Polyphthienoyl trehalose, glycolipids specific for virulent strains of the tubercle bacillus

Phthienoic acids constitute a family of dextro-rotary odd-numbered unsaturated fatty acids isolated exclusively from virulent strains of human and bovine tubercle bacilli. In the bacterial cell they are not free and a search for their linked form in complex wall lipids of Mycobacterium tuberculosis (strain Canetti) showed that they esterified trehalose. Structural elucidation of the major phthienoyl trehalose showed the occurrence of five acyl residues located at 2, 2', 3', 4 and 6' positions of trehalose. The acyl substituents were mainly 2,4,6-trimethyl tetracos-2-enoic acid (C27 phthienoic acid) accompanied by its homologs. In addition to these branched fatty acids, straight-chain C16 and C18 acyls composed about 20% of the substituents. The proposed structure is a new one, both for the mycobacterial-specific glycolipid and for the substituted positions on trehalose. Other minor acyl trehaloses were detected in M. tuberculosis (strain Canetti), differing from the major component by the occurrence of an additional hydroxy fatty acid (3-hydroxy-2,4,6-trimethyl tetracosanoic acid) or by the number of acyl substituents. The major glycolipid presented a weak activity in vitro on mitochondrial oxidative phosphorylation. These glycolipids and phthienoic acids could serve as virulence indicators.     

Antigenic glycolipids of Mycobacterium fortuitum based on trehalose acylated with 2-methyloctadec-2-enoic acid

Three characteristic glycolipids were identified in extracts of 10 representative strains of Mycobacterium fortuitum. The two most polar major lipids were shown to be antigens with strong reactions against homologous antiserum and sera raised against Mycobacterium tuberculosis H37Rv. Both of these lipids were shown to be acyl trehaloses, containing substantial amounts of 2-methyloctadec-2-enoic acid in addition to straight-chain and monounsaturated acids. These lipids correspond to 'mycoside F', lipids previously identified by infra-red spectroscopy in extracts of Myco. fortuitum.     

Biochemical function of msl5 (pks8 plus pks17) in Mycobacterium tuberculosis H37Rv: biosynthesis of monomethyl branched unsaturated fatty acids

We show that the disruption of one of the mycocerosic acid synthase (mas)-like genes, msl5 (pks8 plus pks17) in Mycobacterium tuberculosis H37Rv generates a mutant incapable of producing monomethyl branched unsaturated C(16) to C(20) fatty acids that are minor constituents of acyltrehaloses and sulfolipids. The msl5 mutation did not cause any significant change in the acyl lipid composition and also did not affect growth in culture, in mouse alveolar macrophage cell line MH-S, or in the murine lung.     

Significance and taxonomic value of iso and anteiso monoenoic fatty acids and branded beta-hydroxy acids in Desulfovibrio desulfuricans.

The fatty acids obtained from extractable lipids of the anaerobic sulfate bacterium Desulfovibrio desulfuricans were identified. Saturated and monoenoic iso (C15-C19) and anteiso (C15, C17) fatty acids and saturated normal (C14-C18) and monoenoic normal (C16, C18) fatty acids were shown to be shown to be present by capillary gas chromatography-mass spectrometry. Iso and anteiso beta-hydroxy fatty acids were analyzed as trimethylsilyl ethers in the same way. The position of methyl branches in the monoenoic fatty acids was determined from characteristic fragment ions in the mass spectra of their methyl esters. Disilyloxy methyl esters, prepared by derivatization of the mono unsaturated methyl esters and analyzed by capillary gas chromatography-mass spectrometry, provided the position of double bonds. The monoenoic fatty acids identified in this way were normal (delta7-C16:1, delta9-C16:1, delta9-C18:1, delta11-C18:1), iso (delta7-C15:1, delta9-C16:1, delta9-C17:1, delta11-C18:1, delta11-C19:1), and anteiso (delta7-C15:1, delta9-C17:1). Iso delta9-C17:1 fatty acid is present as the major component. The occurrence of these monoenoic fatty acids in this bacterium is of taxonomical importance.     

