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Abstract

toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB between Leu and Gly and the naturally occurring variant TcdB between Leu and Gly. The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB) and 62 761 Da (TcdB)] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da (TcdB) and 206 245 Da (TcdB)] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.

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2005-01-01
2024-11-02
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References

  1. Barth H., Pfeifer G., Hofmann F., Meier E., Benz R., Aktories K. 2001; Low pH-induced formation of ion channels by Clostridium difficile toxin B in target cells. J Biol Chem 276:10670–10676 [CrossRef]
    [Google Scholar]
  2. Borriello S. P., Wren B. W., Hyde S., Seddon S. V., Sibbons P., Krishna M. M., Tabaqchali S., Manek S., Price A. B. 1992; Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile . Infect Immun 60:4192–4199
    [Google Scholar]
  3. Castagliuolo I., Riegler M. F., Valenick L., LaMont J. T., Pothoulakis C. 1999; Saccharomyces boulardii protease inhibits the effects of Clostridium difficile toxins A and B in human colonic mucosa. Infect Immun 67:302–307
    [Google Scholar]
  4. Chaves-Olarte E., Florin I., Boquet P., Popoff M., Eichel-Streiber C. v., Thelestam M. 1996; UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii . J Biol Chem 271:6925–6932 [CrossRef]
    [Google Scholar]
  5. Chaves-Olarte E., Freer E., Parra A., Guzman-Verri C., Moreno E., Thelestam M. 2003; R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile . J Biol Chem 278:7956–7963 [CrossRef]
    [Google Scholar]
  6. Eichel-Streiber C. v., Boquet P., Sauerborn M., Thelestam M. 1996; Large clostridial cytotoxins – a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol 4:375–382 [CrossRef]
    [Google Scholar]
  7. Falnes P. Ø., Sandvig K. 2000; Penetration of protein toxins into cells. Curr Opin Cell Biol 12:407–413 [CrossRef]
    [Google Scholar]
  8. Florin I., Thelestam M. 1983; Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts. Biochim Biophys Acta 763:383–392 [CrossRef]
    [Google Scholar]
  9. Florin I., Thelestam M. 1986; Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B. Microb Pathog 1:373–385 [CrossRef]
    [Google Scholar]
  10. Frisch C., Gerhard R., Aktories K., Hofmann F., Just I. 2002; The complete receptor-binding domain of Clostridium difficile toxin A is required for endocytosis. Biochem Biophys Res Commun 300:706–711
    [Google Scholar]
  11. Fryling C., Ogata M., FitzGerald D. 1992; Characterization of a cellular protease that cleaves Pseudomonas exotoxin. Infect Immun 60:497–502
    [Google Scholar]
  12. Gordon V. M., Leppla S. H. 1994; Proteolytic activation of bacterial toxins: role of bacterial and host cell proteases. Infect Immun 62:333–340
    [Google Scholar]
  13. Gordon V., Klimpel K. R., Arora N., Henderson M. A., Leppla S. H. 1995; Proteolytc activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases. Infect Immun 63:82–87
    [Google Scholar]
  14. Hartmuth K., Urlaub H., Vornlocher H.-P., Will C. L., Gentzel M., Wilm M., Lührmann R. 2002; Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method. Proc Natl Acad Sci U S A 99:16719–16724 [CrossRef]
    [Google Scholar]
  15. Hofmann F., Busch C., Prepens U., Just I., Aktories K. 1997; Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin. J Biol Chem 272:11074–11078 [CrossRef]
    [Google Scholar]
  16. Jean F., Thomas L., Molloy S. S., Liu G., Jarvis M. A., Nelson J. A., Thomas G. 2000; A protein-based therapeutic for human cytomegalovirus infection. Proc Natl Acad Sci U S A 97:2864–2869 [CrossRef]
    [Google Scholar]
