Identification, sequencing, and expression of Mycobacterium leprae superoxide dismutase, a major antigen.

The gene encoding a major 28-kilodalton antigen of Mycobacterium leprae has now been sequenced and identified as the enzyme superoxide dismutase (SOD) on the basis of the high degree of homology with known SOD sequences. The deduced amino acid sequence shows 67% homology with a human manganese-utilizing SOD and 55% homology with the Escherichia coli manganese-utilizing enzyme. The gene is not expressed from its own promoter in E. coli but is expressed from its own promoter in Mycobacterium smegmatis. The amino acid sequences of epitopes recognized by monoclonal antibodies against the 28-kilodalton antigen have been determined.

The specific 18-kilodalton antigen of Mycobacterium leprae is present in Mycobacterium habana and functions as a heat-shock protein.

A mAb previously thought to be specific for Mycobacterium leprae has been found to cross-react with a cultivable mycobacterium, Mycobacterium habana TMC5135. The epitope is present on a protein of identical molecular mass (18 kDa) in both species. When M. habana is subjected to heat shock, expression of the protein is significantly increased, whereas other forms of environmental stress do not increase its expression. Since immunization of mice with M. habana results in protection against infection with M. leprae, the possibility of using a molecular genetic approach to investigate the role of this protein in protective immunity is raised.

Analysis of variation in batches of armadillo-derived Mycobacterium leprae by immunoblotting.

Several batches of cell-free extracts of armadillo-derived Mycobacterium leprae were analyzed by SDS-PAGE and by immunoblotting with monoclonal antibodies. The presence or absence of protease inhibitors had a profound effect on the protein antigens, particularly the 65-kDa antigen. In the absence of protease inhibitors, there were both quantitative and qualitative differences between the different batches of M. leprae extracts.

Partial nucleotide sequence of 16S ribosomal RNA isolated from armadillo-grown Mycobacterium leprae.

Ribosomal RNA (rRNA) was isolated from Mycobacterium leprae recovered from infected tissue of the Nine-banded Armadillo, and nucleotide sequences near the 3' end of the 16S species were determined by primer extension in the presence of dideoxynucleotides. Previously published data for bacterial 16S rRNAs show a pattern of conserved and non-conserved sequences that fit a common secondary structure. Our data for M. leprae fits this general pattern.

Heterologous expression of the 65-kilodalton antigen of Mycobacterium leprae and murine T-cell responses to the gene product.

The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned from a lambda gt11 clone into the high-copy-number plasmid pUC8. Escherichia coli containing these recombinants produced large amounts of the antigen, which was purified by polyacrylamide gel electrophoresis in the presence of urea. The ability of E. coli to recognize the mycobacterial promoter was confirmed by constructing additional clones in which the gene is flanked by transcriptional terminators from phage fd. A similar approach was used to demonstrate the expression of this gene in Streptomyces lividans. Mice immunized with killed M. leprae showed cell-mediated immune reactivity to the purified 65-kilodalton protein which stimulated both in vitro lymphoproliferative and in vivo delayed-type hypersensitivity responses.