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.

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.

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.

Crossreactivity between Mycobacterium leprae and various actinomycetes and related organisms.

Serological crossreactivity was analyzed between M. leprae and strains of various species of Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, and related organisms. M. leprae shares antigens with most of these organisms, and sera from patients with lepromatous leprosy contain antibodies against them. The results demonstrate that M. leprae shares more antigens with the mycobacteria than with strains of the other tested genera, thus supporting the view that the leprosy organism belongs to the genus Mycobacterium. One precipitinogen (designated p beta) was found to be common to M. leprae and the streptomycetes, and sera from patients with lepromatous leprosy contain antibodies against this antigen.

Immunodiffusion analyses of some diphtheroid organisms isolated from patients with leprosy.

Eight strains of diphtheroid bacteria isolated from patients with leprosy were analyzed by immunodiffusion, using precipitation systems representing various species of Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, and related organisms. The analyses showed that six of the eight strains shared several antigens with representatives of these four genera. The largest number of shared precipitinogens was revealed when the corynebacterial precipitation systems were used, thus indicating that these organisms either belong to, or are closely related to the genus Corynebacterium. This assumption was further supported by the fact that the ribosomal precipitinogen beta--earlier demonstrated in mycobacteria but not in corynebacteria--was not found in the diphtheroid strains. Other ribosomal antigens were, however, revealed to be common to the diphtheroid organisms and mycobacteria. Further, the reaction between sera from patients with lepromatous leprosy and the diphtheroid strains was analyzed, very few and faint precipitates being demonstrated. It is concluded that the presence of anti-beta antibodies in leprosy sera is, most likely, not a result of the presence of diphtheroid organisms in the patients.