  17. Johnson S., Gerding D. 1997; Clostridium difficile -associated diarrhea. Clin Infect Dis 26:1027–1036
    [Google Scholar]
  18. Kariazova L. K., Montal M. 2003; Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nat Struct Biol 10:13–17 [CrossRef]
    [Google Scholar]
  19. Krieglstein K. G., Henschen A. H., Weller U., Habermann E. 1991; Limited proteolysis of tetanus toxin. Relation to activity and identification of cleavage sites. Eur J Biochem 202:41–51 [CrossRef]
    [Google Scholar]
  20. Lencer I. L., Constable C., Moe S. 7 other authors 1997; Proteolytic activation of cholera toxin and Escherichia coli labile toxin by entry into epithelial cells. J Biol Chem 272:15562–15568 [CrossRef]
    [Google Scholar]
  21. Molloy S. S., Bresnahan P. A., Leppla S. H., Klimpel K. R., Thomas G. 1992; Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402
    [Google Scholar]
  22. Moos M., Eichel-Streiber C. v. 2000; Purification and evaluation of large clostridial cytotoxins that inhibit small GTPases Rho and Ras subfamilies. Methods Enzymol 325:114–125
    [Google Scholar]
  23. Oh K. J., Senzel L., Collier R. J., Finkelstein A. 1999; Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain. Proc Natl Acad Sci U S A 96:8467–8470 [CrossRef]
    [Google Scholar]
  24. Olsnes S., Wesche J., Falnes P. Ø. 1999; Binding, uptake, routing and translocation of toxins with intracellular sites of action. In Comprehensive Sourcebook of Bacterial Protein Toxins pp 73–93 Edited by Alouf J. E, Freer J. H. London: Academic Press;
    [Google Scholar]
  25. Pfeifer G., Schirmer J., Leemhuis J., Busch C., Meyer D. K., Aktories K., Barth H. 2003; Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. J Biol Chem 278:44535–44541 [CrossRef]
    [Google Scholar]
  26. Qa'Dan M., Spyres L. M., Ballard J. D. 2000; pH-induced conformational changes in Clostridium difficile toxin B. Infect Immun 68:2470–2474 [CrossRef]
    [Google Scholar]
  27. Qa'Dan M., Spyres L. M., Ballard J. D. 2001; pH-enhanced cytopathic effect of Clostridium difficile lethal toxin. Infect Immun 69:5487–5493 [CrossRef]
    [Google Scholar]
  28. Qa'Dan M., Ramsey M., Daniel J., Spyres L. M., Safiejko-Mroczka B., Ortiz-Leduc W., Ballard J. D. 2002; Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells. Cell Microbiol 4:425–434 [CrossRef]
    [Google Scholar]
  29. Sauerborn M., Hegenbarth S., Laufenberg-Feldmann R., Leukel P., Eichel-Streiber C. v. 1994; Monoclonal antibodies discriminating between Clostridium difficile toxins A and B. Zentralbl Bakteriol suppl 24:510–511
    [Google Scholar]
  30. Shevchenko A., Wilm M., Vorm O., Mann M. 1996; Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858 [CrossRef]
    [Google Scholar]
  31. Soehn F., Wagenknecht-Wiesner A., Leukel P., Kohl M., Weidman M., Eichel-Streiber C. v., Braun V. 1998; Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864 – implications for transcription, expression and enzymatic activity of toxins A and B. Mol Gen Genet 258:222–232 [CrossRef]
    [Google Scholar]
  32. Thelestam M., Chaves-Olarte E. 2000; Cytotoxic effects of the Clostridium difficile toxins. Curr Top Microbiol Immunol 250:85–96
    [Google Scholar]
  33. Thelestam M., Florin I., Chaves-Olarte E. 1997; Clostridium difficile toxins. In Bacterial Toxins, Tools in Cell Biology and Pharmacology pp 141–158 Edited by Aktories K. Weinheim: Chapman & Hall;
    [Google Scholar]
  34. Wagenknecht-Wiesner A., Weidman M., Braun V., Leukel P., Moos M., Eichel-Streiber C. v. 1997; Delineation of the catalytic domain of Clostridium difficile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment. FEMS Microbiol Lett 152:109–116 [CrossRef]
    [Google Scholar]
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