Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis:Immunological analysis and evidence of glycosylation

The gene encoding a 19-kDa antigen from Mycobacterium tuberculosis was expressed as a recombinant protein in the rapid-growing species Mycobacterium smegmatis. The recombinant antigen was expressed at a level approximately ninefold higher than in M. tuberculosis and, like the native antigen, was found in the pellet fraction after high-speed centrifugation of bacterial extracts. The 19-kDa antigen in crude bacterial extracts, and the purified recombinant antigen, bound strongly to concanavalin A, indicating the possibility of posttranslational glycosylation. The recombinant antigen stimulated T-cell proliferation in vitro when added to assays either in the form of whole recombinant bacteria or as a purified protein. Homologous expression of mycobacterial antigens in a rapid-growing mycobacterial host may be particularly useful for the immunological characterization of proteins which are subject to posttranslational modification

The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins.

Eight monoclonal antibodies (MAbs) directed against the 14,000-molecular-weight (14K) antigen of Mycobacterium tuberculosis reacted specifically with mycobacteria of the M. tuberculosis complex. The nucleotide sequence of the gene encoding the 14K antigen was determined by using recombinant DNA clones isolated from lambda gt11 and cosmid libraries of the M. tuberculosis genome. The DNA sequence of the 14K protein gene coded for a polypeptide of 144 amino acids with a calculated molecular mass of 16,277 Da. The 14K antigen has a marked homology with proteins belonging to the alpha-crystallin family of low-molecular-weight heat shock proteins, which includes the 18K antigen of M. leprae. The eight MAbs recognized at least four distinct epitopes localized within the following three regions of the 14K protein: amino acids 10 to 92 (MAbs F67-8 and F67-16), amino acids 41 to 92 (F159-1 and F159-11), and amino acids 41 to 144 (F23-41, F24-2, F23-49, and TB68)

Recognition of peptide epitopes of the 16,000 MW antigen of Mycobacterium tuberculosis by murine T cells

The T-cell repertoire to a prominent immunogen of Mycobacterium tuberculosis has been investigated on the assumption that differences in epitope specificity could influence the protective and pathogenic host reactions. Proliferative responses of lymph node and spleen cells to overlapping peptides, spanning the entire sequence of the 16,000 MW protein antigen were analysed in C57BL/10 and B10.BR mice. Following footpad priming and in vitro challenge with homologous peptide, 12 out of the 14 peptides tested were found to be immunogenic. However, only two peptides of residues 31-40 and 71-91 stimulated strong proliferative responses of T cells from mice which had been presensitized with either killed or live M. tuberculosis organisms; another three peptides were only weakly stimulatory. These epitopes have been immunodominant in both H-2b and H-2k mouse strains, indicating the genetically permissive nature of their recognition. Furthermore, both major immunodominant epitopes were found to be species specific for the M. tuberculosis complex and therefore potentially suitable for the early diagnosis of tuberculous infection