title: Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells.
abstract: Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (approximately 65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Otto-Krayer-Haus, Albertstrasse 25, D-79104 Freiburg, Germany.
authors.forename: Gunther
authors.initials: G
authors.lastname: Pfeifer
authors.forename: Jörg
authors.initials: J
authors.lastname: Schirmer
authors.forename: Jost
authors.initials: J
authors.lastname: Leemhuis
authors.forename: Christian
authors.initials: C
authors.lastname: Busch
authors.forename: Dieter K
authors.initials: DK
authors.lastname: Meyer
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; C031815:toxB protein, Clostridium difficile; D005982:Glutathione Transferase