Nagahama M, Itohayashi Y, Hara H, Higashihara M, Fukatani Y, Takagishi T, Oda M, Kobayashi K, Nakagawa I, Sakurai J.
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-β-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.