1. Johannes-Gutenberg Universität Mainz, Institut für medizinische Mikrobiologie and Hygiene, Hochhaus am Augustusplatz, 55131 Mainz, Germany
2. Johannes-Gutenberg Universität Mainz, Institut für Immunologie, Hochhaus am Augustusplatz, 55131 Mainz, Germany
3. University of Maribor, Faculty of Medicine, Slomskov trg 15 and Institute of Public Health Maribor, Prvomajska 1, 2000 Maribor, Slovenia
4. Justus-Liebig Universität Giessen, Rudolf-Buchheim-Institut für Pharmakologie, Frankfurter Strasse 107, 35392 Giessen, Germany
5. ProteoSys AG, Carl-Zeiss-Strasse 51, 55129 Mainz, Germany
6. These authors contributed equally to this work.

Correspondence to: Hansjörg Schild²Christoph von Eichel-Streiber¹ Correspondence and requests for materials should be addressed to C.v.E.-S. (Email: veichel@uni-mainz.de) or H.S. (Email: schild@uni-mainz.de).

Abstract

Clostridium difficile, the causative agent of nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis, possesses two main virulence factors: the large clostridial cytotoxins A and B. It has been proposed that toxin B is cleaved by a cytosolic factor of the eukaryotic target cell during its cellular uptake. Here we report that cleavage of not only toxin B, but also all other large clostridial cytotoxins, is an autocatalytic process dependent on host cytosolic inositolphosphate cofactors. A covalent inhibitor of aspartate proteases, 1,2-epoxy-3-(p-nitrophenoxy)propane, completely blocked toxin B function on cultured cells and was used to identify its catalytically active protease site. To our knowledge this is the first report on a bacterial toxin that uses eukaryotic signals for induced autoproteolysis to deliver its toxic domain into the cytosol of target cells. On the basis of our data, we present an integrated model for the uptake and inositolphosphate-induced activation of toxin B.

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