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 beta-mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into approximately 250/210- and approximately 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.

Highlights

  • Responsible for the diseases induced by C. difficile are two exotoxins, toxin A and B, that are produced by the pathogen [3,4,5]

  • More recently the autocatalytic cleavage of toxin B has been proposed on the basis of a putative inherent aspartate protease activity, which is increased in the presence of InsP6 [18]

  • MALDI-TOF mass spectrometric analysis revealed that cleavage occurred at the N-terminal region of the toxins where the toxin is suggested to be processed [16, 17]

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Summary

The abbreviations used are

InsP6, myo-inositol hexakisphosphate; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight. Auto-catalytic Cleavage of C. difficile Toxins mM L-glutamate, 100 units/ml penicillin, and 100 ␮g/ml streptomycin (PAN Biotech GmbH, Aidenbach, Germany). The cells were trypsinized and reseeded three times a week. (Invitrogen) and TG1 bacteria (Stratagene, La Jolla, CA) were cultivated in LB broth (Luria/Miller; Carl Roth GmbH, Karlsruhe, Germany) at 37 °C if not otherwise mentioned. Sense (cgcggatccatgagtttagttaatagaaaac) and CDB-955-NotI-antisense (stop) (cgcggccgcttattcgtgtgtagtatctaaatt) were purchased from Apara Bioscience GmbH (Freiburg, Germany). Dithiothreitol (DTT) and polyvinylidene difluoride membrane were from Carl Roth. GmbH, ␤-mercaptoethanol, iodoacetamide, InsP6, trypsin, and thrombin were purchased from Sigma-Aldrich; benzamidine-Sepharose 6B and glutathione-Sepharose 4B were from GE. Anti-Rac monoclonal antibody Clone 23A8 reacting with glucosylated and nonglucosylated Rac was from Upstate (Millipore GmbH, Eschborn, Germany); anti-Rac monoclonal antibody clone 102 recognizing only nonglucosylated Rac

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