Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis

Abstract

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anionexchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol-and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum. MATERIALS AND METHODS Antigen preparation. Short-term culture filtrate (ST-CF), which is highly enriched in proteins actively secreted by M. tuberculosis, was produced with minor modifications as described previously (9). M. tuberculosis H37Rv was grown on an orbital shaker at 37ЊC in modified Sauton medium. The bacteria were removed from the cultures by filtration (Milliguard housing system [Millipore]) after 7 days of growth, and the culture supernatant was concentrated (ϫ100) on YM3 membrane (Amicon, Danvers, Mass.), resulting in protein concentrations ranging from 5 to 20 mg/ml. Three-to 5-week-old culture filtrates from stationary cultures of M. tuberculosis H37Rv were prepared as d...

Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis

“…tuberculosis and BCG, including the Myco. tuberculosis 38-kD [24], MPB70 [25] and the 10-kD ESAT-6 proteins [26]. A combination of these Myco.…”

Enzyme-linked immunospot assay for interferon-gamma in the diagnosis of tuberculous pleurisy

“…The 6-kDa early-secreted antigenic target (ESAT-6) and culture filtrate protein 10 (CFP10) are antigens preferentially found in Mycobacterium tuberculosis and virulent Mycobacterium bovis, but not in M. bovis bacille Calmette-Guérin vaccine or most environmental mycobacteria [20][21][22]. It has been shown that ESAT-6 and CFP10 can stimulate peripheral blood mononuclear cells (PBMCs) from patients with tuberculosis to secrete specific IFN-c. An ex vivo enzyme-linked immunospot (ELISPOT) test employing these antigens and detecting IFN-csecreting T-cells in peripheral blood [23][24][25][26][27] and bronchoalveolar lavage fluid [28] has been found useful in diagnosing latent and active tuberculosis.…”

Association of ESAT-6/CFP-10-induced IFN-γ, TNF-α and IL-10 with clinical tuberculosis: evidence from cohorts of pulmonary tuberculosis patients, household contacts and community controls in an endemic setting

“…Several studies have suggested that ESAT-6, CFP-10 and/or ESAT-6/CFP-10, alone or in combination with other proteins or BCG, induce strong protective T cell immunity during Mtb infection, especially in a murine model of TB [11,13,14,[27][28][29]. These reports have led to extensive research to realize the potential of these proteins as vaccine candidates [29][30][31][32][33][34].…”