1887

Abstract

, a human pathogen, produces two very large protein toxins, A and B (250–600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3–8 kb chromosomal DNA fragments of strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2·5–7·0 kb inserts representing a 10 kb region of the genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30–170 kDa) in strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300–600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.

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1989-01-01
2024-04-20
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References

  1. Banno Y., Kobayashi T., Kono K. 1984; Biochemical characterization and biological actions of two toxins (D-l and D-2) from Clostridium difficile. Review of Infectious Diseases 6:11–20
    [Google Scholar]
  2. Bartlett J.G., Chang T.W., Gurwitt M. 1978; Antibiotic associated pseudomembranous colitis due to toxin producing clostridia. New England Journal of Medicine 298:531–534
    [Google Scholar]
  3. Chang T.W., Sullivan N.M., Wilkins T.D. 1986; Insusceptibilityof fetal intestinal mucosa and fetal cells to Clostridium difficile toxins. Acta pharma-cologica sinica 7:448–453
    [Google Scholar]
  4. Eichel-Streiber C.v., Harperath U., Bosse D., Hadding U. 1987; Purification of two high molecular weight toxins of Clostridium difficile which are antigenetically related. Microbial Pathogenesis 2:307–318
    [Google Scholar]
  5. Eisel U., Jarausch W., Goretzki K., Henschen A., Engels J., Weller U., Hudel M., Haber-Mann E., Niemann H. 1986; Tetanus toxin: primary structure, expression in E. coli and homology with botulinum toxins. EMBO Journal 5:2495–2502
    [Google Scholar]
  6. Engers H.D., Houba V., Bennedsen J., Buchanan T.M., Chaparas S.D., Kadival G., Closs O., David J.R., Van Embden J.D.A., Godal T., Mustafa S.A., Ivanyi J., Young D.B., Kaufmann S.H.E., Khomenko A.G., Kolk A.H.J., Kubin M., Louis J.A., Minden P., Shinnick T.M., Trnka L., Young R.A. 1986; Results of a World Health Organization-sponsored workshop to characterize antigens recognized by mycobacterium-specific monoclonal antibodies. Infection and Immunity 51:718–720
    [Google Scholar]
  7. Fairweather N.F., Lyness V.A., Pickard D.J., Allen G., Thomson R.O. 1986; Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli. Journal of Bacteriology 165:21–27
    [Google Scholar]
  8. George R.H. 1986; The carrier state: Clostridium difficile. Journal of Antimicrobial Chemotherapy 18: (Suppl. A) 47–58
    [Google Scholar]
  9. Gottschalk G., Andreesen J.R., Hippe H. 1981; The genus Clostridium. In The Prokaryotes, a Handbook on the Habitats, Isolation and Identification of Bacteria pp. 1767–1803 Starr H., Callely A.G. Edited by London: Academic Press;
    [Google Scholar]
  10. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166:557–580
    [Google Scholar]
  11. Haymerle J., Herz G.B., Frank R., Stanley K.K. 1986; Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy. Nucleic Acids Research 14:8615–8624
    [Google Scholar]
  12. Howley P.M., Israel M.A., Law M.F., Martin M.A. 1979; A rapid method for detecting and mapping homology between heterologous DNAs. Journal of Biological Chemistry 254:4876–4883
    [Google Scholar]
  13. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  14. Laughon B.E., Viscidi R.P., Gdovin S.L., Yolken R.H., Bartlett J.G. 1984; Enzyme immunoassays for detection of Clostridium difficile toxins A and B in fecal specimens. Journal of Infectious Diseases 149:781–788
    [Google Scholar]
  15. Libby J.M., Wilkins T.D. 1982; Production of antitoxins to two toxins of Clostridium difficile and immunological comparison of the toxins by crossneutralisation studies. Infection and Immunity 35:374–376
