变形链球菌lmrB外排子对感受态形成的调控作用及机制研究

Streptococcus mutans is one of the principal cariogenic dental biofilm inhabitants. Its establishment and virulence are tightly integrated with the development of genetic competence. Competence affords S. mutans the ability to internalize exogenous DNA, thus contributing to the survival and virulence traits evolution of bacterial populations, such as biofilm formation, antimicrobial tolerance and persistence. Control the pathways regulating competence development may reduce cariogenic ability. However, the key genes involved with competence development of S. mutans UA159 have yet to be characterized. Our previous microarray study showed the high expression level of lmrB during competence development, which encodes putative efflux pump. Further studies revealed that efflux pump inhibitor had a negative effect on competence development. Data presented in our preliminary study demonstrated that lmrB efflux pump played a crucial role in competence development, while the mechanism remained unclear. This project will examine the function of lmrB encoding product and its transcriptional regulation through mutation of the upstream regulatory gene SMU747c. The effect of lmrB efflux pump on S. mutans competence development and virulence will also be examined. The development of competence in S. mutans is initiated by small molecule signal CSP and activated by sigma factor ComX. We will focus on expression level of the promoter of CSP-encoding gene（comC）and the transporter related gene（comAB）after lmrB mutation, as well as the expression of downstream critical proteins( ComX and ComYA) of competence development pathways. This study will provide evidence for establishing lmrB efflux pump as useful targets for therapeutics to diminish the virulence of S. mutans

变形链球菌是龋病的主要病原菌，它在生长过程中可形成感受态进而摄取并整合外源 DNA，促进毒力因子进化，有利于致龋和生存能力的提高。 抑制调控感受态形成信号通路的某一环节可降低其致龋能力，但目前对参与调控感受态形成基因的认识仍不全面。本课题组研究发现lmrB（SMU.745）在感受态形成过程中高表达，生物信息学分析提示lmrB编码产物为可疑外排子；进一步研究显示外排子抑制剂可阻碍感受态形成。研究结果提示lmrB外排子可能参与调控感受态形成，但具体机制尚不清楚。本项目拟通过构建其上游调控基因（SMU747c）突变株，明确lmrB编码产物的外排功能和表达调控；检测lmrB外排子对感受态形成和毒力因子表达的影响；探讨lmrB外排子对感受态形成信号肽合成及转运相关基因启动子表达的影响，和感受态形成调控通路关键蛋白ComX及ComYA表达的影响，为确立lmrB外排子为龋病防治的新靶点提供科学依据。

变形链球菌是龋病的主要病原菌，它在生长过程中可形成感受态进而摄取并整合外源 DNA，促进毒力因子进化，有利于致龋和生存能力的提高。 抑制调控感受态形成信号通路的某一环节可降低其致龋能力，但目前对参与调控感受态形成基因的认识仍不全面。本课题组前期研究发现lmrB（SMU.745）在感受态形成过程中高表达，生物信息学分析提示lmrB编码产物为可疑外排子，研究结果提示lmrB外排子可能参与调控感受态形成，但具体机制尚不清楚。本项目通过构建将lmrB基因克隆至大肠杆菌后研究大肠杆菌的外排功能，发现其外排能力增加，证实了lmrB编码产物的功能。未明确lmrB编码产物在变形链球菌中的作用，项目进一步构建变形链球菌lmrB基因缺失突变株，发现突变株的多项毒力因子表达发生改变，提示lmrB可作为龋病防治的潜在靶点。为明确其临床应用可行性，课题组对常用的四代外排子抑制剂进行筛选，发现利血平可抑制变形链球菌外排功能，调控毒力因子的表达。研究为确立lmrB外排子为龋病防治的新靶点提供了科学依据和理论基础。