Clostridium perfringens Epsilon Toxin Induces a Rapid Change of Cell Membrane Permeability to Ions and Forms Channels in Artificial Lipid Bilayers

Laetitia Petit,Elke Maier,Maryse Gibert,Michel R Popoff,Roland Benz

doi:10.1074/jbc.m010412200

Laetitia Petit, Elke Maier + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m010412200

Copy DOI

Abstract

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., Laurent-Winter, 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.

Highlights

Epsilon toxin is synthesized by Clostridium perfringens types B and D
Because epsilon toxin interacts with the cell membrane and has apparently no intracellular activity, its mechanism of action could consist in pore formation, as it is the case for many hemolysins
C. perfringens Epsilon Toxin Forms Pores Permeable to Ions and propidium iodide (PI) in MDCK Cells—As previously reported, the in vivo experiments suggest that epsilon toxin forms a complex in the membranes of the MDCK cells and does not enter their cytoplasm

Summary

EXPERIMENTAL PROCEDURES

Materials—Epsilon toxin was purified as previously described [14]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide were from Sigma. Each fluorescent probe was added to 45 ␮l of lysed cells to a final concentration of 1 ␮M for CD222 (Kϩ assay) or 10 ␮M for sodium green, calcium green, and MQAE (ClϪ assay). The fluorescence obtained by washing MDCK cells with MOPS-glucose and lysing with 1% (w/v) Triton X-100 addition was considered as the 0% base line, and the fluorescence of MDCK cells incubated with serum-free DMEM containing 0.2% (w/v) Triton X-100 for 5 min at 37 °C in the absence of epsilon toxin and washed with MOPS-glucose and lysed with 1% (w/v) Triton X-100 was considered as an activity of 100%. Native and activated C. perfringens epsilon toxin were added from concentrated stock solutions to the aqueous phase bathing membranes in the black state. Zerocurrent membrane potential measurements were performed by establishing a salt gradient across membranes containing 100 to 1000 epsilon toxin channels as they have been described earlier [17]

RESULTS

Selectivity permeability ratio pS

Salt c

DISCUSSION