Immunological and functional characterization of Mycobacterium leprae protein antigens: an overview

A major focus of leprosy research in the last 10 years has been the identification and characterization of antigens of Mycobacterium leprae that interact with antibodies and T cells of the host's immune response. Through the combined efforts of many different laboratories, a substantial number of protein antigens have been identified and characterized. In this MicroReview we present an updated list of M. leprae protein antigens, and, with emphasis on recent developments, summarize what is known regarding their functional and immunological features.     

Immunophyletic relationships between actinomycetales and eubacteriales determined by examinations of their cytoplasmic antigens.

Cytoplasms obtained from a total of 205 different strains of Actinomycetales, Eubacteriales, Lower Fungi, and Protozoa were examined against selected anti-cytoplasm antisera using the immunodiffusion and counterimmunoelectrophoresis procedures. Immunological relationships between the different genera have been revealed and, in respect to Eubacteriales, the possible immunophyletic determinants have been discerned. Cytoplasms of Enterobacter, Clostridium, Staphylococcus, and Diplococcus were found to retain the significant linkage determinants between large groups of Eubacteriales and Actinomycetales. Evolutionary implications of the findings are discussed.     

A quantitative view on Mycobacterium leprae antigens by proteomics

Leprosy is an ancient disease and the focus of the researchers' scrutiny for more than a century. However, many of the molecular aspects related to transmission, virulence, antigens and immune responses are far from known. Initially, the implementation of recombinant DNA library screens raised interesting antigen candidates. Finally, the availability of Mycobacterium leprae genomic information showed an intriguing genome reduction which is now largely used in comparative genomics. While predictive in silico tools are commonly used to identify possible antigens, proteomic approaches have not yet been explored fully to study M. leprae biology. Quantitative information obtained at the protein level, and its analysis as part of a complex system, would be a key feature to be used to help researchers to validate and understand many of such in silico predictions. Through a re-analysis of data from a previous publication of our group, we could easily tackle many questions regarding antigen prediction and pseudogene expression. Several well known antigens are among the quantitatively dominant proteins, while several major proteins have not been explored as antigens. We argue that combining proteomic approaches together with bioinformatic workflows is a required step in the characterization of important pathogens.     

Enumeration of Mycobacterium leprae using real-time PCR

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.     

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.     

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae

Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene.     

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.     

Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, encodes the enzyme serine hydroxymethyltransferase, has been cloned in Escherichia coli, over-expressed and purified the protein product (mlSHMT) for folding and stability studies under various denaturating conditions. The recombinant mlSHMT exists as homo-dimer of molecular mass about 90 kDa under physiological conditions . The studies on catalytic properties of mlSHMT show that the enzyme catalyzes the H(4)-folate dependent retro-aldol cleavage of L-serine, however, D-alanine dependent transaminase activity was absent in the enzyme. Further analysis of the enzyme kinetics for hydroxymethyltransferase reaction for mlSHMT demonstrates a comparable K(m) value for L-serine to SHMTs from other sources but significantly lower catalytic efficiency (k(cat)/K(m)). The mlSHMT is resistant to alkaline denaturation and exist as apo-dimer up to pH 10.5. Urea and guanidinium chloride induces dissociation of mlSHMT dimer into monomer at low denaturant concentrations, and leads to loss of enzymatic activity.     

Local comparison of the genomes of Mycobacterium tuberculosis and Mycobacterium leprae using the polymerase chain reaction

Abstract A gene fusion between the Saccharomyces cerevisiae actin gene promoter and the cDNA of the Fusarium solani f. sp. pisi pelA gene has been constructed. This expression cassette has been introduced into the industrial wine yeast strain T₇₃. The resulting recombinant strain is able to secrete active PELA enzyme into the culture medium. In preliminary microvinification experiments the wine produced by this pectinolytic strain is indistinguishable from wine produced using the non-transformed strain on the basis of the chemical analyses. Large scale fermentations need to be carried out in order to assess the effects on filtrability.     

