Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics

• Published in: Microbiology (Society for General Microbiology) Vol. 151; no. 7; pp. 2411 - 2419
• Main Authors: Sinha, Sudhir, Kosalai, K, Arora, Shalini, Namane, Abdelkader, Sharma, Pawan, Gaikwad, Anil N, Brodin, Priscille, Cole, Stewart T
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.07.2005; Society for General Microbiology
• Subjects: Bacterial Proteins - chemistry; Bacteriology; Biological and medical sciences; Electrophoresis, Gel, Two-Dimensional; Fundamental and applied biological sciences. Psychology; Humans; Membrane Proteins - analysis; Membrane Proteins - chemistry; Membrane Proteins - immunology; Microbiology; Mycobacterium leprae; Mycobacterium tuberculosis; Mycobacterium tuberculosis - chemistry; Mycobacterium tuberculosis - immunology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Proteome; Proteomics - instrumentation; Proteomics - methods; T-Lymphocytes - immunology; Asia; India; Membrane protein; Mycobacterial infection; Infection; Tuberculosis; Immunogenicity; Mycobacterium tuberculosis; Mycobacteriales; Proteomics; Bacteriosis; Mycobacteriaceae; Bacteria; Actinomycetes; Two dimensional electrophoresis; Bioinformatics; Mass spectrometry; ELISA assay
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.27799-0

1 Division of Drug Target Discovery and Development, Biochemistry Block, Central Drug Research Institute, Post Box no. 173, Lucknow 226001, India 2 PF-3 Protéomique, Génopole, Institut Pasteur, Paris, France 3 International Centre for Genetic Engineering and Biotechnology, New Delhi, India 4 Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France Correspondence Sudhir Sinha sinhas{at}lycos.com Membrane-associated proteins of Mycobacterium tuberculosis 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 M. tuberculosis 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 M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae , 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. Abbreviations: AP, aqueous phase; CEGE, continuous-elution gel electrophoresis; 1-DGE, 1-D gel electrophoresis; 2-DGE, 2-D gel electrophoresis; DP, detergent phase; SI, stimulation index; TX-114, Triton X-114 A table of protein identification data is available as supplementary material with the online version of this paper.