Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.
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-gamma 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.
Sinha
Sudhir
S
Division of Drug Target Discovery and Development, Biochemistry Block, Central Drug Research Institute, Post Box no. 173, Lucknow 226001, India.
Kosalai
K
K
Division of Drug Target Discovery and Development, Biochemistry Block, Central Drug Research Institute, Post Box no. 173, Lucknow 226001, India.
Arora
Shalini
S
Division of Drug Target Discovery and Development, Biochemistry Block, Central Drug Research Institute, Post Box no. 173, Lucknow 226001, India.
Namane
Abdelkader
A
PF-3 Protéomique, Génopole, Institut Pasteur, Paris, France.
Sharma
Pawan
P
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Gaikwad
Anil N
AN
Division of Drug Target Discovery and Development, Biochemistry Block, Central Drug Research Institute, Post Box no. 173, Lucknow 226001, India.
Brodin
Priscille
P
Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France.
Cole
Stewart T
ST
Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France.
D001426:Bacterial Proteins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000276:immunology; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D020543:Proteome