Immunostimulatory activity of major membrane protein-II from Mycobacterium leprae

We examined the antigenicity of an immunomodulatory protein, major membrane protein (MMP)-II, from Mycobacterium leprae, since host defense against M. leprae largely depends on adaptive immunity. Both unprimed and memory T cells from healthy individuals were stimulated by autologous MMP-II-pulsed monocyte-derived dendritic cells (DCs) to produce IFN-gamma. The DC-mediated IFN-gamma production was dependent on the expression of MHC, CD86, and MMP-II antigens. Memory T cells from paucibacillary (PB) leprosy more extensively responded to MMP-II-pulsed DCs than T cells from healthy individuals, while comparable IFN-gamma was produced by unprimed T cells. Memory T cells from multibacillary leprosy, which are normally believed to be anergic, were activated similarly to those from healthy individuals by MMP-II-pulsed DCs. These results suggest that memory T cells from PB leprosy are primed with MMP-II prior to the manifestation of the disease, and MMP-II is highly antigenic in terms of activation of adaptive immunity.     

Characterization of Mycobacterium leprae cell wall-associated proteins with the use of T lymphocyte clones

Development of a vaccine against leprosy depends on the identification of Ag that stimulate cell-mediated immune responses. We have previously demonstrated that cell wall proteins of Mycobacterium leprae are highly immunogenic. By using human cell wall-specific T cell clones we have begun to characterize soluble proteins that integrate into the cell wall skeleton. T cells from leprosy lesions were expanded with IL-2 in vitro yet retained specificity to Ag of the insoluble cell wall core (CWC) in vitro, indicating that T cells had been activated by CWC Ag in vivo. A cell wall protein-peptidoglycan complex and cell wall protein preparations lacking carbohydrates and lipids from CWC retained T cell reactivity. To identify immunogenic protein component(s) of cell wall protein, T cell lines were established to cell walls and tested against M. leprae proteins separated by SDS-PAGE and transferred to nitrocellulose. Greatest T cell reactivity was observed to proteins of Mr 7 kDa, 16 kDa, and 28 kDa. T cell clones reactive with 7-kDa and 16-kDa Ag from gels failed to respond to proteins of other Mr separated under either reducing or nonreducing conditions, indicating that these molecules are not subunits of larger proteins and may represent monomeric units polymerized into cell walls. The approaches described herein for characterization of immunodominant T cell Ag of M. leprae may be useful for study of T cell Ag in cell walls of bacterial pathogens of man.     

Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein

To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.     

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate

Abstract The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti- M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weigh ( M _r) M. leprae protein (MLP) with a subunit M _r of 22000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae . The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis . It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed.     

Continued proteomic analysis of Mycobacterium leprae subcellular fractions

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.     

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.     

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.     

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.     

Evaluation of major membrane protein-II as a tool for serodiagnosis of leprosy

As serodiagnosis is the easiest way of diagnosing a disease, the utility of Mycobacterium leprae-derived major membrane protein-II (MMP-II), one of the immuno-dominant antigens, in the serodiagnosis of leprosy was examined. The percent positivity by an enzyme-linked immunosorbent assay for anti-MMP-II antibody was 82.4% for multi-bacillary leprosy, and the specificity of the test was 90.1%. For pauci-bacillary leprosy where cell-mediated immunity predominates, 39.0% showed positive results. These percentage values were significantly higher than these values obtained for existing phenolic glycolipid-I based methods, suggesting that MMP-II antibody detection would facilitate the diagnosis of leprosy.     

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.     

Mechanisms of Mycobacterium leprae-specific T-cell deficiency in lepromatous leprosy

Patients suffering from lepromatous leprosy fail to develop an efficient cell-mediated immunity towards Mycobacterium leprae, the causative agent. The mechanism of such a specific T-cell tolerance to the bacillus remains a key question in the pathophysiology of leprosy. Macrophages do not show any intrinsic defect in phagocytizing and killing M. leprae or in presenting antigen to helper T-cells. On the other hand, M. leprae-reactive helper T-cells do persist in lepromatous patients, but their activation appears to prevented by active suppressor mechanisms, involving both suppressor T-cells and macrophages. The target of this specific suppression could be the interleukin 2-producing T-cell subset. A better molecular definition of M. leprae antigens, both by monoclonal antibodies and T-cell clones, should open new perspectives for further analysis of the regulation of immune responses to M. leprae.     

GM-CSF-mediated T-cell activation by macrophages infected with recombinant BCG that secretes major membrane protein-II of Mycobacterium leprae

The potential of Mycobacterium bovis Bacillus Calmette–Guerin (BCG) needs to be augmented to efficiently activate CD4⁺ T cells through macrophages. Mycobacterium leprae -derived recombinant major membrane protein (MMP)-II induced GM-CSF production from macrophages. A recombinant BCG-SM that secretes MMP-II more efficiently produced GM-CSF and activated interferon (IFN)-γ-producing CD4⁺ T cells than did vector control BCG when infected with macrophages. The T-cell activation by BCG-SM was dependent on the GM-CSF production by macrophages. Interleukin (IL)-10 production by macrophages stimulated with M. leprae was inhibited in a GM-CSF-dependent manner when the precursor monocytes were infected with BCG-SM. BCG inducing GM-CSF production was effective in macrophage-mediated T-cell activation partially through IL-10 inhibition.     

