PURIFICATION AND CHARACTERIZATION OF A LOW-MOLECULAR-MASS T-CELL ANTIGEN SECRETED BY MYCOBACTERIUM-TUBERCULOSIS

A novel immunogenic antigen, the 6-kDa early secretory antigenic targe t (ESAT-6), from short-term culture filtrates of Mycobacterium tubercu losis was purified by hydrophobic interaction chromatography and anion -exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoele ctric focusing, and each of these components separated into three spot s ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfat e-polyacrylamide gel electrophoresis. The apparent differences in mole cular masses or pIs of these isoforms were not due to posttranslationa l glycosylation. The molecular weight of the purified native protein w as determined by applying gel filtration and nondenaturing polyacrylam ide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized b y the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressin g a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fr agment of the mycobacterial DNA insert was sequenced. The structural g ene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be align ed with the 10 amino-terminal amino acids derived from sequence analys es of the native protein, N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amin o acid analysis of the antigen was in good agreement with the DNA sequ ence-deduced amino acid composition. Thus, the heterogeneities observe d 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 immunological ly active in that both elicited a high release of gamma interferon fro m T cells isolated from memory-immune mice challenged with M. tubercul osis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions, Int erspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not be longing to the M. tuberculosis complex, reactivity was observed in Myc obacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.