rnc基因时空性调控gtf和dex影响变异链球菌生物膜致龋性的分子机制研究

Extracellular polysaccharide is important virulence factor of S. mutans for its cariogenicity. Our preliminary study found that in the different growth stages of S. mutans, the rnc gene had significantly different effects on extracellular polysaccharide production. Another study confirmed that: RNase III protein encoded by rnc gene, can regulate the expression level and activity of extracellular proteins in bacteria by a different mechenism (including Sec pathway) at different stages of bacterial growth. The Sec pathway is utilised by S. mutans to secrete virulence factor proteins such as Gtfs and Ftf, and the level of Sec is influenced significantly by biofilm formation. Thereby, the applicant proposes a novel hypothesis that in different growth phases, rnc gene, through different molecular mechanisms, regulates the expression and activity of extracellular polysaccharides' related genes gtfs and dex, and that Sec pathway may be involved in this regulation. In this study, some gene deletion mutant strains and complementary strains will be constructed, combined with related technologies such as reporter gene, Western-blot, protein reorganization and EMSA, to study in the different stages of biofilm formation ①the molecular biological mechanism of rnc gene's spatial and temporal regulation on the expression of gtfs and dex. ②The role of Sec pathway played in such a spatial and temporal regulation mechanism.The results will help us to clarify the molecular biological mechanism of the rnc gene in affecting the production of extracellular polysaccharides in S. mutans, and to find a novel way to undermine or decrease the formation of extracellular polysaccharide to reduce the cariogenic virulence of S. mutans. It will have important theoretical and application value in caries prevention.

胞外多糖是变异链球菌(S. mutans)重要的致龋毒力因子。rnc基因可以差异性调控胞外多糖的形成。在其他细菌中，rnc被证实在不同生长阶段，通过不同通路（包括Sec路径）调控胞外蛋白表达、活性与分泌。Sec路径是变异链球菌毒力因子分泌的重要通路，其表达受生物膜状态调控。申请者由此提出变异链球菌rnc基因通过时序性调控gtfs和dex影响胞外多糖形成、Sec路径可能参与rnc对胞外多糖相关蛋白分泌调控的新思路。课题拟构建多种基因缺陷与互补株，使用报告基因、Western-blot、蛋白重组与EMSA等技术，分析生物膜形成不同阶段①rnc对gtfs、dex时序性调控的机制②探讨Sec路径在rnc时序性调控gtfs和dex中的作用。有助于阐明S. mutans生物膜形成不同阶段胞外多糖形成的分子生物学机制，对寻求干扰胞外多糖，降低S. mutans的致龋毒力的防龋途径有重要理论和应用价值。

背景：变异链球菌是主要的致龋菌、rnc基因可影响变异链球菌致龋性，但作用机制未明。.主要内容：1构建变异链球菌rnc缺失(Smurnc)及过表达突变株(Smurnc+)；2对比野生株与突变株生物膜形成、生物膜活力、ldh活性；3 对比三菌株在不同培养阶段gftB/C/D、dexA及 secA的表达；4蔗糖对三菌株gftB/C/D、dexA与secA表达的影响；5生物膜状态对gftB/C/D、dexA及secA表达的影响；6 secA与gtfB/C/D、dexA的相互关系；7 rnc对gbpB基因表达的影响； 8野生株与突变株凝聚性与疏水性的差异。.重要结果：1成功构建rnc缺失(Smurnc)及过表达突变株(Smurnc+)；2 rnc缺失导致变异链球菌无法离开蔗糖形成生物膜，并显著降低生物膜活性与ldh活性；3不同培养时间，胞外多糖相关基因表达有显著变化，12h时gftB/C/D与dexA表达水平显著上调；4 细菌培养8h，secA基因表达明显高于其他时间；5 添加1%蔗糖，gtfB/D表达下调，gtfC表达上调，dexA上调不明显；6 生物膜状态下gtfC/D、dexA与secA表达上调，gtfB表达明显下调；8h时secA表达与dexA、gtfB/D表达水平一致，12h时dexA、gtfB/D表达显著上调，secA上调幅度不如前者；7无蔗糖悬浮状态，gbpB表达显著下调，添加蔗糖其表达显著上调，尤以生物膜状态中上调最显著；8 rnc缺失突变株凝聚性与疏水性较UA159升高。.关键数据及意义：1 Smurnc致龋性明显降低，有望成为防龋治疗的替代菌株；2胞外多糖相关基因表达具有明显时相性；3 Smurnc培养初期，secA表达显著上调，说明rnc对secA负向调控；4蔗糖可以影响胞外多糖相关基因的表达，对于不同基因影响不同；5生物膜中gtfC/D、dexA、gbpB与secA表达均上调，说明rnc缺失后Smurnc生物膜的形成与这些基因有关；6在不同时期，secA与胞外多糖相关基因的表达呈现不同的比例关系，说明secA对其调控具有时相性；7 rnc缺失后，gbpB表达显著下调，说明rnc正向调控gbpB，可能是Smurnc无法在不含蔗糖时形成生物膜的原因；8 rnc缺失突变株凝聚性与疏水性较UA159升高，生长过程中为凝聚状生长，减少界面触点，影响细菌粘附。