1887

Abstract

Production of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the /, / and genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of A. The / expression level was about 100-fold less than those of the / and genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the /, / and genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 °C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 °C. High temperature did inhibit transcription, since expression levels of the /, / and genes were similar in cultures at 37 and 44 °C. However, incubation at 44 °C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In E, which contains no gene related to , the / and genes were also expressed during the transition phase, and no protease activation at 44 °C was evident.

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2006-03-01
2020-03-29
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References

  1. Bradshaw M, Dineen S. S, Maks N. D, Johnson E. A. 2004; Regulation of neurotoxin complex expression in Clostridium botulinum strains 62A, Hall A- hyper , and NCTC2916. Anaerobe 10:321–333[CrossRef]
    [Google Scholar]
  2. Broussolle V, Alberto F, Shearman C. A, Mason D. R, Botella L, Nguyen-The C, Peck M. W, Carlin F. 2002; Molecular and physiological characterization of spore germination in Clostridium botulinum and Clostridium sporogenes . Anaerobe8:89–100[CrossRef]
    [Google Scholar]
  3. Call J. E, Cooke P. H, Miller A. J. 1995; In situ characterization of Clostridium botulinum neurotoxin synthesis and export. J Appl Bacteriol79:257–263[CrossRef]
    [Google Scholar]
  4. Chen F, Kuziemko G. M, Stevens R. C. 1998; Biophysical characterization of the stability of the 150-kilodalton botulinum toxin, the nontoxic component, and the 900-kilodalton botulinum toxin complex species. Infect Immun66:2420–2425
    [Google Scholar]
  5. DasGupta B. R. 1989; The structure of the botulinum neurotoxins. In Botulinum Neurotoxin and Tetanus Toxin pp 53–67 Edited by Simpson L. L.. San Diego: Academic Press;
    [Google Scholar]
  6. DasGupta B. R, Sugiyama H. 1972; Role of protease in natural activation of Clostridium botulinum neurotoxin. Infect Immun6:587–590
    [Google Scholar]
  7. Dekleva M. L, DasGupta B. R. 1989; Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease. Biochem Biophys Res Commun162:767–772[CrossRef]
    [Google Scholar]
  8. Dekleva M. L, DasGupta B. R. 1990; Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into di-chain form. J Bacteriol172:2498–2503
    [Google Scholar]
  9. Dineen S. S, Bradshaw M, Karasek C. E, Johnson E. A. 2004; Nucleotide sequence and transcriptional analysis of the type A2 neurotoxin gene cluster in Clostridium botulinum . FEMS Microbiol Lett235:9–16[CrossRef]
    [Google Scholar]
  10. Dupuy B, Sonenshein A. L. 1998; Regulated transcription of Clostridium difficile toxin genes. Mol Microbiol27:107–120[CrossRef]
    [Google Scholar]
  11. Dupuy B, Mani N, Katayama S, Sonenshein A. L. 2005; Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor. Mol Microbiol55:1196–1206
    [Google Scholar]
  12. Fu F. N, Sharma S. K, Singh B. R. 1997; A protease-resistant novel hemagglutinin purified from type A Clostridium botulinum . J Protein Chem17:53–60
    [Google Scholar]
  13. Fujinaga Y, Inoue K, Watanabe S, Yokota K, Hirai Y, Nagamachi E, Oguma K. 1997; The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig intestine, leading to the efficient absorption of the toxin. Microbiology143:3841–3847[CrossRef]
    [Google Scholar]
  14. Fujinaga Y, Inoue K, Nomura T, Sasaki J, Marvaud J. C, Popoff M. R, Kozaki S, Oguma K. 2000; Identification and characterization of functional subunits of Clostridium botulinum A progenitor toxin involved in binding to intestinal microvilli and erythrocytes. FEBS Lett467:179–183[CrossRef]
    [Google Scholar]
  15. Garnier T, Cole S. T. 1988; Studies of UV-inducible promoters from Clostridium perfringens in vivo and in vitro . Mol Microbiol2:607–614[CrossRef]
    [Google Scholar]
  16. Hatheway C. L. 1989; Bacterial sources of clostridial neurotoxins. In Botulinum Neurotoxin and Tetanus Toxin pp 3–24 Edited by Simpson L. L.. San Diego: Academic Press;
    [Google Scholar]
  17. Hatheway C. L. 1993; Clostridium botulinum and other clostridia that produce botulinum neurotoxin. In Clostridium Botulinum: Ecology and Control in Foods pp 3–20 Edited by Hauschild A. H. W., Dodds K. L.. New York: Marcel Dekker;
    [Google Scholar]
  18. Henderson I, Whelan S. M, Davis T. O, Minton N. P. 1996; Genetic characterization of the botulinum toxin complex of Clostridium botulinum strain NCTC2916. FEMS Microbiol Lett140:151–158[CrossRef]
    [Google Scholar]
  19. Henderson I, Davis T, Elmore M, Minton N. 1997; The genetic basis of toxin production in Clostridium botulinum and Clostridium tetani. In The Clostridia: Molecular Biology and Pathogenesis pp 261–294 Edited by Rood I.. New York: Academic Press;
