Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 May;65(5):1824–1829. doi: 10.1128/iai.65.5.1824-1829.1997

Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F. tularensis.

M Ericsson 1, I Golovliov 1, G Sandström 1, A Tärnvik 1, A Sjöstedt 1
PMCID: PMC175224  PMID: 9125567

Abstract

The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characterized, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed. The cpn10 and cpn60 genes were amplified by PCR with degenerate oligonucleotides derived from the N-terminal sequence of the two proteins. The sequence analysis revealed the expected two open reading frames, encoding proteins with estimated Mrs of 10,300 and 57,400. The deduced amino acid sequences closely resembled Cpn10 and Cpn60 proteins of other prokaryotes. The genes constituted a bicistronic operon, the cpn10 gene preceding the cpn60 gene. Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma70-dependent but not of sigma32-dependent promoters of Escherichia coli were found. The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sigma32-dependent promoters. Primer extension analysis disclosed one and the same transcription start, irrespective of the presence or absence of heat or oxidative stress. After separation of lysates of the F. tularensis LVS organism by two-dimensional gel electrophoresis, DnaK, Cpn60, and Cpn10 were extracted and used as antigens in T-cell tests. When compared to those from nonvaccinated individuals, T cells from individuals previously vaccinated with live F. tularensis LVS showed an increased proliferative response to DnaK and Cpn60 but not to Cpn10. The present data will facilitate further studies of the involvement of the heat shock proteins in protective immunity to tularemia.

Full Text

The Full Text of this article is available as a PDF (317.3 KB).

