Abstract
Mycobacterium "habana" strain TMC 5135, which has been proposed as a vaccine against both leprosy and tuberculosis, is considered to be a strain of serotype I of the recognized species Mycobacterium simiae. We have now shown that each of these strains possesses characteristic polar glycopeptidolipids (GPL) which are sufficiently different to allow unequivocal strain identification. Thin layer chromatographic analysis demonstrated that M. habana synthesizes a family of apolar GPLs and three distinct polar GPLs (pGPL-I to -III) which exhibited migration patterns different from those of M. simiae serotype I (pGPL-Sim). Using a combination of chemical, mass spectrometric, and proton-NMR analyses, the GPLs from M. habana were determined to be based on the same generic structure as those from the M. avium complex, namely N-fatty acyl-D-Phe-(O-saccharide)-D-allo-Thr-D-Ala-L-alaninyl-O-m onosaccharide. The de-O-acetylated apolar GPLs contain a 3-O-Me-6-deoxy-Tal attached to the allo-Thr and either a 3-O-Me-Rha or a 3,4-di-O-Me-Rha attached to the alaninol. In the pGPLs, oligosaccharides were found to be attached to the allo-Thr. The oligoglycosyl alditol reductively released from the least polar pGPL-I was fully characterized as L-Fucp alpha 1 in --7 with 3-(6-O-Me)-D-Glcp beta 1 in --7 with 3-(4-O-Me)-L-Rhap alpha 1 in --7 with 3-L-Rhap alpha 1 in --7 with 2-(3-O-Me)-6-deoxy-Tal. In pGPl-II and -III, the terminal Fuc residue is further 3-O-methylated and 4-O-substituted with an additional 2,4-di-O-Me-D-GlcA and 4-O-Me-D-GlcA, respectively. The corresponding oligosaccharide from pGPL-Sim was shown to be of identical molecular weight to pGPL-II but terminating with a 3,4-di-O-Me-GlcA. Enzyme-linked immunosorbent assay-based serological studies using anti-M. habana and anti-M. simiae sera against whole cells and purified pGPLs firmly established the polar GPLs as important antigens and indicated that the terminal epitopes L-Fuc-, 2,4-di-O-Me-D-GlcA, and 4-O-Me-D-GlcA uniquely present in pGPL-I, -II, and -III, respectively, confer sufficient specificity for the identification of M. habana as a distinct serotype of M. simiae.
Highlights
Leprosy and tuberculosis are grave diseases with large worldwide prevalences (Raviglione et al, 1995; Noordeen, 1994)
Species or strainspecific glycolipids structurally based on the same prototype mycoside C have since been identified in other mycobacterial species such as Mycobacterium peregrinum, Mycobacterium senegalense, Mycobacterium porcinum, and Mycobacterium butyricum (Lopez-Marin et al, 1991, 1992, 1993; Besra et al, 1994; Khoo et al, 1995) in these species the GPLs are mainly of the apolar types without the haptenic oligosaccharide appendages which characterize the M. avium serotypes
The GPLs isolated from M. habana strain TMC 5135 and characterized in this study closely resemble those of the M
Summary
Mycobacteria—M. habana strain TMC 5135 was obtained from Dr N. Glycolipids were identified in side bands cut from the plates, using ␣-naphthol-sulfuric acid, and corresponding iodine vapor-stained bands on the main part of each chromatographic plate were extracted from scraped off silica gel with chloroform/methanol (2:1 by volume) or chloroform/methanol/water (65: 25:4 by volume). They were repurified by TLC until homogeneous. Glycolipid extracts or purified polar GPLs were suspended in water by vigorous vortexing or by sonication, applied to ELISA plates at 0.1 ml/well (containing 1– 4 g of lipid) and dried onto the plates at 37 °C. For experiments involving GPL from M. avium serotype 19, sera at 1/200 dilution were absorbed with 20 and 40 g/ml of glycolipid suspension for 66 h before being added to wells containing 1 g of glycolipid
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