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Volume 151, Issue 7

Immunogenic membrane-associated proteins of revealed by proteomics Free

Abstract

Membrane-associated proteins of offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the proteome. Functional orthologues of 73 of these proteins were also present in , suggesting their relative importance. Bioinformatics predicted that as many as 73 % of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN- production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

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Keyword(s): 1-DGE, 1-D gel electrophoresis , 2-DGE, 2-D gel electrophoresis , AP, aqueous phase , CEGE, continuous-elution gel electrophoresis , DP, detergent phase , SI, stimulation index and TX-114, Triton X-114
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2005-07-01
2024-04-19
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References

  1. Arand M., Friedberg T., Oesch F. 1992; Colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins. Anal Biochem 207:73–75 [CrossRef]
     [Google Scholar]
  2. Asselineau C., Asselineau J., Laneelle G., Laneelle M. A. 2002; The biosynthesis of mycolic acids by mycobacteria: current and alternative hypotheses. Prog Lipid Res 41:501–523 [CrossRef]
     [Google Scholar]
  3. Barksdale L., Kim K. S. 1977; Mycobacterium . Bacteriol Rev 41:217–372
     [Google Scholar]
  4. Blum H., Beirer H., Gross H. J. 1987; Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electophoresis 8:93–99 [CrossRef]
     [Google Scholar]
  5. Bordier C. 1981; Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256:1604–1607
     [Google Scholar]
  6. Brennan P. J., Nikaido H. 1995; The envelope of mycobacteria. Annu Rev Biochem 64:29–63 [CrossRef]
     [Google Scholar]
  7. Broquet A. H., Thomas G., Masliah J., Trugnan G., Bachelet M. 2003; Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J Biol Chem 278:21601–21606 [CrossRef]
     [Google Scholar]
  8. Caccamo N., Milano S., Di Sano C., Cigna D., Ivanyi J., Krensky A. M., Dieli F., Salerno A. 2002; Identification of epitopes of Mycobacterium tuberculosis 16-kDa protein recognized by human leukocyte antigen-A*0201 CD8+ T lymphocytes. J Infect Dis 186:991–998 [CrossRef]
     [Google Scholar]
  9. Cole S. T., Brosch R., Parkhill J. 39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
     [Google Scholar]
  10. Cole S. T., Eiglmeier K., Parkhill J. 41 other authors 2001; Massive gene decay in the leprosy bacillus. Nature 409:1007–1011 [CrossRef]
     [Google Scholar]
  11. Driessen A. J., Manting E. H., van der Does C. 2001; The structural basis of protein targeting and translocation in bacteria. Nat Struct Biol 8:492–498 [CrossRef]
     [Google Scholar]
  12. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. 1982; Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97–102 [CrossRef]
     [Google Scholar]
  13. Futai M., Noumi T., Maeda M. 1989; ATP synthase (H+-ATPase): results by combined biochemical and molecular biological approaches. Annu Rev Biochem 58:111–136 [CrossRef]
     [Google Scholar]
  14. Galeva N., Altermann M. 2002; Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomic analysis of rat liver microsomes: cytochromes P450 and other membrane proteins. Proteomics 2:713–722 [CrossRef]
     [Google Scholar]
  15. Gorg A., Obermaier C., Boguth G., Harder A., Scheibe B., Wildgruber R., Weiss W. 2000; The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037–1053 [CrossRef]
     [Google Scholar]
  16. Gu S., Chen J., Dobos K. M., Bradbury E. M., Belisle J. T., Chen X. 2003; Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain. Mol Cell Proteomics 2:1284–1296 [CrossRef]
     [Google Scholar]
  17. Iborra S., Soto M., Carrion J., Nieto A., Fernandez E., Alonso C., Requena J. M. 2003; The Leishmania infantum acidic ribosomal protein P0 administered as a DNA vaccine confers protective immunity to Leishmania major infection in BALB/c mice. Infect Immun 71:6562–6572 [CrossRef]
     [Google Scholar]
  18. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132 [CrossRef]
     [Google Scholar]
  19. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
     [Google Scholar]
  20. Lalvani A., Nagvenkar P., Udwadia Z. 7 other authors 2001; Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians. J Infect Dis 183:469–477 [CrossRef]
     [Google Scholar]
  21. Lehninger A. L. 1975 Biochemistry , 2nd edn. New York: Worth Publishers;
     [Google Scholar]
