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

Abstract

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.

Escherichia coli virulence factors

“…At first VTx do not cross the intestinal epithelium though bovine crypt enterocytes express the Gb3 receptor (Hoey et al, 2002). Indeed after endocytosis the intracellular trafficking localizes VTx in lysosomes leading to abrogation of transcytosis (Hoey et al, 2003). Moreover the Gb3 receptor is not expressed on endothelial cells of cattle (Pruimboom-Brees et al, 2000;Hoey et al, 2002).…”

The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer

“…Microlaser Technologies, Bernried, Germany) by following manufacturer's protocols. To detect Gb 3 in cancer epithelial cells, the LCM collected cells from two cases of nonmetastatic and two cases of metastatic colon cancers or from Caco-2 cells infected either with small inhibitory Gb 3 or scRNA were subjected to HPTLC as described in detail (Supporting Materials and Methods) (18)(19)(20).…”

Escherichia coli O157:H7-secreted cytotoxins are toxic to enterocytes and increase Escherichia coli O157:H7 colonization of jejunum and descending colon in cattle

“…There is very little information available on the impact of EHEC O157:H7-secreted cytotoxins or Shiga toxins on epithelial cells in the intestinal tract of cattle. Shiga toxin 1 has binding sites in the jejunum, ileum, cecum and colon, but the toxin is not toxic to primary cultures of colonocytes, and is rapidly taken up and degraded by the crypt cells (Hoey et al 2002(Hoey et al , 2003. Shiga toxins are toxic to lymphocytes (Ferens and Hovde 2000) and heart endothelial cells (ValdiviesoGarcia et al 1996) from cattle and have sublethal effects on lymphocytes (Menge et al 2004;Ferens and Hovde 2007) and fibroblasts (Ferens and Hovde 2007).…”