Mycolipenates and mycolipanolates of trehalose from mycobacterium tuberculosis

Analysis of the lipids of Mycobacterium tuberculosis, by thin-layer chromatography, revealed the presence of two families of novel glycolipids each having two closely-related members but differing widely in polarity. The least and most polar families of lipids were characterized from M. tuberculosis strains C and H37Rv, respectively; all were based on trehalose, the least polar pair of glycolipids having more long-chain substituents than the more polar pair. The acyl substituents of the least polar of the four glycolipids were mainly straight-chain C16 and C18 acids and 2,4,6-trimethyltetracos-2-enoic (C27-mycolipenic) acid, and the second least polar glycolipid contained major amounts of 3-hydroxy-2,4,6-trimethyltetracosanoic (C27-mycolipanolic) acid in addition to these non-hydroxylated acids. The relatively polar pair of glycolipids were analysed together and released mainly straight-chain C16 and C18 acids, C27-mycolipanolic acid, minor amounts of C25- and C27-mycolipenic acids and major proportions of an acid having the chromatographic properties of 2,4-dimethyldocosanoic acid. The most polar pair of glycolipids co-chromatographed with glycolipid antigens previously detected in Mycobacterium bovis BCG.     

Liquid chromatography/mass spectrometry of fatty acids as their anilides

The mass spectra of a series of saturated (C16:0-C30:0) and unsaturated (C16:1, C18:1, C18:2, C18:3) fatty acids have been recorded as their anilides using liquid chromatography/mass spectrometry with the atmospheric-pressure-ionization interface system. The spectra show an intense peak for (molecule + H)+ ion in each case. The liquid chromatographic/mass spectrometric separation was performed on a reverse phase column using a solvent system of methanol alone or methanol + 2-propanol. This method seemed promising for application to both qualitative and quantitative micro-analysis of fatty acids including very long chain fatty acids.     

Sporulation in Bacillus subtilis is independent of membrane fatty acid composition.

Growth and sporulation of a Bacillus subtilis mutant deficient in branched fatty acid synthesis (gene symbol bfmB) were examined. The mutant, which produces an acyl-coenzyme A:acyl carrier protein transacylase with reduced affinity for branched fatty acid primers, could grow in media containing any one of a wide range of low-molecular-weight fatty acids having branched, cyclic, saturated, or unsaturated carbon chains. The fatty acid composition of cellular lipids depended on the compound used to support growth. Cultures of the bfmB mutant grown in the presence of 3-methylcrotonate contained an unusually high fraction (73%) of straight-chain fatty acids in the cellular lipids. The mutant sporulated with any one of the precursors of branched fatty acids in the medium; isolated spores contained mainly this branched fatty acid and only 10% or less straight-chain fatty acids regardless of the straight-chain fatty acid content of vegetative cells. Exceptional were spores grown in the presence of cyclobutane-carboxylic acid, which contained 28% straight-chain fatty acids. The branched fatty acid composition of spores could be modified greatly by changing the supply of precursors in the medium.     

Characterization of the fatty acid synthetase system of Curtobacterium pusillum

Curtobacterium pusillum contains 11-cyclohexylundecanoic acid as a major component of cellular fatty acids. A trace amount of 13-cyclohexyltridecanoic acid is also present. Fatty acids other than omega-cyclohexyl fatty acids present are 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, 13-methylpentadecanoic, n-hexadecanoic, 15-methylhexadecanoic, 14-methylhexadecanoic, and n-heptadecanoic acids. The fatty acid synthetase system of this bacterium was studied. Various 14C-labeled precursors were added to the growth medium and the incorporation of radioactivity into cellular fatty acids was analyzed. Sodium [14C]acetate and [14C]glucose were incorporated into almost all species of cellular fatty acids, the incorporation into 11-cyclohexylundecanoic acid being predominant. [14C]Isoleucine was incorporated into 12-methyltetradecanoic and 14-methylhexadecanoic acids: [14C]leucine into 13-methyltetradecanoic and 15-methylhexadecanoic acids; and [14C]valine into 14-methylpentadecanoic acid. [14C]-Shikimic acid was incorporated almost exclusively into omega-cyclohexyl fatty acids. The fatty acid synthetase activity of the crude enzyme preparation of C. pusillum was reconstituted on the addition of acyl carrier protein. This synthetase system required NADPH and preferentially utilized cyclohexanecarbonyl-CoA as a primer. The system was also able to use branched- and straight-chain acyl-CoAs with 4 to 6 carbon atoms effectively as primers but was unable to use acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the system produced straight-chain fatty acids. The results imply that the specificity of the initial acyl-CoA:acyl carrier protein acyltransferase dictates the structure of fatty acids synthesized and that the enzymes catalyzing the subsequent chain-elongation reactions do not have the same specificity restriction.     

