1887

Abstract

An unknown cytotoxin was identified in the culture supernatant of type C. The cytotoxin, named TpeL, which was purified using mAb-based affinity chromatography, had a lethal activity of 62 minimum lethal dose (MLD) mg in mice and a cytotoxic activity of 6.2×10 cytotoxic units (CU) mg in Vero cells. The nucleotide sequence of TpeL was determined. The entire ORF had a length of 4953 bases, and the same nucleotide sequence was not recorded in the GenBank/EMBL/DDBJ databases. The molecular mass calculated from the deduced amino acid sequence was 191 kDa, and a signal peptide region was not found within the ORF. The deduced amino acid sequence exhibited 30–39 % homology to toxins A (TcdA) and B (TcdB), lethal toxin (TcsL) and alpha-toxin (TcnA). The amino acid sequence of TpeL is shorter than these toxins, and the homologous region was located at the N-terminal site. Eighteen strains of types A, B and C were surveyed for the presence of the gene by PCR. The gene was detected in all type B (one strain) and C strains (five strains), but not in any type A strains (12 strains). TpeL was detected in culture filtrates of the five type C strains by dot-blot analysis, but not in the type B strain. It was concluded that TpeL is a novel toxin similar to the known large clostridial cytotoxins. Furthermore, the data indicated that TpeL is produced by many type C strains.

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2007-04-01
2021-08-05
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References

