Cellular vacuolation induced by Clostridium perfringens epsilon-toxin.

The epsilon-toxin of Clostridium perfringens forms a heptamer in the membranes of Madin-Darby canine kidney cells, leading to cell death. Here, we report that it caused the vacuolation of Madin-Darby canine kidney cells. The toxin induced vacuolation in a dose-dependent and time-dependent manner. The monomer of the toxin formed oligomers on lipid rafts in membranes of the cells. Methyl-ß-cyclodextrin and poly(ethylene glycol) 4000 inhibited the vacuolation. Epsilon-toxin was internalized into the cells. Confocal microscopy revealed that the internalized toxin was transported from early endosomes (early endosome antigen 1 staining) to late endosomes and lysosomes (lysosomal-associated membrane protein 2 staining) and then distributed to the membranes of vacuoles. Furthermore, the vacuolation was inhibited by bafilomycin A1, a V-type ATPase inhibitor, and colchicine and nocodazole, microtubule-depolymerizing agents. The early endosomal marker green fluorescent protein-Rab5 and early endosome antigen 1 did not localize to vacuolar membranes. In contrast, the vacuolar membranes were specifically stained by the late endosomal and lysosomal marker green fluorescent protein-Rab7 and lysosomal-associated membrane protein 2. The vacuoles in the toxin-treated cells were stained with LysoTracker Red DND-99, a marker for late endosomes and lysosomes. A dominant negative mutant of Rab7 prevented the vacuolization, whereas a mutant form of Rab5 was less effective. These results demonstrate, for the first time, that: (a) oligomers of epsilon-toxin formed in lipid rafts are endocytosed; and (b) the vacuoles originating from late endosomes and lysosomes are formed by an oligomer of epsilon-toxin.

Oligomerization of Clostridium perfringens epsilon-toxin is dependent upon membrane fluidity in liposomes.

Clostridium perfringens epsilon-toxin binds to receptors on MDCK cells and forms a heptamer in membranes. The mechanism behind the oligomerization of epsilon-toxin was studied using carboxyfluorescein (CF)-loaded liposomes composed of various phosphatidylcholines (PCs). The toxin caused CF to leak from liposomes in a dose-dependent manner. The toxin-induced leakage of CF, binding of the toxin to liposomes, and formation of a functional oligomer increased as the phase-transition temperature (Tm) of the PC used in the liposomes decreased. Surface plasmon resonance analysis using an HPA sensorchip (BIAcore) also revealed that the binding of the toxin to liposomes increased with a decrease in the Tm of the PC used in liposomes. The oligomer that was formed in 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID)-treated liposomes was labeled, indicating that it inserts into a hydrophobic region. Furthermore, the rate of epsilon-toxin-induced CF leakage was enhanced by treatment with phosphatidylethanolamine or diacylglycerol, which is known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. We show that membrane fluidity in the liposome plays an important role in the binding of the toxin to liposomes, insertion into the hydrophobic region in the bilayer of liposomes, and the assembly process in the bilayer.