Synthesis and physical properties of phosphatidylcholines containing ω-cyclohexyl fatty acyl chains

Phosphatidylcholines (PCs) with cyclohexyl fatty acids as acyl chains were synthesized from 11-cyclohexyl and 13-cyclohexyl fatty acids and their physical properties were examined. The thermotropic behavior and barrier function of liposomal membranes formed from these PCs were studied. These PCs showed about 10°C lower gel-to-liquid crystalline phase transition temperatures (T_c) than the corresponding straight-chain PCs. The properties of mixtures of these cyclohexyl acyl PCs with straight-chain PCs were rather different from those observed with mixtures of straight-chain PCs. Cyclohexyl fatty acyl PCs showed barrier functions even above the T_c unlike the corresponding straight-chain PCs. These results indicate significant differences between the overall packing of cyclohexyl fatty acyl PCs and of the corresponding straight-chain PCs both in the gel state and in the liquid crystalline state. The significance of these cyclohexyl acyl chains in polar lipids, which are abundant in the thermophilic acidophilic bacterium, Bacillus acidocaldarius, is discussed.     

Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregation

Cell wall lipids of Mycobacterium tuberculosis containing multiple methylbranched fatty acids play critical roles in pathogenesis and thus offer targets for new antimycobacterial drugs. Mycocerosicacid synthase gene (mas) encodes the enzyme that produces one class of such acids. Seven mas-like genes (msls) were identified in the genome. One of them, msl3, originally annotated as two separate genes, pks 3 and pks 4, is now shown to constitute a single open reading frame, which encodes a 220.3 kDa protein. Msl3 was disrupted using a phage mediated delivery system and the gene replacement in the mutant was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Biochemical analysis showed that the msl3 mutant does not produce mycolipanoic acids and mycolipenic(phthienoic) acids, the major constituents of polyacyl trehaloses and thus lacks this cell wall lipid, but synthesizes all of the other classes of lipids. The absence of the major acyl chains that anchor the surface-exposed acyltrehaloses causes a novel growth morphology; the cells stick to each other, most probably via the intercellular interaction between the exposed hydrophobic cell surfaces, manifesting a bead-like growth morphology without affecting the overall growth rate.     

Composition of human serum sphingomyelins

Serum sphingomyelins were analyzed by argentation chromatography of the corresponding ceramide diacetates. Six subfractions were obtained. Three of them contained 4-sphingenines in combination with saturated, trans-, or cis-monoenoic fatty acids; the remaining three contained sphingadienine, also in combination with saturated, trans-, or cis-monoenoic fatty acids. Palmitic acid was the principal fatty acid combined with 4-sphingenines, while nervonic acid was the principal fatty acid combined with sphingadienine. About 4% of the total fatty acids of sphingomyelin were trans-monoenoic. They were comprised of many positional isomers of straight-chain C(22-24) compounds. The cis-monoenoic acids made up 33% of the total acids and consisted of almost pure nervonic acid. The rest of the acids were saturated. The 4-sphingenines contained small amounts of iso-C(18) and anteiso-C(19) compounds in addition to the straight-chain C(16-18) bases.     

Liver fatty acid binding protein expression enhances branched-chain fatty acid metabolism

Although liver fatty acid binding protein (L-FABP) is known to enhance uptake and esterification of straight-chain fatty acids such as palmitic acid and oleic acid, its effects on oxidation and further metabolism of branched-chain fatty acids such as phytanic acid are not completely understood. The present data demonstrate for the first time that expression of L-FABP enhanced initial rate and average maximal oxidation of [2,3-3H] phytanic acid 3.5- and 1.5-fold, respectively. This enhancement was not due to increased [2,3-3H] phytanic acid uptake, which was only slightly stimulated (20%) in L-FABP expressing cells after 30 min. Similarly, L-FABP also enhanced the average maximal oxidation of [9,10-3H] palmitic acid 2.2-fold after incubation for 30 min. However, the stimulation of L-FABP on palmitic acid oxidation nearly paralleled its 3.3-fold enhancement of uptake. To determine effects of metabolism on fatty acid uptake, a non-metabolizable fluorescent saturated fatty acid, BODIPY-C16, was examined by laser scanning confocal microscopy (LSCM). L-FABP expression enhanced uptake of BODIPY-C16 1.7-fold demonstrating that L-FABP enhanced saturated fatty acid uptake independent of metabolism. Finally, L-FABP expression did not significantly alter [2,3-3H] phytanic acid esterification, but increased [9,10-3H] palmitic acid esterification 4.5-fold, primarily into phospholipids (3.7-fold) and neutral lipids (9-fold). In summary, L-FABP expression enhanced branched-chain phytanic acid oxidation much more than either its uptake or esterification. These data demonstrate a potential role for L-FABP in the peroxisomal oxidation of branched-chain fatty acids in intact cells.     

