Activation of human neutrophils by mycobacterial phenolic glycolipids.

The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells.

Lipid domains of mycobacteria studied with fluorescent molecular probes.

The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non-tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non-covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl-arabinogalactan-peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high-resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.

Fast atom bombardment mass spectrometry of mycobacterial phenolic glycolipids.

Fast atom bombardment mass spectra were successfully recorded for intact glycosylphenolphthiocerol dimycocerosates (phenolic glycolipids, PGLs) from Mycobacterium kansasii, M. leprae, M. tuberculosis, M. marinum, M. bovis and M. haemophilum. Characteristic fragment ions from the loss of the oligosaccharide moiety and one of the long-chain multimethyl-branched mycocerosic acids were observed in most cases. A tandem mass spectrometric experiment was carried out on the PGL from M. tuberculosis, revealing the type of mycocerosic acids esterified to individual homologues. Mass spectra of homologues separated by reversed-phase high-performance liquid chromatography gave information on the substitution pattern in certain cases. The potential of matrix-assisted laser desorption ionization spectroscopy was demonstrated by a successful analysis of the PGL from M. tuberculosis.

Identification of the leprosy bacillus and related mycobacteria by analysis of mycocerosate profiles.

Members of the phthiocerol dimycocerosate family of waxes were extracted from Mycobacterium bovis BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans and a skin biopsy from a leprosy patient. The waxes were degraded by alkaline hydrolysis and the mycocerosic acids converted to pentafluorobenzyl ester. Profiles of the esters, recorded using electron-capture gas-chromatography, gave characteristic profiles for the mycocerosates from M. leprae but those from M. bovis, M. tuberculosis and M. kansasii were superficially similar. The mycocerosate profiles from M. marinum and M. ulcerans were similar, but distinct from the others. Selected ion monitoring negative ion-chemical ionisation gas chromatography-mass spectrometry of of the pentafluorobenzyl esters allowed the analysis of mycocerosate isomers not revealed on gas chromatography alone. M. bovis and M. tuberculosis had similar profiles of C29, C30 and C32 mycocerosates; and additional C33 component was also present in M. kansasii. The mycocerosates from M. marinum and M. ulcerans were C27, C29 and C30 and those from M. leprae were distinct in having C29, C30, C32, C33 and C34 components. These methods have excellent potential for use in the detection of mycobacterial disease by direct analysis of infected tissue without prior cultivation of the causative agent.

Structural elucidation and antigenicity of a novel phenolic glycolipid antigen from Mycobacterium haemophilum.

The structure of a novel antigenic glycolipid that distinguishes the opportunistic pathogen Mycobacterium haemophilum from all other mycobacteria was established by a series of degradation reactions leading to products that were analyzed by gas/liquid chromatography-mass spectrometry. The complete structure of the oligosaccharide unit was determined as 2,3-di-O-CH3-alpha-L-Rhap(1----2)3-O-CH3-alpha-L-Rhap(1----4 )-2,3-di-O-CH3-alpha-L-Rhap(1----. The lipid portion of the phenolic glycolipid was composed of two component phenolphthiocerols differing by two methylene groups, as determined by analysis of their per-O-trideuteriomethylated derivatives. The diol unit of the phenolphthiocerols has a threo relative configuration. The absolute stereochemistry of the asymmetric centers of the phenolphthiocerols is uncertain, but the centers are probably 3R, 4S, 9R, and 11R as found for phthiocerol A from Mycobacterium tuberculosis. The hydroxyl functions of the branched glycolic chain are esterified to a complex mixture of multi-methyl branched mycocerosic acids, C27, C30, C32, C34, and C37 with molecular weights (as methyl esters) of 424, 466, 494, 522, and 564, respectively. The stereochemistry of the methyl branches of the mycocerosates have R absolute configuration. The glycolipid is highly antigenic and appears to be specific for M. haemophilum. There are intriguing similarities between the product from M. haemophilum and the well-known phenolic glycolipid I of Mycobacterium leprae, a matter that is discussed.

Characteristic new members of the phthiocerol and phenolphthiocerol families from Mycobacterium ulcerans.

Diacyl phthiodiolone A and phenolphthiodiolone A lipids were isolated from two strains of Mycobacterium ulcerans. The diol units of the phthiodiolone A and phenolphthiodiolone A components were shown to have erythro stereochemistry by infrared spectroscopy and proton nuclear magnetic resonance of an acetal derivative. This stereochemistry is shared only by related diols from M. marinum, the diols from M. bovis, M. kansasii, M. leprae and M. tuberculosis having threo stereochemistry.

Comparative studies of antigenic glycolipids of mycobacteria related to the leprosy bacillus.

The leprosy bacillus, Mycobacterium leprae, is a member of a small group of mycobacteria comprising the species Mycobacterium bovis, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium ulcerans and related taxa. This relationship is based on the similarity of the characteristic lipid types in the cell envelope. Mycobacterium leprae produces a phenolic glycolipid antigen which is species specific. This communication reports a comparison of the specificity of the lipid antigens of other members of this group of mycobacteria. Mycobacterium kansasii, in accordance with previous studies, produces phenolic glycolipid and trehalose-based lipooligosaccharide antigens which do not cross react with antisera raised against other mycobacteria. The phenolic glycolipid and an uncharacterised polar glycolipid, with the properties of a lipooligosaccharide, from Mycobacterium marinum are also shown to be specific antigens. An acylated trehalose glycolipid antigen from Mycobacterium tuberculosis H37Rv reacts strongly with antisera raised against the same strain and sera from eight out of ten tuberculosis patients. The phenolic glycolipid antigen, isolated only from Mycobacterium tuberculosis "Canetti" variants, did not react with antisera raised against the type strain, Mycobacterium tuberculosis H37Rv, although it had been shown previously to react with sera from tuberculosis patients. It is apparent that there are populations of the tubercle bacillus which differ in the lipid antigens expressed on their cell surface.

