Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. II. Presence of a novel dideoxyhexose

A novel O-methyl-2,6-dideoxyhexose was isolated from the major phenolic glycolipid (previously called mycoside A) of Mycobacterium kansasii. Its molecular weight (162) was determined by gas chromatography-mass spectrometry analysis (chemical ionization with ammonia as reactant gas) of its underivatized reducing form. The methoxyl group was located by electron impact-mass spectrometry of its alditol acetate. The configuration was established by 1H NMR of its peracetylated derivative. The structure 2,6-dideoxy-4-O-methyl-arabino-hexopyranose is proposed for this new sugar. Evidence is also presented that the phenolic glycolipid previously called mycoside A is an antigen of M. kansasii since it reacts with rabbit antisera raised against whole M. kansasii.     

A characteristic phenolic glycolipid antigen from Mycobacterium haemophilum

A characteristic phenolic glycolipid was detected, by thin layer chromatography, in non-polar lipid extracts of nine representative strains of Mycobacterium haemophilum . The lipid was a specific antigen that reacted strongly with serum raised against whole cells of M. haemophilum. Sera from six other mycobacterial strains were tested but only that from Mycobacterium kansasii give a weak reaction.     

A novel phenolic glycolipid from Mycobacterium leprae possibly involved in immunogenicity and pathogenicity.

A phenolic glycolipid was obtained in high amounts (2% of dry weight) from Mycobacterium leprae isolated from infected armadillo liver. Infrared and nuclear magnetic resonance spectroscopy showed that it is closely related to "mycoside A" from Mycobacterium kansasii and is therefore a glycosylphenolic phthiocerol diester. The crucial difference between the two products is in the composition of the attached trisaccharide. Gas-liquid chromatography-mass spectroscopy showed that the product from M. kansasii is composed of 2,4-di-O-methylrhamnose, 2-O-methylrhamnose, and 2-O-methylfucose, whereas that from M. leprae contains 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, and 3,6-di-O-methylglucose. The distinct composition of the oligosaccharide segment of the glycolipid from M. leprae may make it useful for the chemical and serological differentiation of this organism from other mycobacteria. Surprisingly large quantities (2.2 mg/g of dry liver) of the glycolipid were also found in infected liver residue freed of M. leprae, suggesting that it may be responsible for the electron-transparent "foam" surrounding the organism in infected lepromatous tissue.     

Absolute configuration of the unique 2,6-dideoxy-4-O-methyl-arabino-hexopyranose of the major phenolic glycolipid antigen from Mycobacterium kansasii

The antigenicity and the structure of the major phenolic glycolipid from Mycobacterium kansasii have been established. A monoacetylated tetrasaccharide structure was proposed for the oligosaccharide moiety in which the distal sugar, unique in nature, corresponds to 2,6-dideoxy-4-O-methyl-alpha-arabinohexopyranose. Its terminal position in the oligosaccharide part confers to this residue a key role in the antigen-antibody interaction. By improvement of the methanolysis procedure, this new kind of sugar was obtained in higher amounts than by hydrolysis of the glycolipid. Its 1H-NMR spectrum is presented and its optical rotatory power measurement agrees with a D absolute configuration while the deoxyhexoses involved in the glycolipid tetrasaccharide structure present the L absolute configuration.     

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.     

Carbohydrate epitope structural elucidation by 1H-NMR spectroscopy of a new Mycobacterium kansasii phenolic glycolipid antigen.

The complete primary structure of the carbohydrate moiety of a new phenolic glycolipid antigen namely PheGl K-IV from Mycobacterium kansasii was successfully established from only one- and two-dimensional 1H-NMR data. Among the scalar two-dimensional techniques, correlated spectroscopy with a 45 degree mixing pulse and phase-sensitive double-quantum-filtered correlated spectroscopy were selected, combined with two-dimensional dipolar techniques (nuclear Overhauser effect). These techniques using milligram of quantities native PheGl K-IV allowed the following monoacetylated tetrasaccharide to be proposed for its carbohydrate part: 4-O-Me-alpha-Manp-(1----3)-4-O-Ac-2-O-Me-alpha-Fucp-(1----3) -2-O-Me-alpha-Rhap- (1----3)-2,4-di-O-Me-alpha-Rhap. The PheGl K-IV shares, with the other phenolic glycolipids isolated from M. kansasii (K-I, K-II), a common core assigned to the lipid aglycone glycosylated by the monoacetylated trisaccharide part. It differs in the structure of the distal monosaccharide residue.     

Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. I. Evidence for tetrasaccharide structure of the oligosaccharide moiety

Data from the literature indicate the presence, in the Mycobacterium kansasii wall, of a phenolic glycolipid called mycoside A. A tentative trisaccharide structure was proposed for the oligosaccharide part, whereas the aglycone was found to correspond to a phenol phthiocerol residue esterified by two mycocerosic acids. In the present work, structural information mainly arising from fast atom bombardment-mass spectrometry, tandem mass spectrometry, and 1H NMR of native and chemically degraded phenolic glycolipid indicates a tetrasaccharide structure for the inherent oligosaccharide. This structure is now determined as: 2,6-dideoxy-4-O-methyl-arabino-hexopyranosyl(1 alpha----3)2-O-methyl-4-O- acetylfucopyranosyl(1 alpha----3)2-O-methyl-rhamnopyranosyl(1 alpha----3)2-4-di- O-methylrhamnopyranosyl-1 alpha----phenolglycol. Evidence for the structure of the dideoxyhexose, unique in mycobacteria, is presented in the following paper (Fournié, J.-J., Rivière, M., Papa, F., and Puzo, G. (1987) J. Biol. Chem. 262, 3180-3184).     

Structural and immunological properties of the phenolic glycolipids from Mycobacterium gastri and Mycobacterium kansasii.

Mycobacterial species-specific antigens belong to the three following classes: phenolic glycolipids (Phe Gl), acyltrehalose-containing lipooligosaccharides and polar glycopeptidolipids. These antigens have been chemically defined and alkali-labile epitopes were found to characterize the lipooligosaccharide antigen type. In the present study the major Mycobacterium kansasii phenolic glycolipid epitope namely Phe Gl K-I was delineated as the distal monoacetylated disaccharidic residue: 2,6-dideoxy-4-O-methyl-alpha-D-arabino-hexopyranosyl-(1----3)-2-O-methyl -4-O- acetyl-alpha-L-fucopyranose. This acetoxy group is required for K-I epitope recognition demonstrating that alkali-labile epitopes also occur in the phenolic glycolipid antigen class. Using immunoelectron microscopy, the Phe Gl K-I epitope was localized around the electron-transparent layer on the M. kansasii cell-wall surface. Furthermore, two new phenolic glycolipids namely Phe Gl K-III and Phe Gl K-IV were discovered in minute amounts. They were purified and characterized by their retention time in direct-phase column HPLC. These molecules are also M. kansasii antigens, whose epitopes differ from that of Phe Gl K-I. The complete family of phenolic glycolipids Phe Gl K-I, K-II, K-III and K-IV was found in both rough and smooth variants of both M. kansasii and Mycobacterium gastri species.     

Synthesis and immunoreactivity of neoglycoproteins containing the trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae

The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.     

Further specific extracellular phenolic glycolipid antigens and a related diacylphthiocerol from Mycobacterium leprae

Mycobacterium leprae in infected armadillo tissue produces extracellular phthiocerol-containing lipids in amounts well in excess of the bacterial mass. The principal component (1.38 mg in 1 g of liver, wet weight, containing 3.7 X 10(10) M. leprae bacilli) consists of a mixture of two phthiocerol homologs, 3-methoxyl-4-methyl-9, 11-dihydroxyoctacosane and 3-methoxyl-4-methyl-9, 11-dihydroxytriacontane, (formula: see text); in which the hydroxyl functions are acylated by a mixture of three 'mycocerosic acids': 2,4,6,8-tetramethylhexacosanoate, 2,4,6,8-tetramethyloctacosanoate, and 2,4,6,8-tetramethyltriacontanoate. The structures were established by saponification of the native lipid, direct probe electron impact- or chemical ionization-mass spectrometry of the phthiocerol or its permethylated derivative, and gas-liquid chromatography-electron impact-mass spectrometry of the methyl esters of the fatty acids. In addition to the previously reported M. leprae-specific triglycosylphenolicdiacyl phthiocerol (Hunter, S. W., Fujiwara, T., and Brennan, P. J. (1982) J. Biol. Chem. 257, 15072-15078), the extracellular products contain small amounts (about 60 micrograms/g of infected liver, wet weight) of two other phenolic glycolipids, one of which (Phenolic Glycolipid III) has been structurally elucidated, (formula: see text); assuming certain enantiomeric configurations for the sugar substituents; the R-acyl functions are identical with those in the diacylphthiocerol. Phenolic Glycolipid-III reacts in enzyme-linked immunosorbent assays with sera from patients with leprosy and with rabbit antisera raised against whole M. leprae. The phthiocerol-containing lipids may be synonymous with the electron transparent capsules of M. leprae, and their unreactive state may confer on them the role of passive protectors of the bacillus.     

N-acylkansosamine. A novel N-acylamino sugar from the trehalose-containing lipooligosaccharide antigens of Mycobacterium kansasii

A novel N-acylamino sugar was isolated from the antigenic trehalose-containing lipooligosaccharides IV-VII of Mycobacterium kansasii. The native reducing sugar, its O-acetyl derivative, the methylglycoside, the O-acetylated alditol, and the de-N-acylated N-, O-acetylated alditol were all examined by high resolution 1H NMR, 13C NMR, direct probe and gas-liquid chromatography-mass spectrometry in both the chemical ionization and electron impact modes, and by high resolution mass spectrometry. The dideoxy sugar had a formula weight of 277, an empirical formula of C12H23NO6, C- and O-methyl substituents, and a N-methoxypropionyl branch. Upon alkaline hydrolysis, methoxypropionic acid was released and shown to correspond to the synthetic compound by gas chromatography and chemical ionization and electron impact mass spectrometry. The structure 4,6-dideoxy-2-O-methyl-3-C-methyl-4-(2'-methoxypropionamido)-alpha and beta-L-manno-hexopyranose, with the trivial name N-acylkansosamine, is proposed. The sugar is present in the more polar, highly antigenic lipooligosaccharides and is regarded as exclusive to M. kansasii and as its primary cell wall immunodeterminant.     

Chemical synthesis of the trisaccharide unit of the species-specific phenolic glycolipid from Mycobacterium leprae

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, the haptenic trisaccharide of the Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) antigen, and related trisaccharides, were synthesized by allylation of O-2 of benzyl 4-O-benzyl-alpha-L-rhamnopyranoside using phase-transfer catalysis, methylation of the product, deallylation, and coupling to O-(2,4-di-O-acetyl-3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3- di-O-methyl-L-rhamnopyranosyl bromide or related disaccharides. Anomeric mixtures of the trisaccharide derivatives were separated by preparative t.l.c., deacetylated, and hydrogenolyzed, to give the pure trisaccharides. It had already been demonstrated that only those trisaccharides containing an intact, terminal 3,6-di-O-methyl-beta-D-glucopyranosyl unit are effective in inhibiting the specific binding between PGL-I and anti-PGL-I immunoglobulin M antibodies in human lepromatous leprosy sera.     

Recognition of novel glycolipid antigens from smooth variants of Mycobacterium tuberculosis

A major polar and three minor slightly less polar glycolipids were identified in extracts of two smooth (Canetti) strains of Mycobacterium tuberculosis. Immunostaining on thin-layer chromatograms and enzyme-linked immunosorbent assay (ELISA) of purified lipids demonstrated that the major and the two most polar of the minor glycolipids are potent antigens, reacting with homologous antisera and also with that raised against the type strain (H37Rv).     

A new glycolipid from Mycobacterium avium--Mycobacterium intracellulare complex.

From a nonpolar lipid fraction of Mycobacterium avium--Mycobacterium intracellulare complex cell mass, a new glycolipid was obtained, which was shown to be 5-mycoloyl-beta-arabinofuranosyl-(1-->2)-5-mycoloyl-alpha-ar abinofuranosyl- (1-->1')-glycerol. When examined by TLC, all the 12 strains of this species tested, including clinical isolates, were found to contain this glycolipid. But the glycolipid was not detected in Mycobacterium bovis BCG or Mycobacterium tubrculosis H37Rv.     

Suppression of monocyte oxidative response by phenolic glycolipid I of Mycobacterium leprae

Mycobacterium leprae synthesizes a unique phenolic glycolipid (PGL-I) in abundant quantities. We studied the effect of PGL-I on the generation of superoxide anion (O2-) by stimulated human monocytes. Peripheral blood monocytes pretreated with PGL-I released less O2- when stimulated with M. leprae than did control monocytes. Monocytes pretreated with dimycocerosyl phthiocerol, mycoside A of Mycobacterium kansasii, or mycoside B of Mycobacterium microti, on the other hand, released O2- in quantities comparable to control monocytes in response to M. leprae stimulation. Monocyte O2- release in response to other stimuli of the oxidative metabolic burst, such as PMA, zymosan, Mycobacterium bovis Bacille Calmette-Guérin, or M. kansasii, was unaffected by lipid pretreatment. These findings demonstrate that PGL-I has a direct effect on monocyte O2- generation in response to M. leprae and suggest that PGL-I is a modulator of phagocytic cell function.     