Structure and antigenicity of the phosphorylated lipopolysaccharide antigens from the leprosy and tubercle bacilli

A family of major arabinose- and mannose-containing phosphorylated lipopolysaccharides was isolated from Mycobacterium leprae and Mycobacterium tuberculosis. The only antigenic member of the family, lipoarabinomannan (LAM)-B, was purified by anion exchange and gel filtration chromatography in detergent and recovered in large quantities (15 mg/g of bacteria). It yielded a broad diffuse band on polyacrylamide gel electrophoresis but appeared homogeneous by this criterion and gel filtration. Besides arabinose and mannose, it contained glycerol and a polyol phosphate and was acylated by lactate, succinate, palmitate, and 10-methyloctadecanoate. The phosphate was released by alkalinolysis and identified by thin layer chromatography and gas chromatography-mass spectrometry as myoinositol 1-phosphate. Thus, the group-specific "arabinomannan" of the genus Mycobacterium in the native state is acylated, contains the substituents of phosphatidylinositol, and is apparently membrane associated. LAM-B is one of the dominant immunogens of the leprosy bacillus reacting readily with antibodies from lepromatous leprosy patients and monoclonal antibodies in plate and nitrocellulose enzyme-linked immunosorbent assay and on electrophoretic immunoblots. It is immunologically cross-reactive with a like product from M. tuberculosis. LAM-B is clearly the pervasive "glycoprotein" antigen of the leprosy bacillus and may be the long sought lipoteichoic acid-like polymer of Mycobacterium with a role in cell wall physiology, macrophage recognition, and perhaps an involvement in cross-protective immunity.     

Advances in the diagnosis and treatment of leprosy

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nerves. Over recent years, many important advances have been made in developing molecular diagnostics, in identifying highly effective drugs and designing multidrug regimens for treatment, and in unravelling the genomic structure and functions of the leprosy bacillus. Using the new information about specific sequences of M. leprae, several gene probes and gene amplification systems for confirming diagnosis and monitoring treatment have been developed. Among these, polymerase chain reaction (PCR)-based methods have been useful in confirming the diagnosis in paucibacillary leprosy (where few bacilli are present). RNA-targeting systems for monitoring the progress of treatment, in situ hybridisation techniques for analysing specimens with nonspecific histological features, and molecular methods for direct detection of rifampicin/dapsone resistance are other major technological advances with immense applied value. Several effective regimens for the treatment of leprosy have been developed, which include rifampicin, clofazimine and dapsone as core drugs. Although these regimens are generally satisfactory, limitations in terms of persisting activity and late reactions/relapses in paucibacillary leprosy, and persistence of dead and/or live organisms in multibacillary forms of the disease, have been observed.     

Cloning and Expression of Mycobacterium tuberculosis and Mycobacterium leprae Dihydropteroate Synthase in Escherichia coli

The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.     

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.     

Polymerase chain reaction for the detection of Mycobacterium leprae

A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium. This PCR appears to meet the criteria of specificity and sensitivity required for a useful tool in epidemiology and eventually for the control of leprosy.     

Immunogold labeling method for Mycobacterium leprae-specific phenolic glycolipid in glutaraldehyde-osmium-fixed and Araldite-embedded leprosy lesions

Phenolic glycolipid (PGL)-I, a Mycobacterium leprae-specific antigen currently used for serodiagnosis of preclinical leprosy, has thus far not been localized subcellularly in leprosy bacilli and their host cells. In this study, we developed an immunogold-labeling technique for qualitative identification of PGL-I sites in glutaraldehyde-osmium-fixed and Araldite-embedded M. leprae and host macrophages in human skin biopsies. Such "hard-fixed," plastic-embedded skin and nerve biopsies from patients with varying cell-mediated immunity to leprosy are amply available worldwide. Our method involves etching of plastic sections with H2O2, incubation with swine serum to eliminate nonspecific labeling, and long (22 hr) incubation at room temperature with monoclonal antibodies to PGL-I. Gold labeling was seen predominantly on cell walls of M. leprae, in vacuolar spaces of bacillated phagolysosomes, and occasionally on the cytoplasm and cell membrane of M. leprae. Host macrophage cytoplasm was labeled very infrequently. This technique allows studies on possibly persisting antigenic PGL-I in multibacillary leprosy patients during or after multidrug therapy. The method may also prove useful for subcellular localization of specific bacterial lipids in other mycobacterial diseases, including tuberculosis.     

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.