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Epsilon-toxin is produced by. Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K ؉ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37°C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37°C was more stable than that formed at 4°C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in sheep and other animals which causes heavy economic losses (24). It is synthesized as a relatively inactive prototoxin (296 amino acids) which is converted to a highly active mature protein by proteolytic removal of a basic N-terminal peptide (13 amino acids) (3, 16).Epsilon-toxin is lethal and dermonecrotic. It has been reported to increase intestinal permeability (4), to cause kidney damage (9), to elevate blood pressure (27, 39), and to cause contraction of isolated rat ileum (40). A basic property of epsilon-toxin is that it increases vascular permeability. The toxin binds to vascular endothelial cells and causes severe vascular damage and edema in various organs (brain, heart, lung, and kidney) (5, 9). It was reported that the major pathological changes caused by enterotoxemia appear to occur in the brain (12). Moreover, it was shown that labeled toxin specifically accumulates in the brains of mice after intravenous injection and that the lethal activity of the toxin depends on its specific binding in the brain, probably to a sialoglycoprotein (28,29). Certain amino acids of epsilon-toxin, such as histidine, tryptophan, and aspartic or glutamic acid have been found to be essential for its biological activity (35)(36)(37)(38).Recently, it has been found that epsilon-toxin has 20 and 27% identity with Mtx2 and Mtx3 toxins, respectively, of Bacillus sphaericus (22, 43) and 26.5% identity with the open reading frame c53 of Bacillus thuringiensis. However, the mechanism of action of these mosquitocidal toxins is unknown.The Madin-Darby canine kidney (MDCK) cell line was found to be susceptible to epsilon-toxin. The alteration of cell viability determined by the conversion of 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium to a water-soluble formazan by the mitochondrial cytochrome systems or by the neutral red assay (an indicator of lysosomal integrity) was correlated with the lethal activity of epsilon-toxin in mice (21, 30). The cytotoxic effects were found to be very rapid (2.5 min) and potentiated by EDTA (21). The morphological effects of epsilon-toxin on cells included a condensation of the nucleus and a progressive swelling of the cells (13). The permeability of polarized MD...

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Epsilon toxin is a potent toxin produced by Clostridium perfringens types B and D, which are responsible for a rapidly fatal enterotoxemia in animals. One of the main properties of epsilon toxin is the production of edema. We have previously found that epsilon toxin causes a rapid swelling of Madin-Darby canine kidney cells and that the toxin does not enter the cytosol and remains associated with the cell membrane by forming a large complex (Petit, L., Gibert, M., Gillet, D., LaurentWinter, C., Boquet, P., and Popoff, M. R. (1997) J. Bacteriol. 179, 6480 -6487). Here, we report that epsilon toxin induced in Madin-Darby canine kidney cells a rapid decrease of intracellular K ؉ , and an increase of Cl ؊ and Na ؉ , whereas the increase of Ca 2؉ occurred later. The entry of propidium iodide that was correlated with the loss of cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test indicates that epsilon toxin formed large pores. In artificial lipid bilayers, epsilon toxin caused current steps with a single-channel conductance of 60 pS in 100 mM KCl, which represented general diffusion pores. The channels were slightly selective for anions, but cations could also penetrate. Epsilon toxin formed wide and water-filled channels permeable to hydrophilic solutes up to a molecular mass of at least 1 kDa, which probably represents the basic mechanism of toxin action on target cells.

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