Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Nov;174(21):6928–6937. doi: 10.1128/jb.174.21.6928-6937.1992

Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae.

Y Pannekoek 1, J P van Putten 1, J Dankert 1
PMCID: PMC207372  PMID: 1400243

Abstract

Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenyl-Sepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pI of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein.

Full text

PDF
6928

Images in this article

6932Image
on p.6932
6932Image
on p.6932
6933Image
on p.6933
6933Image
on p.6933
6933Image
on p.6933
6934Image
on p.6934
6934Image
on p.6934

Selected References

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

  1. Ballard S. A., Faine S., Adler B. Purification and characterization of a protein antigen from Leptospira interrogans serovar hardjo, common to a wide range of bacteria. J Gen Microbiol. 1990 Sep;136(9):1849–1857. doi: 10.1099/00221287-136-9-1849. [DOI] [PubMed] [Google Scholar]
  2. Bavoil P., Stephens R. S., Falkow S. A soluble 60 kiloDalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL. Mol Microbiol. 1990 Mar;4(3):461–469. doi: 10.1111/j.1365-2958.1990.tb00612.x. [DOI] [PubMed] [Google Scholar]
  3. Bochkareva E. S., Lissin N. M., Girshovich A. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature. 1988 Nov 17;336(6196):254–257. doi: 10.1038/336254a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Brener D., DeVoe I. W., Holbein B. E. Increased virulence of Neisseria meningitidis after in vitro iron-limited growth at low pH. Infect Immun. 1981 Jul;33(1):59–66. doi: 10.1128/iai.33.1.59-66.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Britigan B. E., Cohen M. S., Sparling P. F. Gonococcal infection: a model of molecular pathogenesis. N Engl J Med. 1985 Jun 27;312(26):1683–1694. doi: 10.1056/NEJM198506273122606. [DOI] [PubMed] [Google Scholar]
  7. Brown M. R., Williams P. The influence of environment on envelope properties affecting survival of bacteria in infections. Annu Rev Microbiol. 1985;39:527–556. doi: 10.1146/annurev.mi.39.100185.002523. [DOI] [PubMed] [Google Scholar]
  8. Buchanan T. M., Hildebrandt J. F. Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein. Infect Immun. 1981 Jun;32(3):985–994. doi: 10.1128/iai.32.3.985-994.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Buchner J., Schmidt M., Fuchs M., Jaenicke R., Rudolph R., Schmid F. X., Kiefhaber T. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry. 1991 Feb 12;30(6):1586–1591. doi: 10.1021/bi00220a020. [DOI] [PubMed] [Google Scholar]
  10. Cerrone M. C., Ma J. J., Stephens R. S. Cloning and sequence of the gene for heat shock protein 60 from Chlamydia trachomatis and immunological reactivity of the protein. Infect Immun. 1991 Jan;59(1):79–90. doi: 10.1128/iai.59.1.79-90.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheng M. Y., Hartl F. U., Martin J., Pollock R. A., Kalousek F., Neupert W., Hallberg E. M., Hallberg R. L., Horwich A. L. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 1989 Feb 16;337(6208):620–625. doi: 10.1038/337620a0. [DOI] [PubMed] [Google Scholar]
  12. Clark V. L., Knapp J. S., Thompson S., Klimpel K. W. Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections. Microb Pathog. 1988 Nov;5(5):381–390. doi: 10.1016/0882-4010(88)90038-1. [DOI] [PubMed] [Google Scholar]
  13. Cohen M. S., Sparling P. F. Mucosal infection with Neisseria gonorrhoeae. Bacterial adaptation and mucosal defenses. J Clin Invest. 1992 Jun;89(6):1699–1705. doi: 10.1172/JCI115770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. De Bruyn J., Bosmans R., Turneer M., Weckx M., Nyabenda J., Van Vooren J. P., Falmagne P., Wiker H. G., Harboe M. Purification, partial characterization, and identification of a skin-reactive protein antigen of Mycobacterium bovis BCG. Infect Immun. 1987 Jan;55(1):245–252. doi: 10.1128/iai.55.1.245-252.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. De Moreno M. R., Smith J. F., Smith R. V. Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie blue-silver stain procedure. Anal Biochem. 1985 Dec;151(2):466–470. doi: 10.1016/0003-2697(85)90206-4. [DOI] [PubMed] [Google Scholar]
  16. Dorward D. W., Garon C. F., Judd R. C. Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J Bacteriol. 1989 May;171(5):2499–2505. doi: 10.1128/jb.171.5.2499-2505.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dunn S. D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. doi: 10.1016/0003-2697(86)90207-1. [DOI] [PubMed] [Google Scholar]
