| The heat-stable enterotoxin b (STb) produced by enterotoxigenic Escherichia coli is a causative agent of diarrheal disease in weaning piglets. The mature STb enterotoxin corresponds to a 48 amino acid peptide (5,2 kDa) composed of two antiparallel alpha-helices stabilized by two disulfide bonds that are essential for enterotoxicity. Since its discovery, epidemiological, physicochemical and genetic aspects of STb have been well studied. However, little is known about its mechanism of action resulting in secretion. Recently, it has been shown that STb enterotoxin interacted with sulfatides and that these glycolipids seemed to be essential for the biological activity of the toxin. In order to clarify the role of the sulfatides in the mechanism of action of STb, the interaction between STb and pure sulfatide and also the interaction of STb with cultured cells, were studied.; A quantitative method in microtiter plate was first developed, to evaluate the chemical and physical characteristics of the interaction of STb with glycolipids. The results indicated that STb has a strong specificity for sulfatides and that this binding is dose-dependent and saturable. The epitope recognized by STb was the terminal sulfated galactose, present on sulfatides. The interaction between these two molecules is of low affinity (KD : 2--6 +/- 1,5 muM).; Then, the chemical nature of the sulfatides present in the porcine jejenum was demonstrated. A preceding study indicated the presence of molecules harboring a sulfated galactose epitope recognized by STb in the pig intestine. However, there was no certitude on the exact nature of these molecules. Therefore, mass spectrometry (LC/MS) was used to study the exact nature of the molecules. A lipid, extracted from the pig jejunum epithelial cells, was identified as sulfatides and the interaction of STb with these sulfatides was demonstrated.; Finally, a cellular model (in vitro) was developed to replace the animal model (in vivo) and to facilitate the study of the biological activity of the STb toxin. In presence of STb, it was observed that the CHO cell line absorbed 50% more trypan blue than the control, after two hours of incubation. Statistical analysis demonstrated the possibility to use the trypan blue intake by CHO cells as a cellular model for determination of the biological activity of STb.; In conclusion, STb toxin interacts with sulfatides present at the surface of pig jejunum epithelial cells. The toxin recognizes the sulfated galactose present on these molecules and binds to it with low affinity. The presence of sulfatide is essential for the trypan blue intake caused by STb on cultured cells. Taken together, to date, these data suggest that sulfatide may be a docking molecule that allows STb to interact with membrane lipids present on the cell surface. |
