Low pH-induced formation of ion channels by Clostridium difficile toxin B in target cells

Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis, inactivates Rho GTPase 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 less than or equal to 5.2) induced uptake of toxin B into the cytosol even in the presence of bafilomycin A1. Toxin B increased Rb-86(+) release when preloaded Chinese hamster ovary cells were exposed to low pH (pH less than or equal to 5.6) for 5 min. Release of Rb-86(+) 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 Rb-86(+) 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 Rb-86(+) 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.