  18. Filip C., Fletcher G., Wulff J. L., Earhart C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol. 1973 Sep;115(3):717–722. doi: 10.1128/jb.115.3.717-722.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
  20. Goloubinoff P., Gatenby A. A., Lorimer G. H. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature. 1989 Jan 5;337(6202):44–47. doi: 10.1038/337044a0. [DOI] [PubMed] [Google Scholar]
  21. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  22. Hendrix R. W. Purification and properties of groE, a host protein involved in bacteriophage assembly. J Mol Biol. 1979 Apr 15;129(3):375–392. doi: 10.1016/0022-2836(79)90502-3. [DOI] [PubMed] [Google Scholar]
  23. Hoffman P. S., Houston L., Butler C. A. Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells. Infect Immun. 1990 Oct;58(10):3380–3387. doi: 10.1128/iai.58.10.3380-3387.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Houston L. S., Cook R. G., Norris S. J. Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli. J Bacteriol. 1990 Jun;172(6):2862–2870. doi: 10.1128/jb.172.6.2862-2870.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnston K. H., Holmes K. K., Gotschlich E. C. The serological classification of Neisseria gonorrhoeae. I. Isolation of the outer membrane complex responsible for serotypic specificity. J Exp Med. 1976 Apr 1;143(4):741–758. doi: 10.1084/jem.143.4.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Keevil C. W., Davies D. B., Spillane B. J., Mahenthiralingam E. Influence of iron-limited and replete continuous culture on the physiology and virulence of Neisseria gonorrhoeae. J Gen Microbiol. 1989 Apr;135(4):851–863. doi: 10.1099/00221287-135-4-851. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lindquist S., Craig E. A. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677. doi: 10.1146/annurev.ge.22.120188.003215. [DOI] [PubMed] [Google Scholar]
  29. Mietzner T. A., Luginbuhl G. H., Sandstrom E., Morse S. A. Identification of an iron-regulated 37,000-dalton protein in the cell envelope of Neisseria gonorrhoeae. Infect Immun. 1984 Aug;45(2):410–416. doi: 10.1128/iai.45.2.410-416.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Morrison R. P., Belland R. J., Lyng K., Caldwell H. D. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein. J Exp Med. 1989 Oct 1;170(4):1271–1283. doi: 10.1084/jem.170.4.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morrison R. P., Lyng K., Caldwell H. D. Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus-specific 57-kD protein. J Exp Med. 1989 Mar 1;169(3):663–675. doi: 10.1084/jem.169.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  33. Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 1989 Sep 14;341(6238):125–130. doi: 10.1038/341125a0. [DOI] [PubMed] [Google Scholar]
  34. Parsons N. J., Kwaasi A. A., Patel P. V., Martin P. M., Smith H. Association of resistance of Neisseria gonorrhoeae to killing by human phagocytes with outer-membrane proteins of about 20 kilodaltons. J Gen Microbiol. 1985 Mar;131(3):601–610. doi: 10.1099/00221287-131-3-601. [DOI] [PubMed] [Google Scholar]
  35. Reading D. S., Hallberg R. L., Myers A. M. Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor. Nature. 1989 Feb 16;337(6208):655–659. doi: 10.1038/337655a0. [DOI] [PubMed] [Google Scholar]
  36. Schlossman D. M., Schmid S. L., Braell W. A., Rothman J. E. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):723–733. doi: 10.1083/jcb.99.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schryvers A. B., Morris L. J. Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis. Infect Immun. 1988 May;56(5):1144–1149. doi: 10.1128/iai.56.5.1144-1149.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Steinmetz I., Rheinheimer C., Hübner I., Bitter-Suermann D. Genus-specific epitope on the 60-kilodalton Legionella heat shock protein recognized by a monoclonal antibody. J Clin Microbiol. 1991 Feb;29(2):346–354. doi: 10.1128/jcm.29.2.346-354.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tamada H., Ohta T., Hamamoto T., Otawara-Hamamoto Y., Yanagi M., Hiraiwa H., Hirata H., Kagawa Y. Gene structure of heat shock proteins 61KDa and 12KDa (thermophilic chaperonins) of thermophilic bacterium PS3. Biochem Biophys Res Commun. 1991 Aug 30;179(1):565–571. doi: 10.1016/0006-291x(91)91408-5. [DOI] [PubMed] [Google Scholar]
  40. Taylor H. R., Maclean I. W., Brunham R. C., Pal S., Whittum-Hudson J. Chlamydial heat shock proteins and trachoma. Infect Immun. 1990 Sep;58(9):3061–3063. doi: 10.1128/iai.58.9.3061-3063.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Terlesky K. C., Tabita F. R. Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides. Biochemistry. 1991 Aug 20;30(33):8181–8186. doi: 10.1021/bi00247a013. [DOI] [PubMed] [Google Scholar]