The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis

Multidrug-resistant tuberculosis is a major global health emergency. Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis. The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen. A variety of multiple methyl-branched fatty acids are among such unique lipids. We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids. One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Thin-layer and radio gas-chromatographic analyses of lipids derived from [1-14C]propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta- and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by two-dimensional thin-layer chromatographic analysis of lipids derived from [1-14C]propionic acid and 35SO4(-2). With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M. tuberculosis.     

A single column packing for the gas-liquid chromatographic separation of various biologically important compounds.

The use of a single, commercially available column packing, TabsorbR, is described for the g.l.c. separation of a large number of different compounds. The resolution of the homologous members of the following series of compounds was achieved: (1) saturated fatty acids (C1-C18), (2) normal aliphatic saturated dicarboxylic acids (C2-C14), (3) normal aliphatic saturated alcohols (C1-C24), (4) normal aliphatic saturated amines (C1-C12), (5) the common amino acids except arginine, histidine and cysteine, (6) aliphatic hydrocarbons (C10-C20) and (7) monosaccharides. It should be noted that twenty-two monosaccharides including three hexosamines and two anhydrohexoses, could be resolved as alditol acetates in a single run. In addition, galacturonic, glucuronic and iduronic acids could be separated from one another as their 1,4-lactones. The resolution achieved in these series of compounds was found to be consistent and highly reproducible. It is of further interest that certain isomers of the higher fatty acids and hydrocarbons with one double bond could also be separated from the normal and saturated compounds, respectively. The applicability of "Tabsorb" for the g.l.c. separation, although noted above to be considerably broad, is by far not yet exhausted. These procedures which form the basis for the quantitative determinations of the various compounds studied as demonstrated by analysis of glycopeptides for neutral hexoses and proteins for the amino acids, can readily be adapted to preparative methods. From the biochemical point of view "Tabsorb" is an extremely versatile column packing in that it can be used for the identification of many of the common building blocks of natural products.     

Skin surface lipids of the mole Scalopus aquaticus

Skin surface lipids of the mole Scalopus aquaticus were found to consist principally of squalene (70%), wax esters (15%), and sterol esters (5%), together with small amounts of triglycerides, free fatty acids, free fatty alcohols, and free sterols. Analysis of the fatty acids occurring free and as wax esters and sterol esters showed these to consist of approximately equal amounts of saturated and monounsaturated compounds. The saturated fatty acids consisted predominantly of odd-carbon anteiso and even-carbon straight-chain compounds, with minor amounts of even-carbon iso-branched chains. The unsaturated fatty acids had double bond positions that would have been produced by delta 9-desaturation of C14, C16 and C18 straight chain saturated precursors. Both the free and the esterified fatty alcohols had chain structures corresponding with those of the fatty acids but of somewhat greater average chain length. Discovery of a major proportion of squalene in the sebum of this animal extends the number of non-human species that have this characteristic to four, all of which inhabit a damp environment, suggesting that squalene conveys some biological advantage under these conditions.     

Distribution of molecular species of sphingomyelins in different parts of bovine digestive tract.

Sphingomyelins were isolated from mucosal layers of bovine rennet stomach, duodenum, jejunoileum, and colon ascendens. The ceramides obtained after phospholipase degradation were characterized by thin-layer chromatography, mass spectrometry, and gas-liquid chromatography. The main ceramide group from all regions consisted of dihydroxy long-chain bases and normal fatty acids. Sphingosine was the predominant base in all these fractions, and only in rennet stomach were smaller amounts of the C17 and C20 homologs present. Normal saturated C16, C18, C22, and C24 fatty acids were most abundant. In rennet stomach there was in addition a ceramide group having dihydroxy long-chain bases in combination with hydroxy fatty acids. Sphingosine was the predominant long-chain base and the fatty acids were 2-hydroxy C16, C22, C23, and C24. From jejunoileum three minor ceramide fractions were isolated; these consisted of phytosphingosine and normal fatty acids C22-C24), sphingosine and 2-hydroxy fatty acids (C16-C24), and phytosphingosine and 2-hydroxy fatty acids (C22-C24), respectively. No branched paraffin chains were found in significant amounts. Sphingomyelins with trihydroxy long-chain bases and 2-hydroxy fatty acids found in jejunoileum were also detected in bovine kidney and have not been demonstrated before. These sphingomyelins from both kidney and jejunoileum showed a preferential combination of trihydroxy bases and fatty acids with very long chains (C22-C24).     

Diglycosyl diacylglycerol of Mycobacterium tuberculosis.

