Immunity to leprosy. II. Genetic control of murine T cell proliferative responses to Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured after immunization of mice at the base of the tail with antigen and challenging lymphocytes from draining lymph nodes in culture with M. leprae. This T cell response to M. leprae has been compared in 18 inbred strains of mice. C57BL/10J mice were identified as low responder mice. The congenic strains B10.M and B10.Q were found to be high responders, whereas B10.BR and B10.P were low responders. F1 (B10.M X C57BL/10J) and F1 (B10.Q X C57BL/10J) hybrid mice were found to be low responders, similar to the C57BL/10J parent, indicating that the low responsive trait is dominant. Whereas B10.BR mice were shown to be low responders to M. leprae, B10.AKM and B10.A(2R) were clearly high responders, indicating that the H-2D region influences the magnitude of the T cell proliferative response. Gene complementation within the H-2 region was evident. Genes outside the H-2 region were also shown to influence the response to M. leprae. C3H/HeN were shown to be high responder mice, whereas other H-2k strains, BALB.K, CBA/N, and B10.BR, were low responders. Gene loci that influence the T cell proliferation assay have been discussed and were compared to known background genes which may be important for the growth of intracellular parasites. Because mycobacteria are intracellular parasites for antigen-presenting cells, genes that affect bacterial growth in these cells will also influence subsequent immune responses of the host.     

Nucleotide sequence of the Mycobacterium leprae katG region.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.     

Computational genome analyses of metabolic enzymes in Mycobacterium leprae for drug target identification

Leprosy is an infectious disease caused by Mycobacterium leprae. M. leprae has undergone a major reductive evolution leaving a minimal set of functional genes for survival. It remains non-cultivable. As M. leprae develops resistance against most of the drugs, novel drug targets are required in order to design new drugs. As most of the essential genes mediate several biosynthetic and metabolic pathways, the pathway predictions can predict essential genes. We used comparative genome analysis of metabolic enzymes in M. leprae and H. sapiens using KEGG pathway database and identified 179 non-homologues enzymes. On further comparison of these 179 non-homologous enzymes to the list of minimal set of 48 essential genes required for cell-wall biosynthesis of M. leprae reveals eight common enzymes. Interestingly, six of these eight common enzymes map to that of peptidoglycan biosynthesis and they all belong to Mur enzymes. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy and thus targeting these enzymes is a step towards facilitating the search for new antibiotics.     

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.     

Sequence and immunogenicity of the 70-kDa heat shock protein of Mycobacterium leprae

The gene encoding the Mycobacterium leprae 70-kDa heat shock protein has been isolated from a cosmid library using a fragment of the clone JKL2. Southern blot analysis of a positive clone identified a 4.4-kb fragment containing the entire coding region of the gene plus 2.4 kb upstream. Sequencing revealed the gene to encode a 621-amino acid protein, bearing 56% identity with the Escherichia coli dnaK gene product and 47% and 46% identity with the human and Caenorhabditis elegans hsp70, respectively. Comparison with the C-terminal 203 amino acids of the Mycobacterium tuberculosis 71-kDa Ag yielded 70% identity. Recombinant M. leprae p70 was produced in E. coli as a fusion protein (rp70f) with a portion of the schistosomal glutathione-S-transferase, using the expression vector, pGEX-2T. Cleavage with thrombin resulted in the release of a 70.0-kDa protein (rp70c) from the glutathione-S-transferase. Examination of the proteins by immunoblotting demonstrated that anti-M. leprae mAb, L7, and sera from lepromatous leprosy patients bound to both the cleaved and fusion proteins. We compared the T cell reactivity of the M. leprae recombinant proteins with that of mAb affinity-purified bacille Calmette-Guerin (BCG) 70-kDa Ag using proliferation assays. PBMC of BCG vaccinees responded to both M. leprae cleaved and fusion p70, though more subjects responded to the rp70c (18 of 20) than to rp70f (13 of 20). Responses were generally higher to rp70c than to rp70f, however all responses to the M. leprae recombinant proteins were lower than to mAb affinity-purified BCG p70. Thus, the M. leprae 70-kDa heat shock protein elicits T and B cell responses in subjects exposed to mycobacteria, despite its homology with the human hsp70.     

