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 agen ts of antibiotic-associated diarrhea and pseudomembranous colitis, inactiva tes Rho GTPase by glucosylation, Here we studied the uptake and membrane in teraction of the toxin with eukaryotic target cells. Bafilomycin A1, which prevents acidification of endosomal compartments, blocked the cellular upta ke of toxin B in Chinese hamster ovary cells cells. Extracellular acidifica tion (pH less than or equal to 5.2) induced uptake of toxin B into the cyto sol even in the presence of bafilomycin A1. Toxin B increased Rb-86(+) rele ase when preloaded Chinese hamster ovary cells were exposed to low pH (pH l ess than or equal to 5.6) for 5 min. Release of Rb-86(+) depended on the co ncentration of toxin B and on the pH of the extracellular medium. An antibo dy directed against the holotoxin prevented channel formation, whereas an a ntibody against the N-terminal enzyme domain was without effect. The N-term inally truncated toxin B fragment consisting of amino acids 547-2366 increa sed Rb-86(+) efflux when cells were exposed to low pH, Toxin B also induced pH-dependent channel formation in artificial lipid bilayer membranes. Clos tridium sordellii lethal toxin, another member of the family of large clost ridial cytotoxins, also induced increased Rb-86(+) release at low pH, The r esults suggest that large clostridial cytotoxins including C. difficile tox in B and C, sordellii lethal toxin undergo structural changes at low pH of endosomes that are accompanied by membrane insertion and channel formation.