c-di-GMP通过趋化调节蛋白CheB／CheR调控钩端螺旋体运动  

Cyclic diguanylate （c-di-GMP） regulates the motility of Leptospira interrogans via chemotaxis regulatory proteins CheB/CheR

作　　者：孔亮亮[1] 肖国辉[1] 袁富迪 严杰[1] 林旭瑷[1] 

机构地区：[1]浙江大学医学院病原生物学系,杭州310058

出　　处：《中华微生物学和免疫学杂志》2017年第7期521-526,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81271781）

摘　　要：目的问号钩端螺旋体（简称钩体）是引起钩体病的重要病原体，人类通过带菌动物尿液污染的疫水或疫土而感染，趋化和运动系统在钩体感染过程中发挥重要作用。钩体基因组序列分析表明其中有数个趋化调节蛋白CheB和CheR的编码基因，但其具体的调控机制未知。方法Real-timePCR检测了钩体基因组中5个基因在钩体感染宿主细胞过程中mRNA的水平变化：T-A克隆了CheB和CheR的编码基因片段并构建了原核表达系统；检测了c-di-GMP浓度变化对CheB和CheR表达菌株表型的影响。结果在感染细胞60min后，cheBl的mRNA水平显著提高，cheB3也有一定程度的提高，而cheB2和cheR则无明显差异；成功构建了5个基因的原核表达系统，其中重组CheBl、CheB3和CheR2蛋白使大肠杆菌运动能力明显增强；在加入二鸟苷酸环化酶抑制剂和磷酸二酯酶抑制剂后，各表达菌株形成菌落大小与对照相比无明显不同，但CheB2菌落形态与对照相比出现明显褶皱。结论CheB和CheR可影响大肠杆菌的swarming运动能力，并受胞内c．di，GMP浓度的影响。Objective To investigate the regulatory effects of cyclic diguanylate （c-di-GMP） sig- naling on CheB and CheR, which were chemotaxis regulatory proteins relating to the motility of Leptospira in- terrogans. Methods Real-time PCR was used to determine the expression of cheBI, cheB2, cheB3, cheRl and cheR2 genes at mRNA level during Leptospira interrogans infection. Fragments of these genes were am- plified and cloned into the expression vector pET-28a, respectively, to construct the prokaryotic expression system for them. Colony morphologies of Escherichia coli （E. coli） strains that overexpressed the target genes were observed to determine the regulatory effects of c-di-GMP on CheB and CbeR. Results The expression of cheB1 gene at mRNA level increased 60 min after infection and reached the peak at 90 min. Compared with the control group, the expression of cheB3 gene at mRNA level were up-regulated, while no significant difference in the expression of cheB2 and cheR genes was observed 60 min after infection. The prokaryotic expression system for the five genes was successfully constructed and the purified proteins were obtained. CheB1, CheB3 and CheR2 improved the motility of E. coli, but that was inhibited by the inhibitor of digua- nylate cyclase （DGC） or phosphodiesterase （PDE）. Conclusion CheB and CheR regulate the swarming motility of E. coli and are affected by intracellular c-di-GMP.

关 键 词：钩端螺旋体 趋化 CheB CheR 环二鸟苷酸 

分 类 号：R383.1[医药卫生—医学寄生虫学]