Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity

Martina Egerer, Torsten Giesemann, Thomas Jank, Karla J. Fullner Satchell, Klaus Aktories*

*Corresponding author for this work

Research output: Contribution to journalArticle

135 Scopus citations

Abstract

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 β-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.

Original languageEnglish (US)
Pages (from-to)25314-25321
Number of pages8
JournalJournal of Biological Chemistry
Volume282
Issue number35
DOIs
StatePublished - Aug 31 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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