    [Google Scholar]
  20. Humeau Y, Doussau F, Grant N. J, Poulain B. 2000; How botulinum and tetanus neurotoxins block neurotransmitter. Biochimie82:427–446[CrossRef]
    [Google Scholar]
  21. Inoue K, Fujinaga Y, Watanabe T, Ohyama T, Takeshi K, Moriishi K, Nakajima H, Inoue K, Oguma K. 1996; Molecular composition of Clostridium botulinum type A progenitor toxins. Infect Immun64:1589–1594
    [Google Scholar]
  22. Jovita M. R, Collins M. D, East A. K. 1998; Gene organization and sequence determination of the two botulinum neurotoxin gene clusters in Clostridium botulinum . Curr Microbiol36:226–231[CrossRef]
    [Google Scholar]
  23. Karlsson S, Dupuy B, Mukherjee K, Norin E, Burman L. G, Akerlund T. 2003; Expression of Clostridium difficile toxins A and B and their sigma factor TcdD is controlled by temperature. Infect Immun71:1784–1793[CrossRef]
    [Google Scholar]
  24. Kimura B, Kawasaki S, Nakano H, Hujii T. 2001; Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish. Appl Environ Microbiol67:206–218[CrossRef]
    [Google Scholar]
  25. Krieglstein K. G, DasGupta B. R, Henschen A. H. 1994; Covalent structure of botulinum neurotoxin A: location of sulfhydryl bridges and identification of C-termini of light and heavy chains. J Protein Chem13:49–57[CrossRef]
    [Google Scholar]
  26. Lövenklev M, Holst E, Borch E, Radstrom P. 2004a; Relative neurotoxin gene expression in clostridium botulinum type B, determined using quantitative reverse transcription-PCR. Appl Environ Microbiol70:2919–2927[CrossRef]
    [Google Scholar]
  27. Lövenklev M, Artin I, Hagberg O, Borch E, Holst E, Radström P. 2004b; Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B. Appl Environ Microbiol70:2928–2934[CrossRef]
    [Google Scholar]
  28. Mani N, Dupuy B. 2001; Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A98:5844–5849[CrossRef]
    [Google Scholar]
  29. Marvaud J. C, Eisel U, Binz T, Niemann H, Popoff M. R. 1998a; tetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to botR . Infect Immun66:5698–5702
    [Google Scholar]
  30. Marvaud J. C, Gibert M, Inoue K, Fujinaga V, Oguma K, Popoff M. R. 1998b; botR/A is a positive regulator of botulinum neurotoxin and associated non-toxin protein genes in Clostridium botulinum A. Mol Microbiol29:1009–1018[CrossRef]
    [Google Scholar]
  31. McGrath S, Dooley J. S, Haylock R. W. 2000; Quantification of Clostridium botulinum toxin gene expression by competitive reverse transcription-PCR. Appl Environ Microbiol66:1423–1428[CrossRef]
    [Google Scholar]
  32. Meunier F. A, Herreros J, Schiavo G, Poulain B, Molgo J. 2002; Molecular mechanism of action of botulinal neurotoxins and the synaptic remodeling they induce in vivo at the skeletal neuromuscular junction. In Handbook of Neurotoxicology pp 305–347 Edited by Massaro J.. Totowa, NJ: Humana Press;
    [Google Scholar]
  33. Oguma K, Inoue K, Fujinaga Y, Yokota K, Watanabe T, Ohyama T, Takeshi K, Inoue K. 1999; Structure and function of Clostridium botulinum progenitor toxin. J Toxicol18:17–34
    [Google Scholar]
  34. Popoff M. R, Marvaud J. C. 1999; Structural and genomic features of clostridial neurotoxins. In The Comprehensive Sourcebook of Bacterial Protein Toxins pp 174–201 Edited by Alouf J. E., Freer J. H.. London: Academic Press;
    [Google Scholar]
  35. Porfirio Z, Prado S. M, Vancetto M. D. C, Fratelli F, Alves E. W, Raw I, Fernandes B. L, Camargo A. C. M, Lebrun I. 1997; Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin. J Appl Microbiol83:678–684[CrossRef]
    [Google Scholar]
  36. Quinn C. P, Minton N. P, Dürre P.. 2001; Clostridial neurotoxins. In Clostridia pp 211–250 Edited by Bahl H.. Weinheim: Wiley-VCH;
    [Google Scholar]
  37. Raffestin S, Dupuy B, Marvaud J. C, Popoff M. R. 2005; BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani . Mol Microbiol55:235–249
    [Google Scholar]
  38. Schiavo G, Matteoli M, Montecucco C. 2000; Neurotoxins affecting neuroexocytosis. Physiol Rev80:717–766
    [Google Scholar]
  39. Sharma S. K, Fu F. N, Singh B. R. 1999; Molecular properties of a hemagglutinin purified from type A Clostridium botulinum . J Protein Chem18:29–38[CrossRef]
    [Google Scholar]
  40. Sharma S. K, Ramzan M. A, Singh B. R. 2003; Separation of the components of type A botulinum neurotoxin complex by electrophoresis. Toxicon41:321–331[CrossRef]
    [Google Scholar]
  41. Siegel L. S, Metzger J. F. 1979; Toxin production by Clostridium botulinum type A under various fermentation conditions. Appl Environ Microbiol38:606–611
    [Google Scholar]
  42. Tavallaie M, Chenal A, Gillet D, Raffestin S, Popoff M. R, Marvaud J. C. 2004; Interaction between the two subdomains of the C-terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies. FEBS Lett572:299–306[CrossRef]
    [Google Scholar]
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