Analysis of dimycocerosates of glycosylphenolphthiocerols in the identification of some clinically significant mycobacteria.

Extracts of representative mycobacteria were examined by thin-layer chromatography for glycosylphenolphthiocerol dimycocerosates. The glycolipid typical of Mycobacterium bovis was also found in Mycobacterium africanum and Mycobacterium microti, but it was absent in Mycobacterium bovis AN 5. Mycobacterium gastri strains contained a glycolipid which was chromatographically similar to that in Mycobacterium kansasii. Representatives of Mycobacterium marinum produced a distinct glycolipid type, and one strain had major amounts of a more polar variant. The sugar moieties of purified lipids, including that from Mycobacterium leprae, were identified by thin-layer chromatography of methyl glycosides in acid methanolysates.

The free lipids of Mycobacterium leprae harvested from experimentally infected nine-banded armadillos.

The free lipids of a sample of Mycobacterium leprae were extracted by a procedure designed to produce separate non-polar and polar fractions. The composition of these lipids was analysed semi-quantitatively by five special thin-layer chromatographic systems covering the total range of mycobacterial lipid polarities. In order of increasing polarity, the major lipids were dimycocerosates of phthiocerol A, phthiocerol B and phthiodiolone A, glycosyl phenolphthiocerol dimycocerosates and phospholipids, including monoacylphosphatidylinositol di- and pentamannosides. The diacylated forms of these latter lipids, found in most mycobacteria, were not present. The composition of the free lipids of the leprosy bacillus, surveyed over the total polarity range for the first time, showed that the patterns were particularly related to those of Mycobacterium bovis, Mycobacterium kansasii and Mycobacterium marinum.

Quantitative comparison of the mycolic and fatty acid compositions of Mycobacterium leprae and Mycobacterium gordonae.

The mycolic and fatty acids of three samples each of Mycobacterium leprae and Mycobacterium gordonae were compared. Acids released by whole-organism alkaline hydrolysis were converted to 4-nitrobenzyl esters and mycolic acids were further derivatized to t-butyldimethylsilyl ethers. Thin-layer chromatography of the derivatized long-chain extracts showed that all three M. leprae preparations contained so-called alpha-mycolates and ketomycolates but that the M. gordonae samples had a methoxymycolate in addition to the above types. Silica gel normal-phase high-performance liquid chromatography of the total mycolic acid derivatives confirmed the lack of detectable amounts of methoxymycolates in M. leprae and reverse-phase chromatography of the individual mycolate types demonstrated the homogeneity of the chain lengths of the mycolic acids in each species. Non-hydroxylated fatty acid 4-nitrobenzyl esters were transformed to methyl esters and examined by gas chromatography. Tuberculostearic (10-methyloctadecanoic) acid was a major component of the lipids of all three M. leprae preparations but it was absent in one M. gordonae strain and a very minor component in the other representatives of this latter species. On the basis of fatty and mycolic acid compositions, therefore, a previously suggested close relationship between M. leprae and M. gordonae was not supported.

Characterization of Mycobacterium leprae by lipid analysis.

The lipid composition of the leprosy bacillus, harvested from experimentally infected nine-banded armadillos, strongly supports it status as a distinct species of the genus Mycobacterium. Phthiocerol dimycocerosate waxes and glycosylated phenophthiocerol dimycocerosates are distinct from those characterised from a number of other mycobacteria. The polar lipids of a single isolate lack diacylated forms of phosphatidylinositol di- and pentamannosides, lipids usually found in most mycobacteria. A simple mycolic acid pattern composed of alpha-mycolates and ketomycolates is characteristic of most preparations of M. leprae.

Isolation of a characteristic phthiocerol dimycocerosate from Mycobacterium leprae.

A characteristic mycobacterial wax, phthiocerol dimycocerosate, has been isolated from liver of armadillos experimentally infected with Mycobacterium leprae. The structure of this wax is generally similar to that produced by Mycobacterium tuberculosis, but the homologous phthiocerol and the mycocerosic acid components from M. leprae are significantly different from those of M. tuberculosis.

The mycolic acids of Mycobacterium leprae harvested from experimentally infected nine-banded armadillos.

Mycolic acid methyl esters were prepared from defatted cells of armadillo-derived Mycobacterium leprae and analysed by thin-layer and high-performance liquid chromatography, proton magnetic resonance spectrometry and mass spectroscopy. The first type of mycolic acid characterized was an "alpha-mycolate" having two cis-cyclopropane rings, a 78-carbon main component and an overall size-range of 72 to 83 carbons. Ketomycolates, with an 83-carbon main component, were the only other type of mycolate isolated; the major 79- to 87-carbon series of ketomycolates apparently contained a single trans-cyclopropane and the minor 80- to 86-carbon series had a cis-cyclopropane function.