title: Cell penetration of diphtheria toxin. Reduction of the interchain disulfide bridge is the rate-limiting step of translocation in the cytosol.
abstract: The pathway of cell penetration of diphtheria toxin (DT) was studied in Vero cells by following the kinetics of uptake, reduction, degradation, and sub-cellular distribution of 125I-DT in the absence or presence of bafilomycin A1 (baf-A1), a powerful inhibitor of the endosomal H(+)-ATPase. After a lag phase of 4 min, DT, bound to Vero cells, reached an acidic intracellular compartment, where about one-third of it underwent a transition to a state competent for subsequent reduction and membrane translocation. After further 4 min, this DT fraction was reduced in a baf-A1-insensitive reaction and DT-A, the intracellularly active protomer of DT, was immediately released in the cytosol. The present data indicate that cell-mediated reduction of the toxin is the rate-determining step of the DT cell intoxication process. Subcellular fractionation showed that DT underwent the low pH-driven conformational change in an early endosome, distinct from the subsequent endosomal compartment where reduction took place. DT-B remained endosome-bound and was proteolyzed at low pH as well as the portion of DT which was not reduced after the exposure to low pH in early endosomes.
authors.affiliation: Centro Consiglio Nazionale delle Richerche Biomembrane, Università di Padova, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Papini
authors.forename: R
authors.initials: R
authors.lastname: Rappuoli
authors.forename: M
authors.initials: M
authors.lastname: Murgia
authors.forename: C
authors.initials: C
authors.lastname: Montecucco
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport; D003600:Cytosol / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018942:Macrolides; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D014709:Vero Cells / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D004167:Diphtheria Toxin; D004220:Disulfides; D018942:Macrolides; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases