fliY基因在空肠弯曲菌致病性相关趋化和定植中的作用

Role of fliY gene in pathogenicity-associated chemotaxis and colonization of Campylobacter jejuni

目的:构建空肠弯曲菌(Campylobacter jejuni)鞭毛马达开关蛋白FliY编码基因(fliY)敲除突变株,了解FliY蛋白控制细菌鞭毛运动的作用及其与细菌趋化和定植的关系。方法:采用pBluescript-Ⅱ-SK质粒构建用于敲除空肠弯曲菌NCTC11168株fliY基因的自杀质粒。根据同源交换原理,利用自杀质粒构建空肠弯曲菌fliY基因敲除突变株(fliY-)。采用PCR、PCR产物测序与Western Blot,对fliY-突变株进行鉴定。采用体外菌落迁徙试验、细菌趋化试验以及小鼠空肠定植试验,检测fliY-突变株趋化和定植能力。结果:fliY-突变株生长曲线与野生株相似,但PCR与测序及Western Blot结果显示,fliY-突变株中fliY基因已被敲除。与野生株比较,fliY-突变株半固体平板上菌落明显较小(P<0.05),对脱氧胆酸钠和牛胆汁趋化能力显著下降(P<0.05),在感染小鼠空肠组织标本中的细菌数(CFU)也明显减少(P<0.05)。结论:空肠弯曲菌fliY基因功能与调控鞭毛运动作用有关,从而对细菌趋化和定植过程产生重大影响。

Objective： To construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization. Methods： The plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant（fliY） in Campylobacterjejuni strain NCTCll168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice. Results： The fliY- mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that thefliY gene in fliY- mutant was deleted. Compared to the wild-type strain,the colonies of fliY- mutant on semisolid plate were much smaller（ P 〈 0.05 ）, the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated （ P 〈 0.05 ）, and the number of fliY mutant （CFU） in jejunal tissue specimens of the infected mice was significantly decreased （ P 〈 0.05 ）. Conclusion： The function of C. jejuni fliY gene refers to controlling flagellar movement,which is involved in bacterial chemotaxis and colonization.