  42. Thole J. E., Keulen W. J., De Bruyn J., Kolk A. H., Groothuis D. G., Berwald L. G., Tiesjema R. H., van Embden J. D. Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in escherichia coli K-12. Infect Immun. 1987 Jun;55(6):1466–1475. doi: 10.1128/iai.55.6.1466-1475.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thole J. E., van Schooten W. C., Keulen W. J., Hermans P. W., Janson A. A., de Vries R. R., Kolk A. H., van Embden J. D. Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG. Infect Immun. 1988 Jun;56(6):1633–1640. doi: 10.1128/iai.56.6.1633-1640.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Viitanen P. V., Lorimer G. H., Seetharam R., Gupta R. S., Oppenheim J., Thomas J. O., Cowan N. J. Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring. J Biol Chem. 1992 Jan 15;267(2):695–698. [PubMed] [Google Scholar]
  46. Vodkin M. H., Williams J. C. A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli. J Bacteriol. 1988 Mar;170(3):1227–1234. doi: 10.1128/jb.170.3.1227-1234.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weel J. F., Hopman C. T., van Putten J. P. Bacterial entry and intracellular processing of Neisseria gonorrhoeae in epithelial cells: immunomorphological evidence for alterations in the major outer membrane protein P.IB. J Exp Med. 1991 Sep 1;174(3):705–715. doi: 10.1084/jem.174.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weel J. F., Hopman C. T., van Putten J. P. In situ expression and localization of Neisseria gonorrhoeae opacity proteins in infected epithelial cells: apparent role of Opa proteins in cellular invasion. J Exp Med. 1991 Jun 1;173(6):1395–1405. doi: 10.1084/jem.173.6.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weel J. F., Hopman C. T., van Putten J. P. Stable expression of lipooligosaccharide antigens during attachment, internalization, and intracellular processing of Neisseria gonorrhoeae in infected epithelial cells. Infect Immun. 1989 Nov;57(11):3395–3402. doi: 10.1128/iai.57.11.3395-3402.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weel J. F., van Putten J. P. Fate of the major outer membrane protein P.IA in early and late events of gonococcal infection of epithelial cells. Res Microbiol. 1991 Nov-Dec;142(9):985–993. doi: 10.1016/0923-2508(91)90009-y. [DOI] [PubMed] [Google Scholar]
  51. Weel J. F., van Putten J. P. Ultrastructural localization of gonococcal antigens in infected epithelial cells as visualized by post-embedding immuno-electronmicroscopy. Microb Pathog. 1988 Mar;4(3):213–222. doi: 10.1016/0882-4010(88)90071-x. [DOI] [PubMed] [Google Scholar]
  52. Weinberg E. D. Iron and infection. Microbiol Rev. 1978 Mar;42(1):45–66. doi: 10.1128/mr.42.1.45-66.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. West S. E., Sparling P. F. Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production. Infect Immun. 1985 Feb;47(2):388–394. doi: 10.1128/iai.47.2.388-394.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Witt K., Veale D. R., Finch H., Penn C. W., Sen D., Smith H. Resistance of Neisseria gonorrhoeae grown in vivo to ingestion and digestion by phagocytes of human blood. J Gen Microbiol. 1976 Oct;96(2):341–350. doi: 10.1099/00221287-96-2-341. [DOI] [PubMed] [Google Scholar]
  55. Woods M. L., 2nd, Bonfiglioli R., McGee Z. A., Georgopoulos C. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress. Infect Immun. 1990 Mar;58(3):719–725. doi: 10.1128/iai.58.3.719-725.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Young D., Lathigra R., Hendrix R., Sweetser D., Young R. A. Stress proteins are immune targets in leprosy and tuberculosis. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4267–4270. doi: 10.1073/pnas.85.12.4267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zeilstra-Ryalls J., Fayet O., Georgopoulos C. The universally conserved GroE (Hsp60) chaperonins. Annu Rev Microbiol. 1991;45:301–325. doi: 10.1146/annurev.mi.45.100191.001505. [DOI] [PubMed] [Google Scholar]
  58. van Eden W., Thole J. E., van der Zee R., Noordzij A., van Embden J. D., Hensen E. J., Cohen I. R. Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature. 1988 Jan 14;331(6152):171–173. doi: 10.1038/331171a0. [DOI] [PubMed] [Google Scholar]
  59. van Putten J. P. Iron acquisition and the pathogenesis of meningococcal and gonococcal disease. Med Microbiol Immunol. 1990;179(6):289–295. doi: 10.1007/BF00189607. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES