Biogenesis of the mycobacterial cell wall and the site of action of ethambutol.

The effect of ethambutol (EMB) is primarily on polymerization steps in the biosynthesis of the arabinan component of cell wall arabinogalactan (AG) of Mycobacterium smegmatis. Inhibition of the synthesis of the arabinan of lipoarabinomannan (LAM) occurred later, and thus in the cases of AG and LAM, the polymerization of D-arabinofuranose apparently involves separate pathways. While the synthesis of these arabinans was normal in an EMB-resistant isogeneic strain, the addition of EMB to the resistant strain resulted in partial inhibition of the synthesis of the arabinan of LAM and the emergence of a novel, truncated form of LAM, indicating partial susceptibility of the resistant gene(s) and providing a new intermediate in the LAM biosynthetic sequence.

Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis.

Mycolic acids represent a major constituent of the mycobacterial cell wall complex, which provides the first line of defense against potentially lethal environmental conditions. Slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis modify their mycolic acids by cyclopropanation, whereas fast-growing saprophytic species such as Mycobacterium smegmatis do not, suggesting that this modification may be associated with an increase in oxidative stress experienced by the slow-growing species. We have demonstrated the transformation of the distal cis double bond in the major mycolic acid of M.

A new interpretation of the structure of the mycolyl-arabinogalactan complex of Mycobacterium tuberculosis as revealed through characterization of oligoglycosylalditol fragments by fast-atom bombardment mass spectrometry and 1H nuclear magnetic resonance spectroscopy.

Previous structural analysis of small oligosaccharide fragments had allowed the recognition of several small structural motifs within arabinogalactan, the dominant cell was structural polysaccharide of Mycobacterium tuberculosis. To determine how these motifs are connected to one another to form the complete polymer, oligosaccharide fragments containing up to 26 glycosyl residues were released by gentle acid hydrolysis of the per-O-methylated arabinogalactan, converted to fully per-O-alkylated oligoglycosylalditols, and purified by high-performance liquid chromatography, and the molecular weights and alkylation patterns of the resultant oligoglycosyl fragments were determined by fast atom bombardment mass spectrometry. The results, combined with previous studies, allowed further understanding of the intricate structural features of the nonreducing ends of arabinogalactan.

Identification of the apparent carrier in mycolic acid synthesis.

The mycolic acids are large (C70-90) alpha-alkyl, beta-hydroxy fatty acids and are the major determinants of the mycobacterial cell wall's impermeable barrier. The biosynthesis of mycolic acids is barely understood (they are probably the products of specialized elongation and Claisen-type condensation), and yet their synthesis is the site of action of several mainline antituberculosis drugs. We describe the isolation from Mycobacterium smegmatis and the full characterization of a 6-O-mycolyl-beta-D-mannopyranosyl-1-monophosphoryl-3,7,11,15,19,23 ,27- heptamethyl-(2Z,6E,10E)-octacosatrien-1-ol .

Stereochemistry of 2,4-dimethyleicos-2-enoate from the pyruvylated glycolipid of Mycobacterium smegmatis.

The absolute stereochemistry of 2,4-dimethyleicos-2-enoic acid, isolated from the pyruvylated glycolipid of Mycobacterium smegmatis, has been determined. The two enantiomers of methyl 2,4-dimethyleicos-2-enoate were synthesised for the first time but could not be separated by gas chromatography on cyclodextrin phases. (E)-2-Methyloctadec-2-enoate, an intermediate in the synthesis, is a characteristic component of acyl trehalose glycolipids from Mycobacterium fortuitum.

Characterization of the specific antigenicity of representatives of M. senegalense and related bacteria.

Representative strains of M. senegalense and an unusual strain, labelled M. farcinogenes (M280) were examined by thin-layer chromatography for the presence of characteristic surface glycolipids.

New pyruvylated, glycosylated acyltrehaloses from Mycobacterium smegmatis strains, and their implications for phage resistance in mycobacteria.

Phage resistance and apparent lysogenization of Mycobacterium smegmatis due to infection with mycobacteriophage D29 results in the emergence of new variations of the pyruvylated, acylated trehaloses described by Saadat and Ballou, J. Biol. Chem.

Trehalose-containing lipooligosaccharides of Mycobacterium gordonae: presence of a mono-O-methyltetra-O-acyltrehalose "core" and branching in the oligosaccharide backbone.

Past evidence has indicated that Mycobacterium gordonae, as isolated from soil and as an occasional opportunistic pathogen, exists as a serocomplex. We now demonstrate that the basis of seroreactivity and diversity is a novel series of alkali-labile, trehalose-containing lipooligosaccharides (LOS). The structures from two strains were established by per-O-methylation, partial acid hydrolysis, infrared and high-field NMR spectroscopy, electron-impact MS, and fast atom bombardment/mass spectrometry of the native lipooligosaccharides and hydrolysis products.

Synthesis of methyl (Z)-tetracos-5-enoate and both enantiomers of ethyl (E)-6-methyltetracos-4-enoate: possible intermediates in the biosynthesis of mycolic acids in mycobacteria.

The high molecular weight 2-alkyl-3-hydroxy mycolic acids are key structural components of the cell envelope of pathogenic mycobacteria, such as Mycobacterium tuberculosis. A prime target for action would be the initial stages where the biosynthetic pathways diverge from those of ordinary fatty acids. It has been postulated that the pathway for the alpha-mycolates, without oxygen functions in addition to the hydroxy-acid unit, appears to diverge from (Z)-tetracos-5-enoic acid.

Stimulation of mycolic acid biosynthesis by incorporation of cis-tetracos-5-enoic acid in a cell-wall preparation from Mycobacterium smegmatis.

Mycolic acids are high molecular weight hydroxy fatty acids which are a covalently linked part of the cell wall structure of all mycobacteria and their biosynthetic pathways offer potential drug targets. Three good candidates, cis-tetracos-5-enoic acid and R or S trans-6-methyl-tetracos-4-enoic acids, for the key initial intermediates where mycolic acid biosynthesis might diverge from other metabolic pathways, were tested as possible substrates. A cell-wall preparation from Mycobacterium smegmatis, capable of mycolic acid synthesis, was developed to investigate the possible incorporation of these, and other 16 to 24 carbon acids into mycolic acids.

Further structural definition of a new family of glycopeptidolipids from Mycobacterium xenopi.

The highly antigenic surface glycolipids of serovars of the Mycobacterium avium complex are glycopeptidolipids of the general structure [formula: see text] However, it has recently been shown [Rivière, M., & Puzo, G. (1991) J.

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.

An integrated procedure for the direct detection of characteristic lipids in tuberculosis patients.

An integrated method is described for the sensitive detection of tuberculostearic, mycocerosic and mycolic acids in infected materials from tuberculosis patients. Tuberculostearic acid is analysed by two-dimensional gas chromatography of pentafluorobenzyl esters, the key component being switched from a short non-polar column to a high resolution polar column with final electron capture detection. Mycocerosic acids are identified by simple one-dimensional electron capture gas chromatography of pentafluorobenzyl esters, with the use of negative-ion chemical ionisation gas chromatography in indecisive cases.

Structural elucidation of a novel family of acyltrehaloses from Mycobacterium tuberculosis.

Analysis of the lipids of Mycobacterium tuberculosis H37Rv, by both normal- and reverse-phase thin-layer chromatography, revealed a series of novel glycolipids based on 2,3-di-O-acyltrehalose. The structures of these acylated trehaloses were elucidated by a combination of gas chromatography-mass spectrometry, 1H, 13C, two-dimensional 1H-1H, and 1H-13C nuclear magnetic resonance spectrometry. The fatty acyl substituents were mainly of three types: saturated straight-chain C16-C19 acids; C21-C25 "mycosanoic acids"; and C24-C28 "mycolipanolic acids.

Characterization of the specific antigenicity of Mycobacterium fortuitum.

Mycobacterium fortuitum, biovar, fortuitum, the cause of serious skin and soft-tissue infections, can be differentiated from M. fortuitum, biovar. peregrinum, and other rapidly growing opportunistic mycobacteria by the presence of a unique antigenic glycolipid.

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.

Characterisation of phenolic glycolipids from Mycobacterium marinum.

The phenolic glycolipids from two strains of Mycobacterium marinum have been isolated and characterised. The glycolipids from M. marinum MNC 170 were principally glycosides of diacyl C37, C39 and C41 phenolphthiocerols A, but in M.

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.

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.