Chemical synthesis of the trisaccharide unit of the species-specific phenolic glycolipid from Mycobacterium leprae

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, the haptenic trisaccharide of the Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) antigen, and related trisaccharides, were synthesized by allylation of O-2 of benzyl 4-O-benzyl-alpha-L-rhamnopyranoside using phase-transfer catalysis, methylation of the product, deallylation, and coupling to O-(2,4-di-O-acetyl-3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3- di-O-methyl-L-rhamnopyranosyl bromide or related disaccharides. Anomeric mixtures of the trisaccharide derivatives were separated by preparative t.l.c., deacetylated, and hydrogenolyzed, to give the pure trisaccharides. It had already been demonstrated that only those trisaccharides containing an intact, terminal 3,6-di-O-methyl-beta-D-glucopyranosyl unit are effective in inhibiting the specific binding between PGL-I and anti-PGL-I immunoglobulin M antibodies in human lepromatous leprosy sera.     

Synthesis and immunoreactivity of neoglycoproteins containing the trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae

The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.     

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.     

Structure and antigenicity of the major specific glycolipid antigen of Mycobacterium leprae

Earlier we reported on the presence of a specific phenolic glycolipid (Phenolic Glycolipid-I) in Mycobacterium leprae, and in infected armadillo tissues (Hunter, S. W., and Brennan, P. J. (1981) J. Bacteriol. 147, 728-735). It had an inherent oligosaccharide, composed of 3-O-Me-rhamnose, 2,3-di-O-Me-rhamnose, and 3,6-di-O-Me-glucose, glycosidically linked to the phenol substituent. The structure of the oligosaccharide has now been determined, by partial acid hydrolysis, permethylation, 1H NMR, and 13C NMR as: 3,6-di-O-Me-Glcp(1 beta leads to 4)2,3-di-O-Me-Rhap(1 alpha leads to 2)3-O-Me-Rhap1 alpha leads to phenol (assuming that the glucose substituent is in the D-enantiomeric configuration, and the two methylated rhamnoses are L). Acid hydrolysis of deacylated Phenolic glycolipid-I yielded a phenolic phthiocerol "core," and mass spectrometry and proton NMR of the permethylated core suggested the following structure: (formula, see text) Combined gas-liquid chromatography-mass spectrometry showed three tetramethyl branched "mycocerosic" acids, C30, C32 and C34, with molecular weights (as methyl esters) of 466, 494, and 522, respectively. These are esterified to the hydroxyl functions of the branched glycolic chain. Evidence is also presented that the glycolipid is immunologically active, reacting with rabbit antisera to M. leprae and with sera from lepromatous leprosy patients.     

Purification and characterization of a 36 kDa antigen of Mycobacterium leprae

A 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M. tuberculosis, as well as a species-specific epitope (recognized by MAb F47-9). Different treatments of the 36 kDa antigen suggested it to be largely protein in nature; the amino acid composition of 81% of the antigen was determined. A majority of sera from leprosy patients contained antibodies recognizing the 36 kDa antigen.     

In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen.

The heteropolysaccharide chains of enterobacterial common antigen (ECA) are made up of linear trisaccharide repeat units with the structure----3)-alpha-D-Fuc4NAc-(1----4)- beta-D-ManNAcA-(1----4)-alpha-D-GlcNAc-(1----, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The assembly of these chains involves lipid-linked intermediates, and both GlcNAc-pyrophosphorylundecaprenol (lipid I) and ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) are intermediates in ECA biosynthesis. In this study we demonstrated that lipid II serves as the acceptor of Fuc4NAc residues in the assembly of the trisaccharide repeat unit of ECA chains. Incubation of Escherichia coli membranes with UDP-GlcNAc, UDP-[14C]ManNAcA, and TDP-[3H]Fuc4NAc resulted in the synthesis of a radioactive glycolipid (lipid III) that contained both [14C]ManNAcA and [3H]Fuc4NAc. The oligosaccharide moiety of lipid III was identified as a trisaccharide by gel-permeation chromatography, and the in vitro synthesis of lipid III was dependent on prior synthesis of lipids I and II. Accordingly, the incorporation of [3H]Fuc4NAc into lipid III from the donor TDP-[3H]Fuc4NAc was dependent on the presence of both UDP-GlcNAc and UDP-ManNAcA in the reaction mixtures. In addition, the in vitro synthesis of lipid III was abolished by tunicamycin. Direct conversion of lipid II to lipid III was demonstrated in two-stage reactions in which membranes were initially incubated with UDP-GlcNAc and UDP-[14C]ManNAcA to allow the synthesis of radioactive lipid II. Subsequent addition of TDP-Fuc4Nac to the washed membranes resulted in almost complete conversion of radioactive lipid II to lipid III. The in vitro synthesis of lipid III was also accompanied by the apparent utilization of this lipid intermediate for the assembly of ECA heteropolysaccharide chains. Incubation of membranes with UDP-[3H]GlcNAc, UDP-ManNAcA, and TDP-Fuc4NAc resulted in the apparent incorporation of isotope into ECA polymers, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In addition, the in vitro incorporation of [3H]Fuc4NAc into ECA heteropolysaccharide chains was demonstrated with ether-treated cells that were prepared from delta rfbA mutants of Salmonella typhimurium. These mutants are defective in the synthesis of TDP-Fuc4NAc; as a consequence, they are also defective in the synthesis of lipid III and they accumulate lipid II. Accordingly, incubation of ether-permeabilized cells of delta rfbA mutants with TDP-[3h]Fuc4NAc resulted in the incorporation of isotope into both lipid III and ECA heteropolysaccharide chains.     

