Shiga Toxins 1 and 2 Translocate Differently across Polarized Intestinal Epithelial Cells

Abstract

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen that causes hemolytic-uremic syndrome. Following ingestion, STEC cells colonize the intestine and produce Shiga toxins (Stx), which appear to translocate across the intestinal epithelium and subsequently reach sensitive endothelial cell beds. STEC cells produce one or both of two major toxins, Stx1 and Stx2. Stx2-producing STEC is more often associated with disease for reasons as yet undetermined. In this study, we used polarized intestinal epithelial cells grown on permeable filters as a model to compare Stx1 and Stx2 movement across the intestinal epithelium. We have previously shown that biologically active Stx1 is able to translocate across cell monolayers in an energy-dependent, saturable manner. This study demonstrates that biologically active Stx2 is also capable of movement across the epithelium without affecting barrier function, but significantly less Stx2 crossed monolayers than Stx1. Chilling the monolayers to 4°C reduced the amount of Stx1 and Stx2 movement by 200-fold and 20-fold respectively. Stx1 movement was clearly directional, favoring an apical-to-basolateral translocation, whereas Stx2 movement was not. Colchicine reduced Stx1, but not Stx2, translocation. Monensin reduced the translocation of both toxins, but the effect was more pronounced with Stx1. Brefeldin A had no effect on either toxin. Excess unlabeled Stx1 blocks the movement of125I-Stx1. Excess Stx2 failed to have any effect on Stx1 movement. Our data suggests that, despite the many common physical and biochemical properties of the two toxins, they appear to be crossing the epithelial cell barrier by different pathways.

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

“…This represents a profound fundamental difference that could have major significance in the differing pathogenicity of EHEC in these hosts. Although human intestinal epithelium lacks Gb3, this remains an important interface with VT as in vitro studies (Philpott et al, 1997;Hurley et al, 1999) have shown that VT is actively transported across these cells without disrupting cellular integrity. VT is thereby delivered to the basal surface of the epithelium where it gains access to receptor-positive cells including endothelium (leading to vascular disruption) and macrophages (evoking inflammatory mediators).…”

Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture

“…Caco‐2 and T84 epithelial cells were grown on collagen‐coated Transwell filter inserts (6.5 mm diameter, 0.4 µm pore size; Costar) as described previously (Hurley et al ., 1999), and TEER was monitored with an EVOM resistance reader (WPI). Cells were differentiated for 7–12 days until stable TEER values of 500 (Caco‐2) and 1800 (T84) Ω cm 2 were obtained indicating establishment of epithelial barrier function.…”

Shiga toxins and apoptosis

“…The characteristic intestinal damage caused by STEC includes hemorrhage and edema in the lamina propria with focal necrosis and neutrophil infiltration. Recently, it was shown that Stx1 and Stx2 are translocated across polarized intestinal epithelial monolayers via transcellular and paracellular mechanisms [19,20]. In this manner, Stxs may gain access to and damage colonic blood vessels.…”

“…These cell types respond differently to Stxs. For example, T84 cells, a human intestinal epithelial cell line, lack the toxin glycolipid receptors and are not killed by Stxs in vitro, yet are capable of toxin translocation from apical to basolateral surfaces [19]. Bovine crypt intestinal epithelial cells, in contrast, express Gb 3 but escape killing by routing the toxins to lysosomes rather than to the ER [32].…”