Comprehensive analysis of bacterial factors involved in enterohemorrhagic E. coli infection to human colon epithelial cells for a preventive approach

我们以前的研究表明，肠出血性E。大肠埃希菌（EHEC）在体外具有侵袭正常人结肠上皮细胞的能力，这种侵袭依赖于宿主细胞骨架的作用。本研究旨在阐明参与入侵潜力的细菌蛋白质。在各种粘附相关分子（如内膜素、tir或III型分泌系统）中存在缺陷的突变体的试验未导致侵袭的显著减少。然而，鞭毛相关基因的一个意外突变体显示出显著的入侵能力丧失。该突变体鞭毛的缺陷表现为不能移动的和不能与11-7抗体反应，并通过二维电泳和飞行时间质谱分析证实了鞭毛蛋白的缺失。目前已经构建了一种新的鞭毛蛋白突变体，并正在研究中。接下来，利用乳酸杆菌属物种探索了正常植物群中的益生菌对EHEC侵袭潜力的影响。供试菌株中，只有L. rhamnosus能显著抑制EHEC对上皮细胞的侵袭。另一方面，EHEC的粘附和定殖不受任何测试的EHEC菌株的影响。关于可能的机制，EHEC和宿主细胞的活力不受影响的存在下，鼠李糖乳杆菌。在某些受体上的简单竞争是不可能的，因为对EHEC侵袭的抑制作用严格依赖于活的L。而用条件培养基或杀死的鼠李糖乳杆菌则不能观察到。L.与其他菌株相比，鼠李糖乳杆菌对结肠上皮细胞系显示出突出的粘附潜力，这表明鼠李糖乳杆菌与宿主细胞的亲和相互作用可能调节负责EHEC侵袭的细胞内事件。

Our previous studies showed that enterohemorrhagic E. coil (EHEC) has a potential to invade to normal human colon epithelial cells in vitro, which is dependent on the action of cytoskeleton of host cells. The present investigation was conducted to elucidate the bacterial proteins involved in the invasive potential. The trial with mutants, which were defective in various adhesion-related molecules such as intimin, tir or type III secretion systems, resulted in no significant reduction of invasion. An unintended mutant, however, of flagella-related gene showed a remarkable inability of invasion. The defect of flagella in this mutant was suggested by being not mobile and not reactive to 11-7 antibody, and was confirmed by the absence of flagellin protein on 2-D electrophoresis and TOF-MS analysis. A novel mutant with flagellin protein has now been constructed and under investigation.Next, the effect of probiotic bacteria in normal flora on the invasive potential of EHEC was explored employing Lactobacillus species. Among tested, only L. rhamnosus showed a significant inhibition of EHEC invasion to epithelial cells. On the other hand, the adhesion and colonization of EHEC was not affected by any of the lactobacillus strains tested. Concerning the possible mechanisms, the viabilities of EHEC and host cell were not affected by the presence of L.rhamnosus. Simple competitions at certain receptors were unlikely because the suppressive effect on EHEC invasion was strictly dependent on viable L. rhamnosus and could not be observed with the conditioned medium or killed L.rhamnosus. The fact that L. rhamnosus showed outstanding adhering potential to the colon epithelial cell line, compared with other strains, suggested that an avid interaction of L.rhamnosus to the host cell might be modulating intracellular events responsible for the invasion of EHEC.