霍乱弧菌DsbA蛋白通过增强MSHA的表达促进生物被膜的形成

VcDsbA enhances bioflim formation by stimulating MSHA expression in Vibrio cholerae

【目的】阐明霍乱弧菌DsbA蛋白(VcDsbA)在生物被膜形成过程中的作用。【方法】采用Overlapping PCR的方法构建VcdsbA基因敲除质粒p MH524;采用同源重组和基因克隆的方法对霍乱弧菌dsbA(vc0034)基因进行敲除和回补;通过结晶紫染色实验,比较野生株(WT)、dsbA突变株(ΔdsbA)和回补菌株(CΔdsbA)的生物被膜形成差异;用荧光素酶基因作为报告基因分析与生物被膜形成相关的甘露糖敏感血凝素纤毛合成蛋白(Mannose-sensitive hemagglutinin,pili biogenesis protein,MSHA)在霍乱弧菌WT、ΔdsbA和CΔdsbA中表达水平的区别。【结果】成功构建霍乱弧菌dsbA基因缺失突变株和回补株;与WT相比,ΔdsbA生物被膜形成能力显著下降,且msh操纵子的表达水平显著降低。【结论】VcDsbA可能通过影响其它调控因子直接或间接增强霍乱弧菌MSHA的生物合成,从而促进霍乱弧菌生物被膜的形成。本文为进一步研究DsbA在霍乱弧菌生物被膜形成中的调控机制奠定了基础。

[Objective] Biofilm plays an important role during the infection cycle of Vibrio cholerae. In this study, we try to demonstrate the role of VcDsbA in the biofilm formation of V. cholerae. [Methods] By making the Vcdsb A inframe knock-out construct, the vcdsb A null mutant（ΔdsbA） strain was obtained. And the complemented strain（CΔdsbA） was constructed by transferring a plasmid-coded Vcdsb A expressed under the control of arabinose to ΔdsbA strain. Crystal violet staining assay was used to analyze the biofilm formation in the wild-type（WT）, ΔdsbA and CΔdsbA strains. V. cholerae strains containing msh promoter lux CDABE transcriptional fusion were used to analyze the transcriptional level. [Results] The ΔdsbA and CΔdsbA strains were constructed successfully. Biofilm formation analysis shows that the ability of biofilm formation of ΔdsbA was significantly reduced compared with WT, whereas CΔdsbA could form even stronger biofilm than WT does. Luminescence expression by Pmsh shows that VcDsbA enhanced msh expression. VcDsbA enhances the biofilm formation of V. cholerae by involving in the regulation of msh expression level. VcDsbA up-regulates msh expression probably through helping the folding of a msh expression activator. [Conclusion] VcDsbA plays an important role in the biofilm formation of V. chlerae, which makes the bacteria better survive in their living niche.