    [Google Scholar]
  16. Lyerly D.M., Saum K.E., Macdonald D.K., Wilkins T.D. 1985a; Effects of Clostridium difficile toxins given intragastrically to animals. Infection and Immunity 47:349–352
    [Google Scholar]
  17. Lyerly D.M., Phelps C.J., Wilkins T.D. 1985b; Monoclonal and specific polyclonal antibodies for immunoassay of Clostridium difficile toxin A. Journal of Clinical Microbiology 21:12–14
    [Google Scholar]
  18. Lyerly D.M., Phelps C.J., Toth J., Wilkins T.D. 1986a; Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies. Infection and Immunity 54:70–76
    [Google Scholar]
  19. Lyerly D.M., Roberts M.D., Phelps C.J., Wilkins T.D. 1986b; Purification and properties of toxins A and B of Clostridium difficile. FEMS Microbiology Letters 33:31–35
    [Google Scholar]
  20. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Messing J. 1979; A multipurpose cloning system based on the single-stranded DNA bacteriophage Ml 3. In Recombinant DNA Technical Bulletin, part 2, pp. 43–48 NIH Publication no.79-99
    [Google Scholar]
  22. Middlebrook J.L., Dorland R.B. 1984; Bacterial toxins: cellular mechanisms of action. Microbiological Reviews 48:199–221
    [Google Scholar]
  23. Miura K.I. 1967; Preparation of bacterial DNA by phenol-pH9-RNase method. Methods in Enzymology 12:543–545
    [Google Scholar]
  24. Muldrow L.L., Ibeanu G.C., Lee N.I., Bose N.K., Johnson J. 1987; Molecular cloning of Clostridium difficile toxin A gene fragment in Agtll. FEBS Letters 213:249–253
    [Google Scholar]
  25. Pothoulakis C., Barone L.M., Ely R., Faris B., Clark M.E., Franzblau C., Lamont J.T. 1986; Purification and properties of Clostridium difficile cytotoxin B. FEMS Microbiology Letters 33:31–35
    [Google Scholar]
  26. Reed K.C., Mann D.A. 1985; Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Research 13:7207–7221
    [Google Scholar]
  27. Rigby P.W.J., Diekman M., Rhodes C., Berg P. 1977; Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. Journal of Molecular Biology 113:237–251
    [Google Scholar]
  28. Rihn B., Scheftel J.M., Girardot R. 1984; A new purification procedure for Clostridium difficile enterotoxin. Biochemical and Biophysical Research Communications 124:690–695
    [Google Scholar]
  29. Southern E.M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503–517
    [Google Scholar]
  30. Stanley K.K. 1983; Solubilization and immunedetection of β-galactosidase hybrid proteins carrying foreign antigenic determinants. Nucleic Acids Research 11:4077–4092
    [Google Scholar]
  31. Stanley K.K., Luzio J.P. 1984; Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO Journal 3:1429–1434
    [Google Scholar]
  32. Sullivan N.M., Pellett S., Wilkins T.D. 1982; Purification and characterization of toxins A and B of Clostridium difficile. Infection and Immunity 35:1032–1040
    [Google Scholar]
  33. Timmis K.N., Montenegro M.A., Bulling E., Chakraborty T., Sanyal S. 1984 Genetics of Toxin Synthesis in Pathogenic Gram-negative Enteric Bacteria, pp. 13–27 London: Academic Pressy;
    [Google Scholar]
  34. Volkaert G. 1987; A systematic approach to chemical DNA sequencing by subcloning into pGV451 and derived vectors. Methods in Enzymology 155:231–251
    [Google Scholar]
  35. Yanisch-Perron C., Vieria J., Messing J. 1985; Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the MBmp 18 and pUC19 vectors. Gene 33:103–119
    [Google Scholar]
  36. Young R.A., Bloom B.R., Grosskinsky C.M., Ivanyi J., Thomas D. 1985; Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proceedings of the National Academy of Sciences of the United States of America 82:2583–2587
    [Google Scholar]
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