title: Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells.
abstract: Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.
authors.affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 64110, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Chao
authors.forename: L A
authors.initials: LA
authors.lastname: Dreyfus
mesh_terms: D001427:Bacterial Toxins / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000502:physiology; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000502:physiology
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; C034151:heat stable toxin (E coli); D015638:Cytochalasin D; D019204:GTP-Binding Proteins