1887

Abstract

Summary

After sonic disintegration of cells, intracellular toxin A was purified to homogeneity by thyroglobulin affinity chromatography (TGAC) followed by anion-exchange (Mono Q) by fast protein liquid chromatography (FPLC). High haemagglutinating (HA) activity was detected in TGAC-unbound fractions (2/50 μl), but not in TGAC thermal eluates (2/50 μl). The low HA titre of the thermal eluates was markedly increased to 2/50 μl) after dialysis against 0.02 M Tris-HCI (pH 7.5). A disparity in the position of the peaks containing cytotoxic and HA activity was observed in the first Mono Q-FPLC step. Intracellular toxin A without HA activity was obtained by a second Mono Q-FPLC step. The M of the intracellular toxin A was estimated by polyacrylamide gel electrophoresis (PAGE) to be 580 kDa under non-denaturing conditions. The minimum doses of the toxin causing cytotoxicity, mouse lethality and enterotoxicity were 0.83 ng, 8.7 ng and 5 μg, respectively.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/00222615-38-1-69
1993-01-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/38/1/medmicro-38-1-69.html?itemId=/content/journal/jmm/10.1099/00222615-38-1-69&mimeType=html&fmt=ahah

References

  1. Sullivan NM, Pellett S, Wilkins TD. Purification and characterization of toxins A and B of Clostridium difficile. Infect Immun 1982; 35:1032–1040
    [Google Scholar]
  2. Banno Y, Kobayashi T, Kono H, Watanabe K, Ueno K, Nozawa Y. Biochemical characterization and biologic actions of two toxins (D-l and D-2) from Clostridium difficile. Rev Infect Dis 1984; 6:Suppl 1S11–S20
    [Google Scholar]
  3. Lyerly DM, Roberts MD, Phelps CJ, Wilkins TD. Purification and properties of toxins A and B of Clostridium difficile. FEMS Microbiol Lett 1986; 33:31–35
    [Google Scholar]
  4. Lyerly DM, Krivan HC, Wilkins TD. Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1988; 1:1–18
    [Google Scholar]
  5. Borriello SP, Welch AR, Barclay FE, Davies HA. Mucosal association by Clostridium difficile in the hamster gastrointestinal tract. J Med Microbiol 1988; 25:191–196
    [Google Scholar]
  6. Seddon SV, Hemmingway I, Borriello SP. Hydrolytic enzyme production by Clostridium difficile and its relationship to toxin production and virulence in the hamster model. J Med Microbiol 1990; 31:169–174
    [Google Scholar]
  7. Borriello SP, Davies HA, Barclay FE. Detection of fimbriae amongst strains of Clostridium difficile. FEMS Microbiol Lett 1988; 49:65–67
    [Google Scholar]
  8. Davies HA, Borriello SP. Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity. Microbial Pathogen 1990; 9:141–146
    [Google Scholar]
  9. Krivan HC, Wilkins TD. Purification of Clostridium difficile toxin A by affinity chromatography on immobilized thyroglobulin. Infect Immun 1987; 55:1873–1877
    [Google Scholar]
  10. Kamiya S, Reed PJ, Borriello SP. Purification and characterization of Clostridium difficile toxin A by bovine thyroglobulin affinity chromatography and dissociation in denaturing conditions with or without reduction. J Med Microbiol 1989; 30:69–77
    [Google Scholar]
  11. Nakamura S, Mikawa M, Nakashio S. et al. Isolation of Clostridium difficile from the feces and the antibody in sera of young and elderly adults. Microbiol Immunol 1981; 25:345–351
    [Google Scholar]
  12. Krivan HC, Clark GF, Smith DF, Wilkins TD. Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Galαl-3Galβl-4GlcNAc. Infect Immun 1986; 53:573–581
    [Google Scholar]
  13. De SN, Chatterje DN. An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J Pathol Bacteriol 1953; 66:559–562
    [Google Scholar]
  14. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–254
    [Google Scholar]
  15. Kamiya S, Borriello SP. A non-haemagglutinating form of Clostridium difficile toxin A. J Med Microbiol 1992; 36:190–197
    [Google Scholar]
  16. Aronsson B, Grauström M, Möllby R, Nord CE. Enzyme immunoassay for detection of Clostridium difficile toxins A and B in patients with antibiotic-associated diarrhoea and colitis. Eur J Clin Microbiol 1985; 4:102–107
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/00222615-38-1-69
Loading
/content/journal/jmm/10.1099/00222615-38-1-69
Loading

Data & Media loading...

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