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. 1995 Sep;63(9):3309–3315. doi: 10.1128/iai.63.9.3309-3315.1995

The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin.

F Betsou 1, P Sebo 1, N Guiso 1
PMCID: PMC173456  PMID: 7642260

Abstract

The adenylate cyclase-hemolysin of Bordetella pertussis consists of a cell-invasive N-terminal adenylate cyclase domain linked to a C-terminal RTX hemolysin containing extensive glycine-rich repeats. The toxin is an essential virulence factor required in the initial stages of infection. Adenylate cyclase-hemolysin was also shown to be a potent vaccinating antigen inducing protection against B. pertussis colonization of the mouse respiratory tract. This protective activity depends on a posttranslational fatty-acylation modification. We used a set of deletion derivatives of the recombinant adenylate cyclase-hemolysin to localize the protective epitopes on the 1,706-residue toxin. We show that specific anti-adenylate cyclase-hemolysin antibodies present in the sera of B. pertussis-infected mice and humans are directed predominantly against the modification-and-repeat portion of the toxin, contained in the last 800 residues of the adenylate cyclase-hemolysin. These antibodies appear to recognize conformational epitopes present only in a structure formed by the intact C-terminal half of the toxin. There was no correlation between the capacity of the truncated adenylate cyclase-hemolysin derivatives to induce both toxin-neutralizing antibodies upon immunization of mice and protective immunity. However, only the truncated proteins which were recognized by the sera of infected mice and humans and which had their last 800 residues intact had the capacity to induce protection of mice against colonization by B. pertussis. This indicates that the structure of the modification-and-repeat region of adenylate cyclase-hemolysin is critical for its protective activity.

