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. 1995 Dec;102(3):626–634. doi: 10.1111/j.1365-2249.1995.tb03863.x

Serological distinction of integral plasma membrane proteins as a class of mycobacterial antigens and their relevance for human T cell activation.

J Mehrotra 1, D Bisht 1, V D Tiwari 1, S Sinha 1
PMCID: PMC1553371  PMID: 8536383

Abstract

This study pertains to classification and antigenic analysis of mycobacterial plasma membrane proteins in relation to human T cell proliferative responses, using a 'fast grower' Mycobacterium fortuitum as model. Membrane vesicles, prepared by sonication and differential centrifugation, were subjected to biphasic Triton X-114 extraction for isolation of integral peripheral (aqueous phase) proteins. Neither protein pool showed any appreciable overlap serologically. SDS-PAGE showed five prominent bands in peripheral and three in the integral protein pool, whereas immunoblotting with rabbit antisera identified only two major antigens (60 and 67 kD) in the former and five (24, 34, 42, 51 and 54 kD) in the latter. ELISA with a panel of anti-mycobacterial MoAbs revealed that nine out of 12 previously known antigens were present in the peripheral protein pool. Only two of them (33 and 40 kD) were additionally detected amongst integral proteins. The membrane-associated immunosuppressive moiety lipoarabinomannan was semiquantitatively located in aqueous phase. In bulk T cell proliferation assays, seven out of 10 subjects belonging to a 'responder' background (BT-BB leprosy patients and healthy contacts) showed high responses for Myco. fortuitum antigens. Proliferative response with integral proteins was comparable to that with whole membrane, but it was significantly higher (P < 0.0005) than the response with peripheral proteins. The distinction and relevance of integral membrane proteins as a class of mycobacterial antigens make them worthy of consideration in a subunit vaccine design.

