title: Cellular vacuolation induced by Clostridium perfringens epsilon-toxin.
abstract: 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.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan. nagahama@ph.bunri-u.ac.jp
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
authors.forename: Yukari
authors.initials: Y
authors.lastname: Itohayashi
authors.forename: Hideki
authors.initials: H
authors.lastname: Hara
authors.forename: Masahiro
authors.initials: M
authors.lastname: Higashihara
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Fukatani
authors.forename: Teruhisa
authors.initials: T
authors.lastname: Takagishi
authors.forename: Masataka
authors.initials: M
authors.lastname: Oda
authors.forename: Keiko
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Ichiro
authors.initials: I
authors.lastname: Nakagawa
authors.forename: Jun
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000633:toxicity*; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure*; D007700:Kinetics; D052119:Lysosomal-Associated Membrane Protein 2 / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D021962:Membrane Microdomains / Q000187:drug effects / Q000648:ultrastructure; D008870:Microtubules / Q000187:drug effects; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D050257:Tubulin Modulators / Q000494:pharmacology; D014617:Vacuoles / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure*; D033921:Vesicular Transport Proteins / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D001427:Bacterial Toxins; D015415:Biomarkers; C039811:Clostridium perfringens epsilon-toxin; D052119:Lysosomal-Associated Membrane Protein 2; D050505:Mutant Proteins; D050257:Tubulin Modulators; D033921:Vesicular Transport Proteins; D047392:beta-Cyclodextrins; C093846:early endosome antigen 1; C108732:methyl-beta-cyclodextrin; D000090802:rab7 GTP-Binding Proteins; D011092:Polyethylene Glycols; D020691:rab GTP-Binding Proteins