Molecular drug susceptibility testing and genotyping of Mycobacterium leprae strains from South America.

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

Molecular drug susceptibility testing and genotyping of Mycobacterium leprae strains from South America

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

Mycobacterium leprae phenolglycolipid-1 expressed by engineered M. bovis BCG modulates early interaction with human phagocytes.

The species-specific phenolic glycolipid 1 (PGL-1) is suspected to play a critical role in the pathogenesis of leprosy, a chronic disease of the skin and peripheral nerves caused by Mycobacterium leprae. Based on studies using the purified compound, PGL-1 was proposed to mediate the tropism of M. leprae for the nervous system and to modulate host immune responses. However, deciphering the biological function of this glycolipid has been hampered by the inability to grow M. leprae in vitro and to genetically engineer this bacterium. Here, we identified the M. leprae genes required for the biosynthesis of the species-specific saccharidic domain of PGL-1 and reprogrammed seven enzymatic steps in M. bovis BCG to make it synthesize and display PGL-1 in the context of an M. leprae-like cell envelope. This recombinant strain provides us with a unique tool to address the key questions of the contribution of PGL-1 in the infection process and to study the underlying molecular mechanisms. We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses. PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation. Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells.

Comparative genomic and phylogeographic analysis of Mycobacterium leprae.

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38x coverage) and NHDP63 (46x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy.

From genome-based in silico predictions to ex vivo verification of leprosy diagnosis.

The detection of hundreds of thousands of new cases of leprosy every year suggests that transmission of Mycobacterium leprae infection still continues. Unfortunately, tools for identification of asymptomatic disease and/or early-stage M. leprae infection (likely sources of transmission) are lacking. The recent identification of M. leprae-unique genes has allowed the analysis of human T-cell responses to novel M. leprae antigens. Antigens with the most-promising diagnostic potential were tested for their ability to induce cytokine secretion by using peripheral blood mononuclear cells from leprosy patients and controls in five different areas where leprosy is endemic; 246 individuals from Brazil, Nepal, Bangladesh, Pakistan, and Ethiopia were analyzed for gamma interferon responses to five recombinant proteins (ML1989, ML1990, ML2283, ML2346, and ML2567) and 22 synthetic peptides. Of these, the M. leprae-unique protein ML1989 was the most frequently recognized and ML2283 the most specific for M. leprae infection/exposure, as only a limited number of tuberculosis patients responded to this antigen. However, all proteins were recognized by a significant number of controls in areas of endemicity. T-cell responses correlated with in vitro response to M. leprae, suggesting that healthy controls in areas where leprosy is endemic are exposed to M. leprae. Importantly, 50% of the healthy household contacts and 59% of the controls in areas of endemicity had no detectable immunoglobulin M antibodies to M. leprae-specific PGL-I but responded in T-cell assays to >or=1 M. leprae protein. T-cell responses specific for leprosy patients and healthy household contacts were observed for ML2283- and ML0126-derived peptides, indicating that M. leprae peptides hold potential as diagnostic tools. Future work should concentrate on the development of a sensitive and field-friendly assay and identification of additional peptides and proteins that can induce M. leprae-specific T-cell responses.

Are variable-number tandem repeats appropriate for genotyping Mycobacterium leprae?

Comparative genomics analysis of the Tamil Nadu strain of Mycobacterium leprae has uncovered several polymorphic sites with potential as epidemiological tools. In this study we compared the stability of two different markers of genomic biodiversity of M. leprae in several biopsy samples isolated from the same leprosy patient. The first type comprises five different variable-number tandem repeats (VNTR), while the second is composed of three single nucleotide polymorphisms (SNP). Contrasting results were obtained, since no variation was seen in the SNP profiles of M. leprae from 42 patients from 7 different locations in Mali whereas the VNTR profiles varied considerably. Furthermore, since variation in the VNTR pattern was seen not only between different isolates of M. leprae but also between biopsy samples from the same patient, these VNTR may be too dynamic for use as epidemiological markers for leprosy.

Towards an immunodiagnostic test for leprosy.

