无乳链球菌VicRK双组分信号转导系统的功能分析

Both the nature of the regulon and the physiological role of the two component signal transdution system VicRK have remained uncharacterized in Streptococcus agalatiae. VicRK is highly conserved in and specific to the low-G+C content gram-positive bacteria.Because it is essential for viability in most of these bacteria by controlling cell wall metabolism and virulence factor expression, VicRK has been suggested as a prime antimicrobial target. In recent years, Streptococcus agalatiae has been recognized as one of the most serious bacterial diseases in tilapia culture and usually causes high mortality and lasts a long period of time.Hainan, as the main area of tilapia culture in China, has caused heavy losses. So, the VicRK is a new hope for the control of this dangerous disease. In this study, Bioinformatic assay and chromatin immunoprecipitation-sequencing (ChIP-Seq) assay will be performed to characterize the recognizing binding sequences and binding sites of VicR in the whole genome, and then the candidate genes which are directly regulated by VicR near binding regions will be predicted by detailed DNA motif analysis and confirmed by Gel mobility shift (GMSA）. Digital gene expression profiling RNA-Seq(quantification) [DGE RNA-Seq(quantification) ] assay will be applied to survey and figure out the expression direction of above modulated genes by VicR, and proved by RT-qPCR. Then, the VicRK regulation mechanism of murein biosynthesis and virulence factor expression will be predicted by combining all the above data and gene Go analysis. Last, the considerable potential as new target for antibiotic and vaccine development will be evaluated and uncovered in Streptococcus agalatiae by phenotype analysis of vicRK mutants.This study will enhance our understanding of the VicRK funciton in Streptococcus agalatiae and help us to develop an effective, novel strategy to eliminate this important potential bacterial agent of tilapias.

VicRK是少数低G+C值革兰氏阳性菌中高度保守的关键性双组分信号转导系统。该系统因具有调控细胞壁代谢和毒力等重要功能而成为现代病原菌防治中的有效靶标。无乳链球菌是近些年来引起我国南方罗非鱼大量死亡的主要病原，海南作为罗非鱼的主产区而损失严重。因此，在当前该菌致病机理不明，存在强耐药性问题和缺乏有效疫苗的情况下,利用VicRK可为该病原防治带来新的希望。目前,尚未发现有关无乳链球菌VicRK的功能研究。本课题拟采用生物信息学、蛋白质－DNA互作、数字基因表达谱分析等现代研究方法揭示VicRK系统在无乳链球菌基因组上的结合位点及其对下游调控基因的表达调节，并通过对这些调控基因的功能诠释，以及突变菌株的表型分析，阐述VicRK在无乳链球菌细胞壁代谢和在致病过程中的双重调控功能，评价其在无乳链球菌弱毒疫苗株开发和新型抗生素筛选中的可利用价值,为高危难防的鱼类无乳链球菌病害防治探索新的方向。

无乳链球菌作为近年来罗非鱼感染死亡的主要病因，对海南水产养殖业影响严重。在防治方面，抗生素使用所引起的该菌耐药性正呈逐年上升，药物残留所产生的环境与健康问题也日益加重，因此，极需建立更有效的免疫手段以实现对该菌的安全高效防治。研究发现，细菌作为单细胞生物，其所具有的双组分系统是应对复杂多变外界环境的重要感应应答系统，其中VicRK双组分系统因在调控致病菌细胞壁合成与毒力基因表达等关键功能中发挥重要作用，在阐述细菌致病机制及实现免疫防治等具有研究价值。.本课题从无乳链球菌菌株分离收集、基因组测序、现场快速检测方法建立、构建快速基因编辑体系和突变体部分表型分析等进行了探究，研究成果如下：共分离4株人源、0株牛源和14株罗非鱼源无乳链球菌。其中人源无乳链球菌血清型和MLST血清型相互差异明显，而鱼源菌株全部为Ia血清型和ST7基因型。在耐药性方面，鱼源菌株皆具有多重耐药性，但对依诺沙星、四环素和复方新诺明不具抗性；通过采用重测序技术，发现E0菌株的全基因组大小为 2.058913Mb，与参考基因组GD201008-001相差4,200bp，缺失基因为细胞分裂相关基因ftsK和三个未知功能的蛋白基因。通过以vicRK和pcsB为靶基因进行LAMP技术设计，研究结果显示，以pcsB为靶基因所设计的LAMP方法最为忠实，具有较好的灵敏性、准确性和稳定性。比传统PCR和实时定量PCR方法相比，采用浊度仪灵敏度可提高100倍，采用SYBR或HNB可视化检测可提高10倍；通过采用Lambda red和CRIPSR构建双质粒基因编辑系统，该系统的特点可以同时对两靶点进行基因编辑，实现点突变、基因插入和基因替换等，对于单链DNA的小片段重组，其效率在90%以上，对于双链DNA的大片段重组，其效率也在60%以上；结合CRISPR和膜泡的方法可以在自然转化的条件下对无乳链球菌进行清除；采用sacB基因的反向筛选，构建vicR的缺失突变体，研究发现，胞内VicR含量下降可造成细胞生活力严重受损；采用VicR与6His标签的融合表达，不影响该蛋白与调节基因pcsB启动子的体外结合，为体内的ChIP分析提供研究基础。发表论文9篇（2篇SCI）；申请专利2项；培养研究生5名。