A diglycosyl diacylglycerol was isolated from Mycobacterium tuberculosis, and its structure was established by a combination of methylation analysis, 1H nuclear magnetic resonance, and fast atom bombardment-mass spectrometry. It is a 1,2-diacyl-[beta-D-glucopyranosyl(1"----6')-beta-D-glucopyranosyl(1'---- 3)]- sn-glycerol and exists in at least five molecular species differing in fatty acyl substituents. The major constituent fatty acids were identified as iso- and anteisopentadecanoate, iso- and n-hexadecanoate, and iso- and anteisoheptadecanoate. Although glycosyl diacylglycerols are common membrane components of gram-positive bacteria, this report represents the first substantial evidence for the presence of a glycosyl diacylglycerol within a member of the Mycobacterium genus. Although the glycolipid is not a major component of M. tuberculosis, it reacts readily in enzyme-linked immunosorbent assay against rabbit antibodies raised against whole bacteria and thus may be useful for the serodiagnosis of tuberculosis.     

The uropygiols: identification of the unsaponifiable constituent of a diester wax from chicken preen glands

The chief lipid fraction in the uropygial gland excretion of the domestic hen is a diester wax. The saponifiable fraction of this wax consists of saturated normal C(10)-C(20) fatty acids. The unsaponifiable fraction consists of a series of three homologous compounds, which have been named the uropygiols and identified by mass spectrometry, gas-liquid chromatography, and periodate cleavage as 2,3-n-alkanediols containing 22-24 carbon atoms. The native diols were shown to consist of about equal amounts of the threo and erythro isomers. Records of analyses of the natural products as well as related synthetic compounds are shown.     

Identification of two mammalian reductases involved in the two-carbon fatty acyl elongation cascade

The de novo synthesis of fatty acids occurs in two distinct cellular compartments. Palmitate (16:0) is synthesized from acetyl-CoA and malonyl-CoA in the cytoplasm by the enzymes acetyl-CoA carboxylase 1 and fatty acid synthase. The synthesis of fatty acids longer than 16 carbons takes place in microsomes and utilizes malonyl-CoA as the carbon source. Each two-carbon addition requires four sequential reactions: condensation, reduction, dehydration, and a final reduction to form the elongated fatty acyl-CoA. The initial condensation reaction is the regulated and rate-controlling step in microsomal fatty acyl elongation. We previously reported the cDNA cloning and characterization of a murine long chain fatty acyl elongase (LCE) . Overexpression of LCE in cells resulted in the enhanced addition of two-carbon units to C12-C16 fatty acids, and evidence was provided that LCE catalyzed the initial condensation reaction of long chain fatty acid elongation. The remaining three enzymes in the elongation reaction have not been identified in mammals. Here, we report the identification and characterization of two mammalian enzymes that catalyze the 3-ketoacyl-CoA and trans-2,3-enoyl-CoA reduction reactions in long and very long chain fatty acid elongation, respectively.     

Molecular motions and dynamics of a diunsaturated acyl chain in a lipid bilayer: implications for the role of polyunsaturation in biological membranes.

The nature and dynamics of the motions of a diunsaturated fatty acyl chain in a lipid bilayer were examined using a comprehensive simulation program for 2H NMR line shapes developed by Wittebort et al. [Wittebort, R. J., Olejniczak, E. T., & Griffin, R. G. (1987) J. Chem. Phys. 36, 5411-5420]. A motional model in which the isolinoleoyl chain (18:2 delta 6,9) adopts two conformations consistent with the low energy structures proposed for 1,4-pentadiene [Applegate, K. R., & Glomset, J. A. (1986) J. Lipid Res. 27, 658-680], but undergoes a rapid jump between these states, is sufficient to account for the experimentally observed quadrupolar couplings, the 2H-2H and 1H-2H dipolar couplings, the longitudinal relaxation times, and the changes in the average conformation of the chain that occur with a variation in temperature. The jump motion originates via rotations about the C7-C8 and the C8-C9 carbon bonds and leads to the low order parameters assigned to the C8 methylene segment (0.18) and the C9-C10 double bond (0.11). In contrast, the C6-C7 double bond, which is not involved in the two-site jump, characterized by a relatively large order parameter (0.56). Fatty acyl chains containing three or more double bonds likely cannot undergo the same jump motion and consequently will be highly ordered structures. Correlation times for diffusion of the molecular long axis of the diunsaturated acyl chain about the bilayer normal (approximately 10(-10) s) and for the local jump motion (approximately 10(-10) s) were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)