变异链球菌致龋毒力关键转录调节因子的鉴定及作用机制研究

Streptococcus mutans is generally recognized as one of the most important causative factors of dental caries. It has been well demonstrated that S. mutans possesses classic cariogenic virulence factors, including potent acidogenicity and aciduricity, and is able to produce extracellular polysaccharides which mediate cellular adherence and facilitate multi-species biofilm formation. However, the molecular mechanism of the virulence regulation of S. mutans is not clear. Although certain global regulators with potential association with the cariogenicity of S. mutans have been identified, the arbitrary hypothesis-driven monogene mutation approach failed to locate the key regulators associated with its virulent phenotypes, and the network through which these regulators orchestrate to modulate the cariogenic virulence of this bacterium is not well established. Therefore, in order to elucidate the sophisticated molecular network by which the cariogenic virulence of S. mutans is regulated, we propose a systemic approach by constructing a mutants library of S. mutans with each single transcriptional regulator deletion. The mutant strains will then be screened using both in vitro and in vivo animal models, and the key transcriptional factors that are closely associated with the cariogenicity of S. mutans will be identified. Further molecular level studies, including ChIP-seq and transcriptome microarray will be performed on those identified mutants in order to anchor target sequences and downstream genes that involved with the virulence regulation of S. mutans. An intricate network of virulence modulation with cross-talks between several classic pathways is expected, and those important nodes at the intersections of pathways will be exploited as potential targets for virulence inhibition. We believe findings from this proposal will not only further our knowledge about the intricate regulation of S. mutans cariogenicity at transcriptional level, but may also provide potential target(s) for novel approach to dental caries management.

变异链球菌是人类龋病最密切的相关菌，其产酸、耐酸能力，粘附能力，合成细胞内外多糖和形成生物膜的能力是经典的毒力表型。目前，变异链球菌致龋毒力表达相关调控因子及作用机制尚不清楚，现有的针对变异链球菌个别宏观转录调控因子的单基因突变研究，难以深入认识其致龋毒力调控模式和关键位点。针对这一科学问题，本课题拟通过系统突变手段，构建变异链球菌转录调节因子单基因突变体库，通过体外模型和龋病动物模型筛选鉴定调控变异链球菌致龋毒力的关键转录调节因子，进一步通过生物信息学和高通量测序技术，确定鉴定因子的结合序列位点及其调控下游靶基因的分子机制，最终建立变异链球菌致龋毒力相关调控网络。研究结果将有助于我们从分子水平深入认识变异链球菌致龋毒力的调控模式及其致龋机制，为龋病防治新技术的研发提供候选靶标，具有重大的理论意义和应用价值。

变异链球菌与人类龋病的关系最为密切，而基因表达调控在其致龋毒力调控机制中发挥着非常重要的作用，但毒力表达相关调控因子的调控模式与调控网络尚不清楚。为解决这一科学问题，本课题围绕变异链球菌毒力表达调控的的关键基因，首先对变异链球菌标准株的代谢调控展开研究。通过系统突变手段，构建了变异链球菌转录调节因子单基因突变体库，并通过体外模型及龋病动物模型依据基因功能分组筛选出调控变异链球菌致龋毒力关键因子。在此基础上，发现编码c-di-AMP合成酶的cdaA，编码细胞分裂蛋白FtsZ抑制剂的ezrA和GntR家族转录调节因子ltsR三个关键基因。分别对各关键基因展开深入研究发现，cdaA参与调控变异链球菌细菌细胞壁稳定性及氧应激反应，影响细菌生物膜中EPS合成及致龋毒力；ezrA对变异链球菌生长分裂、细胞形态及生物膜形成调控具有重要作用；ltsR通过下调半乳糖代谢和转运相关基因调节变异链球菌生物膜胞外多糖的代谢。其次，以变异链球菌耐氟菌株为对象，研究变异链球菌氟耐受的调控模式。运用全基因鸟枪测序法发现了耐氟菌株中的基因变异，发现操纵子mut基因启动子上的单个SNP(-44A →C)就足以导致下游氟转运基因表达量的上调和变异链球菌耐氟能力的增强，氟转运体的调控可能是变异链球菌获得耐氟性的一种有效方式。另外，基于变异链球菌致龋毒力调控关键因子的发现及现有防龋技术存在的问题，对于变异链球菌重要的毒力因子Gtfs，运用计算机虚拟筛选技术筛选出一种对Gtf催化活性有抑制作用的小分子喹喔啉亚胺类化合物，并在体外和动物模型中均验证了其显著效果。本课题发现了一系列有望作为龋病抗毒力治疗的潜在分子靶点，发现细菌对抗龋药物耐受机制，并突破性开发了靶向药物，为龋病生态防治提供新的模式。.