In addition to multidrug therapy, elimination of leprosy requires improved diagnostic methods. Using a comparative genomics approach, 17 potential protein antigens (MLP) that are restricted to Mycobacterium leprae, or of limited distribution, were produced and tested for antigen-specific immune responses on leprosy patients, healthy contacts of leprosy patients, and tuberculosis patients in Mali and Bangladesh, as well as on non-endemic controls. T-cell antigenicity of MLP was confirmed by IFN-gamma production in whole-blood assays with the highest responses observed in paucibacillary leprosy patients and healthy contacts. Four MLP behaved well in both countries and induced significantly different responses between the study groups. Peptides carrying T cell epitopes from one of the antigens gave promising results in restimulation assays in mice and immune responses were not influenced by prior exposure to BCG or environmental mycobacteria. This study provides the immunological framework for the development of a specific, peptide-based immunodiagnostic test for leprosy.

Antigen discovery: a postgenomic approach to leprosy diagnosis.

Leprosy is an infectious, neurodegenerative disease of humans caused by Mycobacterium leprae. Despite effective control programs, the incidence of leprosy remains stubbornly high, suggesting that transmission may be more common than expected. The rationale of this work was to use bioinformatics and comparative genomics to identify potentially antigenic proteins for diagnostic purposes. This approach defined three classes of proteins: those restricted to M. leprae (class I), those present in M. leprae with orthologues in other organisms besides mycobacteria (class II), and exported or surface-exposed proteins (class III). Twelve genes (two class I, four class II, and six class III proteins) were cloned in Escherichia coli, and their protein products were purified. Six of these proteins were detected in cell extracts of M. leprae by immunoblotting. The immunogenicity of each recombinant protein was then investigated in leprosy patients by measuring the reactivity of circulating antibody and gamma interferon (IFN-gamma) responses in T-cell restimulation assays. Several class II and class III proteins were recognized by circulating antibodies. Importantly, most class II proteins elicited IFN-gamma responses that were significantly stronger than those produced by previously identified antigens. Among them, two class II proteins, ML0308 and ML2498, showed marked humoral and cellular immunogenicity, therefore providing promising candidates for the diagnosis of both tuberculoid and lepromatous forms of leprosy.

On the origin of leprosy.

Leprosy, a chronic human disease with potentially debilitating neurological consequences, results from infection with Mycobacterium leprae. This unculturable pathogen has undergone extensive reductive evolution, with half of its genome now occupied by pseudogenes. Using comparative genomics, we demonstrated that all extant cases of leprosy are attributable to a single clone whose dissemination worldwide can be retraced from analysis of very rare single-nucleotide polymorphisms. The disease seems to have originated in Eastern Africa or the Near East and spread with successive human migrations. Europeans or North Africans introduced leprosy into West Africa and the Americas within the past 500 years.

On the origin of leprosy

Leprosy, a chronic human disease with potentially debilitating neurological consequences, results from infection with Mycobacterium leprae. This unculturable pathogen has undergone extensive reductive evolution, with half of its genome now occupied by pseudogenes. Using comparative genomics, we demonstrated that all extant cases of leprosy are attributable to a single clone whose dissemination worldwide can be retraced from analysis of very rare single-nucleotide polymorphisms. The disease seems to have originated in Eastern Africa or the Near East and spread with successive human migrations. Europeans or North Africans introduced leprosy into West Africa and the Americas within the past 500 years.

Isolation of three Mycobacterium ulcerans strains resistant to rifampin after experimental chemotherapy of mice

By use of a murine model for Buruli ulcer, Mycobacterium ulcerans was found to be susceptible to rifampin, with the MIC being 0.5 to 1 micro g/ml. Three mutants were isolated after rifampin monotherapy. Two were resistant to rifampin at 8 micro g/ml, and one was resistant to rifampin at 32 micro g/ml. The mutants harbored Ser416Phe mutations and His420Tyr mutations in the rpoB gene, and these mutations have also been found to be responsible for rifampin resistance in the leprosy and tubercle bacilli. The results indicate that while rifampin may be active against M. ulcerans, it should never be used as monotherapy in humans.

Isolation of three Mycobacterium ulcerans strains resistant to rifampin after experimental chemotherapy of mice.

By use of a murine model for Buruli ulcer, Mycobacterium ulcerans was found to be susceptible to rifampin, with the MIC being 0.5 to 1 micro g/ml. Three mutants were isolated after rifampin monotherapy. Two were resistant to rifampin at 8 micro g/ml, and one was resistant to rifampin at 32 micro g/ml. The mutants harbored Ser416Phe mutations and His420Tyr mutations in the rpoB gene, and these mutations have also been found to be responsible for rifampin resistance in the leprosy and tubercle bacilli. The results indicate that while rifampin may be active against M. ulcerans, it should never be used as monotherapy in humans.