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

我们前期的研究表明肠出血性大肠杆菌（EHEC）在体外具有侵入正常人结肠上皮细胞的潜力，这依赖于宿主细胞的细胞骨架的作用。目前的研究是为了阐明细菌蛋白参与侵袭电位。突变体在各种黏附相关分子（如内膜素、tir或III型分泌系统）中存在缺陷，其试验结果没有显著减少侵袭。然而，鞭毛相关基因的意外突变显示出明显的入侵能力不足。该突变体的鞭毛不活动，对11-7抗体无反应，并通过二维电泳和TOF-MS分析证实了鞭毛蛋白缺失。一种新的鞭毛蛋白突变体已经被构建并正在研究中。接下来，以乳酸菌为研究对象，探讨正常菌群中益生菌对肠出血性大肠杆菌侵袭潜力的影响。在实验中，只有鼠李糖对肠出血性大肠杆菌侵袭上皮细胞有明显的抑制作用。另一方面，肠出血性大肠杆菌的粘附和定植不受任何乳杆菌菌株的影响。从可能的机制来看，鼠李糖的存在并未影响肠出血性大肠杆菌和宿主细胞的活力。由于对肠出血性大肠杆菌入侵的抑制作用严格依赖于活鼠李糖，而在条件培养基或杀死鼠李糖中无法观察到，因此某些受体不太可能发生简单的竞争。与其他菌株相比，鼠李糖乳杆菌对结肠上皮细胞系表现出出色的粘附潜力，这表明鼠李糖乳杆菌与宿主细胞的强烈相互作用可能调节了与肠出血性大肠杆菌侵袭有关的细胞内事件。

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.