1887

Abstract

The toxins A and B are primarily responsible for symptoms of associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of that lacks the toxin genes and . TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a toxoid vaccine.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.066712-0
2013-07-01
2022-01-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/7/1254.html?itemId=/content/journal/micro/10.1099/mic.0.066712-0&mimeType=html&fmt=ahah

References

  1. Antunes A., Camiade E., Monot M., Courtois E., Barbut F., Sernova N. V., Rodionov D. A., Martin-Verstraete I., Dupuy B.( 2012). Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile. Nucleic Acids Res 40:10701–10718 [View Article][PubMed]
    [Google Scholar]
  2. Barth H., Pfeifer G., Hofmann F., Maier 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 [View Article][PubMed]
    [Google Scholar]
  3. Braun V., Hundsberger T., Leukel P., Sauerborn M., von Eichel-Streiber C.( 1996). Definition of the single integration site of the pathogenicity locus in Clostridium difficile. Gene 181:29–38 [View Article][PubMed]
    [Google Scholar]
  4. Busch C., Hofmann F., Selzer J., Munro S., Jeckel D., Aktories K.( 1998). A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J Biol Chem 273:19566–19572 [View Article][PubMed]
    [Google Scholar]
  5. Chumbler N. M., Farrow M. A., Lapierre L. A., Franklin J. L., Haslam D., Goldenring J. R., Lacy D. B.( 2012). Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessing-independent mechanism. PLoS Pathog 8:e1003072 [View Article][PubMed]
    [Google Scholar]
  6. Dineen S. S., Villapakkam A. C., Nordman J. T., Sonenshein A. L.( 2007). Repression of Clostridium difficile toxin gene expression by CodY. Mol Microbiol 66:206–219 [View Article][PubMed]
    [Google Scholar]
  7. Egerer M., Giesemann T., Jank T., Satchell K. J. F., Aktories K.( 2007). Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity. J Biol Chem 282:25314–25321 [View Article][PubMed]
    [Google Scholar]
  8. Fang A., Gerson D. F., Demain A. L.( 2009). Production of Clostridium difficile toxin in a medium totally free of both animal and dairy proteins or digests. Proc Natl Acad Sci U S A 106:13225–13229 [View Article][PubMed]
    [Google Scholar]
  9. Genisyuerek S., Papatheodorou P., Guttenberg G., Schubert R., Benz R., Aktories K.( 2011). Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B. Mol Microbiol 79:1643–1654 [View Article][PubMed]
    [Google Scholar]
  10. Gerhard R., Nottrott S., Schoentaube J., Tatge H., Olling A., Just I.( 2008). Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells. J Med Microbiol 57:765–770 [View Article][PubMed]
    [Google Scholar]
  11. Giesemann T., Jank T., Gerhard R., Maier E., Just I., Benz R., Aktories K.( 2006). Cholesterol-dependent pore formation of Clostridium difficile toxin A. J Biol Chem 281:10808–10815 [View Article][PubMed]
    [Google Scholar]
  12. Gonçalves C., Decré D., Barbut F., Burghoffer B., Petit J.-C.( 2004). Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile. J Clin Microbiol 42:1933–1939 [View Article][PubMed]
    [Google Scholar]
  13. Govind R., Dupuy B.( 2012). Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE. PLoS Pathog 8:e1002727 [View Article][PubMed]
    [Google Scholar]
  14. Heap J. T., Pennington O. J., Cartman S. T., Carter G. P., Minton N. P.( 2007). The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70:452–464 [View Article][PubMed]
    [Google Scholar]
  15. Heap J. T., Pennington O. J., Cartman S. T., Minton N. P.( 2009). A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78:79–85 [View Article][PubMed]
    [Google Scholar]
  16. Heap J. T., Cartman S. T., Kuehne S. A., Cooksley C., Minton N. P.( 2010). ClosTron-targeted mutagenesis. Methods Mol Biol 646:165–182 [View Article][PubMed]
    [Google Scholar]
  17. Hussain H. A., Roberts A. P., Mullany P.( 2005). Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Δerm) and demonstration that the conjugative transposon Tn916ΔE enters the genome of this strain at multiple sites. J Med Microbiol 54:137–141 [View Article][PubMed]
    [Google Scholar]
  18. Jank T., Aktories K.( 2008). Structure and mode of action of clostridial glucosylating toxins: the ABCD model. Trends Microbiol 16:222–229 [View Article][PubMed]
    [Google Scholar]
  19. Jank T., Giesemann T., Aktories K.( 2007a). Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. Glycobiology 17:15R–22R [View Article][PubMed]
    [Google Scholar]
  20. Jank T., Giesemann T., Aktories K.( 2007b). Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding. J Biol Chem 282:35222–35231 [View Article][PubMed]
    [Google Scholar]
  21. Ke N., Wang X., Xu X., Abassi Y. A.( 2011). The xCELLigence system for real-time and label-free monitoring of cell viability. Methods Mol Biol 740:33–43 [View Article][PubMed]
