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. 1995 Oct;63(10):4003–4010. doi: 10.1128/iai.63.10.4003-4010.1995

Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

A Laqueyrerie 1, P Militzer 1, F Romain 1, K Eiglmeier 1, S Cole 1, G Marchal 1
PMCID: PMC173562  PMID: 7558311

Abstract

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome. The N-terminal identity or homology of the M. tuberculosis and M. bovis BCG purified molecules and their similar global and deduced amino acid compositions demonstrated the perfect correspondence between the molecular and chemical analyses. The presence of a high percentage of proline (21.7%) was confirmed and explained the apparent higher molecular mass (45/47 kDa) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulting from the increased rigidity of molecules due to proline residues.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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  1. Andersen P. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun. 1994 Jun;62(6):2536–2544. doi: 10.1128/iai.62.6.2536-2544.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes P. F., Bloch A. B., Davidson P. T., Snider D. E., Jr Tuberculosis in patients with human immunodeficiency virus infection. N Engl J Med. 1991 Jun 6;324(23):1644–1650. doi: 10.1056/NEJM199106063242307. [DOI] [PubMed] [Google Scholar]
  3. Content J., de la Cuvellerie A., De Wit L., Vincent-Levy-Frébault V., Ooms J., De Bruyn J. The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis. Infect Immun. 1991 Sep;59(9):3205–3212. doi: 10.1128/iai.59.9.3205-3212.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DUBOS R. J., PIERCE C. H., SCHAEFER W. B. Antituberculous immunity induced in mice by vaccination with living cultures of attenuated tubercle bacilli. J Exp Med. 1953 Feb 1;97(2):207–220. doi: 10.1084/jem.97.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dannenberg A. M., Jr Delayed-type hypersensitivity and cell-mediated immunity in the pathogenesis of tuberculosis. Immunol Today. 1991 Jul;12(7):228–233. doi: 10.1016/0167-5699(91)90035-R. [DOI] [PubMed] [Google Scholar]
  6. Espitia C., Mancilla R. Identification, isolation and partial characterization of Mycobacterium tuberculosis glycoprotein antigens. Clin Exp Immunol. 1989 Sep;77(3):378–383. [PMC free article] [PubMed] [Google Scholar]
  7. Furthmayr H., Timpl R. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem. 1971 Jun;41(2):510–516. doi: 10.1016/0003-2697(71)90173-4. [DOI] [PubMed] [Google Scholar]
  8. Higgins C. F., Gallagher M. P., Mimmack M. L., Pearce S. R. A family of closely related ATP-binding subunits from prokaryotic and eukaryotic cells. Bioessays. 1988 Apr;8(4):111–116. doi: 10.1002/bies.950080406. [DOI] [PubMed] [Google Scholar]
  9. Jacobs W. R., Jr, Kalpana G. V., Cirillo J. D., Pascopella L., Snapper S. B., Udani R. A., Jones W., Barletta R. G., Bloom B. R. Genetic systems for mycobacteria. Methods Enzymol. 1991;204:537–555. doi: 10.1016/0076-6879(91)04027-l. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Lefford M. J. Transfer of adoptive immunity to tuberculosis in mice. Infect Immun. 1975 Jun;11(6):1174–1181. doi: 10.1128/iai.11.6.1174-1181.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol. 1985;39:615–648. doi: 10.1146/annurev.mi.39.100185.003151. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Pal P. G., Horwitz M. A. Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect Immun. 1992 Nov;60(11):4781–4792. doi: 10.1128/iai.60.11.4781-4792.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Romain F., Augier J., Pescher P., Marchal G. Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions only in guinea pigs immunized with living mycobacteria. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5322–5326. doi: 10.1073/pnas.90.11.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Romain F., Laqueyrerie A., Militzer P., Pescher P., Chavarot P., Lagranderie M., Auregan G., Gheorghiu M., Marchal G. Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria. Infect Immun. 1993 Feb;61(2):742–750. doi: 10.1128/iai.61.2.742-750.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schorey J. S., Li Q., McCourt D. W., Bong-Mastek M., Clark-Curtiss J. E., Ratliff T. L., Brown E. J. A Mycobacterium leprae gene encoding a fibronectin binding protein is used for efficient invasion of epithelial cells and Schwann cells. Infect Immun. 1995 Jul;63(7):2652–2657. doi: 10.1128/iai.63.7.2652-2657.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R., Jr Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol. 1990 Nov;4(11):1911–1919. doi: 10.1111/j.1365-2958.1990.tb02040.x. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Thierry D., Chavarot P., Marchal G., Le Thi K. T., Ho M. L., Nguyen N. L., Le N. V., Ledru S., Fumoux F., Guesdon J. L. Mycobacterium tuberculosis strains unidentified using the IS6110 probe can be detected by oligonucleotides derived from the Mt308 sequence. Res Microbiol. 1995 May;146(4):325–328. doi: 10.1016/0923-2508(96)81055-2. [DOI] [PubMed] [Google Scholar]
  23. WEISS D. W., DUBOS R. J. Antituberculous immunity induced in mice by vaccination with killed tubercle bacilli or with a soluble bacillary extract. J Exp Med. 1955 Mar 1;101(3):313–330. doi: 10.1084/jem.101.3.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wieles B., van Agterveld M., Janson A., Clark-Curtiss J., Rinke de Wit T., Harboe M., Thole J. Characterization of a Mycobacterium leprae antigen related to the secreted Mycobacterium tuberculosis protein MPT32. Infect Immun. 1994 Jan;62(1):252–258. doi: 10.1128/iai.62.1.252-258.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Williamson M. P. The structure and function of proline-rich regions in proteins. Biochem J. 1994 Jan 15;297(Pt 2):249–260. doi: 10.1042/bj2970249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zhang Y., Heym B., Allen B., Young D., Cole S. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature. 1992 Aug 13;358(6387):591–593. doi: 10.1038/358591a0. [DOI] [PubMed] [Google Scholar]

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