title: Pyknotic cell death induced by Clostridium difficile TcdB: chromatin condensation and nuclear blister are induced independently of the glucosyltransferase activity.
abstract: TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation.
authors.affiliation: Institute of Toxicology, Medical School Hannover, Hannover, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Wohlan
authors.forename: Sebastian
authors.initials: S
authors.lastname: Goy
authors.forename: Alexandra
authors.initials: A
authors.lastname: Olling
authors.forename: Sangar
authors.initials: S
authors.lastname: Srivaratharajan
authors.forename: Helma
authors.initials: H
authors.lastname: Tatge
authors.forename: Harald
authors.initials: H
authors.lastname: Genth
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D016923:Cell Death; D002460:Cell Line; D002462:Cell Membrane / Q000473:pathology; D002467:Cell Nucleus / Q000473:pathology*; D002843:Chromatin / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D006031:Glycosylation; D022781:Hepatocytes / Q000187:drug effects / Q000473:pathology; D006657:Histones / Q000378:metabolism; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D009685:Nuclear Envelope / Q000473:pathology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017382:Reactive Oxygen Species / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002843:Chromatin; D006657:Histones; D050505:Mutant Proteins; D017382:Reactive Oxygen Species; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases; D020741:rho GTP-Binding Proteins