致病区tcdC基因在艰难梭菌致病过程中的作用及机制研究

TcdA and B toxins in Clostridium difficile are two important factors leading to diseases in organism. The expression of tcdA and B genes is regulated by many factors located in the pathogenicity locus (PaLoc). So far, it has been still debatable whether tcdC gene is regarded as a negative regulator, and the mechanism of tcdC gene is still unclear. We hypothesize that the expression of tcdA and B toxin genes could be regulated by tcdC gene under the assistance of co-factors. In this proposal, the C. difficile ZJCDC1 isolate carrying a new tcdC mutant gene and expressing toxins in a low level, the hypervirulent strain (NAP1), and the standard strain (VPI10463) are chosen as the targets. Four recombinant strains will be prepared through the Tn 916 clostridial transferable shuttle vector. The concentration of TcdA and B toxin and PaLoc gene transcription level in the recombinant strains will be quantitatively measured by using the real-time cell analysis system, the real-time PCR assay, respectively. Thus, the role of tcdC gene for regulating tcdA and B gene will be illustrated through analysis of data. Meanwhile, three tcdC genes with different gene sequences will be expressed, and the corresponding antibodies will be prepared respectively. The factors cooperated with tcdC protein in the hypervirulent strain will be sought by using co-immunoprecipitation in order to further analyze the biological mechanism of tcdC gene. Through implementing this project, the function of tcdC gene will be clarified in the regulation of PaLoc gene. Furthermore, the pathogenic mechanism of tcdC gene will be further disclosed in the course of pathogenesis of C. difficile.

艰难梭菌tcdA和B毒素是机体致病的重要因子, 其基因表达在致病区（PaLoc）中受多个因子调控。目前tcdC基因是否具有负调节功能尚存争论、在致病过程中的作用机制仍不明确。课题组推测tcdC基因能在协同因子的辅助下调控毒素基因表达。本课题将前期获得的携带新型tcdC基因但毒素低表达的分离株（ZJCDC1）、强产毒株（NAP1）和标准菌株（VPI10463）作为研究对象，以Tn916梭菌穿梭质粒为载体制备四种tcdC基因重组菌。采用实时细胞分析和荧光PCR技术分别测定重组菌的毒素浓度和PaLoc基因转录水平，通过数据分析阐明tcdC基因在tcdA和B基因表达中的作用。同时表达三种tcdC基因并制备相应抗体，采用免疫共沉淀技术寻找毒素高表达株tcdC蛋白的协同因子，阐明其作用机制。通过本课题的实施，将明确tcdC基因在PaLoc基因调控中的作用，并进一步揭示其在艰难梭菌致病过程中的作用机制。

艰难梭菌tcdA和B毒素是机体致病的重要因子,其基因表达在致病区（PaLoc）中受多个因子调控。目前tcdC基因是否具有负调节功能尚存争论、在致病过程中的作用机制仍不明确。采用RPF144梭菌穿梭质粒为载体，构建ZJCDC1、NAP1和VPI10463三种tcdC重组质粒，分别导入VPI10463中制备三种tcdC 基因重组菌。通过实时细胞分析和荧光PCR分别测定重组菌的毒素浓度和PaLoc 基因转录水平。同时表达三种tcdC 基因并制备相应抗体，采用免疫共沉淀寻找产毒株tcdC 蛋白的协同因子。将ZJCDC1传代培养，毒力测定、mRNA检测、全基因组测序及动物实验。. 成功制备了特异性识别艰难梭菌TcdC蛋白的多克隆抗体，为后续分析tcdC蛋白的表达特性、研究tcdC基因功能与作用机制奠定基础。ZJCDC1毒素检测阴性，传代后毒素检测阳性。将ZJCDC1进行全基因测序，发现mmgC基因序列第197位点由G突变为A。ZJCDC1未表达tcdA与tcdB mRNA，mmgC基因突变株表达tcdA与tcdB mRNA，表明mmgC基因能够调控tcdA与tcdB mRNA的转录及表达。小鼠实验证明mmgC基因突变株能引起艰难梭菌感染。通过对三种tcdC基因重组菌功能研究发现tcdC基因的突变未造成艰难梭菌毒素表达的显著性差异（P > 0.05）.经设计重组接合子验证，转入tcdC基因重组菌的tcdC蛋白表达明显升高（P < 0.01），但从mRNA转录水平、毒素蛋白表达水平上并未有显著性差异（P > 0.05）。. 通过本项目的研究，首次证明了tcdC 基因在艰难梭菌感染致病过程中不是艰难梭菌毒素表达的负调控因子，不能调控菌体毒素基因的表达，同时并无协同作用因子参与tcdC 基因的调控。mmgC基因可能对艰难梭菌毒力基因的转录与表达具有调控作用，但调节机制不明确，需要进一步的实验验证。