title: Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.
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.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität, D-79104 Freiburg, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Egerer
authors.forename: Torsten
authors.initials: T
authors.lastname: Giesemann
authors.forename: Thomas
authors.initials: T
authors.lastname: Jank
authors.forename: Karla J Fullner
authors.initials: KJ
authors.lastname: Satchell
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016360:Clostridioides difficile / Q000201:enzymology*; D004229:Dithiothreitol / Q000737:chemistry; D004768:Enterotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007295:Inositol Phosphates / Q000737:chemistry; D007460:Iodoacetamide / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D007295:Inositol Phosphates; D011994:Recombinant Proteins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases; D010447:Peptide Hydrolases; D020741:rho GTP-Binding Proteins; D005033:Ethylmaleimide; D004229:Dithiothreitol; D007460:Iodoacetamide