title: Cyclophilin-facilitated membrane translocation as pharmacological target to prevent intoxication of mammalian cells by binary clostridial actin ADP-ribosylated toxins.
abstract: Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 μM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.
authors: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.
authors: Katharina
authors: K
authors: Ernst
authors: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.
authors: Simon
authors: S
authors: Langer
authors: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.
authors: Eva
authors: E
authors: Kaiser
authors: Institute of Biophysics, University of Ulm, 89081 Ulm, Germany.
authors: Christian
authors: C
authors: Osseforth
authors: Institute of Biophysics, University of Ulm, 89081 Ulm, Germany.
authors: Jens
authors: J
authors: Michaelis
authors: Department of Anaerobic Bacteria, Pasteur Institute, 75724 Paris, France.
authors: Michel R
authors: MR
authors: Popoff
authors: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, 79104 Freiburg, Germany.
authors: Carsten
authors: C
authors: Schwan
authors: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, 79104 Freiburg, Germany.
authors: Klaus
authors: K
authors: Aktories
authors: Max Planck Research Unit for Enzymology of Protein Folding Halle, 06120 Halle (Saale), Germany.
authors: Viktoria
authors: V
authors: Kahlert
authors: Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany.
authors: Miroslav
authors: M
authors: Malesevic
authors: Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany. Electronic address: cordelia.schiene-fischer@biochemtech.uni-halle.de.
authors: Cordelia
authors: C
authors: Schiene-Fischer
authors: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany. Electronic address: holger.barth@uni-ulm.de.
authors: Holger
authors: H
authors: Barth
mesh_terms: D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000633:toxicity; D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000633:toxicity; D001905:Botulinum Toxins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000633:toxicity; D002522:Chlorocebus aethiops; D021983:Cyclophilins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport / Q000187:drug effects; D014709:Vero Cells; D000097322:Peptidyl-Prolyl Isomerase D
chemical_list: D036002:ADP Ribose Transferases; D000199:Actins; D000244:Adenosine Diphosphate; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001905:Botulinum Toxins; D021983:Cyclophilins; D016572:Cyclosporine; C050737:iota toxin, Clostridium perfringens; C410164:actin-specific ADP-ribosyltransferase, Clostridium; C114456:PPID protein, human; D000097322:Peptidyl-Prolyl Isomerase D; C096324:botulinum toxin type C
keywords: PPIase; bacterial protein toxin; cellular uptake; cyclophilin 40; membrane transport