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Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.

ABSTRACT: Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.

SUBMITTER: Weldingh K

PROVIDER: S-EPMC108378 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

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Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.

Weldingh K K Rosenkrands I I Jacobsen S S Rasmussen P B PB Elhay M J MJ Andersen P P

Infection and immunity 19980801 8

Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their cap ...[more]

PMID: 9673225

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Project description: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 alpha-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.

Project description:Despite being the subject of intensive research, tuberculosis, caused by Mycobacterium tuberculosis, remains at present the leading cause of death from an infectious agent. Secreted and cell wall proteins interact with the host and play important roles in pathogenicity. These proteins are explored as candidate diagnostic markers, potential drug targets or vaccine antigens, and more recently special attention is being given to the role of their post-translational modifications. With the purpose of contributing to the proteomic and glycoproteomic characterization of this important pathogen, we performed a shotgun analysis of culture filtrate proteins of M. tuberculosis based on a liquid nano-HPLC tandem mass spectrometry and a label-free spectral counting normalization approach for protein quantification. We identified 1314 M. tuberculosis proteins in culture filtrate and found that the most abundant proteins belong to the extracellular region or cell wall compartment, and that the functional categories with higher protein abundance factor were virulence, detoxification and adaptation, and cell wall and cell processes. We could identify a group of proteins consistently detected in previous studies, most of which were highly abundant proteins. In culture filtrate, 140 proteins were predicted to contain one of the three types of bacterial N-terminal signal peptides. Besides, various proteins belonging to the ESX secretion systems, and to the PE and PPE families, secreted by the type VII secretion system using nonclassical secretion signals, were also identified. O-glycosylation was identified in 46 proteins, many of them lipoproteins and cell wall associated proteins. Finally, we provide proteomic evidence for 33 novel O-glycosylated proteins, aiding to the glycoproteomic characterization of relevant antigenic membrane and exported proteins. These findings are expected to collaborate with the research on pathogen derived biomarkers, virulence factors and vaccine candidates, and to provide clues to the understanding of the pathogenesis and survival strategies adopted by M. tuberculosis.

Dataset Information

Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.

Publications

Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.

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