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J. Biol. Chem., Vol. 282, Issue 35, 25314-25321, August 31, 2007

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Auto-catalytic Cleavage of Clostridium difficile Toxins A and B Depends on Cysteine Protease Activity^*

Martina Egerer¹, Torsten Giesemann¹, Thomas Jank, Karla J. Fullner Satchell, and Klaus Aktories²

From the Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität, D-79104 Freiburg, Germany and the Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

The action of Clostridium difficile toxins A and B depends on processing and translocation of the catalytic glucosyltransferase domain into the cytosol of target cells where Rho GTPases are modified. Here we studied the processing of the toxins. Dithiothreitol and -mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into 250/210- and 63-kDa fragments. The 63-kDa fragment was identified by mass spectrometric analysis as the N-terminal glucosyltransferase domain. This cleavage was blocked by N-ethylmaleimide or iodoacetamide. Exchange of cysteine 698, histidine 653, or aspartate 587 of toxin B prevented cleavage of full-length recombinant toxin B and of an N-terminal fragment covering residues 1–955 and inhibited cytotoxicity of full-length toxin B. Dithiothreitol synergistically increased the effect of myo-inositol hexakisphosphate, which has been reported to facilitate auto-cleavage of toxin B (Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., Schild, H., and Von Eichel-Streiber, C. (2007) Nature 446, 415–419). N-Ethylmaleimide blocked auto-cleavage induced by the addition of myo-inositol hexakisphosphate, suggesting that cysteine residues are essential for the processing of clostridial glucosylating toxins. Our data indicate that clostridial glucosylating cytotoxins possess an inherent cysteine protease activity related to the cysteine protease of Vibrio cholerae RTX toxin, which is responsible for auto-cleavage of glucosylating toxins.

Received for publication, April 11, 2007 , and in revised form, June 22, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Projects GI684 and AK6/16, by the research group Klinische Infektiologie Freiburg, TP 1a, and by United States Public Health Services Grant AI051490 from the National Institutes of Health (to K. J. F. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Otto-Krayer-Haus, Albert-strasse 25, D-79104 Freiburg, Germany. Tel.: 49-761-2035301; Fax: 49-761-2035311; E-mail: Klaus.Aktories{at}pharmakol.uni-freiburg.de.

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