Abstract
Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis, inactivates Rho GTPases by glucosylation. Here we studied the uptake and membrane interaction of the toxin with eukaryotic target cells. Bafilomycin A1, which prevents acidification of endosomal compartments, blocked the cellular uptake of toxin B in Chinese hamster ovary cells cells. Extracellular acidification (pH </= 5.2) induced uptake of toxin B into the cytosol even in the presence of bafilomycin A1. Toxin B increased (86)Rb(+) release when preloaded Chinese hamster ovary cells were exposed to low pH (pH </= 5.6) for 5 min. Release of (86)Rb(+) depended on the concentration of toxin B and on the pH of the extracellular medium. An antibody directed against the holotoxin prevented channel formation, whereas an antibody against the N-terminal enzyme domain was without effect. The N-terminally truncated toxin B fragment consisting of amino acids 547-2366 increased (86)Rb(+) efflux when cells were exposed to low pH. Toxin B also induced pH-dependent channel formation in artificial lipid bilayer membranes. Clostridium sordellii lethal toxin, another member of the family of large clostridial cytotoxins, also induced increased (86)Rb(+) release at low pH. The results suggest that large clostridial cytotoxins including C. difficile toxin B and C. sordellii lethal toxin undergo structural changes at low pH of endosomes that are accompanied by membrane insertion and channel formation.
Highlights
The large clostridial cytotoxins from Clostridium difficile toxin A (308 kDa) and toxin B (269 kDa) are major virulence factors of antibiotic-associated diarrhea and the causative agents of pseudomembranous colitis [1,2,3,4]
The results suggest that large clostridial cytotoxins including C. difficile toxin B and C. sordellii lethal toxin undergo structural changes at low pH of endosomes that are accompanied by membrane insertion and channel formation
The data are in line with the recent report that toxin B escapes from an acidic endosomal compartment into the cytoplasm [19]
Summary
Materials—Cell culture medium was from Biochrom (Berlin, Germany), and fetal calf serum was from PAN Systems (Aidenbach, Germany). The toxin B fragment CDB1–546 (catalytic domain of C. diffcile toxin B (amino acids 1–546)) was cloned, expressed, and purified as described previously [22], and a monoclonal antibody (anti-CDB1–546) was produced against this fragment. Cell Culture and Toxin Translocation Assay—CHO1-K1, HT-29, Caco-2, and Vero cells were cultivated in tissue culture flasks at 37 °C and 5% CO2 in Ham’s F-12/Dulbecco’s modified Eagle’s medium (DMEM) (1:1) containing 5% heat-inactivated (30 min, 56 °C) fetal calf serum, 2 mM L-glutamate, 100 units/ml penicillin, and 100 g/ml streptomycin. Membranes were formed from a 1% solution of asolectin or diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in n-decane
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