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Autocatalytic cleavage of Clostridium difficile toxin B

Nature volume 446, pages 415–419 (2007)Cite this article

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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Figure 1: Purified cytosolic splenocyte extract in the in vitro TcdB cleavage assay and mass spectrometry.
Figure 2: In vitro cleavage of LCTs by purified splenocyte extracts or inositolhexaphosphate.
Figure 3: An integrated model of events necessary for the cellular cytotoxic action of TcdB.

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Acknowledgements

We thank H. Müller, J. Forsch, A. Malotta, N. Robertz and E. Scholz for technical assistance; M. Moos, C. Srokowski and C. Tertilt for critically discussing the manuscript; and A. Lange for his advice in affinity purification of TcdB. Special thanks to M. Popoff for providing C. sordellii TcsL of high quality. This work was supported by grants from the Stiftung Innovation Rheinland-Pfalz and Deutsche Forschungs Gemeinschaft (to C.v.E.-S.). S.T. and H.S. were supported by the Sonderforschungsbereich ‘Invasion und Persistenz bei Infektionen’, the Hochschulbauförderungsgesetz Program (H.S.), and the Immunology Cluster of Excellence (ICE) at the University of Mainz (H.S.). M.R. was supported by an EMBO grant. C.v.E.-S. acknowledges the support of the University of Mainz for offering additional laboratory space in the Verfügungsgebäude für Forschung und Entwicklung.

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Author notes

  1. Jessica Reineke and Stefan Tenzer: These authors contributed equally to this work.

Authors and Affiliations

  1. Johannes-Gutenberg Universität Mainz, Institut für medizinische Mikrobiologie and Hygiene, Hochhaus am Augustusplatz, 55131 Mainz, Germany,

    Jessica Reineke, Oliver Hasselmayer & Christoph von Eichel-Streiber

  2. Johannes-Gutenberg Universität Mainz, Institut für Immunologie, Hochhaus am Augustusplatz, 55131 Mainz, Germany,

    Stefan Tenzer & Hansjörg Schild

  3. University of Maribor, Faculty of Medicine, Slomskov trg 15 and Institute of Public Health Maribor, Prvomajska 1, 2000 Maribor, Slovenia,

    Maja Rupnik

  4. Justus-Liebig Universität Giessen, Rudolf-Buchheim-Institut für Pharmakologie, Frankfurter Strasse 107, 35392 Giessen, Germany,

    Andreas Koschinski

  5. ProteoSys AG, Carl-Zeiss-Strasse 51, 55129 Mainz, Germany,

    André Schrattenholz

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Correspondence to Hansjörg Schild or Christoph von Eichel-Streiber.

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Reineke, J., Tenzer, S., Rupnik, M. et al. Autocatalytic cleavage of Clostridium difficile toxin B. Nature 446, 415–419 (2007). https://doi.org/10.1038/nature05622

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