Hoey D E Elaine, Sharp Linda, Currie Carol, Lingwood Clifford A, Gally David L, Smith David G E
Department of Medical Microbiology, University of Edinburgh, Edingburgh, Scotland, UK.
Cell Microbiol. 2003 Feb;5(2):85-97. doi: 10.1046/j.1462-5822.2003.00254.x.
Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceramide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells contrasts with the absence of Gb3 on human intestinal epithelium in vivo. Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithelial cells. Sub-cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abrogation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl-beta-cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor-positive cells that resist verocytotoxicity. These studies demonstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans.
志贺毒素（VTs）是肠出血性大肠杆菌（EHEC）的重要毒力因子，EHEC是一类与人类严重疾病后遗症相关的细菌。VTs强大的细胞毒性活性在致病性中很重要，会导致表达受体Gb3（球三糖神经酰胺）的细胞死亡。EHEC，尤其是O157:H7血清型，在无疾病的情况下经常定植于储存宿主（如牛）中，然而，该宿主中无毒力的基础尚不清楚。本研究的目的是评估VT与肠上皮之间的相互作用，肠上皮代表宿主与肠道微生物之间的主要界面。牛肠上皮细胞在原代细胞培养中体外表达Gb3，与肠道黏膜的原位免疫组织学观察结果一致，Gb3特异性定位于增殖的隐窝细胞。这些细胞中受体的表达与人类肠上皮体内缺乏Gb3形成对比。尽管有受体表达，但VT对牛上皮细胞没有细胞毒性活性。VT的亚细胞定位表明，这种毒素被内质网排除在外，但定位于溶酶体，这与细胞毒性的消除相对应。用甲基-β-环糊精处理原代细胞培养物不会影响VT的细胞内运输，这表明这些细胞中的Gb3与脂筏无关，而是随机分布在膜中。Gb3异构体、膜分布和VT运输的组合与其他抗志贺细胞毒性的受体阳性细胞的观察结果相关。这些研究表明，肠上皮是VT相互作用中的一个重要决定因素，对EHEC在储存宿主和人类中感染的不同后果具有重要意义。
