Fur调控霍乱弧菌生物膜形成和TCP合成的分子机制研究

项目名称： Fur调控霍乱弧菌生物膜形成和TCP合成的分子机制研究

项目编号： No.81471917

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 高鹤

作者单位： 中国疾病预防控制中心传染病预防控制所

项目金额： 72万元

中文摘要： Fur作为整体调控子，不仅控制霍乱弧菌铁代谢，维持细胞内铁平衡，还可能通过调节环二鸟苷酸（c-di-GMP）代谢、生物膜形成、毒素共调菌毛（toxin coregulated pili, TCP）合成，使霍乱弧菌得以在宿主肠道内定殖并致病。本研究在系统分析野生株、fur突变株、回补株的肠毒性、细胞毒性、细胞黏附能力、肠道定殖能力、生物膜形成等表型的基础上，采用RNA-seq比较转录组分析，鉴定体内感染条件下受Fur直接调控的靶基因，组成Fur调控元。采用Lux报告基因融合实验、引物延伸实验、凝胶阻滞实验、DNase I足迹实验等技术，分析Fur对关键靶基因（c-di-GMP代谢酶基因vieA、cdgD和VCA0560、毒力相关基因tcpP）转录调控的精细分子机制，探究Fur调节生物膜形成和TCP合成的分子机制，更深入的理解Fur在霍乱弧菌致病中的作用。

中文关键词： 霍乱弧菌；Fur；生物膜；TCP；转录调控

英文摘要： Fur is a well-charaterized transcription factor responsible for control of iron uptake and homeostasis in Vibrio cholerae. In the present study, V. cholerae Fur is hypothesized to regulate c-di-GMP metabolism, biofilm formation, and TCP (toxin coregulated pili ) biosynthesis to facilitate colonization to host intestine. The phenotypes, including intestinal toxicity, cell toxicity, cell adhesion, intestinal colonization and biofilm formation, of the wild-type V. cholerae, its isogenic fur mutant and the complemented mutant will be characterized, and then RNA-seq-based comparative transcriptomics analysis will be carried out to identify the fur regulon which is composed of a collection of direct Fur targets. Subsequent combined use of Lux reporter fusion, primer extension, gel mobility shift, DNase I footprinting assays will lead to elucidation of the detailed mechanisms of Fur-dependent expression of c-di-GMP metabolism genes vieA, cdgD and VCA0560 as well as virulence-related gene tcpP, which will promote us to gain deeper understanding of regulation of biofilm formation, TCP biosynthesis, and pathogenicity by Fur in Vibrio cholerae.

英文关键词： Vibrio cholerae;Fur;Biofilm;TCP;Transcriptional regulation