  1. Altschul S. F., Madden T. L., Zhang J., Zhang Z., Miller W., Lipman D. J., Schäffer A. A. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
    [Google Scholar]
  2. Amimoto K., Sasaki O., Isogai M., Kitajima T., Oishi E., Okada N., Yasuhara H. 1998; The protective effect of Clostridium novyi type B alpha-toxoid against challenge with spores in guinea pigs. J Vet Med Sci 60:681–685 [CrossRef]
    [Google Scholar]
  3. Amimoto K., Oishi E., Yssuhara H., Sasaki O., Katayama S., Kitajima T., Izumida A., Hirahara T. 2001; Protective effects of Clostridium sordellii LT and HT toxoids against challenge with spores in guinea pigs. J Vet Med Sci 63:879–883 [CrossRef]
    [Google Scholar]
  4. Ball D. W., Van Tassell R. L., Roberts M. D., Hahn P. E., Lyerly D. M., Wilkins T. D. 1993; Purification and characterization of alpha-toxin produced by Clostridium novyi type A. Infect Immun 61:2912–2918
    [Google Scholar]
  5. Bendtsen J. D., Nielsen H., von Heijne G., Brunak S. 2004; Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795 [CrossRef]
    [Google Scholar]
  6. Busch C., Hofmann F., Gerhard R., Aktories K. 2000a; Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases. J Biol Chem 275:13228–13234 [CrossRef]
    [Google Scholar]
  7. Busch C., Hofmann F., Aktories K., Schömig K. 2000b; Characterization of the catalytic domain of Clostridium novyi alpha-toxin. Infect Immun 68:6378–6383 [CrossRef]
    [Google Scholar]
  8. Ciesla W. P. Jr, Bobak D. A. 1998; Clostridium difficile toxins A and B are cation-dependent UDP-glucose hydrolases with differing catalytic activities. J Biol Chem 273:16021–16026 [CrossRef]
    [Google Scholar]
  9. Duffy L. K., McDonel J. L., McClane B. A., Kurosky A. 1982; Clostridium perfringens type A enterotoxin: characterization of the amino-terminal region. Infect Immun 38:386–388
    [Google Scholar]
  10. Faust C., Ye B., Song K. P. 1998; The enzymatic domain of Clostridium difficile toxin A is located within its N-terminal region. Biochem Biophys Res Commun 251:100–105 [CrossRef]
    [Google Scholar]
  11. Felsenstein J. 1985; Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 [CrossRef]
    [Google Scholar]
  12. Gibert M., Jolivet-Reynaud C., Popoff M. R. 1997; Beta2-toxin, a novel toxin produced by Clostridium perfringens . Gene 203:65–73 [CrossRef]
    [Google Scholar]
  13. Herholz C., Miserez R., Nicolet J., Frey J., Popoff M., Gibert M., Gerber H., Straub R. 1999; Prevalence of beta2-toxigenic Clostridium perfringens in horses with intestinal disorders. J Clin Microbiol 37:358–361
    [Google Scholar]
  14. 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]
  15. Jolivet-Reynaud C., Cavaillon J. M., Alouf J. E. 1982; Selective cytotoxicity of Clostridium perfringens delta-toxin on rabbit leukocytes. Infect Immun 38:860–864
    [Google Scholar]
  16. Katayama S., Dupuy B., Daube G., China B., Cole S. T. 1996; Genome mapping of Clostridium perfringens strains with I- Ceu I shows many virulence genes to be plasmid-borne. Mol Gen Genet 251:720–726
    [Google Scholar]
  17. Kato H., Kato N., Watanabe K., Iwai N., Nakamura H., Yamamoto T., Suzuki K., Kim S. M., Chong Y., Wasito E. B. 1998; Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR. J Clin Microbiol 36:2178–2182
    [Google Scholar]
  18. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  19. Lawrence G., Walker P. D. 1976; Pathogenesis of enteritis necroticans in Papua New Guinea. Lancet 17:125–126
    [Google Scholar]
  20. Manteca C., Daube G., Jauniaux T., Linden A., Pirson V., Detilleux J., Ginter A., Coppe P., Kaeckenbeeck A., Mainil J. G. 2002; A role for the Clostridium perfringens β 2 toxin in bovine enterotoxaemia?. Vet Microbiol 86:191–202 [CrossRef]
    [Google Scholar]
  21. McDonel J. L., McClane B. A. 1981; Highly sensitive assay for Clostridium perfringens enterotoxin that uses inhibition of plating efficiency of Vero cells grown in culture. J Clin Microbiol 13:940–946
    [Google Scholar]
  22. Mollby R., Holme T. 1976; Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D. J Gen Microbiol 96:137–144 [CrossRef]
    [Google Scholar]
  23. Nagahama M., Kihara A., Miyawaki T., Mukai M., Sakaguchi Y., Ochi S., Sakurai J. 1999; Clostridium perfringens β -toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity. Biochim Biophys Acta 145497–105 [CrossRef]
    [Google Scholar]
  24. Nagahama M., Hayashi S., Morimitsu S., Sakurai J. 2003; Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells. J Biol Chem 278:36934–36941 [CrossRef]
    [Google Scholar]
  25. Nakamura S., Ogura H., Tanaka J., Tanabe N., Yamakawa K., Hatano M., Nishida S. 1984; Difference in susceptibility of various cell cultures to cytotoxic culture filtrates of Clostridium sordellii . Microbiol Immunol 28:493–497 [CrossRef]
    [Google Scholar]
  26. Niilo L. 1987; Toxigenic characteristics of Clostridium perfringens type C in enterotoxemia of domestic animals. Can J Vet Res 51:224–228
    [Google Scholar]
  27. O'Brien D. K., Melville S. B. 2004; Effects of Clostridium perfringens alpha-toxin (PLC) and perfringolysin O (PFO) on cytotoxicity to macrophages, on escape from the phagosomes of macrophages, and on persistence of C. perfringens in host tissues. Infect Immun 72:5204–5215 [CrossRef]
    [Google Scholar]
  28. Popoff M. R. 1987; Purification and characterization of Clostridium sordellii lethal toxin and cross-reactivity with Clostridium difficile cytotoxin. Infect Immun 55:35–43
    [Google Scholar]
  29. Reed L. J., Muench H. 1938; A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493–496
    [Google Scholar]
  30. Roehm N. W., Rodgers G. H., Hatfield S. M., Glasebrook A. L. 1991; An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods 142:257–265 [CrossRef]
    [Google Scholar]
  31. Rupnik M., Dupuy B., Fairweather N. F., Gerding D. N., Johnson S., Just I., Lyerly D. M., Popoff M. R., Rood J. I. & other authors 2005; Revised nomenclature of Clostridium difficile toxins and associated genes. J Med Microbiol 54:113–117 [CrossRef]
    [Google Scholar]
  32. Saitou N., Nei M. 1987; The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
    [Google Scholar]
  33. Shimizu T., Ohtani K., Hirakawa H., Ohshima K., Yamashita A., Shiba T., Ogasawara N., Hattori M., Kuhara S., Hayashi H. 2002; Complete genome sequence of Clostridium perfringens , an anaerobic flesh-eater. Proc Natl Acad Sci U S A 99:996–1001 [CrossRef]
    [Google Scholar]
  34. Singh U., Mitic L. L., Wieckowski E. U., Anderson J. M., McClane B. A. 2001; Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells. J Biol Chem 276:33402–33412 [CrossRef]
    [Google Scholar]
  35. Springer S., Selbitz H. J. 1999; The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine. FEMS Immunol Med Microbiol 24:333–336 [CrossRef]
    [Google Scholar]
  36. Thelestam M., Möllby R. 1975; Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. Infect Immun 12:225–232
    [Google Scholar]
  37. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [CrossRef]
    [Google Scholar]
  38. Titball R. W., Hunter S. E., Martin K. L., Morris B. C., Shuttleworth A. D., Rubidge T., Anderson D. W., Kelly D. C. 1989; Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens . Infect Immun 57:367–376
    [Google Scholar]
  39. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76:4350–4354 [CrossRef]
    [Google Scholar]
  40. Tsutsui K., Minami J., Matsushita O., Katayama S., Taniguchi Y., Nakamura S., Nishioka M., Okabe A. 1995; Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi . J Bacteriol 177:7164–7170
    [Google Scholar]
  41. Tweten R. K. 2001; Clostridium perfringens beta toxin and Clostridium septicum alpha toxin: their mechanisms and possible role in pathogenesis. Vet Microbiol 82:1–9 [CrossRef]
    [Google Scholar]
  42. von Eichel-Streiber C., Sauerborn M., Kuramitsu H. K. 1992; Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases. J Bacteriol 174:6707–6710
    [Google Scholar]
  43. Waters M., Savoie A., Garmory H. S., Bueschel D., Popoff M. R., Songer J. G., Titball R. W., McClane B. A., Sarker M. R. 2003; Genotyping and phenotyping of beta2-toxigenic Clostridium perfringens fecal isolates associated with gastrointestinal diseases in piglets. J Clin Microbiol 41:3584–3591 [CrossRef]
    [Google Scholar]
  44. Weiss K. F., Strong D. H. 1967; Some properties of heat-resistant and heat-sensitive strains of Clostridium perfringens . I. Heat resistance and toxigenicity. . J Bacteriol 93:21–26
    [Google Scholar]
  45. Yoo H. S., Lee S. U., Park K. Y., Park Y. H. 1997; Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR. J Clin Microbiol 35:228–232
    [Google Scholar]
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