  22. Lopez M., Sly L. M., Luu Y., Young D., Cooper H., Reiner N. E. 2003; The 19-kDa Mycobacterium tuberculosis protein induces macrophage apoptosis through Toll-like receptor-2. J Immunol 170:2409–2416 [CrossRef]
     [Google Scholar]
  23. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. 1978; A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210 [CrossRef]
     [Google Scholar]
  24. Matsuoka M., Nomaguchi H., Yukitake H., Ohara N., Matsumoto S., Mise K., Yamada T. 1997; Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by immunization with ribosomal fraction and culture filtrate from Mycobacterium bovis BCG. Vaccine 15:1214–1217 [CrossRef]
     [Google Scholar]
  25. McIntosh T. J., Vidal A., Simon S. A. 2003; Sorting of lipids and transmembrane peptides between detergent-soluble bilayers and detergent-resistant rafts. Biophys J 85:1656–1666 [CrossRef]
     [Google Scholar]
  26. Mehrotra J., Bisht D., Tiwari V. D., Sinha S. 1995; Serological distinction of integral plasma membrane proteins as a class of mycobacterial antigens and their relevance for human T cell activation. Clin Exp Immunol 102:626–634
     [Google Scholar]
  27. Mehrotra J., Mittal A., Dhindsa M. S., Sinha S. 1997; Fractionation of mycobacterial integral membrane proteins by continuous elution SDS-PAGE reveals the immunodominance of low molecular weight subunits for human T cells. Clin Exp Immunol 109:446–450 [CrossRef]
     [Google Scholar]
  28. Mehrotra J., Mittal A., Rastogi A. K., Jaiswal A. K., Bhandari N. K., Sinha S. 1999; Antigenic definition of plasma membrane proteins of Bacillus Calmette-Guérin: predominant activation of human T cells by low-molecular-mass integral proteins. Scand J Immunol 50:411–419 [CrossRef]
     [Google Scholar]
  29. Nigou J., Gilleron M., Puzo G. 2003; Lipoarabinomannans: from structure to biosynthesis. Biochimie 85:153–166 [CrossRef]
     [Google Scholar]
  30. Ortiz-Ortiz L., Solarolo E. B., Bojalil L. F. 1971; Delayed hypersensitivity to ribosomal protein from BCG. J Immunol 107:1022–1026
     [Google Scholar]
  31. Pessolani M. C., Smith D. R., Rivoire B., McCormick J., Hefta S. A., Cole S. T., Brennan P. J. 1994; Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae . J Exp Med 180:319–327 [CrossRef]
     [Google Scholar]
  32. Santoni V., Molloy M., Rabilloud T. 2000; Membrane proteins and proteomics: un amour impossible?. Electrophoresis 21:1054–1070 [CrossRef]
     [Google Scholar]
  33. Schmidt F., Donahoe S., Hagens K., Mattow J., Schaible U. E., Kaufmann S. H., Aebersold R., Jungblut P. R. 2004; Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology. Mol Cell Proteomics 3:24–42
     [Google Scholar]
  34. Shaw M. M., Riederer B. M. 2003; Sample preparation for two-dimensional gel electrophoresis. Proteomics 3:1408–1417 [CrossRef]
     [Google Scholar]
  35. Shevchenko A., Wilm M., Vorm O., Mann M. 1996; Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858 [CrossRef]
     [Google Scholar]
  36. Shin B. K., Wang H., Yim A. M. 9 other authors 2003; Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J Biol Chem 278:7607–7616 [CrossRef]
     [Google Scholar]
  37. Sigler K., Hofer M. 1997; Biotechnological aspects of membrane function. Crit Rev Biotechnol 17:69–86 [CrossRef]
     [Google Scholar]
  38. Sinha S., Arora S., Kosalai K., Namane A., Pym A. S., Cole S. T. 2002; Proteome analysis of the plasma membrane of Mycobacterium tuberculosis . Comp Funct Genomics 3:470–483 [CrossRef]
     [Google Scholar]
  39. Snider D. E. Jr, Castro K. G. 1998; The global threat of drug-resistant tuberculosis. N Engl J Med 338:1689–1690 [CrossRef]
     [Google Scholar]
  40. Tekaia F., Gordon S. V., Garnier T., Brosch R., Barrell B. G., Cole S. T. 1999; Analysis of the proteome of Mycobacterium tuberculosis in silico . Tuber Lung Dis 79:329–342 [CrossRef]
     [Google Scholar]
  41. Wessel D., Flugge U. I. 1984; A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143 [CrossRef]
     [Google Scholar]
  42. Yuan Y., Crane D. D., Simpson R. M., Zhu Y., Hickey M. J., Sherman D. R., Barry C. E. III 1998; The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc Natl Acad Sci U S A 95:9578–9583 [CrossRef]
     [Google Scholar]
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Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics
Microbiology 151, 2411 (2005); https://doi.org/10.1099/mic.0.27799-0
/content/journal/micro/10.1099/mic.0.27799-0
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