Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 Jul;3(7):1595–1601. doi: 10.1002/j.1460-2075.1984.tb02016.x

IgA protease of Neisseria gonorrhoeae: isolation and characterization of the gene and its extracellular product.

R Halter, J Pohlner, T F Meyer
PMCID: PMC557564  PMID: 6430698

Abstract

Gonococcal virulence is thought to rely on multiple characteristics including the production of an extracellular protease specific for human IgA1. Using a sensitive filter assay we have isolated an Escherichia coli clone which harbours the gene of Neisseria gonorrhoeae MS11 IgA protease on a multicopy number plasmid. This clone secrets IgA protease activity to an extent similar to that of the parental MS11 strain. By exonucleolytic digestion of the cloned insert we obtained a fragment of 4.6 kb which could not be shortened further without loss of IgA protease expression. Compared with the cloned IgA protease gene from N. gonorrhoeae F62, this minimal gene segment shows marked differences in the arrangement of restriction sites. We suppose that these differences determine strain-specific variations of N. gonorrhoeae IgA proteases and also affect the secretory properties of the enzyme when produced in E. coli. A novel purification procedure developed for IgA protease of N. gonorrhoeae allowed us to correlate the enzyme activity with a distinct protein band in SDS acrylamide gels. By comparison with the enzyme prepared from the E. coli clone, we identified a 105-kd protein as the extracellular form of gonococcal IgA protease.

Full text

PDF
1595

Images in this article

1596Fig. 1.
on p.1596
1597Fig. 3.
on p.1597
1598Fig. 4.
on p.1598
1599Fig. 6.
on p.1599

Selected References

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

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bricker J., Mulks M. H., Plaut A. G., Moxon E. R., Wright A. IgA1 proteases of Haemophilus influenzae: cloning and characterization in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1983 May;80(9):2681–2685. doi: 10.1073/pnas.80.9.2681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Catlin B. W. Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica in chemically defined media and the use of growth requirements for gonococcal typing. J Infect Dis. 1973 Aug;128(2):178–194. doi: 10.1093/infdis/128.2.178. [DOI] [PubMed] [Google Scholar]
  6. Frangione B., Wolfenstein-Todel C. Partial duplication in the "hinge" region of IgA 1 myeloma proteins. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3673–3676. doi: 10.1073/pnas.69.12.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. GAREN A., LEVINTHAL C. A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta. 1960 Mar 11;38:470–483. doi: 10.1016/0006-3002(60)91282-8. [DOI] [PubMed] [Google Scholar]
  8. Goebel W., Hedgpeth J. Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1290–1298. doi: 10.1128/jb.151.3.1290-1298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. Kilian M., Thomsen B. Antigenic heterogeneity of immunoglobulin A1 proteases from encapsulated and non-encapsulated Haemophilus influenzae. Infect Immun. 1983 Oct;42(1):126–132. doi: 10.1128/iai.42.1.126-132.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Koomey J. M., Falkow S. Nucleotide sequence homology between the immunoglobulin A1 protease genes of Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae. Infect Immun. 1984 Jan;43(1):101–107. doi: 10.1128/iai.43.1.101-107.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Koomey J. M., Gill R. E., Falkow S. Genetic and biochemical analysis of gonococcal IgA1 protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7881–7885. doi: 10.1073/pnas.79.24.7881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kornfeld S. J., Plaut A. G. Secretory immunity and the bacterial IgA proteases. Rev Infect Dis. 1981 May-Jun;3(3):521–534. doi: 10.1093/clinids/3.3.521. [DOI] [PubMed] [Google Scholar]
  15. Kratzin H., Altevogt P., Kortt A., Ruban E., Hilschmann N. Zur Strukturregel der Antikörper. Die Primärstruktur eines monoklonalen IgA1-Immunoglobulins (Myelomprotein Tro), V. Die Anordnung der tryptischen Peptide und die Diskussion der vollständigen Primärstruktur der H-Kette. Hoppe Seylers Z Physiol Chem. 1978 Dec;359(12):1717–1745. [PubMed] [Google Scholar]
  16. 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]
  17. Levy S. B. R factor proteins synthesized in Escherichia coli minicells: incorporation studies with different R factors and detection of deoxyribonucleic acid-binding proteins. J Bacteriol. 1974 Dec;120(3):1451–1463. doi: 10.1128/jb.120.3.1451-1463.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meyer T. F., Mlawer N., So M. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell. 1982 Aug;30(1):45–52. doi: 10.1016/0092-8674(82)90010-1. [DOI] [PubMed] [Google Scholar]
  19. Milazzo F. H., Delisle G. J. Immunoglobulin A proteases in gram-negative bacteria isolated from human urinary tract infections. Infect Immun. 1984 Jan;43(1):11–13. doi: 10.1128/iai.43.1.11-13.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mulks M. H., Kornfeld S. J., Plaut A. G. Specific proteolysis of human IgA by Streptococcus pneumoniae and Haemophilus influenzae. J Infect Dis. 1980 Apr;141(4):450–456. doi: 10.1093/infdis/141.4.450. [DOI] [PubMed] [Google Scholar]
  21. Mulks M. H., Plaut A. G., Feldman H. A., Frangione B. IgA proteases of two distinct specificities are released by Neisseria meningitidis. J Exp Med. 1980 Nov 1;152(5):1442–1447. doi: 10.1084/jem.152.5.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  23. Plaut A. G., Gilbert J. V., Artenstein M. S., Capra J. D. Neisseria gonorrhoeae and neisseria meningitidis: extracellular enzyme cleaves human immunoglobulin A. Science. 1975 Dec 12;190(4219):1103–1105. doi: 10.1126/science.810892. [DOI] [PubMed] [Google Scholar]
  24. Plaut A. G., Gilbert J. V., Wistar R., Jr Loss of antibody activity in human immunoglobulin A exposed extracellular immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis. Infect Immun. 1977 Jul;17(1):130–135. doi: 10.1128/iai.17.1.130-135.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Plaut A. G. The IgA1 proteases of pathogenic bacteria. Annu Rev Microbiol. 1983;37:603–622. doi: 10.1146/annurev.mi.37.100183.003131. [DOI] [PubMed] [Google Scholar]
  26. Salit I. E., Blake M., Gotschlich E. C. Intra-strain heterogeneity of gonococcal pili is related to opacity colony variance. J Exp Med. 1980 Mar 1;151(3):716–725. doi: 10.1084/jem.151.3.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  28. Stern A., Nickel P., Meyer T. F., So M. Opacity determinants of Neisseria gonorrhoeae: gene expression and chromosomal linkage to the gonococcal pilus gene. Cell. 1984 Jun;37(2):447–456. doi: 10.1016/0092-8674(84)90375-1. [DOI] [PubMed] [Google Scholar]
  29. Switzer R. C., 3rd, Merril C. R., Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem. 1979 Sep 15;98(1):231–237. doi: 10.1016/0003-2697(79)90732-2. [DOI] [PubMed] [Google Scholar]
  30. Williams R. C., Gibbons R. J. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science. 1972 Aug 25;177(4050):697–699. doi: 10.1126/science.177.4050.697. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES