NOVEL O-METHYLATED TERMINAL GLUCURONIC-ACID CHARACTERIZES THE POLAR GLYCOPEPTIDOLIPIDS OF MYCOBACTERIUM-HABANA STRAIN TMC-5135

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. W e have now shown that each of these strains possesses characteristic p olar glycopeptidolipids (GPL) which are sufficiently different to allo w unequivocal strain identification. Thin layer chromatographic analys is 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). Usin g a combination of chemical, mass spectrometric, and proton-NMR analys es, the GPLs from M. habana were determined to be based on the same ge neric structure as those from the M. avium complex, namely N-fatty ide )-D-allo-Thr-D-Ala-L-alaninyl-O-monosaccharide. The de-O-acetylated ap olar GPLs contain a 3-O-Me-6-deoxy-Tal attached to the allo-Thr and ei ther a 3-O-Me-Rha or a 3,4-di-O-Me-Rha attached to the alaninol. In th e pGPLs, oligosaccharides were found to be attached to the allo-Thr. T he oligoglycosyl alditol reductively released from the least polar pGP L-I was fully characterized as L-Fucp alpha 1-->3-(6-O-Me)-D-Glcp beta 1-->3-(4-O-Me)-L-Rhap alpha 1-->3-L-Rhap alpha 1-->2-(3-O-Me)-6-deoxy -Tal. In pGPL-II and -III, the terminal Fuc residue is further 3-O-met hylated 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 pG PL-Sim was shown to be of identical molecular weight 60 pGPL-II but te rminating with a 3,4-di-O-Me-GlcA. Enzyme-linked immunosorbent assay-b ased serological studies using anti-M. habana and anti-M. simiae sera against whole cells and purified pGPLs firmly established the polar GP Ls as important antigens and indicated that the terminal epitopes L-Fu c-, 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 iden tification of M. habana as a distinct serotype of M. simiae.