EHECO157∶H7粘附Caco-2细胞缺陷突变株的构建

Construction of mutants of enterohemorrhagic Escherichia coli O157∶H7 deficient in adherence to caco-2 cells

目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞株,观察Caco-2单层细胞中的微菌落数量和m in i-Tn5Km2插入位点。结果:经诱变,共分离到3组粘附缺陷突变株,1组完全丧失了粘附力,2组粘附力减弱,3组的粘附力更弱。1组和3组拥有LEE岛上多基因的转座子插入,而2组m in i-Tn5Km2的插入位点在LEE岛之外。结论:O157Sakai的肠道粘附能力可能与LEE岛的III型分泌系统有关。

Objective：To construct an EHEC O157：H7 mutant which deficient in adherence to Caco -2 Cells in order to obtain a tool useful for researching the adherence mechanism in vitro and in vivo. Methods：An EHEC O157：H7 strain （O157Sakai） was mutagenized by mini -TnSKm2, where Km refers to kanamycin resistance, and 4 677 insertion mutants were screened for their ability to form microcolonies （ MC ） on Caco - 2 cells. The deficient adherent mutants were divided into three groups and the sits of mini -Tn5Km2 were detected. Results：Mutants of 1 to 3 groups were obtained. Their adherence abilities to Caco -2 cells were loss, weak and more weak. The sites of mini - Tn5Km2 were detected to be inside of LEE in 1 and 3 groups, meanwhile the sits of mini -TnSKm2 were detected to be outside of LEE in 2 group mutant. Concluslon：EHEC encodes additional adherenceassociated loci and that the type Ⅲ secreted proteins are involved in the initial diffuse adherence, while the intimin -tir interaction is required for the subsequent development of MC.