霍乱弧菌DegS蛋白通过cAMP-CRP-RpoS信号途径调控细菌动力的分子机制研究

鞭毛介导的细菌动力是霍乱弧菌建立肠道定植与感染的关键环节，是霍乱分子发病学的重要研究内容。DegS是一种丝氨酸蛋白酶，其在σE应激反应中发挥重要作用。本课题组首次发现DegS蛋白与霍乱弧菌的动力调控存在密切联系，但其分子机制并不清楚。基于前期DegS蛋白研究基础，提出了“DegS通过cAMP-CRP-RpoS信号途径调控鞭毛基因表达，进而调控细菌动力，参与霍乱弧菌在肠内定植感染过程”的工作假说。本项目拟通过构建ΔdegS+cyaA过表达菌、ΔdegS+cyaA+Δcrp菌、ΔdegS+rpoS过表达菌及ΔdegS+degS+ΔrpoS菌等模型，证明cAMP-CRP-RpoS信号途径在DegS调控细菌动力中的作用；联合EMSA、DNaseI足迹实验及半乳糖苷酶报告实验等，证明RpoS通过直接调控鞭毛基因的转录而调控细菌动力。预期研究结果可为丰富霍乱弧菌动力调节机制提供新的实验依据。

Flagella-mediated bacterial motility play a key role in the process of intestinal colonization and infection by Vibrio cholerae, and is an important research content of molecular pathology of cholera.DegS is a serine protease that plays an important role in the σE stress response.For the first time, we found that DegS protease is closely related to the motility regulation of Vibrio cholerae, but its molecular mechanism is not yet clear. Based on the previous research foundation of DegS, we proposed the hypothesis that "the DegS protease regulates the expression of the flagellar genes through cAMP-CRP-RpoS signaling pathway, which regulates bacterial motility and participates in the process of colonization of Vibrio cholerae in the intestine". This project aims to construct a series of model including ΔdegS+cyaA overexpression strains, ΔdegS+cyaA +Δcrp strians, ΔdegS+rpoS overexpression strains and ΔdegS+degS+ΔrpoS strains to demonstrate the role of cAMP-CRP-RpoS signaling pathway in DegS regulation of bacterial motility. Combined EMSA assay，DNase I footprint assay,and galactosidase reporter assay to demonstrate that RpoS regulates bacterial motility by directly regulating transcription of flagellin genes. The expected research results can provide a new experimental basis for enriching the motility regulation mechanism of Vibrio cholerae.