Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry

Abstract

A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were generated. In total, 205 protein spots were observed. The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins. Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry. Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation. Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an ␣-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M. tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B. This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex. Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences. A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis. Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M. tuberculosis KatG catalase/peroxidase. Thus, the detailed mapping of M. tuberculosis proteins, combined with state-of-the-art analytical techniques such as mass spectrometry, provides a basis for further analysis and rapid identification of biologically relevant molecules.

Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis‐infected and culture filtrate protein‐treated macrophages

“…Interestingly, we found 29 M.tb proteins on these exosomes. There are approximately 205 M.tb proteins in the culture supernatant [15] so our findings that 29 of these including ESAT-6 and the 85 complex are trafficked to MVBs and exosomes suggest an efficient transport mechanism. However, why these specific M.tb proteins are targeted is not known.…”

“…The vast majority of mycobacterial proteins identified on the exosomes are known/predicted to be secreted [15] as summarized in Table 1. Most of the proteins are likely secreted via the SecA secretory system but some such as ESAT-6 are released through the newly described type VII secretion system present in mycobacteria [16].…”

Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling

“…a-Crystallin spots showing increased intensity on gels of the starved cultures are therefore species with differing pI and molecular weights compared with the most abundant forms. Different species of this protein at lower molecular weights and differing pIs have been observed previously (Sonnenberg and Belisle, 1997;Betts et al, 2000) and could result from degradation, post-translational processing or post-translational modification, such as phosphorylation.…”

“…Tig is a chaperone-like protein homologous to the Bacillus subtilis trigger factor, thought to be involved in protein export. The 45 kDa antigen and MPT64 are both found in culture filtrates of M. tuberculosis (Sonnenberg and Belisle, 1997). The 45 kDa antigen is a glycoprotein (Dobos et al, 1996), which was identified as being recognized by antibodies and producing a delayed-type hypersensitivity (DTH) response in immunized guinea pigs (Romain et al, 1993).…”

Identification of a secreted superoxide dismutase in Mycobacterium avium ssp. paratuberculosis

“…To rule out autolysis as a source of the extracellular Sod, the HSP65, which is commonly used as a marker for autolysis [22], was also measured using the monoclonal antibody IT-13 [12]. No HSP65 protein was identi¢ed in the culture supernatant £uids using this antibody (Fig.…”