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The need for molecular tools for the differentiation of isolates of Mycobacterium leprae, the organism that causes leprosy, is urgent in view of the continuing high levels of new case detection, despite years of aggressive chemotherapy and the consequent reduction in the prevalence of leprosy. The slow onset of leprosy and the reliance on physical examination for detection of disease have restricted the epidemiological tracking necessary to understand and control transmission. Two genetic loci in several isolates of M. leprae have previously been demonstrated to contain variable-number tandem repeats (VNTRs). On the basis of these reports and the availability of the full genome sequence, multiple-locus VNTR analysis for strain typing has been undertaken. A panel of 11 short tandem repeat (STR) loci with repeat units of 1, 2, 3, 6, 12, 18, 21, and 27 bp from four clinical isolates of M. leprae propagated in armadillo hosts were screened by PCR. Fragment length polymorphisms were detected at 9 of the 11 loci by agarose gel electrophoresis. Sequencing of representative DNA products confirmed the presence of VNTRs between isolates. The application of nine new polymorphic STRs in conjunction with automated methods for electrophoresis and size determination allows greater discrimination between isolates of M. leprae and enhances the potential of this technique to track the transmission of leprosy.The World Health Organization and its partners created the Global Alliance for Leprosy Elimination in 1999 with the aim of achieving the elimination of leprosy by the end of the year 2005, a goal originally set for the year 2000 (http://www.who .int/inf-pr-1999/en/pr99-70.html). Leprosy is effectively controlled by a multidrug therapy (MDT) regimen composed of dapsone, rifampin, and clofazimine. However, continuing large numbers of new cases are being detected in areas of the world where the disease is highly endemic, despite the application of the MDT program since 1982 (36). In order to comprehend this rising incidence of leprosy, it is necessary to identify the natural reservoir of Mycobacterium leprae, the route of infection, and the mode of its transmission. It is commonly believed that the human is the host and reservoir of M. leprae and that successful MDT will eliminate leprosy. However, other modes of transmission involving nonhuman reservoirs, such as soil (6, 15), water (20), vegetation (16), animals (including armadillos and sooty mangabeys) (22, 33), and arthropods (fleas, ticks, mosquitoes, and flies), have been suggested, as reviewed by Blake et al. (3). The route of infection is also unknown, although the entry and the exit of the bacteria via the nasal passages have been proposed (9). Methods that specifically detect M. leprae DNA in nasal swabs are being developed with the aim of early detection in populations at the community and village levels and for the monitoring of leprosy transmission (13,23). Typing methods for distinguishing cases of relapse from new infections are also required. Molecular typing wi...

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n ‫؍‬ 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n ‫؍‬ 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.The causative pathogen of leprosy is Mycobacterium leprae. A continued incidence, defying global campaigns to eliminate leprosy even after years of rigorous case finding and the availability of multidrug therapy regimens (28,29,30,31), is attributed to subclinical human and environmental reservoirs of the pathogen (1,8,13). In recent years, molecular strain-typing methodologies have complemented conventional infectious disease epidemiology. With the publication in 2001 of the complete genome sequence of an isolate from Tamil Nadu, India, called the TN strain (4), selection of potential polymorphic genomic markers for strain typing was feasible. The first genetic markers that showed polymorphism were short tandem repeats (STRs) in the M. leprae genome. One was a 6-bp intragenic sequence in the rpoT gene, and the second, a trinucleotide (TTC) repeat element upstream of a pseudogene (17, 23). These sequences exhibit variable numbers of tandem repeats (VNTRs) when sequenced in different isolates. Based on these observations, we short-listed 44 loci (including the rpoT and TTC loci) by in silico analyses of the M. leprae genome and accomplished the screening of 11 STR loci, of which 9 were polymorphic when tested in a small panel of four human isolates derived from passage through armadillos (6). Five were minisatellites (6-to 50-bp repeat units), and four were microsatellites (1-to 5-bp repeat units). Since then, others have also shown that VNTR loci exist in M. lepra...

SummsryThe study of tissue-derived Mycobacterium leprae provides insights to the immunopathology of leprosy and helps identify broad molecular features necessary for mycobacterial parasitism. A major membrane protein (MMP-II) of in vivo-derived M. teprae previously recognized (Hunter, S.W., B. Rivoire, V. Mehra, B.R. Bloom, and P.J. Brennan. 1990. J. Biol. Chem. 265:14065) was purified from extracts of the organism and partial amino acid sequence obtained. This information allowed recognition, within one of the cosmids that encompass the entire M. leprae genome, of a complete gene, bfr, encoding a protein of subunit size 18.2 kD. The amino acid sequence deduced from the major membrane protein II (MMP-II) gene revealed considerable homology to several bacterioferritins. Analysis of the native protein demonstrated the iron content, absorption spectrum, and large native molecular mass (380 kD) of several known bacterioferritins. "][prosy is a chronic disease of the skin and nerves caused by Mycobacterium leprae. Chemotherapy is successful but requires protracted multidrug therapy and the continuing evolution of new drug regimens. The world-wide prevalence of leprosy is now estimated at less than 5 million cases compared with 10-12 million in the mid-1980s (1). Much of this decline is attributed to chemotherapy. Nevertheless, leprosy still represents a major public health problem within regions of Latin America, Africa, and Asia, many of them not amenable to the standard drug regimens. Hence, the development of a vaccine that could contribute to the eradication of the disease remains a goal of some leprosy research programs.The molecular definition of M. leprae is fundamental to an understanding of the physiology and metabolism of the bacillus and the immunoregulatory mechanisms underlying the disease and protection against it. The leprosy bacillus grows intracellularly in cells of the reticuloendothelial system and Schwann cells, and it is so well adapted to this habitat that it has evaded axenic cultivation so far (2). The development of the armadillo as a relatively rich source of the bacillus has allowed extensive study of the physiology of the organism (2) and definition of the chemistry of the highly antigenic glycoconjugates and their roles in pathogenesis (3). Screening of M. leprae genomic libraries with antibodies has also permitted the isolation of genes that encode protein antigens, many of which are related to components of the highly conserved family of stress proteins (4).A landmark in leprosy research that may revolutionize our knowledge of the basis of the obligate parasitism ofM. leprae is the recently initiated "M. leprae genome project" (5). Present achievements from this undertaking include an ordered collection of overlapping clones encompassing the complete chromosome of M. leprae in which 72 loci have been mapped (5) and the complete sequence of 27 cosmids representing approximately one third of the genome (6; Smith, D.R., unpublished data). In an attempt to complement this genetic approac...

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The availability of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.