Contrasting evolutionary rates in the duplicate chaperonin genes of Mycobacterium tuberculosis and M. leprae

A phylogenetic analysis of chaperonin (heat shock protein 60) sequences from prokaryotes and eukaryotes indicated that a single gene duplication event in the common ancestor of Mycobacterium tuberculosis, M. leprae, and Streptomyces albus gave rise to the duplicate chaperonin genes found in these species (designated HSP65 and GroEL in the mycobacterial species). Comparison of rates of synonymous and nonsynonymous nucleotide substitution in different gene regions suggested that the 5' end of the HSP65 gene was homogenized by an ancient recombination event between M. tuberculosis and M. leprae. In S. albus, the two duplicated chaperonin genes have evolved at essentially the same rate. In both M. tuberculosis and M. leprae, however, the GroEL gene has evolved considerably more rapidly at nonsynonymous nucleotide sites than has the HSP65 gene. Because this difference is not seen at synonymous sites, it must be due to a difference in selective constraint on the proteins encoded by the two genes, rather than to a difference in mutation rate. The difference between GroEL and HSP65 is striking in regions containing epitopes recognized by T cells of the vertebrate host; in certain cross-reactive epitopes conserved across all organisms, nonsynonymous sites in GroEL have evolved twice as fast as those in HSP65. It is suggested that these differences are correlated with differences in the way in which the duplicate chaperonins of M. tuberculosis and M. leprae interact with the host immune system.      

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.     

The antibody repertoire to proteins of Mycobacterium leprae. Genetic influences at the antigen and epitope level.

The effects of both H-2 and non-H-2 genes on the antibody response to the proteins of Mycobacterium leprae were investigated. Using a B10 series of congenic mice we found that the repertoire of antibody responses was under H-2 gene control, and that non-H2 genes were also involved. By Western blotting, differences in the number and m.w. of proteins recognised by mice of different genetic background were apparent. Such differences were also reflected in the total antibody response to a soluble extract of M. leprae (M. leprae sonicate), as measured by ELISA. Concentrating on one particular Ag, the 65-kDa heat shock protein, we found that all strains of mice developed antibodies following immunization with the purified recombinant protein, although there was a continuous distribution in the titer of antibodies obtained, with differences between individual strains indicating both H-2 and non-H-2 effects. Using a library of overlapping peptides based on the amino acid sequence of this protein, we have mapped the B cell epitopes in the different strains of mice. H-2 genes had no effect on the structure and number of epitopes recognized, although this was influenced by non-H-2 genes. There was a high level of concordance between actual epitopes recognized and those predicted by calculations of antigenic index, and B cell epitopes were located in similar positions to previously determined T cell epitopes.     

High-level expression of pseudogenes in Mycobacterium leprae

Recent studies have revealed that some RNAs are transcribed from noncoding DNA regions, including pseudogenes, and are functional as riboregulators. We have attempted to assess the gene expression profile throughout the Mycobacterium leprae genome using an array technique. Twelve highly expressed gene regions were identified that show an alteration in expression levels upon infection. Six of these were pseudogenes. Although M. leprae has an exceptional number and proportion of pseudogenes among species, our results suggest that some of the M. leprae pseudogenes are not just 'decayed' genes, but may have a functional role.     

Genomic arrangement of a putative operon involved in maltose transport in the Mycobacterium tuberculosis complex and Mycobacterium leprae

A Mycobacterium bovis gene coding for a putative MalE maltose binding protein was cloned and its full-length sequence determined. Database searches revealed 99.9% identity with IpqY, encoding a putative sugar uptake protein from Mycobacterium tuberculosis strain H37Rv. The deduced protein product showed high sequence similarity to MalE-like proteins from a variety of bacterial species, including Mycobacterium leprae. Analysis of flanking database sequences from M. tuberculosis and M. leprae revealed the presence of malF-, malG- and malK-like genes. Comparison of these mycobacterial sequences with other maltose operons has allowed us to deduce a unique genomic arrangement of the genes involved in the uptake of maltose in members of the Mycobacterium tuberculosis complex and M. leprae.     

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.     

Aminoglycoside 2'-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2 ')-Ic gene from Mycobacterium tuberculosis and the aac(2 ')-Id gene from Mycobacterium smegmatis

The genus Mycobacterium comprises clinically important pathogens such as M. tuberculosis , which has re-emerged as a major cause of morbidity and mortality world-wide especially with the emergence of multidrug-resistant strains. The use of fast-growing species such as Mycobacterium smegmatis has allowed important advances to be made in the field of mycobacterial genetics and in the study of the mechanisms of resistance in mycobacteria. The isolation of an aminoglycoside-resistance gene from Mycobacterium fortuitum has recently been described. The aac(2 ' )-Ib gene is chromosomally encoded and is present in all isolates of M. fortuitum . The presence of this gene in other mycobacterial species is studied here and genes homologous to that of M. fortuitum have been found in all mycobacterial species studied. In this report, the cloning of the aac(2 ' )-Ic gene from M. tuberculosis H37Rv and the aac(2 ' )-Id gene from M. smegmatis mc²155 is described. Southern blot hybridizations have shown that both genes are present in all strains of this species studied to date. In addition, the putative aac(2 ' )-Ie gene has been located in a recent release of the Mycobacterium leprae genome. The expression of the aac(2 ' )-Ic and aac(2 ' )-Id genes has been studied in M. smegmatis and only aac(2 ' )-Id is correlated with aminoglycoside resistance. In order to elucidate the role of the aminoglycoside 2'- N -acetyltransferase genes in mycobacteria and to determine whether they are silent resistance genes or whether they have a secondary role in mycobacterial metabolism, the aac(2 ' )-Id gene from M. smegmatis has been disrupted in the chromosome of M. smegmatis mc²155. The disruptant shows an increase in aminoglycoside susceptibility along with a slight increase in the susceptibility to lysozyme.     

Characterization and taxonomic implications of the rRNA genes of Mycobacterium leprae.

The number of rRNA genes of Mycobacterium leprae was determined by restriction analysis of M. leprae total chromosomal DNA. A single set of rRNA genes was found. This set was subcloned from a cosmid library of M. leprae DNA into pUC13 and was characterized by restriction analysis and hybridization with Escherichia coli rRNA genes. The 16S, 23S, and 5S genes of M. leprae were clustered on a 5.3-kilobase DNA fragment. On one hand, restriction analysis of the set of rRNA genes showed the uniqueness of M. leprae among mycobacteria, but on the other hand, it suggested that M. leprae strains of several origins are very much alike. Quantitative hybridization studies between M. leprae rDNA and total DNA of various bacteria demonstrated a close relatedness between M. leprae and corynebacteria, nocardia, and mycobacteria, especially Mycobacterium tuberculosis.     

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy

Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a spectrum of clinical presentations that correlate with host immune responses. Here we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) versus the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target key immune genes downregulated in L-lep versus T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in Mycobacterium leprae-infected monocytes. By directly downregulating Toll-like receptor 2/1 heterodimer (TLR2/1)-induced CYP27B1 and IL1B expression as well as indirectly upregulating interleukin-10 (IL-10), hsa-mir-21 inhibited expression of the genes encoding two vitamin D-dependent antimicrobial peptides, CAMP and DEFB4A. Conversely, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae. Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D-dependent antimicrobial pathway.     

Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.