艰难梭菌细胞粘附因子参与毒素基因表达调控研究

The intestinal pathogen Clostridium difficile is a leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. In recent years, due to the emergence of hypervirulent epidemic strains, such as ribosome 027, the morbidity and mortality of C.difficile associated disease have increased. C.difficile produces three major virulence factors, toxin A, toxin B and binary toxin (CDT). Our previous study found that C.difficile adhesion to intestinal epithelial cells can up-regulate the expression of three toxin genes. Owing to the cell adhesion process is mediated by a variety of adhesion factors. We hypothesize that the adhesion factors are involved in the regulation of C.difficile toxin genes expression. However, the mechanism of how C.difficile adherent factors regulate the expression of toxin genes is not well defined. In order to explore the question, this study first constructs adhesion mutants of C.difficile by rational gene-targeting ClosTron technology. Then, use unrational gene-targeting transposon mutagenesis to construct a random mutations library of C.difficile and use flow cytometry for rapid screening of novel adhesion defective mutants. After that, foreign gene expression system of C.difficile will be applied to construct complementary strains of corresponding adhesion defective mutants. On this basis, the toxins expression abundance, at mRNA/protein and cell toxin levels, of different adhesion mutants and complementary strains will be studied to clarify the contributions of different adhesion factors to regulation of toxin genes expression. Finally, elucidate the mechanisms of how adhesion factors regulate the expression of C.difficile toxin genes.

艰难梭菌是导致抗生素相关性腹泻、伪膜性结肠炎的主要病因。近年来，随着强毒株的出现（如核糖体027型），其流行性与致死率逐年上升。艰难梭菌主要产生三种毒素，分别为毒素A，毒素B和二元毒素（CDT）。我们前期研究发现，艰难梭菌粘附于肠上皮细胞时，三种毒素基因的表达上调。又由于该细胞粘附过程由多种粘附因子介导，因此我们推测粘附因子参与了艰难梭菌毒素基因的表达调控，但其机制尚不明确。为了探索该问题，本研究首先使用靶向ClosTron失活已知粘附因子基因，构建细胞粘附突变株（理性方法）；然后，利用转座子随机突变技术，构建艰难梭菌随机突变体库（非理性方法），并通过流式细胞仪筛选未知粘附因子基因；接下来，借助已建立的外源基因表达系统构建回补对照菌株。在此基础上，研究不同突变株的毒素基因在表达、蛋白和毒力水平的变化，分析不同粘附因子对毒素基因表达调控的贡献，最终阐明粘附因子调控艰难梭菌毒素基因表达的机制。

艰难梭菌是导致抗生素相关性腹泻、伪膜性结肠炎的主要病因。艰难梭菌黏附于肠壁细胞是其致病过程中的起始和关键步骤，前期研究发现艰难梭菌黏附于细胞表面可以上调毒素基因的表达量。为了研究其潜在机制，我们建立了基于CRISPR-Cpf1的精准、高效和易用的先进基因编辑系统，并为其开发了靶点搜索和引物设计软件。利用该系统实现了艰难梭菌单、多和连续基因编辑，成功构建了∆fur, ∆tetM, ∆ermB1/2, ∆cwp66, ∆tcdA, ∆phiCD630-2和∆PaLoc基因敲除突变株。通过对突变株的生理、生化特征（如细胞壁表面小凸起数量、自溶速率、对酸、碱耐受能力以及对抗生素的耐受能力等）的深入的研究。结果表明，∆cwp66黏附因子突变株表面小突起数量降低、自溶速率和对酸的耐受能力增加，抗生素敏感谱发生了显著的变化。生理、生化特征的改变提示cwp66基因对于艰难梭菌细胞壁的组成至关重要，并可能通过影响艰难梭菌细胞壁的组成增加胞外毒素的释放量和改变抗生素敏感性谱。对该突变株和野生型菌株的转录组对比分析发现，∆cwp66黏附因子突变株黏附能力提高的原因是其他黏附相关基因的表达上调（如CD630_02880, CD630_05140, CD630_03050等）。此外，我们还发现了多种潜在的艰难梭菌黏附因子编码基因。