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. 1990 Jun;58(6):1518–1526. doi: 10.1128/iai.58.6.1518-1526.1990

Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin.

R Antoine 1, C Locht 1
PMCID: PMC258665  PMID: 2341166

Abstract

A Bordetella pertussis expression system was developed to analyze the structure-function relationship, in vivo assembly, and biosynthesis of pertussis toxin. The toxin structural gene was first deleted from the B. pertussis chromosome; into the resulting B. pertussis strain the toxin gene was introduced on a low-copy-number, broad-host-range plasmid. The amount of pertussis toxin produced and secreted with this expression system was in the same order of magnitude as that produced by B. pertussis Tohama I, indicating that although the plasmid may be present in more than one copy per cell, overproduction of the toxin was not achieved in B. pertussis. Expression of mutant pertussis toxin genes in which the codon for Cys-41 was deleted or altered or in which the carboxy-terminal region was deleted showed that both the single intrachain disulfide bond and the carboxy-terminal region of S1 are essential for the stable expression, assembly, and secretion of S1. On the other hand, the B oligomer was efficiently secreted in the culture medium in the absence of the S1 subunit. The secreted B oligomer contained S2, S3, and S4 subunits as evidenced by enzyme-linked immunosorbent assay and was fully functional with respect to haptoglobin binding. Furthermore, the deletion of the hydrophobic carboxy-terminal region has a drastic effect on S1 subunit solubility; however, inclusion of the hydrophobic region was not sufficient for assembly and secretion, indicating that other interactions involving amino acids beyond residue 207 of the S1 subunit are also required.

