产志贺毒素大肠杆菌O18 XZ113 株主要毒力基因eaeA、stx2、ehxA 突变株的构建及其对小鼠的致病作用

[目的]探讨毒力基因eaeA、stx2、ehxA与产志贺毒素大肠杆菌O18致病力的关系.[方法]利用λ-Red重组系统,构建STEC XZ113株eaeA、stx2、ehxA基因缺失突变株并进行一系列生物学特性的研究.[结果]细胞粘附试验表明突变株XZ113△eaeA对HEp-2细胞的粘附能力明显降低；Vero细胞毒素试验表明突变株XZ113 △stx2失去了使Vero细胞发生病变的能力；溶血活性试验表明突变株XZ113△ehxA无法在血平板上产生溶血圈,丢失了溶血能力.回复株在以上表型方面与野生株XZ113一致；与亲本株的体外竞争试验结果表明,突变株竞争力减弱,体内竞争结果表明突变株XZ1 13△eaeA被中度致弱；突变株XZ113 △stx2和突变株XZ113△ehxA被高度致弱.[结论]stx2、ehxA基因在STEC O18 XZ113株的致病过程中发挥着更为重要的作用.

Construction of the XZ113 delta eaeA, XZ113 delta stx2 and XZ113 delta ehxA mutants of STEC O18 XZ113 and their pathogenicity in mice

[Objective] To study the contribution of virulence genes of STEC O18 XZ113 isolate to the pathogenicity in mice.[Methods] The eaeA,stx2 and ehxA knock-out mutants of STEC strain XZ113 were generated using λ-Red recombination system.[Results] Bacterial adherence test showed that the eaeA mutant adhered to HEp-2 cells in a diffuse manner with no microcolony formation.Vero cells assay showed that the stx mutant had no cytotoxicity to Vero cells.Enterohemolytic activity test showed that the ehxA mutant lost the ability to express the enterohemolytic activity.Competition assay between the wild-type strain XZ113 and its mutants in vivo and in vitro showed that all mutants were mildly attenuated in vitro,but in vivo,XZ113△eaeA was moderate attenuated,XZ113△stx2 and XZ113△ehxA were all highly attenuated.[Conclusions] These results indicate that the virulence factors encoded by the stx2 and ehxA genes were important for the pathogenesis of STEC O18 in mice.