双组分系统TCS11在肺炎链球菌万古霉素耐受中的作用机制研究

Streptococcus pneumoniae is one of the main causes for community-acquired pneumonia and a main reason for the death of the children and the elderly. Emergence of vancomycin tolerance strains seriously affects the treatment of related infectious diseases. However, the molecular mechanism of antibiotic tolerance remains to be elucidated. The two-component system TCS11 was related with pneumococcal vancomycin tolerance, and we found that TCS11 gene cluster was ubiquitous in different serotype strains. The genes of TCS11 cluster were co-transcription in the same operon. To elucidate the mechanism of TCS11 regulation, we will construct gene knockout and overexpression strains of pneumococcal two-component system genes, and explore the specific genes regulated by TCS11 through analyzing the growth characteristics and transcriptome sequencing of the strains. To determine its key binding elements, EMSA will be used to analyze the interaction of TCS11 RR with the promoters of its target genes, 5'RACE and the promoter activity analysis will be utilized. Mouse model of pneumococcal pneumoniae will be constructed to verify the role of TCS11 in vancomycin tolerance in vivo. This study would clarify the function and transcriptional regulation mechanism of pneumococcal TCS11 in its vancomycin tolerance, and could be the theoretical basis for the treatment of pneumococcal related diseases.

肺炎链球菌是社区获得性肺炎的主要病原菌，也是导致儿童和老年人死亡的主要诱因。万古霉素耐受菌株的出现，严重影响相关感染性疾病的治疗，但其抗生素耐受的分子机制尚不明确。肺炎链球菌双组分系统TCS11与其万古霉素耐受相关，通过生物信息学分析发现TCS11基因簇在各种血清型菌株中普遍存在，而反转录PCR提示该基因簇相邻基因共同转录形成一个操纵子单元。为深入解析TCS11具体作用机制，本项目拟对肺炎链球菌TIGR4中TCS11各基因进行敲除及过表达，通过分析野生型、敲除和过表达菌株生长特性及其转录组的改变，发掘下游靶基因；采用EMSA实验分析RR蛋白与其靶基因的相互作用，结合5'RACE和启动子活性分析，确定其关键结合元件；构建肺炎链球菌肺炎小鼠模型，体内验证其在万古霉素耐受中的作用。本项目的实施将明确肺炎链球菌TCS11在万古霉素耐受中的作用和调控机制，为肺炎链球菌相关疾病的治疗提供理论基础。

肺炎链球菌可以引起多种感染性疾病，包括非侵袭性的中耳炎和鼻窦炎以及侵袭性的肺炎、菌血症和脑膜炎，是社区获得性肺炎的主要病原，也是导致儿童和老年人死亡的主要诱因。多重耐药菌及抗生素耐受菌株的出现，严重影响相关感染性疾病的治疗，但是其分子机制尚不明确。双组分系统TCS是细菌中广泛存在的调控系统，通常由组氨酸激酶 HK 和调控蛋白 RR 组成，参与细菌耐药、毒力等多种关键途径基因的调控。本项目对肺炎链球菌双组分系统TCS11组成、功能及其转录调控机制进行研究：通过TCS11基因簇同源比对、共转录分析，初步确定其操纵子的组成；通过Δhk11和Δtcs11基因敲除菌株的构建及其生长、抗生素耐受等表型分析，研究TCS11功能；对基因缺失前后菌株，进行转录组测序及荧光定量PCR，筛选差异基因及调控通路，初步解析其调控的下游基因；通过KEGG富集差异基因参与的代谢通路，发现差异基因富集在次级代谢产物合成、氨基酸合成、群体感应系统、双组分系统等通路；在大肠杆菌中表达纯化了RR11蛋白，通过DAP-seq实验，进行RR11蛋白与DNA结合分析，解析了RR11结合序列及其关键基序。项目实施期间，发表SCI论文9篇、英文论著1篇，共计10篇，培养研究生1名。