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Selected References

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

  1. Arciniega J. L., Burns D. L., Garcia-Ortigoza E., Manclark C. R. Immune response to the B oligomer of pertussis toxin. Infect Immun. 1987 May;55(5):1132–1136. doi: 10.1128/iai.55.5.1132-1136.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbieri J. T., Cortina G. ADP-ribosyltransferase mutations in the catalytic S-1 subunit of pertussis toxin. Infect Immun. 1988 Aug;56(8):1934–1941. doi: 10.1128/iai.56.8.1934-1941.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black W. J., Falkow S. Construction and characterization of Bordetella pertussis toxin mutants. Infect Immun. 1987 Oct;55(10):2465–2470. doi: 10.1128/iai.55.10.2465-2470.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N., Cieplak W., Mar V. L., Kaljot K. T., Sato H., Keith J. M. Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope. Science. 1988 Oct 7;242(4875):72–74. doi: 10.1126/science.2459776. [DOI] [PubMed] [Google Scholar]
  5. Burns D. L., Hausman S. Z., Lindner W., Robey F. A., Manclark C. R. Structural characterization of pertussis toxin A subunit. J Biol Chem. 1987 Dec 25;262(36):17677–17682. [PubMed] [Google Scholar]
  6. Burns D. L., Kenimer J. G., Manclark C. R. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect Immun. 1987 Jan;55(1):24–28. doi: 10.1128/iai.55.1.24-28.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burns D. L., Manclark C. R. Adenine nucleotides promote dissociation of pertussis toxin subunits. J Biol Chem. 1986 Mar 25;261(9):4324–4327. [PubMed] [Google Scholar]
  8. Burns D. L., Manclark C. R. Role of cysteine 41 of the A subunit of pertussis toxin. J Biol Chem. 1989 Jan 5;264(1):564–568. [PubMed] [Google Scholar]
  9. Figurski D. H., Pohlman R. F., Bechhofer D. H., Prince A. S., Kelton C. A. Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1935–1939. doi: 10.1073/pnas.79.6.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frank D. W., Parker C. D. Interaction of monoclonal antibodies with pertussis toxin and its subunits. Infect Immun. 1984 Oct;46(1):195–201. doi: 10.1128/iai.46.1.195-201.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  12. Gross R., Rappuoli R. Positive regulation of pertussis toxin expression. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3913–3917. doi: 10.1073/pnas.85.11.3913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hewlett E. L., Sauer K. T., Myers G. A., Cowell J. L., Guerrant R. L. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect Immun. 1983 Jun;40(3):1198–1203. doi: 10.1128/iai.40.3.1198-1203.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hofstra H., Witholt B. Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin. J Biol Chem. 1985 Dec 15;260(29):16037–16044. [PubMed] [Google Scholar]
  15. Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature. 1981 Jul 30;292(5822):413–417. doi: 10.1038/292413a0. [DOI] [PubMed] [Google Scholar]
  16. Hull R. A., Gill R. E., Hsu P., Minshew B. H., Falkow S. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect Immun. 1981 Sep;33(3):933–938. doi: 10.1128/iai.33.3.933-938.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Imaizumi A., Suzuki Y., Ono S., Sato H., Sato Y. Effect of heptakis (2,6-O-dimethyl) beta-cyclodextrin on the production of pertussis toxin by Bordetella pertussis. Infect Immun. 1983 Sep;41(3):1138–1143. doi: 10.1128/iai.41.3.1138-1143.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaslow H. R., Lesikar D. D. Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin. Biochemistry. 1987 Jul 14;26(14):4397–4402. doi: 10.1021/bi00388a031. [DOI] [PubMed] [Google Scholar]
  19. Kaslow H. R., Lim L. K., Moss J., Lesikar D. D. Structure-activity analysis of the activation of pertussis toxin. Biochemistry. 1987 Jan 13;26(1):123–127. doi: 10.1021/bi00375a018. [DOI] [PubMed] [Google Scholar]
  20. Kaslow H. R., Schlotterbeck J. D., Mar V. L., Burnette W. N. Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin. J Biol Chem. 1989 Apr 15;264(11):6386–6390. [PubMed] [Google Scholar]
  21. Katada T., Tamura M., Ui M. The A protomer of islet-activating protein, pertussis toxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch Biochem Biophys. 1983 Jul 1;224(1):290–298. doi: 10.1016/0003-9861(83)90212-6. [DOI] [PubMed] [Google Scholar]
  22. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lee C. K., Roberts A., Perrin S. Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids. Infect Immun. 1989 May;57(5):1413–1418. doi: 10.1128/iai.57.5.1413-1418.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Locht C., Barstad P. A., Coligan J. E., Mayer L., Munoz J. J., Smith S. G., Keith J. M. Molecular cloning of pertussis toxin genes. Nucleic Acids Res. 1986 Apr 25;14(8):3251–3261. doi: 10.1093/nar/14.8.3251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Locht C., Capiau C., Feron C. Identification of amino acid residues essential for the enzymatic activities of pertussis toxin. Proc Natl Acad Sci U S A. 1989 May;86(9):3075–3079. doi: 10.1073/pnas.86.9.3075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Locht C., Cieplak W., Marchitto K. S., Sato H., Keith J. M. Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli. Infect Immun. 1987 Nov;55(11):2546–2553. doi: 10.1128/iai.55.11.2546-2553.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Locht C., Keith J. M. Pertussis toxin gene: nucleotide sequence and genetic organization. Science. 1986 Jun 6;232(4755):1258–1264. doi: 10.1126/science.3704651. [DOI] [PubMed] [Google Scholar]
  29. Marchitto K. S., Munoz J. J., Keith J. M. Detection of subunits of pertussis toxin in Tn5-induced Bordetella mutants deficient in toxin biological activity. Infect Immun. 1987 May;55(5):1309–1313. doi: 10.1128/iai.55.5.1309-1313.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moss J., Stanley S. J., Burns D. L., Hsia J. A., Yost D. A., Myers G. A., Hewlett E. L. Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). J Biol Chem. 1983 Oct 10;258(19):11879–11882. [PubMed] [Google Scholar]
  31. Moss J., Stanley S. J., Watkins P. A., Burns D. L., Manclark C. R., Kaslow H. R., Hewlett E. L. Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents. Biochemistry. 1986 May 6;25(9):2720–2725. doi: 10.1021/bi00357a066. [DOI] [PubMed] [Google Scholar]
  32. Nicosia A., Perugini M., Franzini C., Casagli M. C., Borri M. G., Antoni G., Almoni M., Neri P., Ratti G., Rappuoli R. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4631–4635. doi: 10.1073/pnas.83.13.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pizza M., Bartoloni A., Prugnola A., Silvestri S., Rappuoli R. Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7521–7525. doi: 10.1073/pnas.85.20.7521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stibitz S., Black W., Falkow S. The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene. 1986;50(1-3):133–140. doi: 10.1016/0378-1119(86)90318-5. [DOI] [PubMed] [Google Scholar]
  35. Tamura M., Nogimori K., Murai S., Yajima M., Ito K., Katada T., Ui M., Ishii S. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry. 1982 Oct 26;21(22):5516–5522. doi: 10.1021/bi00265a021. [DOI] [PubMed] [Google Scholar]
  36. Tamura M., Nogimori K., Yajima M., Ase K., Ui M. A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells. J Biol Chem. 1983 Jun 10;258(11):6756–6761. [PubMed] [Google Scholar]
  37. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  38. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun. 1983 Oct;42(1):33–41. doi: 10.1128/iai.42.1.33-41.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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