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Selected References

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

  1. Akins D. R., Purcell B. K., Mitra M. M., Norgard M. V., Radolf J. D. Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity. Infect Immun. 1993 Apr;61(4):1202–1210. doi: 10.1128/iai.61.4.1202-1210.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asano A., Cohen N. S., Baker R. F., Brodie A. F. Orientation of the cell membrane in ghosts and electron transport particles of Mycobacterium phlei. J Biol Chem. 1973 May 25;248(10):3386–3397. [PubMed] [Google Scholar]
  3. Beckman E. M., Porcelli S. A., Morita C. T., Behar S. M., Furlong S. T., Brenner M. B. Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells. Nature. 1994 Dec 15;372(6507):691–694. doi: 10.1038/372691a0. [DOI] [PubMed] [Google Scholar]
  4. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  5. Brennan P. J. The carbohydrate-containing antigens of Mycobacterium leprae. Lepr Rev. 1986 Dec;57 (Suppl 2):39–51. [PubMed] [Google Scholar]
  6. Brodie A. F., Kalra V. K., Lee S. H., Cohen N. S. Properties of energy-transducing systems in different types of membrane preparations from Mycobacterium phlei--preparation, resolution, and reconstitution. Methods Enzymol. 1979;55:175–200. doi: 10.1016/0076-6879(79)55024-1. [DOI] [PubMed] [Google Scholar]
  7. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  8. Chatterjee D., Roberts A. D., Lowell K., Brennan P. J., Orme I. M. Structural basis of capacity of lipoarabinomannan to induce secretion of tumor necrosis factor. Infect Immun. 1992 Mar;60(3):1249–1253. doi: 10.1128/iai.60.3.1249-1253.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chaturvedi V., Sinha S., Girdhar B. K., Katoch K., Bhatia A. S., Sengupta U. Association of mycobacterial-specific and Mycobacterium leprae specific antibody levels with clinical activity in tuberculoid leprosy: a comparative study of three serological enzyme-immunoassays. Lepr Rev. 1991 Jun;62(2):122–133. doi: 10.5935/0305-7518.19910014. [DOI] [PubMed] [Google Scholar]
  10. Coates A. R., Hewitt J., Allen B. W., Ivanyi J., Mitchison D. A. Antigenic diversity of Mycobacterium tuberculosis and Mycobacterium bovis detected by means of monoclonal antibodies. Lancet. 1981 Jul 25;2(8239):167–169. doi: 10.1016/s0140-6736(81)90355-x. [DOI] [PubMed] [Google Scholar]
  11. Convit J., Sampson C., Zúiga M., Smith P. G., Plata J., Silva J., Molina J., Pinardi M. E., Bloom B. R., Salgado A. Immunoprophylactic trial with combined Mycobacterium leprae/BCG vaccine against leprosy: preliminary results. Lancet. 1992 Feb 22;339(8791):446–450. doi: 10.1016/0140-6736(92)91056-e. [DOI] [PubMed] [Google Scholar]
  12. Deres K., Schild H., Wiesmüller K. H., Jung G., Rammensee H. G. In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature. 1989 Nov 30;342(6249):561–564. doi: 10.1038/342561a0. [DOI] [PubMed] [Google Scholar]
  13. Fine P. E., Rodrigues L. C. Modern vaccines. Mycobacterial diseases. Lancet. 1990 Apr 28;335(8696):1016–1020. doi: 10.1016/0140-6736(90)91074-k. [DOI] [PubMed] [Google Scholar]
  14. Gelber R. H., Brennan P. J., Hunter S. W., Munn M. W., Monson J. M., Murray L. P., Siu P., Tsang M., Engleman E. G., Mohagheghpour N. Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae. Infect Immun. 1990 Mar;58(3):711–718. doi: 10.1128/iai.58.3.711-718.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gelber R. H., Mehra V., Bloom B., Murray L. P., Siu P., Tsang M., Brennan P. J. Vaccination with pure Mycobacterium leprae proteins inhibits M. leprae multiplication in mouse footpads. Infect Immun. 1994 Oct;62(10):4250–4255. doi: 10.1128/iai.62.10.4250-4255.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Germain R. N. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell. 1994 Jan 28;76(2):287–299. doi: 10.1016/0092-8674(94)90336-0. [DOI] [PubMed] [Google Scholar]
  17. Hammer J., Valsasnini P., Tolba K., Bolin D., Higelin J., Takacs B., Sinigaglia F. Promiscuous and allele-specific anchors in HLA-DR-binding peptides. Cell. 1993 Jul 16;74(1):197–203. doi: 10.1016/0092-8674(93)90306-b. [DOI] [PubMed] [Google Scholar]
  18. Hunter S. W., Rivoire B., Mehra V., Bloom B. R., Brennan P. J. The major native proteins of the leprosy bacillus. J Biol Chem. 1990 Aug 25;265(24):14065–14068. [PubMed] [Google Scholar]
  19. Kaufmann S. H. Immunity against intracellular bacteria: biological effector functions and antigen specificity of T lymphocytes. Curr Top Microbiol Immunol. 1988;138:141–176. [PubMed] [Google Scholar]
  20. Kaufmann S. H., Young D. B. Vaccination against tuberculosis and leprosy. Immunobiology. 1992 Feb;184(2-3):208–229. doi: 10.1016/S0171-2985(11)80476-6. [DOI] [PubMed] [Google Scholar]
  21. Khanolkar-Young S., Kolk A. H., Andersen A. B., Bennedsen J., Brennan P. J., Rivoire B., Kuijper S., McAdam K. P., Abe C., Batra H. V. Results of the third immunology of leprosy/immunology of tuberculosis antimycobacterial monoclonal antibody workshop. Infect Immun. 1992 Sep;60(9):3925–3927. doi: 10.1128/iai.60.9.3925-3927.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle. 1991 Mar;72(1):1–6. doi: 10.1016/0041-3879(91)90017-m. [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 B. Y., Hefta S. A., Brennan P. J. Characterization of the major membrane protein of virulent Mycobacterium tuberculosis. Infect Immun. 1992 May;60(5):2066–2074. doi: 10.1128/iai.60.5.2066-2074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leopold K., Fischer W. Molecular analysis of the lipoglycans of Mycobacterium tuberculosis. Anal Biochem. 1993 Jan;208(1):57–64. doi: 10.1006/abio.1993.1008. [DOI] [PubMed] [Google Scholar]
  26. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  27. Moreno C., Mehlert A., Lamb J. The inhibitory effects of mycobacterial lipoarabinomannan and polysaccharides upon polyclonal and monoclonal human T cell proliferation. Clin Exp Immunol. 1988 Nov;74(2):206–210. [PMC free article] [PubMed] [Google Scholar]
  28. Murray P. J., Spithill T. W., Handman E. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice. Infect Immun. 1989 Jul;57(7):2203–2209. doi: 10.1128/iai.57.7.2203-2209.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nagao S., Tanaka A. Necrotic inflammatory reaction induced by muramyl dipeptide in guinea pigs sensitized by tubercle bacilli. J Exp Med. 1985 Aug 1;162(2):401–412. doi: 10.1084/jem.162.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pessolani M. C., Smith D. R., Rivoire B., McCormick J., Hefta S. A., Cole S. T., Brennan P. J. Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae. J Exp Med. 1994 Jul 1;180(1):319–327. doi: 10.1084/jem.180.1.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ridley D. S., Jopling W. H. Classification of leprosy according to immunity. A five-group system. Int J Lepr Other Mycobact Dis. 1966 Jul-Sep;34(3):255–273. [PubMed] [Google Scholar]
  32. Shoenfeld Y., Isenberg D. A. Mycobacteria and autoimmunity. Immunol Today. 1988 Jun;9(6):178–182. doi: 10.1016/0167-5699(88)91294-7. [DOI] [PubMed] [Google Scholar]
  33. Sibley L. D., Adams L. B., Krahenbuhl J. L. Inhibition of interferon-gamma-mediated activation in mouse macrophages treated with lipoarabinomannan. Clin Exp Immunol. 1990 Apr;80(1):141–148. doi: 10.1111/j.1365-2249.1990.tb06454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smythe J. A., Coppel R. L., Brown G. V., Ramasamy R., Kemp D. J., Anders R. F. Identification of two integral membrane proteins of Plasmodium falciparum. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5195–5199. doi: 10.1073/pnas.85.14.5195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  37. Vachula M., Holzer T. J., Kizlaitis L., Andersen B. R. Effect of Mycobacterium leprae's phenolic glycolipid-I on interferon-gamma augmentation of monocyte oxidative responses. Int J Lepr Other Mycobact Dis. 1990 Jun;58(2):342–346. [PubMed] [Google Scholar]
  38. Weiss R. On the track of "killer" TB. Science. 1992 Jan 10;255(5041):148–150. doi: 10.1126/science.1553538. [DOI] [PubMed] [Google Scholar]
  39. Wiker H. G., Harboe M. The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol Rev. 1992 Dec;56(4):648–661. doi: 10.1128/mr.56.4.648-661.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Young D. B., Garbe T. R. Lipoprotein antigens of Mycobacterium tuberculosis. Res Microbiol. 1991 Jan;142(1):55–65. doi: 10.1016/0923-2508(91)90097-t. [DOI] [PubMed] [Google Scholar]
  41. Young D. B., Kaufmann S. H., Hermans P. W., Thole J. E. Mycobacterial protein antigens: a compilation. Mol Microbiol. 1992 Jan;6(2):133–145. doi: 10.1111/j.1365-2958.1992.tb01994.x. [DOI] [PubMed] [Google Scholar]
  42. 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]

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