理后生物膜的形态结构变化；分别对口腔粘膜上皮细胞和牙龈成纤维细胞进行MTT实验以评价2-AI/T是否有细胞毒性；建立小鼠变形链球菌致龋模型，将2-AI/T应用到小鼠牙面上以观察其对牙菌斑生物膜的影响；最后,根据前期实验中基因芯片表达结果，围绕有显著性变化的基因对其抗生物膜的机制作进一步探讨。该研究将海洋生物活性物质2-AI/T应用到对口腔变形链球菌致龋生物膜的预防和治疗上，从而为龋病的预防提供新的理论资料和实践参考。

变形链球菌（streptococcus mutans，s mutans）是现今公认的致龋菌，对其生物膜抑制一直是预防和治疗龋病的研究热点。在项目组前期研究中, 以海洋天然产物的衍生物氨基唑类化合物为研究对象，从这些化合物的小分子库中筛选出能选择性抑制变形链球菌致龋生物膜形成的小分子2-氨基咪唑/三唑2-AI/T。 本项目就是在此基础上,进一步通过监测在含有2-AI/T的培养基中，变形链球菌的生长情况，来确定该分子对浮游细菌和生物膜细菌活力的影响；通过激光共聚焦显微镜对比观察经2-AI/T处理后生物膜的形态结构变化；对牙龈成纤维细胞进行MTT实验和对小鼠进行急性毒性实验以评价2-AI/T是否有毒性；建立致龋模型，将2-AI/T应用到动物牙面上以观察其对牙菌斑生物膜及龋病的影响；最后,根据蛋白组分析差异蛋白的表达，并对有显著性变化的蛋白进行mRNA水平的验证。结果体外实验证实其能有效抑制变形链球菌生物膜细胞活力。细胞毒性实验显示该小分子没有细胞毒性，在小鼠体内没有发生急性毒性反应。另外，动物体内实验表明小分子能够有效抑制变形链球菌定植从而减少龋病的发生。本实验采用新型的蛋白质组学技术对其机制研究发现其对变形链球菌核糖体代谢通路与组氨酸代谢通路有显著的影响。该研究将海洋生物活性物质2-AI/T应用到对口腔变形链球菌致龋生物膜的预防和治疗上，从而为龋病的预防提供新的理论资料和实践参考。