Extensive sequence homology between the mycobacterium leprae LSR (12 kDa) antigen and its Mycobacterium tuberculosis counterpart

The Mycobacterium leprae LSR (12 kDa) protein antigen has been reported to mimic whole cell M. leprae in T cell responses across the leprosy spectrum. In addition, B cell responses to specific sequences within the LSR antigen have been shown to be associated with immunopathological responses in leprosy patients with erythema nodosum leprosum. We have in the present study applied the M. leprae LSR DNA sequence as query to search for the presence of homologous genes within the recently completed Mycobacterium tuberculosis genome database (Sanger Centre, UK). By using the BLASTN search tool, a homologous M. tuberculosis open reading frame (336 bp), encoding a protein antigen of 12.1 kDa, was identified within the cosmid MTCY07H7B.25. The gene is designated Rv3597c within the M. tuberculosis H37Rv genome. Sequence alignment revealed 93% identity between the M. leprae and M. tuberculosis antigens at the amino acid sequence level. The finding that some B and T cell epitopes were localized to regions with amino acid substitutions may account for the putative differential responsiveness to this antigen in tuberculosis and leprosy.     

Nitric oxide and Mycobacterium leprae pathogenicity

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.     

The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.     

Deciphering the proteomic profile of Mycobacterium leprae cell envelope

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.     

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.     

Detection of gene mutations related with drug resistance in Mycobacterium leprae from leprosy patients using Touch-Down (TD) PCR

The lack of methods to identify Mycobacterium leprae with the resistance against multi-drugs quickly and specifically has hindered effective chemotherapy against M. leprae infection. To screen M. leprae with resistance against multi-drugs, the Touch-Down (TD)-PCR has been used in this study. Sequences of the folP, rpoA, B, and gyrA, B genes were analyzed for isolates of M. leprae from leprosy patients in Korea. We amplified designated region of several genes in M. leprae involved in drug resistance and could obtain the PCR products of each gene. The mutations in the particular region of folP, rpoB, and gyrB gene were certified by TD-PCR single-stranded conformational polymorphism and DNA sequencing, respectively.     

Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.     

Genes required for mycobacterial growth defined by high density mutagenesis

Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.     

Molecular and immunological analysis of a fibronectin-binding protein antigen secreted by Mycobacterium leprae

By screening a Mycobacterium leprae lambda gt11 genomic DNA library with leprosy-patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin-binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327-amino-acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75-85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55-266 and 266-327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55-266 and 265-327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin-binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.     

Ferricrocin synthesis in Magnaporthe grisea and its role in pathogenicity in rice

Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host–pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea , the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1 , an NRPS gene, and a gene encoding an l -ornithine N5-monooxygenase ( OMO1 ) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N⁵ hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea .     

Identification of specific proteins and peptides in Mycobacterium leprae suitable for the selective diagnosis of leprosy

Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-gamma responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-gamma production, but less specificity, than the peptides. Thirty-five peptides showed IFN-gamma responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-gamma-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.     

DNA isolated from Mycobacterium leprae: genome size, base ratio, and homology with other related bacteria as determined by optical DNA-DNA reassociation

DNA derived from Mycobacterium leprae (grown in armadillos) was isolated, purified, and analyzed spectrophotometrically. The genome size and the guanine-plus-cytosine content of M. leprae were 1.3 x 10(9) and 55.8%, respectively. Among selected strains of mycobacterial, nocardial, and corynebacterial species, Corynebacterium sp. 2628 LB, isolated from a human leprosy patient, showed the highest DNA homology with M. leprae. Of the DNAs derived from mycobacteria, those of M. tuberculosis and M. scrofulaceum showed a comparatively high reassociation with the DNMA of M. liprae.     

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.     

LSHGD: A database for human leprosy susceptible genes

Studies aiming to explore the involvement of host genetic factors to determine susceptibility to develop disease and individual's response to the infection with Mycobacterium leprae have increased in recent years. To address this issue, we have developed a Leprosy Susceptible Human Gene Database (LSHGD) to integrate leprosy and human associated 45 genes by profound literature search. This will serve as a user-friendly and interactive platform to understand the involvement of human polymorphisms (SNPs) in leprosy, independent genetic control over both susceptibility to leprosy and its association with multi-drug resistance of M. leprae. As the first human genetic database in leprosy it aims to provide information about the associated genes, corresponding protein sequences, available three dimensional structures and polymorphism related to leprosy. In conclusion, this will serve as a multifunctional valuable tool and convenient information platform which is freely available at http://www.vit.ac.in/leprosy/leprosy.htm and enables the user to retrieve information of their interest.