New-found phenolic glycolipids in Mycobacterium bovis BCG. Presence of a diglycosylated glycolipid

A crude phenolic glycolipid extract from Mycobacterium bovis bacille Calmette-Guerin (BCG) was fractionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 according to their polarity. The apolar one, which is the most abundant was assigned to the well-known mycoside B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chromatography using a 5 micron Spherisorb column but were only recovered in small amounts (3 mg). A linear gradient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their potential to elucidate the structure at the intact level. Desorption chemical ionization-mass spectrometry allowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molecular formula as C103H192O14. Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two molecules of mycocerosic acid. Using two-dimensional 1H NMR (correlated chemical shift and nuclear Overhauser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap. This is the first report of a diglycosylated phenolic glycolipid in a nonpathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hyperimmunized with M. bovis BCG.     

Chemical synthesis of disaccharides of the specific phenolic glycolipid antigens from Mycobacterium leprae and of related sugars

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3,-di-O-methyl-L -rhamnopyranose, which is the nonreducing disaccharide of the haptenic trisaccharide of the Mycobacterium leprae-specific, phenolic glycolipid I, O-(6-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamn opyranose, the nonreducing end of the specific, phenolic glycolipid III, and the nonhaptenic O-beta-(D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamnopyranose++ +, were synthesized in relatively good yield from 3-O-methyl-D-glucose, or D-glucose, and L-rhamnose via Koenigs-Knorr reactions. These disaccharides can be used as precursors in the synthesis of the trisaccharide unit of phenolic glycolipid I and of neoglycoconjugates suitable for the serodiagnosis of leprosy.     

Fatty Acids of Mycobacterium kansasii

The cellular fatty acids of 35 strains of Mycobacterium kansasii isolated from clinical material were analyzed to establish properties by which we could identify and characterize these acid-fast microorganisms. The fatty acids were extracted from cells grown in liquid synthetic media, and they were analyzed as methyl esters by gas-liquid chromatography. The fatty acid profiles of all strains were similar. They differed from fatty acid profiles of other mycobacteria by their content of a saturated fatty acid with a methyl group at C2.     

Clinical Significance of Mycobacterium kansasii Isolates from Respiratory Specimens

The clinical significance of Mycobacterium kansasii respiratory isolates is uncertain. The aims of this study were to determine the clinical relevance of M. kansasii isolates and to identify the clinical features and outcomes of M. kansasii lung disease. We reviewed the medical records of 104 patients from whom at least one respiratory M. kansasii isolate was obtained from January 2003 to July 2014 at Samsung Medical Center, South Korea. Of these 104 patients, 54 (52%) met the diagnostic criteria for nontuberculous mycobacterial lung disease; among them, 41 (76%) patients received antibiotic treatment for a median time of 15.0 months (interquartile range [IQR], 7.0–18.0 months). The remaining 13 (24%) without overt disease progression were observed for a median period of 24.0 months (IQR, 5.0–34.5 months). Patients with M. kansasii lung disease exhibited various radiographic findings of lung disease, including the fibrocavitary form (n = 24, 44%), the nodular bronchiectatic form (n = 17, 32%), and an unclassifiable form (n = 13, 24%). The fibrocavitary form was more common in patients who received treatment (n = 23, 56%), while the nodular bronchiectatic form was more common in patients with M. kansasii lung disease who did not receive treatment (n = 9, 70%). None of the patients with a single sputum isolate (n = 18) developed M. kansasii disease over a median follow-up period of 12.0 months (IQR, 4.0–26.5 months). In total, 52% of all patients with M. kansasii respiratory isolates exhibited clinically significant disease. Moreover, patients with M. kansasii lung disease displayed diverse radiographic findings in addition to the fibrocavitary form. The nodular bronchiectatic form was more common in patients with M. kansasii lung disease with an indolent clinical course. Thus, since the clinical significance of a single M. kansasii respiratory isolate is not definite, strict adherence to recommended diagnostic criteria is advised.