Further specific extracellular phenolic glycolipid antigens and a related diacylphthiocerol from Mycobacterium leprae

Mycobacterium leprae in infected armadillo tissue produces extracellular phthiocerol-containing lipids in amounts well in excess of the bacterial mass. The principal component (1.38 mg in 1 g of liver, wet weight, containing 3.7 X 10(10) M. leprae bacilli) consists of a mixture of two phthiocerol homologs, 3-methoxyl-4-methyl-9, 11-dihydroxyoctacosane and 3-methoxyl-4-methyl-9, 11-dihydroxytriacontane, (formula: see text); in which the hydroxyl functions are acylated by a mixture of three 'mycocerosic acids': 2,4,6,8-tetramethylhexacosanoate, 2,4,6,8-tetramethyloctacosanoate, and 2,4,6,8-tetramethyltriacontanoate. The structures were established by saponification of the native lipid, direct probe electron impact- or chemical ionization-mass spectrometry of the phthiocerol or its permethylated derivative, and gas-liquid chromatography-electron impact-mass spectrometry of the methyl esters of the fatty acids. In addition to the previously reported M. leprae-specific triglycosylphenolicdiacyl phthiocerol (Hunter, S. W., Fujiwara, T., and Brennan, P. J. (1982) J. Biol. Chem. 257, 15072-15078), the extracellular products contain small amounts (about 60 micrograms/g of infected liver, wet weight) of two other phenolic glycolipids, one of which (Phenolic Glycolipid III) has been structurally elucidated, (formula: see text); assuming certain enantiomeric configurations for the sugar substituents; the R-acyl functions are identical with those in the diacylphthiocerol. Phenolic Glycolipid-III reacts in enzyme-linked immunosorbent assays with sera from patients with leprosy and with rabbit antisera raised against whole M. leprae. The phthiocerol-containing lipids may be synonymous with the electron transparent capsules of M. leprae, and their unreactive state may confer on them the role of passive protectors of the bacillus.     

Detection of Mycobacterium leprae DNA in clinical and environmental samples using serological analysis and PCR

Molecular Biology Reports - Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. This microorganism is inculturable,...     

Species-specific antigens ofMycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas

Cell proteins from the porcine mycoplasmasMycoplasma hyorhinis, M. hyopneumoniae, andM. flocculare have been analyzed by SDS-gel electrophoresis and immunoblotting. The protein profiles ofM. hyopneumoniae andM. flocculare were similar, but the protein profile ofM. hyorhinis was quite different from the others. Antisera prepared against whole cells of the above three mycoplasmas were used in immunoblotting of electrophoretically separated antigens and in enzyme-linked immunosorbent assay. One major antigen, which had a molecular weight of 73 k, was found to be common to all three mycoplasmas. Another major antigen, with a molecular weight of 41 k, was common toM. hyopneumoniae andM. flocculare and may also be present inM. hyorhinis. Several antigens of comparatively high molecular weights (108 k, 102 k, 93 k, 89 k, and 87 k) seemed to be specific forM. hyopneumoniae. Three antisera prepared by immunization of rabbits with immunoprecipitates obtained by crossed immunoelectrophoresis ofM. hyopneumoniae were also used in blotting experiments. One of these antisera was found to be directed against the 73 k antigen common to the three porcine mycoplasmas investigated. The other two antisera were directed againstM. hyopneumoniae-specific antigens with molecular weights of 74 k, 58 k, 45 k, 44 k, and 38 k.     

Chemical synthesis of disaccharides of the specific phenolic glycolipid antigens from Mycobacterium leprae and of related sugars

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3,-di-O-methyl-L -rhamnopyranose, which is the nonreducing disaccharide of the haptenic trisaccharide of the Mycobacterium leprae-specific, phenolic glycolipid I, O-(6-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamn opyranose, the nonreducing end of the specific, phenolic glycolipid III, and the nonhaptenic O-beta-(D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamnopyranose++ +, were synthesized in relatively good yield from 3-O-methyl-D-glucose, or D-glucose, and L-rhamnose via Koenigs-Knorr reactions. These disaccharides can be used as precursors in the synthesis of the trisaccharide unit of phenolic glycolipid I and of neoglycoconjugates suitable for the serodiagnosis of leprosy.     

Peptidoglycan and arabinogalactan of Mycobacterium leprae

The arabinogalactan and peptidoglycan of armadillo-grown Mycobacterium leprae were examined. Within the limits defined by the small amount of material available, the resemblance of these polymers to those of other mycobacteria was confirmed. The polymers were linked by a highly acid-labile bond and the arabinogalactan was itself acid-labile; free arabinose and a variety of oligosaccharides containing both arabinose and galactose, as well as polysaccharide and peptidoglycan, were released by dilute acid. The resonances from anomeric protons in the proton NMR spectrum of the arabinogalactan were similar to those from the arabinogalactan of M. tuberculosis. The composition and structure of the peptidoglycan resembled those of other mycobacteria. The only major difference was the specific replacement of L-alanine by glycine in the peptide of the peptidoglycan.     

High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

Detection of Mycobacterium leprae with the use of the polymerase chain reaction

The review deals with the problem of the detection of the causative agent of lepra in different biological samples with the use of polymerase chain reaction. Special attention is drawn to the characterization of the specificity and sensitivity of different target areas of M. leprae DNA.