Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicity.

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.

Expression of receptors for verotoxin 1 from Escherichia coli O157 on bovine intestinal epithelium.

Human enterohaemorrhagic Escherichia coli (EHEC) infection most commonly arises, either directly or indirectly, from cattle, which act as a reservoir host for these bacteria. In man, EHEC disease can be severe, whereas EHEC do not normally cause disease in cattle. Verotoxins (VTs) are the main virulence factors in human disease but no role for VT has been ascribed in cattle; however, this study shows for the first time that VT receptor is expressed by the bovine intestinal tract. VT bound to crypt epithelial cells of the small (ileum and jejunum) and large (caecum and colon) intestine independently of the animals' age. VT also bound to discrete cell subsets in the bovine kidney and to submucosal lymphoid cells but not to vasculature. Analysis of tissues for isoforms of the VT receptor, Gb3, confirmed the presence of the receptor in the bovine intestinal epithelium and kidney. A distinct pattern of Gb3 receptor isoform mixtures was observed in the bovine kidney. This, together with the general absence of receptors on vasculature, could contribute to the apparent resistance of cattle to systemic effects of VT. Expression of Gb3 on the bovine intestinal epithelium, together with previously described effects, may affect EHEC colonisation in its reservoir hosts and hence the potential for distribution to man.