Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae.

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The availability of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.

Effective vaccination of mice against Mycobacterium leprae with density-gradient subfractions of soluble M. leprae proteins: clues to effective protein epitopes.

It had previously been discovered that intradermal mouse vaccination with a protein fraction of Mycobacterium leprae (called soluble proteins) in Freund's incomplete adjuvant (FIA) resulted in consistent and long-lived protection against M. leprae multiplication from subsequent viable footpad challenges. In this study certain density-gradient subfractions of this soluble protein, but not others, in FIA afforded vaccine protection. The results of this study suggest which M. leprae proteins may be involved in protective immunity, particularly 1-3 kD, 10 kD, 65 kD, and those of higher molecular weight.

Chemical definition, cloning, and expression of the major protein of the leprosy bacillus.

The decline in prevalence of leprosy is not necessarily matched by a fall in incidence, emphasizing the need for new antigens to measure disease transmission and reservoirs of infection. Mycobacterium leprae obtained from armadillo tissues was disrupted and subjected to differential centrifugation to arrive at preparations of cell wall, cytoplasmic membrane, and cytosol. By committing 0.3 g of M. leprae to the task, it was possible to isolate from the cytosol and fully define the major cytosolic protein. Amino-terminus sequencing and chemical and enzymatic cleavage, followed by more sequencing and fast atom bombardment-mass spectrometry of fragments, allowed description of the entire amino acid sequence of a protein of 10,675-Da molecular mass. The sequence derived by chemical means is identical to that deduced previously from DNA analysis of the gene of a 10-kDa protein, a GroES analog. The work represents the first complete chemical definition of an M. leprae protein. PCR amplification of the 10-kDa protein gene, when cloned into Escherichia coli with a pTRP expression vector, allowed production of the recombinant protein. Chemical analysis of the expressed protein demonstrated that it exactly reflected the native protein. The recombinant major cytosolic protein appears to be a promising reagent for skin testing, still probably the most appropriate and pragmatic means of measuring incidence of leprosy.

Relationship between host histones and armadillo-derived Mycobacterium leprae.

A major protein previously recognized as being primarily associated with the cell walls of Mycobacterium leprae, major wall protein (MWP), is now identified as histoprotein H2b based on N-terminal amino-acid sequencing, electrophoretic comparisons, and several other properties. An avid association between several host/armadillo-derived histones and M. leprae was demonstrated. Since such armadillo-derived M. leprae are the basis of several ongoing vaccine trials, a simple procedure that permits the prompt solubilization and quantification of histones in M. leprae preparations is described. The quantity of histones associated with M. leprae is significant, ranging from 0.6 to 4.8 micrograms of histoprotein H2b per mg of bacteria.

Lipoarabinomannan. Multiglycosylated form of the mycobacterial mannosylphosphatidylinositols.

The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.

A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein.

Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.

Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages.

Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy, respectively, produce large quantities of lipoarabinomannan (LAM), a highly immunogenic, cell wall-associated glycolipid. This molecule has been previously reported to be a potent inhibitor of gamma interferon-mediated activation of murine macrophages. Studies of the mechanism by which this mycobacterial glycolipid down-regulates macrophage effector functions provide evidence that LAM acts at several levels and that it can (i) scavenge potentially cytotoxic oxygen free radicals, (ii) inhibit protein kinase C activity, and (iii) block the transcriptional activation of gamma interferon-inducible genes in human macrophage-like cell lines. These results suggest that LAM can inhibit macrophage activation and triggering and cytocidal activity and that it may represent a chemically defined virulence factor contributing to the persistence of mycobacteria within mononuclear phagocytes.

The major native proteins of the leprosy bacillus.

