TRANSLOCATION OF VEROTOXIN-1 ACROSS T84 MONOLAYERS - MECHANISM OF BACTERIAL TOXIN PENETRATION OF EPITHELIUM

Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria as sociated with diarrhea, hemorrhagic colitis, and hemolytic uremic synd rome. Verotoxins (VTs) elaborated by these organisms produce cytopathi c effects on a restricted number of cell types, including endothelial cells lining the microvasculature of the bowel and the kidney. Because human intestinal epithelial cells lack the globotriaosylceramide rece ptor for VT binding, it is unclear how the toxin moves across the inte stinal mucosa to the systemic circulation. The aims of this study were to determine the effects of VT-1 on intestinal epithelial cell functi on and to characterize VT-1 translocation across monolayers of T84 cel ls, an intestinal epithelial cell line. VT-1 at concentrations up to 1 mu g/ml had no effect on the barrier function of T84 monolayers as as sessed by measuring transmonolayer electrical resistance (102 +/- 8% o f control monolayers). In contrast, both VT-positive and VT-negative V TEC bacterial strains lowered T84 transmonolayer resistance (45 +/- 7 and 38 +/- 6% of controls, respectively). Comparable amounts of toxin moved across monolayers of T84 cells, exhibiting high-resistance value s, as monolayers with VTEC-induced decreases in barrier function, sugg esting a transcellular mode of transport. Translocation of VT-1 across T84 monolayers paralleled the movement of a comparably sized protein, horseradish peroxidase. Immunoelectron microscopy confirmed transcell ular transport of VT-1, since the toxin was observed within endosomes and associated with specific intracellular targets, including the Golg i network and endoplasmic reticulum. These data present a mode of VT-1 uptake by toxin-insensitive cells and suggest a general mechanism by which bacterial toxins lacking specific intestinal receptors can penet rate the intestinal epithelial barrier.