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae . M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae . This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 μg ml⁻¹ (35% inhibition), highly significant at 5 μg ml⁻¹ (87% inhibition) and almost total at 10 μg ml⁻¹ (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.     

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.     

Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29-40) and peptide 6 (aa 49-60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.     

Isolation and structural characteristics of a monoclonal antibody-defined cross-reactive phospholipid antigen from Mycobacterium tuberculosis and Mycobacterium leprae.

A low molecular weight antigen of Mycobacterium leprae and other mycobacteria was previously defined in our laboratory by means of IgG2a monoclonal antibody termed L4. The antigen had an apparent molecular mass of 4.5-6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was assumed to be a glycoprotein on the basis of its staining with periodic acid Schiff and sensitivity to periodate treatment. In the present work, the cross-reactive and phospholipidic nature of the antigen, present in Mycobacterium tuberculosis as well as in M. leprae sonicates, was demonstrated and this enabled us to undertake its purification from crude M. tuberculosis phospholipidic extracts. The L4-reactive antigen from M. tuberculosis called L4-PIM, was purified by means of silicic acid high pressure liquid chromatography. Its characterization by gas chromatography and FAB-MS showed the antigen to be the common mycobacterial dimannosylated phosphatidylinositol (PIM2), the structure of which had been previously established by others (Lee, Y. C., and Ballou, C. E. (1964) J. Biol. Chem. 239, 1316-1327; Lee, Y. C., and Ballou, C. E. (1965) J. Biochem. (Tokyo) 4, 1395-1404). Delineation of the L4 epitope on M. tuberculosis L4-PIM revealed the involvement of the axial 2-hydroxyl of the alpha-D-mannosyl residues, without any detectable contribution from the myo-inositol. Consequently, L4 was shown to react with PIM5, the structure of which contains twice the number of epitopes as does PIM2. By using both immunostained thin layer chromatography and indirect enzyme-linked immunosorbent assay, similar L4-PIM epitopes were demonstrated in M. leprae sonicate, thereby explaining the cross-reactive nature of the L4-monoclonal antibody. Antibodies of IgG class directed against M. tuberculosis L4-PIM were detectable in sera from patients with leprosy, but no evidence of T cell reactivity to L4-PIM was obtained. The demonstration of a correlation of anti-L4-PIM IgG and anti-disacharide-conjugated bovine serum albumin IgM antibody titers in the sera of leprosy patients indicates that measurement of antibodies directed against L4-PIM may have the potential to be used as a complementary assay to the disaccharide-conjugated bovine serum albumin test for diagnosis and monitoring of patients undergoing leprosy therapy.     

Truncated structural variants of lipoarabinomannan in Mycobacterium leprae and an ethambutol-resistant strain of Mycobacterium tuberculosis

Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration.     

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.     

The specific 18-kilodalton antigen of Mycobacterium leprae is present in Mycobacterium habana and functions as a heat-shock protein

A mAb previously thought to be specific for Mycobacterium leprae has been found to cross-react with a cultivable mycobacterium, Mycobacterium habana TMC5135. The epitope is present on a protein of identical molecular mass (18 kDa) in both species. When M. habana is subjected to heat shock, expression of the protein is significantly increased, whereas other forms of environmental stress do not increase its expression. Since immunization of mice with M. habana results in protection against infection with M. leprae, the possibility of using a molecular genetic approach to investigate the role of this protein in protective immunity is raised.     

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.