This study addresses a major obstacle to vaccine development for leprosy, the isolation and characterization of the native protein antigens of the leprosy bacillus. Mycobacterium leprae harvested from armadillos was subjected to a simple fractionation protocol to arrive at the three major subcellular fractions, cell walls, cytoplasmic membrane, and soluble cytoplasm. The application of extensive detergent phase separations to membrane fractions allowed removal of lipoarabinomannan and the mannosyl phosphatidylinositols, and the recognition and purification of two major membrane proteins (MMP) of molecular mass 35 kDa (MMP-I) and 22 kDa (MMP-II); recovery of these proteins was about 0.5 mg each per g of M. leprae. MMP-I is N-blocked and is perhaps a lipoprotein. End group analysis on MMP-II indicates a new protein. Three major cytoplasmic proteins (MCP) of molecular mass 14 kDa (MCP-I), 17 kDa (MCP-II), and 28 kDa (MCP-III) were also recognized. MCP-I, the most abundant protein in M. leprae, represents 1% of the bacterial mass. End group analysis of the first 30 residues and immunoblotting studies demonstrate sizeable structural homology to a protein from Mycobacterium tuberculosis but immunological distinctiveness. MCP-I, which also occurs in highly immunogenic peptidoglycan-bound form, is a primary candidate for future vaccine development. The cell walls of M. leprae are also characterized by one major extractable protein, also of molecular mass 17 kDa. Thus the major antigens of the leprosy bacillus, protein and carbohydrate alike, are now nearer to complete definition.

Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis.

The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis.

Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae.

Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.

Isolation and characterization of the highly immunogenic cell wall-associated protein of Mycobacterium leprae.

In a recent study, we demonstrated that certain reactivities crucial to the immune response in leprosy are due to protein associated with the cell wall peptidoglycan "core" of Mycobacterium leprae. We now describe a primary method for the isolation of a highly immunogenic, large molecular-size, cell wall protein (CW-P) complex from M. leprae, freed of soluble proteins, bound mycolates, arabinogalactan, and much of the peptidoglycan. The complex is of apparent relative molecular size 2 x 10(6) to 20 x 10(6) Da, is distinguished by a high content of Ala, Gly, Leu, Asx, and Glx, and some peptidoglycan, and represents up to 7% of the bacterial mass. It is stable to a variety of dissociation and reductive processes and, in accord with its size, is not resolvable by polyacrylamide gel electrophoresis. The mAb to the CW-P complex also react with the heat shock 65-kDa protein of M. leprae. Conversely, antibodies that recognize internal epitopes within the polypeptide chain of the heat shock protein also react with CW-P; however, antibodies that recognize the N and C termini of the 65-kDa protein fail to react with CW-P, and some anti-CW-P mAb do not recognize any of the soluble proteins of M. leprae. Alternate methods to derive the large peptidoglycan-associated protein result in lower yield and less of the associated heat shock protein, implying that the 65-kDa protein may not be crucial to the immunogenicity of the complex. In an accompanying paper, we demonstrate that T cell clones raised to CW-P also selectively recognize soluble proteins, primarily of 7-kDa and 16-kDa size. Thus, the image of the CW-P complex of M. leprae is of a few immunoreactive polypeptides in avid association with a modicum of peptidoglycan to which the 65-kDa polypeptide may be variably attached, perhaps due to involvement in assembly of the complex.

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.

Structure and function of mycobacterial glycolipids and glycopeptidolipids.

Earlier work from this and other laboratories has revealed the presence within Mycobacterium spp. of three classes of glycolipid antigens which we have called the glycopeptidolipids, the lipooligosaccharides and the phenolic glycolipids. Representative structures of each from different species and sub-species have been proposed. More recently, new variants of these antigens and older structures have been analyzed by Fourier transform infrared, NMR, particularly at high temperatures, and, most notably, by fast atom bombardment and Californium desorption mass spectrometry. Extraordinary novelty and diversity were revealed, particularly at the distal non-reducing end of the oligosaccharide chains, marked by the presence of new branched-chain sugars, amino sugars and sugar acids. These epitopes and monoclonal antibodies to them have been used for the critical identification of mycobacteria. In addition, the pure antigens are the basis of specific serological tests for various mycobacterioses. The resurgence of interest in "atypical" mycobacteria stems from their occurrence as opportunistic pathogens in many patients with acquired immunodeficiency syndrome, although they have long been associated with pulmonary and other organ infections. Foremost among these mycobacteria are serovars of the Mycobacterium avium-Mycobacterium intracellulare complex (the M. avium complex). The surface antigens which differentiate these serovars are glycopeptidolipids, related to "mycoside C" and, accordingly, composed of a glycosylated lipopeptide "core", fatty acyl-D-Phe-D-alloThr-D-Ala-L-acanyl-O- (3,4-di-O-methyl-alpha-L-rhamnopyranoside), to which a haptenic oligosaccharide is linked at the threonine substituent; this oligoglycosyl unit is the source of type specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycobacterial lipoarabinomannan inhibits gamma interferon-mediated activation of macrophages.

