Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1991 Oct;59(10):3754–3759. doi: 10.1128/iai.59.10.3754-3759.1991

Purification and characterization of the heat-labile toxin of Bordetella pertussis.

Y L Zhang 1, R D Sekura 1
PMCID: PMC258947  PMID: 1894374

Abstract

A procedure is described for purification of pertussis heat-labile toxin (PEHLT) from cells of Bordetella pertussis. The purification procedure, performed in the cold and in the presence of protease inhibitors, gives 1,350-fold purification with yields of about 60%. The toxin was shown to be a single-chain polypeptide of 140 kDa, pI 6.02. It was completely inactivated by heating at 56 degrees C for 60 min. Rabbit antiserum prepared against PEHLT neutralized the toxin and gave a single precipitin line on immunodiffusion. In immunodiffusion assays, this anti-PEHLT serum did not react with pertussis toxin, filamentous hemagglutinin, or preparations of pertussis adenylate cyclase. Purified PEHLT elicited dermonecrosis and atrophy of the spleen. PEHLT is extraordinarily active; 0.4 X 10(-12) g caused necrotic lesions in newborn mice, and with 18- to 20-g mice the 50% lethal dose was about 11 X 10(-9) g.

Full text

PDF
3757

Images in this article

3756Image
on p.3756
3757Image
on p.3757

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BANERJEA A., MUNOZ J. Antigens of Bordetella pertussis. II. Purification of heat-labile toxin. J Bacteriol. 1962 Aug;84:269–274. doi: 10.1128/jb.84.2.269-274.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bokoch G. M., Katada T., Northup J. K., Hewlett E. L., Gilman A. G. Identification of the predominant substrate for ADP-ribosylation by islet activating protein. J Biol Chem. 1983 Feb 25;258(4):2072–2075. [PubMed] [Google Scholar]
  3. Cabib E., Polacheck I. Protein assay for dilute solutions. Methods Enzymol. 1984;104:415–416. doi: 10.1016/s0076-6879(84)04108-2. [DOI] [PubMed] [Google Scholar]
  4. Confer D. L., Eaton J. W. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science. 1982 Sep 3;217(4563):948–950. doi: 10.1126/science.6287574. [DOI] [PubMed] [Google Scholar]
  5. Cowell J. L., Hewlett E. L., Manclark C. R. Intracellular localization of the dermonecrotic toxin of Bordetella pertussis. Infect Immun. 1979 Sep;25(3):896–901. doi: 10.1128/iai.25.3.896-901.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Endoh M., Amitani M., Nakase Y. Purification and characterization of heat-labile toxin from Bordetella bronchiseptica. Microbiol Immunol. 1986;30(7):659–673. doi: 10.1111/j.1348-0421.1986.tb02992.x. [DOI] [PubMed] [Google Scholar]
  7. Endoh M., Nagai M., Burns D. L., Manclark C. R., Nakase Y. Effects of exogenous agents on the action of Bordetella parapertussis heat-labile toxin on guinea pig skin. Infect Immun. 1990 May;58(5):1456–1460. doi: 10.1128/iai.58.5.1456-1460.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanski E. Invasive adenylate cyclase toxin of Bordetella pertussis. Trends Biochem Sci. 1989 Nov;14(11):459–463. doi: 10.1016/0968-0004(89)90106-0. [DOI] [PubMed] [Google Scholar]
  9. Hewlett E. L., Gordon V. M., McCaffery J. D., Sutherland W. M., Gray M. C. Adenylate cyclase toxin from Bordetella pertussis. Identification and purification of the holotoxin molecule. J Biol Chem. 1989 Nov 15;264(32):19379–19384. [PubMed] [Google Scholar]
  10. Hildebrandt J. D., Sekura R. D., Codina J., Iyengar R., Manclark C. R., Birnbaumer L. Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins. Nature. 1983 Apr 21;302(5910):706–709. doi: 10.1038/302706a0. [DOI] [PubMed] [Google Scholar]
  11. Horiguchi Y., Nakai T., Kume K. Purification and characterization of Bordetella bronchiseptica dermonecrotic toxin. Microb Pathog. 1989 May;6(5):361–368. doi: 10.1016/0882-4010(89)90078-8. [DOI] [PubMed] [Google Scholar]
  12. Kume K., Nakai T., Samejima Y., Sugimoto C. Properties of dermonecrotic toxin prepared from sonic extracts Bordetella bronchiseptica. Infect Immun. 1986 May;52(2):370–377. doi: 10.1128/iai.52.2.370-377.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Livey I., Wardlaw A. C. Production and properties of Bordetella pertussis heat-labile toxin. J Med Microbiol. 1984 Feb;17(1):91–103. doi: 10.1099/00222615-17-1-91. [DOI] [PubMed] [Google Scholar]
  15. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  16. Mann K. G., Fish W. W. Protein polypeptide chain molecular weights by gel chromatography in guanidinium chloride. Methods Enzymol. 1972;26:28–42. doi: 10.1016/s0076-6879(72)26004-9. [DOI] [PubMed] [Google Scholar]
  17. Nakase Y., Endoh M. Bordetella heat-labile toxin: further purification, characterization and mode of action. Dev Biol Stand. 1985;61:93–102. [PubMed] [Google Scholar]
  18. Nakase Y., Takatsu K., Tateishi M., Sekiya K., Kasuga T. Heat-labile toxin of Bordetella pertussis purified by preparative acrylamide gel electrophoresis. Jpn J Microbiol. 1969 Dec;13(4):359–366. doi: 10.1111/j.1348-0421.1969.tb00479.x. [DOI] [PubMed] [Google Scholar]
  19. ONOUE K., KITAGAWA M., YAMAMURA Y. CHEMICAL STUDIES ON CELLULAR COMPONENTS OF BORDETELLA PERTUSSIS. III. ISOLATION OF HIGHLY POTENT TOXIN FROM BORDETELLA PERTUSSIS. J Bacteriol. 1963 Oct;86:648–655. doi: 10.1128/jb.86.4.648-655.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. PENNELL R. B., THIELE E. H. Studies on the fractionation of Hemophilus pertussis extracts. J Immunol. 1951 Jun;66(6):627–633. [PubMed] [Google Scholar]
  21. Parton R. Effect of anti-inflammatory agents on the haemorrhagic response of mouse skin to Bordetella pertussis heat-labile toxin. J Med Microbiol. 1986 May;21(3):265–270. doi: 10.1099/00222615-21-3-265. [DOI] [PubMed] [Google Scholar]
  22. Parton R. Effect of prednisolone on the toxicity of Bordetella pertussis for mice. J Med Microbiol. 1985 Jun;19(3):391–400. doi: 10.1099/00222615-19-3-391. [DOI] [PubMed] [Google Scholar]
  23. ROBBINS K. C., PILLEMER L. Separation of pertussal toxin. Proc Soc Exp Biol Med. 1950 May;74(1):75–78. doi: 10.3181/00379727-74-17814. [DOI] [PubMed] [Google Scholar]
  24. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  25. Sekura R. D., Fish F., Manclark C. R., Meade B., Zhang Y. L. Pertussis toxin. Affinity purification of a new ADP-ribosyltransferase. J Biol Chem. 1983 Dec 10;258(23):14647–14651. [PubMed] [Google Scholar]
  26. Vaitukaitis J., Robbins J. B., Nieschlag E., Ross G. T. A method for producing specific antisera with small doses of immunogen. J Clin Endocrinol Metab. 1971 Dec;33(6):988–991. doi: 10.1210/jcem-33-6-988. [DOI] [PubMed] [Google Scholar]
  27. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES