Molecular Cloning and Immunologic Reactivity of a Novel Low Molecular Mass Antigen of Mycobacterium tuberculosis

• Published in: The Journal of immunology (1950) Vol. 161; no. 5; pp. 2356 - 2364
• Main Authors: Coler, Rhea N, Skeiky, Yasir A. W, Vedvick, Thomas, Bement, Teresa, Ovendale, Pamela, Campos-Neto, Antonio, Alderson, Mark R, Reed, Steven G
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 01-09-1998
• Subjects: Amino Acid Sequence; Animals; Antigens, Bacterial - administration & dosage; Antigens, Bacterial - genetics; Antigens, Bacterial - immunology; Bacterial Proteins; Base Sequence; Blotting, Western; Cloning, Molecular; Culture Media - analysis; Cytokines - metabolism; Genes, Bacterial; Humans; Injections, Subcutaneous; Leukocytes, Mononuclear - immunology; Lymph Nodes - immunology; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Molecular Weight; Mycobacterium avium - immunology; Mycobacterium bovis - immunology; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - immunology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.161.5.2356

Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.