The principal efferent role of the macrophage in acquired resistance to intracellular pathogens depends on activation by T-cell lymphokines, primarily gamma interferon (IFN-gamma). However, mouse macrophages that are heavily burdened with Mycobacterium leprae are refractory to activation by IFN-gamma and are thus severely compromised in their capacity for both enhanced microbicidal and tumoricidal activities. We report here that lipoarabinomannan (LAM), a highly immunogenic lipopolysaccharide that is a prominent component of the cell walls of M. leprae and M. tuberculosis, was a potent inhibitor of IFN-gamma-mediated activation of mouse macrophages in vitro. Inhibition of macrophage activation by LAM required preincubation for approximately 24 h, resulting in uptake of LAM into cytoplasmic vacuoles of macrophages. Intact LAM was necessary to inhibit IFN-gamma-mediated activation, as this property was lost when the acyl side chains were removed from LAM by mild alkaline hydrolysis. In addition, LAM was an abundant constituent of macrophages isolated from lepromatous granulomas of M. leprae-infected nude mice and likely contributed to the defective activation of granuloma macrophages by IFN-gamma.

Immunological significance of Mycobacterium leprae cell walls.

Cell walls of Mycobacterium leprae, prepared by differential solvent extraction, were shown to contain arabinogalactan, mycolates, and peptidoglycan. In addition, amino acid analysis revealed the unexpected presence of large amounts of protein that retained potent immunological reactivity. Purified cell walls stimulated proliferation of T cells from tuberculoid, but not from lepromatous leprosy, patients and elicited delayed-type hypersensitivity skin reactions in guinea pigs and patients sensitized to M. leprae. Analysis of the precursor frequency of antigen-reactive human peripheral T cells revealed that as many cells (approximately equal to 1/6000) proliferate to antigen contained in cell walls as to intact M. leprae. Sequential removal of mycolates and arabinogalactan resulted in a large peptidoglycan-protein complex that retained all the immunological activity. This immunological reactivity and the inherent protein were destroyed by proteolysis. Thus, cell wall protein is a major contributor to cell-mediated immune reactivity to this pathogenic mycobacterium.

Demonstration that the galactosyl and arabinosyl residues in the cell-wall arabinogalactan of Mycobacterium leprae and Mycobacterium tuberculosis are furanoid.

By a complex process involving methylation, partial hydrolysis with acid, reduction with sodium borodeuteride, ethylation, further hydrolysis and reduction, and subsequent capillary gas-liquid chromatography-mass spectrometry of the derived alditol acetates, it was established that the arabinogalactans of Mycobacterium leprae and Mycobacterium tuberculosis contain arabinofuranyosyl and galactofuranosyl residues exclusively. Thus, the covalently bound, highly immunogenic arabinogalactan of mycobacteria, and presumably of other actinomycetes, is highly unusual, in that all of the glycosyl residues are in the furanoid form. Furthermore, by establishing that the galactofuranosyl residues are either 5-, 6-, or 5,6-linked, their linkage pattern was established, and the literature is corrected on this point.

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.

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.

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.

Quantitation of the phenolic glycolipid of Mycobacterium leprae and relevance to glycolipid antigenemia in leprosy.

Chemical and immunologic procedures have been developed for quantitation, in the body fluids of patients with leprosy, of phenolic glycolipid I, the major specific antigen of the leprosy bacillus. Serum samples were extracted with CHCl3/CH3OH and fractionated on columns of silicic acid. Thin-layer chromatography with a sensitivity of about 500 ng allowed detection of the glycolipid in untreated lepromatous and borderline patients, and high-pressure liquid chromatography gave a quantitation of 0.8-3.7 micrograms/ml of serum from four patients. An ELISA-inhibition assay with polyclonal antibodies to glycolipid corroborated these figures. Dot-ELISA on nitrocellulose with polyclonal and monoclonal IgG antibodies allowed for much greater sensitivity (500 pg) and semiquantitative evaluation. Small quantities of glycolipid were present in the urine of patients with lepromatous leprosy. In sera obtained from patients undergoing chemotherapy, the amount of glycolipid declined sooner than did titer of antibody. This experimental approach is applicable to diagnosis of leprosy, bacillary quantification, and standardization of skin-test reagents and vaccines.