Selected References

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

  1. Adams E., Garsia R. J., Hellqvist L., Holt P., Basten A. T cell reactivity to the purified mycobacterial antigens p65 and p70 in leprosy patients and their household contacts. Clin Exp Immunol. 1990 May;80(2):206–212. doi: 10.1111/j.1365-2249.1990.tb05235.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballard S. A., Segers R. P., Bleumink-Pluym N., Fyfe J., Faine S., Adler B. Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni. Mol Microbiol. 1993 May;8(4):739–751. doi: 10.1111/j.1365-2958.1993.tb01617.x. [DOI] [PubMed] [Google Scholar]
  3. Barnes P. F., Mehra V., Rivoire B., Fong S. J., Brennan P. J., Voegtline M. S., Minden P., Houghten R. A., Bloom B. R., Modlin R. L. Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis. J Immunol. 1992 Mar 15;148(6):1835–1840. [PubMed] [Google Scholar]
  4. Bhatnagar N. B., Elkins K. L., Fortier A. H. Heat stress alters the virulence of a rifampin-resistant mutant of Francisella tularensis LVS. Infect Immun. 1995 Jan;63(1):154–159. doi: 10.1128/iai.63.1.154-159.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burke D. S. Immunization against tularemia: analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J Infect Dis. 1977 Jan;135(1):55–60. doi: 10.1093/infdis/135.1.55. [DOI] [PubMed] [Google Scholar]
  6. CHAMBERLAIN R. E. EVALUATION OF LIVE TULAREMIA VACCINE PREPARED IN A CHEMICALLY DEFINED MEDIUM. Appl Microbiol. 1965 Mar;13:232–235. doi: 10.1128/am.13.2.232-235.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. Ericsson M., Tärnvik A., Kuoppa K., Sandström G., Sjöstedt A. Increased synthesis of DnaK, GroEL, and GroES homologs by Francisella tularensis LVS in response to heat and hydrogen peroxide. Infect Immun. 1994 Jan;62(1):178–183. doi: 10.1128/iai.62.1.178-183.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gomez F. J., Allendoerfer R., Deepe G. S., Jr Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis. Infect Immun. 1995 Jul;63(7):2587–2595. doi: 10.1128/iai.63.7.2587-2595.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Görg A., Postel W., Günther S. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 1988 Sep;9(9):531–546. doi: 10.1002/elps.1150090913. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Kaufmann S. H. Heat shock proteins and the immune response. Immunol Today. 1990 Apr;11(4):129–136. doi: 10.1016/0167-5699(90)90050-j. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Lee C., Levin A., Branton D. Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1987 Nov 1;166(2):308–312. doi: 10.1016/0003-2697(87)90579-3. [DOI] [PubMed] [Google Scholar]
  15. McKenzie K. R., Adams E., Britton W. J., Garsia R. J., Basten A. Sequence and immunogenicity of the 70-kDa heat shock protein of Mycobacterium leprae. J Immunol. 1991 Jul 1;147(1):312–319. [PubMed] [Google Scholar]
  16. Mehra V., Bloom B. R., Bajardi A. C., Grisso C. L., Sieling P. A., Alland D., Convit J., Fan X. D., Hunter S. W., Brennan P. J. A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein. J Exp Med. 1992 Jan 1;175(1):275–284. doi: 10.1084/jem.175.1.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mehra V., Sweetser D., Young R. A. Efficient mapping of protein antigenic determinants. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7013–7017. doi: 10.1073/pnas.83.18.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Narberhaus F., Bahl H. Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum. J Bacteriol. 1992 May;174(10):3282–3289. doi: 10.1128/jb.174.10.3282-3289.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Orme I. M., Andersen P., Boom W. H. T cell response to Mycobacterium tuberculosis. J Infect Dis. 1993 Jun;167(6):1481–1497. doi: 10.1093/infdis/167.6.1481. [DOI] [PubMed] [Google Scholar]
  21. Sandström G., Tärnvik A., Wolf-Watz H. Immunospecific T-lymphocyte stimulation by membrane proteins from Francisella tularensis. J Clin Microbiol. 1987 Apr;25(4):641–644. doi: 10.1128/jcm.25.4.641-644.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sandström G., Tärnvik A., Wolf-Watz H., Löfgren S. Antigen from Francisella tularensis: nonidentity between determinants participating in cell-mediated and humoral reactions. Infect Immun. 1984 Jul;45(1):101–106. doi: 10.1128/iai.45.1.101-106.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schmidt A., Schiesswohl M., Völker U., Hecker M., Schumann W. Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis. J Bacteriol. 1992 Jun;174(12):3993–3999. doi: 10.1128/jb.174.12.3993-3999.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Segal G., Ron E. Z. Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure. J Bacteriol. 1993 May;175(10):3083–3088. doi: 10.1128/jb.175.10.3083-3088.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Segal R., Ron E. Z. Regulation and organization of the groE and dnaK operons in Eubacteria. FEMS Microbiol Lett. 1996 Apr 15;138(1):1–10. doi: 10.1111/j.1574-6968.1996.tb08126.x. [DOI] [PubMed] [Google Scholar]
  26. Shinnick T. M. The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol. 1987 Mar;169(3):1080–1088. doi: 10.1128/jb.169.3.1080-1088.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Silva C. L., Lowrie D. B. A single mycobacterial protein (hsp 65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis. Immunology. 1994 Jun;82(2):244–248. [PMC free article] [PubMed] [Google Scholar]
  28. Sjöstedt A., Kuoppa K., Johansson T., Sandström G. The 17 kDa lipoprotein and encoding gene of Francisella tularensis LVS are conserved in strains of Francisella tularensis. Microb Pathog. 1992 Sep;13(3):243–249. doi: 10.1016/0882-4010(92)90025-j. [DOI] [PubMed] [Google Scholar]
  29. Sjöstedt A., Sandström G., Tärnvik A., Jaurin B. Molecular cloning and expression of a T-cell stimulating membrane protein of Francisella tularensis. Microb Pathog. 1989 Jun;6(6):403–414. doi: 10.1016/0882-4010(89)90082-x. [DOI] [PubMed] [Google Scholar]
  30. Sjöstedt A., Sandström G., Tärnvik A., Jaurin B. Nucleotide sequence and T cell epitopes of a membrane protein of Francisella tularensis. J Immunol. 1990 Jul 1;145(1):311–317. [PubMed] [Google Scholar]
  31. Skrodzki E. Investigations on the pathogenesis of tularemia. VII. Attempts to discover F.tularensis toxins. Biul Inst Med Morsk Gdansk. 1968;19(1):69–76. [PubMed] [Google Scholar]
  32. 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]
  33. Surcel H. M. Diversity of Francisella tularensis antigens recognized by human T lymphocytes. Infect Immun. 1990 Aug;58(8):2664–2668. doi: 10.1128/iai.58.8.2664-2668.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sutinen S., Syrjälä H. Histopathology of human lymph node tularemia caused by Francisella tularensis var palaearctica. Arch Pathol Lab Med. 1986 Jan;110(1):42–46. [PubMed] [Google Scholar]
  35. Tärnvik A., Eriksson M., Sandström G., Sjöstedt A. Francisella tularensis--a model for studies of the immune response to intracellular bacteria in man. Immunology. 1992 Jul;76(3):349–354. [PMC free article] [PubMed] [Google Scholar]
  36. Tärnvik A. Nature of protective immunity to Francisella tularensis. Rev Infect Dis. 1989 May-Jun;11(3):440–451. [PubMed] [Google Scholar]
  37. Vordermeier H. M. T-cell recognition of mycobacterial antigens. Eur Respir J Suppl. 1995 Sep;20:657s–667s. [PubMed] [Google Scholar]
  38. Webb R., Reddy K. J., Sherman L. A. Regulation and sequence of the Synechococcus sp. strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin. J Bacteriol. 1990 Sep;172(9):5079–5088. doi: 10.1128/jb.172.9.5079-5088.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Weeratna R., Stamler D. A., Edelstein P. H., Ripley M., Marrie T., Hoskin D., Hoffman P. S. Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60. Infect Immun. 1994 Aug;62(8):3454–3462. doi: 10.1128/iai.62.8.3454-3462.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Witkin S. S., Jeremias J., Toth M., Ledger W. J. Cell-mediated immune response to the recombinant 57-kDa heat-shock protein of Chlamydia trachomatis in women with salpingitis. J Infect Dis. 1993 Jun;167(6):1379–1383. doi: 10.1093/infdis/167.6.1379. [DOI] [PubMed] [Google Scholar]
  41. Yuan G., Wong S. L. Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK. J Bacteriol. 1995 Nov;177(22):6462–6468. doi: 10.1128/jb.177.22.6462-6468.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yuan G., Wong S. L. Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE). J Bacteriol. 1995 Oct;177(19):5427–5433. doi: 10.1128/jb.177.19.5427-5433.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zuber M., Hoover T. A., Dertzbaugh M. T., Court D. L. Analysis of the DnaK molecular chaperone system of Francisella tularensis. Gene. 1995 Oct 16;164(1):149–152. doi: 10.1016/0378-1119(95)00489-s. [DOI] [PubMed] [Google Scholar]
  44. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES