Proteome analysis of the plasma membrane of Mycobacterium tuberculosis

Abstract

The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium.

Mycobacterium tuberculosis GroEL Homologues Unusually Exist as Lower Oligomers and Retain the Ability to Suppress Aggregation of Substrate Proteins

“…35 Moreover, various other reports suggest alternative roles for the GroEL homologues. For example, a GroEL homologue in Buchnera, an intracellular symbiotic bacterium of aphids, is not only a molecular chaperone but also a phosphocarrier protein, suggesting that the protein plays a role in a signal transducing system.…”

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics

“…The membrane of M. tuberculosis H37Rv was isolated and processed as described previously (Sinha et al, 2002). In brief, 3-4-week-old bacterial cultures (in Löwenstein-Jensen medium) were probe-sonicated and centrifuged, initially at 23 000 g to remove unbroken cells and cell-wall debris, and later at 150 000 g to obtain the membrane as sediment, and cytosol as supernatant.…”

“…Membrane-associated proteins were extracted with TX-114, following the protocol of Bordier (1981) as described previously (Mehrotra et al, 1995;Sinha et al, 2002). In brief, pre-condensed TX-114 was added to the membrane (final detergent concentration 2 %, w/v) and the suspension was stirred at 4 uC to get the protein extract in a single phase.…”

“…2-DGE was performed as described previously (Sinha et al, 2002). A solubilization buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 0?3 % DTT, 0?5 % carrier ampholytes 4-7, 40 mM Tris, pH 9?6) recommended for IEF of membrane proteins (Gorg et al, 2000) was used.…”

“…We have previously shown that the extraction of proteins from the isolated membrane of M. tuberculosis with Triton X-114 (TX-114) detergent serves as a useful sample preparatory method (Sinha et al, 2002). This procedure, based on the partitioning of proteins in detergent-and water-rich phases (Bordier, 1981), was found more effective in revealing the putative membrane proteins of the bacillus than an alternative protocol based on extraction with carbonate (Fujiki et al, 1982).…”