Isolation, characterization, molecular cloning and amplification of a species-specific M. leprae antigen

ABSTRACT

A polyclonal serum sample from a lepromatous leprosy (LL) patient, which presented a specific recognition pattern for leprosin, was used to screen a Mycobacterium leprae genomic library constructed with DNA isolated from human lepromas. One clone, designated ML4-1, which expressed a specific antigenic determinant of M. leprae as part of a beta-galactosidase fusion protein, was isolated. The 1.932 bp M. leprae-derived genomic fragment was sequenced, and it had an incomplete open-reading frame shown to code for a 644 amino-acid polypeptide (72.3 kDa). Some partial nucleotide homology to the M. tuberculosis MTCY9C4 cosmid and the M. leprae B1913 cosmid were found. Southern blot assays using the 584 bp Eco RI-Bam HI fragment excised from the ML4-1 clone revealed that this sequence is present only in the M. leprae genome and not in the 24 different mycobacterial DNA tested. Two oligonucleotides based on the genomic sequence were also synthesized and used as amplifiers for a polymerase chain reaction (PCR) test, giving a positive signal exclusively in M. leprae DNA. Furthermore, 32 sequential synthetic peptides, 20 amino-acids long, spanning the entire protein corresponding to the hypothetical ML4-1 clone sequence, were synthesized and evaluated by ELISA. A peptide included in the 221-240 region was significantly recognized by either lepromatous leprosy or healthy tuberculosis contact patient sera. Thus, PCR amplification of this fragment, along with the recognition of its protein sequence by leprosy patient sera, could be a useful tool for a potential diagnostic method in the detection of M. leprae infection in the future.