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Selected References

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  1. Arciniega J. L., Hewlett E. L., Johnson F. D., Deforest A., Wassilak S. G., Onorato I. M., Manclark C. R., Burns D. L. Human serologic response to envelope-associated proteins and adenylate cyclase toxin of Bordetella pertussis. J Infect Dis. 1991 Jan;163(1):135–142. doi: 10.1093/infdis/163.1.135. [DOI] [PubMed] [Google Scholar]
  2. Barry E. M., Weiss A. A., Ehrmann I. E., Gray M. C., Hewlett E. L., Goodwin M. S. Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation. J Bacteriol. 1991 Jan;173(2):720–726. doi: 10.1128/jb.173.2.720-726.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bellalou J., Sakamoto H., Ladant D., Geoffroy C., Ullmann A. Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase. Infect Immun. 1990 Oct;58(10):3242–3247. doi: 10.1128/iai.58.10.3242-3247.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benz R., Maier E., Ladant D., Ullmann A., Sebo P. Adenylate cyclase toxin (CyaA) of Bordetella pertussis. Evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli. J Biol Chem. 1994 Nov 4;269(44):27231–27239. [PubMed] [Google Scholar]
  5. Betsou F., Sebo P., Guiso N. CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin. Infect Immun. 1993 Sep;61(9):3583–3589. doi: 10.1128/iai.61.9.3583-3589.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boehm D. F., Welch R. A., Snyder I. S. Calcium is required for binding of Escherichia coli hemolysin (HlyA) to erythrocyte membranes. Infect Immun. 1990 Jun;58(6):1951–1958. doi: 10.1128/iai.58.6.1951-1958.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Coote J. G. Structural and functional relationships among the RTX toxin determinants of gram-negative bacteria. FEMS Microbiol Rev. 1992 Feb;8(2):137–161. doi: 10.1111/j.1574-6968.1992.tb04961.x. [DOI] [PubMed] [Google Scholar]
  10. Glaser P., Ladant D., Sezer O., Pichot F., Ullmann A., Danchin A. The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol Microbiol. 1988 Jan;2(1):19–30. [PubMed] [Google Scholar]
  11. Glaser P., Sakamoto H., Bellalou J., Ullmann A., Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 1988 Dec 1;7(12):3997–4004. doi: 10.1002/j.1460-2075.1988.tb03288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goodwin M. S., Weiss A. A. Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice. Infect Immun. 1990 Oct;58(10):3445–3447. doi: 10.1128/iai.58.10.3445-3447.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gordon V. M., Young W. W., Jr, Lechler S. M., Gray M. C., Leppla S. H., Hewlett E. L. Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells. J Biol Chem. 1989 Sep 5;264(25):14792–14796. [PubMed] [Google Scholar]
  14. Gray L., Baker K., Kenny B., Mackman N., Haigh R., Holland I. B. A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. J Cell Sci Suppl. 1989;11:45–57. doi: 10.1242/jcs.1989.supplement_11.4. [DOI] [PubMed] [Google Scholar]
  15. Gross M. K., Au D. C., Smith A. L., Storm D. R. Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4898–4902. doi: 10.1073/pnas.89.11.4898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gueirard P., Guiso N. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin. Infect Immun. 1993 Oct;61(10):4072–4078. doi: 10.1128/iai.61.10.4072-4078.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guiso N., Rocancourt M., Szatanik M., Alonso J. M. Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen. Microb Pathog. 1989 Nov;7(5):373–380. doi: 10.1016/0882-4010(89)90040-5. [DOI] [PubMed] [Google Scholar]
  18. Guiso N., Szatanik M., Rocancourt M. Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization. Microb Pathog. 1991 Dec;11(6):423–431. doi: 10.1016/0882-4010(91)90038-c. [DOI] [PubMed] [Google Scholar]
  19. Hackett M., Guo L., Shabanowitz J., Hunt D. F., Hewlett E. L. Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. Science. 1994 Oct 21;266(5184):433–435. doi: 10.1126/science.7939682. [DOI] [PubMed] [Google Scholar]
  20. Hanski E., Farfel Z. Bordetella pertussis invasive adenylate cyclase. Partial resolution and properties of its cellular penetration. J Biol Chem. 1985 May 10;260(9):5526–5532. [PubMed] [Google Scholar]
  21. Hardie K. R., Issartel J. P., Koronakis E., Hughes C., Koronakis V. In vitro activation of Escherichia coli prohaemolysin to the mature membrane-targeted toxin requires HlyC and a low molecular-weight cytosolic polypeptide. Mol Microbiol. 1991 Jul;5(7):1669–1679. doi: 10.1111/j.1365-2958.1991.tb01914.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Issartel J. P., Koronakis V., Hughes C. Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation. Nature. 1991 Jun 27;351(6329):759–761. doi: 10.1038/351759a0. [DOI] [PubMed] [Google Scholar]
  24. Khelef N., Bachelet C. M., Vargaftig B. B., Guiso N. Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins. Infect Immun. 1994 Jul;62(7):2893–2900. doi: 10.1128/iai.62.7.2893-2900.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Khelef N., Sakamoto H., Guiso N. Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection. Microb Pathog. 1992 Mar;12(3):227–235. doi: 10.1016/0882-4010(92)90057-u. [DOI] [PubMed] [Google Scholar]
  26. Khelef N., Zychlinsky A., Guiso N. Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin. Infect Immun. 1993 Oct;61(10):4064–4071. doi: 10.1128/iai.61.10.4064-4071.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ladant D., Brezin C., Alonso J. M., Crenon I., Guiso N. Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay. J Biol Chem. 1986 Dec 5;261(34):16264–16269. [PubMed] [Google Scholar]
  28. Ladant D., Glaser P., Ullmann A. Insertional mutagenesis of Bordetella pertussis adenylate cyclase. J Biol Chem. 1992 Feb 5;267(4):2244–2250. [PubMed] [Google Scholar]
  29. Ladant D., Michelson S., Sarfati R., Gilles A. M., Predeleanu R., Bârzu O. Characterization of the calmodulin-binding and of the catalytic domains of Bordetella pertussis adenylate cyclase. J Biol Chem. 1989 Mar 5;264(7):4015–4020. [PubMed] [Google Scholar]
  30. Ludwig A., Vogel M., Goebel W. Mutations affecting activity and transport of haemolysin in Escherichia coli. Mol Gen Genet. 1987 Feb;206(2):238–245. doi: 10.1007/BF00333579. [DOI] [PubMed] [Google Scholar]
  31. Pradelles P., Grassi J., Chabardes D., Guiso N. Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase. Anal Chem. 1989 Mar 1;61(5):447–453. doi: 10.1021/ac00180a014. [DOI] [PubMed] [Google Scholar]
  32. Rogel A., Schultz J. E., Brownlie R. M., Coote J. G., Parton R., Hanski E. Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme. EMBO J. 1989 Sep;8(9):2755–2760. doi: 10.1002/j.1460-2075.1989.tb08417.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sakamoto H., Bellalou J., Sebo P., Ladant D. Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities. J Biol Chem. 1992 Jul 5;267(19):13598–13602. [PubMed] [Google Scholar]
  34. Sebo P., Glaser P., Sakamoto H., Ullmann A. High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system. Gene. 1991 Jul 31;104(1):19–24. doi: 10.1016/0378-1119(91)90459-o. [DOI] [PubMed] [Google Scholar]
  35. Sebo P., Ladant D. Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the Escherichia coli alpha-haemolysin translocator. Mol Microbiol. 1993 Sep;9(5):999–1009. doi: 10.1111/j.1365-2958.1993.tb01229.x. [DOI] [PubMed] [Google Scholar]
  36. Stainer D. W., Scholte M. J. A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbiol. 1970 Oct;63(2):211–220. doi: 10.1099/00221287-63-2-211. [DOI] [PubMed] [Google Scholar]
  37. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis. J Infect Dis. 1984 Aug;150(2):219–222. doi: 10.1093/infdis/150.2.219. [DOI] [PubMed] [Google Scholar]
  38. Welch R. A. Pore-forming cytolysins of gram-negative bacteria. Mol Microbiol. 1991 Mar;5(3):521–528. doi: 10.1111/j.1365-2958.1991.tb00723.x. [DOI] [PubMed] [Google Scholar]
  39. Wolff J., Cook G. H., Goldhammer A. R., Berkowitz S. A. Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3841–3844. doi: 10.1073/pnas.77.7.3841. [DOI] [PMC free article] [PubMed] [Google Scholar]

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