霍乱弧菌ToxS蛋白间的互作增强ToxR蛋白诱导毒力基因的表达

ToxS homodimerization enhances ToxR acitivating virulence gene expression in Vibrio cholerae

摘要:

[目的]阐明霍乱弧菌ToxR蛋白功能调控的分子机制。[方法]利用巯基捕获（thiol-trapping）的方法分析DsbA蛋白对ToxR周质空间结构域半胱氨酸残基的氧化作用；采用定点突变的方法构建ToxR半胱氨酸突变株（ToxR_C236/293S）；利用荧光素酶基因作为报告基因分析ToxR野生型（ToxR_wt）和半胱氨酸突变体（ToxR_C236/293S）诱导下游基因表达的活性；通过细菌双杂交系统分析ToxR_wt和ToxR_C236/293S蛋白之间、ToxR与ToxS之间以及ToxS之间的相互作用。[结果]ToxR周质空间结构域半胱氨酸残基确实可以被DsbA蛋白氧化，且当ToxR与ToxS共表达时，ToxR诱导ctxAB转录表达的活性显著增强，且在dsbA基因缺失突变株中ToxR诱导ctxAB转录表达的活性更高；成功构建株霍乱弧菌ToxR半胱氨酸突变株（ToxR_C236/293S），在没有ToxS存在的条件下，ToxR_C236/293S诱导毒力基因表达的活性与ToxR_wt相当；细菌双杂交系统分析发现当ToxR与ToxS共转录表达时，ToxS极大增强ToxR蛋白之间的互作；在dsbA基因缺失突变株中，ToxS之间的相互作用显著增强。[结论]ToxR蛋白本身的氧还状态对其诱导毒力基因表达的活性没有影响；ToxS通过增强ToxR形成二聚体的能力从而增强其诱导毒力基因的表达，而DsbA对ToxS蛋白之间的相互作用具有抑制作用，DsbA通过影响ToxS的蛋白互作从而影响ToxR蛋白的功能。本文为进一步阐明霍乱弧菌毒力基因表达调控的分子机制提供重要的理论依据。

Abstract:

[Objective] In this study, we demonstrated the mechanism of ToxR activating virulence gene expression by modifying its protein function in Vibrio cholerae. [Methods] ToxR redox status assay with or without DsbA was analyzed by thiol-trapping assay. ToxR cysteine mutant ToxR_C263/293S was obtained by site-directed mutagenesis. Escherichia coli strains containing ctxAB promoter luxCDABE transcriptional fusion and a plasmid-coded ToxR expressed under the control of arabinose were used to analyze the transcriptional level. ToxR and ToxS proteins interaction was detected by the bacterial two-hybrid system. [Results] The two cysteines in the periplasmic domain of ToxR could be oxidized by DsbA. When ToxR and ToxS were co-transcribed under the control of the same promoter, ToxR activated ctxAB expression to a higher level in E. coli dsbA knock out mutant. However, when ToxR was expressed alone without ToxS, the redox status of ToxR had no effect on its activation of ctxAB expression in E. coli. Bacterial two-hybrid system assay showed that ToxS greatly enhanced ToxR homodimerization regardless of the redox status of ToxR, and DsbA strongly repressed ToxS-ToxS interaction. [Conclusion] The redox status of ToxR has no effect on its activating virulence gene expression and ToxS enhances ToxR activity by promoting ToxR homodimerization. DsbA indirectly affects ToxR activating virulence genes expression by repressing ToxS homodimerization.