SigD因子调控fliC表达在艰难梭菌鞭毛形成中的作用及分子机制研究

Clostridium difficile is one of the most prevalent pathogens associated with antibiotic-related diarrhea. C.difficile flagella have been implicated in contributing to its pathogenesis by promoting biofilm formation and adherence to host cells as well as intestinal colonization. FliC is a major structure component of flagellar filament. However, the mechanism of its regulation is still unclear. In our preliminary experiment, fliC was significantly up-regulated in pathogenic isolates, indicating that fliC could be closely related to clinical pathogenesis of C. difficile. A specific sequence possibly identified by sigD was found located in the upstream of fliC gene. Besides, the expression of fliC was detected to be consistent with sigD, and dramatically decreased in sigD knockout strains. Besides, indicating that fliC might be regulated by sigD through directly binding to the promoter region. In the present study, we will construct fliC and sigD knock-out, reversion and overexpression strains to confirm the relationship between the expression of fliC/sigD and the phenotype associated with flaggellar, including motility, biofilm formation, colonization and adherence ability in vitro and vivo. Then, it needs to be verified that sigD could bind to the promoter region of fliC and active its expression using electrophoretic mobility shift assay, chromatin immunoprecipitation assay and luciferase report assay respectively. Finally, it will be clarified that the formation of flagellar is dependent on the transcriptional regulation of fliC by sigD. The goal of this project is to provide a strong basis for the new solutions for effective prevention and treatment of CDI, and also for the development of vaccines against important target proteins in flagellar formation.

艰难梭菌作为抗生素相关性腹泻的重要病原体，其鞭毛在促生物膜形成与定植粘附等致病过程中起重要作用。FliC是鞭毛形成的关键蛋白，但其表达调控的机制尚不明了。我们在前期实验中发现，艰难梭菌致病性与fliC的高表达密切相关。fliC上游存在可能被调节子sigD识别的序列。此外sigD的表达与fliC一致，sigD敲除菌株fliC表达明显下降，提示sigD有可能通过直接与启动子结合调控fliC的表达。本项目拟首先构建fliC和sigD敲除、回复和过表达菌株，明确sigD分别在鞭毛形成、生物膜形成和体内外定植粘附中的作用；其次通过凝胶阻滞试验、染色质免疫共沉淀和荧光素酶报告系统观察sigD与fliC启动子的结合及对fliC的激活；最后证明艰难梭菌鞭毛的形成依赖于sigD对fliC的转录调控。本项目将为预防和治疗艰难梭菌感染提供新的解决方案，并将为日后针对鞭毛形成的疫苗开发提供有力的实验和理论依据。

艰难梭菌作为抗生素相关性腹泻的重要病原体，其鞭毛在促生物膜形成和致病过程中起着重要的作用。通过体外表达鞭毛蛋白，我们明确了其在促进炎性因子分泌中的作用。在前期研究中，我们推测鞭毛蛋白关键基因fliC的表达可能受上游调节子sigD的表达调控。通过构建sigD基因敲除株，我们发现sigD敲除株fliC表达显著下降，鞭毛运动能力，生物膜形成能力以及菌株在小鼠体内定植能力均明显下降，证实了调节子sigD在调节fliC表达影响鞭毛形成、生物膜形成和体内定植中的作用。同时，我们收集了相同毒素型不同基因型（ST81和ST37）的菌株，比较其生物学表型，发现流行克隆ST81菌株的鞭毛运动能力更强，同时细胞黏附力、抗巨噬细胞杀伤力以及体内定植的竞争力也更强，提示鞭毛的形成及其相关生物表型与菌株克隆的流行也存在密切关系。进一步通过全基因组测序发现，与ST37相比，ST81型菌株虽然存在多种功能基因多态性位点，但鞭毛形成相关基因并未发生明显变化，表明ST81菌株强鞭毛形成能力与基因多态性无明显关系，可能是与上游基因的表达调控密切相关。此外，为了加深对艰难梭菌感染肠道微环境的认知，我们还探究了肠炎合并艰难梭菌感染小鼠的肠道菌群和免疫微环境特征。通过构建小鼠模型，采用16s rRNA肠道菌群多样性测序、流式细胞术和肠组织转录组分析技术，我们发现在肠炎中，艰难梭菌介导的肠道微环境紊乱表现为小鼠狄氏副拟杆菌丰度显著下降和中性粒细胞的浸润，伴随着细胞因子-细胞因子受体作用通路和趋化因子信号通路的显著减弱。综上所述，本项目为针对鞭毛形成的疫苗开发提供了有力的理论依据，也为深入了解艰难梭菌在肠炎中的致病机制打下了实验基础，进而为艰难梭菌感染的防治提供新的思路和解决方案。