    [Google Scholar]
  22. Kuehne S. A., Cartman S. T., Heap J. T., Kelly M. L., Cockayne A., Minton N. P.( 2010). The role of toxin A and toxin B in Clostridium difficile infection. Nature 467:711–713 [View Article][PubMed]
    [Google Scholar]
  23. Li S., Shi L., Yang Z., Feng H.( 2013). Cytotoxicity of Clostridium difficile toxin B does not require cysteine protease-mediated autocleavage and release of the glucosyltransferase domain into the host cell cytosol. Pathog Dis 67:11–18 [View Article][PubMed]
    [Google Scholar]
  24. Libby J. M., Jortner B. S., Wilkins T. D.( 1982). Effects of the two toxins of Clostridium difficile in antibiotic-associated cecitis in hamsters. Infect Immun 36:822–829[PubMed]
    [Google Scholar]
  25. Louie T. J., Miller M. A., Mullane K. M., Weiss K., Lentnek A., Golan Y., Gorbach S., Sears P., Shue Y. K.OPT-80-003 Clinical Study Group( 2011). Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med 364:422–431 [View Article][PubMed]
    [Google Scholar]
  26. Lowy I., Molrine D. C., Leav B. A., Blair B. M., Baxter R., Gerding D. N., Nichol G., Thomas W. D. Jr, Leney M. et al.( 2010). Treatment with monoclonal antibodies against Clostridium difficile toxins. N Engl J Med 362:197–205 [View Article][PubMed]
    [Google Scholar]
  27. Lyerly D. M., Saum K. E., MacDonald D. K., Wilkins T. D.( 1985). Effects of Clostridium difficile toxins given intragastrically to animals. Infect Immun 47:349–352[PubMed]
    [Google Scholar]
  28. Reineke J., Tenzer S., Rupnik M., Koschinski A., Hasselmayer O., Schrattenholz A., Schild H., von Eichel-Streiber C.( 2007). Autocatalytic cleavage of Clostridium difficile toxin B. Nature 446:415–419 [View Article][PubMed]
    [Google Scholar]
  29. Rupnik M., Wilcox M. H., Gerding D. N.( 2009). Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7:526–536 [View Article][PubMed]
    [Google Scholar]
  30. Salcedo J., Keates S., Pothoulakis C., Warny M., Castagliuolo I., LaMont J. T., Kelly C. P.( 1997). Intravenous immunoglobulin therapy for severe Clostridium difficile colitis. Gut 41:366–370 [View Article][PubMed]
    [Google Scholar]
  31. Sambol S. P., Merrigan M. M., Tang J. K., Johnson S., Gerding D. N.( 2002). Colonization for the prevention of Clostridium difficile disease in hamsters. J Infect Dis 186:1781–1789 [View Article][PubMed]
    [Google Scholar]
  32. Sebaihia M., Wren B. W., Mullany P., Fairweather N. F., Minton N., Stabler R., Thomson N. R., Roberts A. P., Cerdeño-Tárraga A. M. et al.( 2006). The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38:779–786 [View Article][PubMed]
    [Google Scholar]
  33. Simor A. E., Bradley S. F., Strausbaugh L. J., Crossley K., Nicolle L. E.SHEA Long-Term-Care Committee( 2002). Clostridium difficile in long-term-care facilities for the elderly. Infect Control Hosp Epidemiol 23:696–703 [View Article][PubMed]
    [Google Scholar]
  34. Sougioultzis S., Kyne L., Drudy D., Keates S., Maroo S., Pothoulakis C., Giannasca P. J., Lee C. K., Warny M. et al.( 2005). Clostridium difficile toxoid vaccine in recurrent C. difficile-associated diarrhea. Gastroenterology 128:764–770 [View Article][PubMed]
    [Google Scholar]
  35. Stephenson K., Lewis R. J.( 2005). Molecular insights into the initiation of sporulation in Gram-positive bacteria: new technologies for an old phenomenon. FEMS Microbiol Rev 29:281–301 [View Article][PubMed]
    [Google Scholar]
  36. Teichert M., Tatge H., Schoentaube J., Just I., Gerhard R.( 2006). Application of mutated Clostridium difficile toxin A for determination of glucosyltransferase-dependent effects. Infect Immun 74:6006–6010 [View Article][PubMed]
    [Google Scholar]
  37. Underwood S., Guan S., Vijayasubhash V., Baines S. D., Graham L., Lewis R. J., Wilcox M. H., Stephenson K.( 2009). Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J Bacteriol 191:7296–7305 [View Article][PubMed]
    [Google Scholar]
  38. von Eichel-Streiber C., Laufenberg-Feldmann R., Sartingen S., Schulze J., Sauerborn M.( 1992). Comparative sequence analysis of the Clostridium difficile toxins A and B. Mol Gen Genet 233:260–268 [View Article][PubMed]
    [Google Scholar]
  39. Whitman C. B., Czosnowski Q. A.( 2012). Fidaxomicin for the treatment of Clostridium difficile infections. Ann Pharmacother 46:219–228 [View Article][PubMed]
    [Google Scholar]
  40. Zemljic M., Rupnik M., Scarpa M., Anderluh G., Palù G., Castagliuolo I.( 2010). Repetitive domain of Clostridium difficile toxin B exhibits cytotoxic effects on human intestinal epithelial cells and decreases epithelial barrier function. Anaerobe 16:527–532 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.066712-0
Loading
/content/journal/micro/10.1099/mic.0.066712-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error