Α-mycolic Acid Research Articles

Conformational Dynamics and Stability of Bilayers Formed by Mycolic Acids from the Mycobacterium tuberculosis Outer Membrane.

Bilayers of mycolic acids (MAs) form the outer membrane of Mycobacterium tuberculosis that has high strength and extremely low permeability for external molecules (including antibiotics). For the first time, we were able to study them using the all-atom long-term molecular dynamic simulations (from 300 ns up to 1.2 μs) in order to investigate the conformational changes and most favorable structures of the mycobacterial membranes. The structure and properties of the membranes are crucially dependent on the initial packing of the α-mycolic acid (AMA) molecules, as well as on the presence of the secondary membrane components, keto- and methoxy mycolic acids (KMAs and MMAs). In the case of AMA-based membranes, the most labile conformation is W while other types of conformations (sU as well as sZ, eU, and eZ) are much more stable. In the multicomponent membranes, the presence of the KMA and MMA components (in the W conformation) additionally stabilizes both the W and eU conformations of AMA. The membrane in which AMA prevails in the eU conformation is much thicker and, at the same time, much denser. Such a packing of the MA molecules promotes the formation of a significantly stronger outer mycobacterial membrane that should be much more resistant to the threatening external factors.

A reversed phase ultra-high-performance liquid chromatography-data independent mass spectrometry method for the rapid identification of mycobacterial lipids

A rapid reversed-phase ultra-high-performance liquid chromatography-high resolution mass spectrometry based mycobacterial lipidomics approach is described. This method enables the separation of various lipid classes including lipids specific to mycobacterial, such as methoxy mycolic acid and α-mycolic acid. Lipid separation occurs during a relatively short runtime of 14 min on a charged surface hybrid C18 column. A high-resolution quadrupole-time of flight mass spectrometer and a data independent acquisition mode allowed for the simultaneous acquisition of the full scan and collision induced dissociation fragmentation. The proposed method provides lipid detection results equivalent to or better than existing methods, but with a faster throughput and an overall higher sensitivity. The reversed-phase ultra-high-performance liquid chromatography-high resolution mass spectrometry method was shown to obtain structural information for lipids extracted from Mycobacterium smegmatis, but the method is applicable to the analysis of lipids from various bacterial and mammalian cell lines.

Expression of mycolic acid in response to stress and association with differential clinical manifestations of tuberculosis.

Extrapulmonary tuberculosis (EPTB), accounting for 10%-20% of all cases of tuberculosis (TB), is known to be determined by host immunity. However, the contribution of bacterial factors to the development of EPTB has not been studied extensively. Mycolic acids are predominant lipids constituting the cell wall of Mycobacterium tuberculosis, and keto-mycolic acid is involved in the synthesis of foamy macrophages that facilitate persistence of mycobacteria. Hence, the present study was performed to gain an insight into variable expression of mycolic acids in clinical isolates of M. tuberculosis under stress. Pansusceptible clinical isolates of M. tuberculosis from patients with lymph node TB (LNTB) (n = 10) and pulmonary TB (PTB) (n = 10) were subjected to sodium dodecyl sulfate (SDS) stress, and the expression of mycolic acid and its biosynthetic genes was compared. Any bias arising due to the genotype of the clinical isolates was ruled out by performing single-nucleotide polymorphism cluster grouping (SCG), wherein no significant difference was observed between the SCG of LNTB or PTB isolates. The expression of α-mycolic acid during the exposure to SDS was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates. Methoxy mycolic acid showed an increased expression in 7/10 (70%) LNTB isolates and 4/10 (40%) PTB isolates. Increased expression of keto-mycolic acid on exposure with SDS was observed in 8/10 (80%) M. tuberculosis LNTB and 3/10 (30%) PTB isolates. Similarly, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro and ex vivo. SCG 3a was the most common SCG observed in 40% (8/20) of the isolates, followed by SCG 3b in 30% (6/20) of the isolates. There was no significant difference between the SCG of LNTB or PTB isolates. The higher expression of keto-mycolic acid in LNTB as against PTB isolates may indicate better survival in LNTB isolates in the presence of stress.

Synthesis of wax esters and related trehalose esters from Mycobacterium avium and other mycobacteria

The synthesis of mycobacterial mycolic acid related wax esters and of their trehalose di- and mono-esters is described. The trehalose dimycolates (TDMs) synthesised activated bone marrow derived dendritic cells (BMDCs) in vitro more strongly than trehalose dibehenate or the trehalose monomycolates (TMMs). The inflammatory effects were similar to those of TDM from either a synthetic keto- or methoxy-mycolic acid, but somewhat stronger than those of a TDM from an α-mycolic acid. In vivo, the effects of one wax ester TDM were similar to those of the methoxy-MA and α-mycolic acid TDMs and trehalose dibehenate.

The synthesis of a single enantiomer of a major α-mycolic acid of M. tuberculosis

We report a synthesis of a single enantiomer of a mycolic acid from Mycobacterium tuberculosis containing a di- cis-cyclopropane. The method can be simply varied to modify the chain lengths or the absolute stereochemistry of either cyclopropane.

The mmaA2 Gene of Mycobacterium tuberculosis Encodes the Distal Cyclopropane Synthase of the α-Mycolic Acid

Infection with Mycobacterium tuberculosis (Mtb) remains a severe global health problem that has prompted an aggressive search for new antibiotic targets and vaccine strategies for this persistent pathogen. Recently, a wide variety of genetic determinants of Mtb pathogenicity have been identified, including several genes involved in the biogenesis of the complex Mtb cell envelope. Among these are the mycolic acid cyclopropane synthases, a family of proteins that modify the major cell envelope lipids of Mtb with a diversity of cyclopropane rings. Despite substantial sequence identity, these proteins catalyze highly specific cyclopropane modifications, including proximal modification of the alpha-mycolate (pcaA) and trans-cyclopropane modification (cmaA2). Here we report the mycolic acid modification function of a third cyclopropane synthase, mmaA2, through the creation and analysis of an M. tuberculosis mmaA2 null mutant. Unexpectedly, mmaA2 is essential for the distal cyclopropane modification of the alpha-mycolate, a function previously attributed to cmaA1. alpha-Mycolates of a cmaA1 null mutant were unaffected, demonstrating that cmaA1 is not required for alpha-mycolate modification. Although fully cyclopropanated methoxymycolates are produced in the mmaA2 mutant, cis-cyclopropanation is impaired, leading to accumulation of unsaturated methoxymycolate derivatives. This study establishes mmaA2 as a distal cyclopropane synthase of the alpha-mycolates of M. tuberculosis and the first cyclopropane synthase to modify both alpha- and oxygenated mycolates. These results expand our knowledge of the biosynthesis of the Mtb cell envelope and will allow further elucidation of the relationship between Mtb pathogenesis and the fine structure of mycolic acids.

Conformational Characterization of α-Mycolic Acid in a Monolayer Film by the Langmuir−Blodgett Technique and Atomic Force Microscopy

Conformational changes of α-mycolic acid (α-MAMAI, isolated from Mycobacterium avium−M. intracellulare complex) in its monolayer at different surface pressures were investigated by using the Langmuir−Blodgett (LB) technique and atomic force microscopy (AFM). Three representative monolayers that correspond to the gas, liquid-expanded, and solid states were prepared and transferred onto freshly cleaved mica. The monolayer LB films of α-MAMAI were examined by AFM in an angstrom resolution in the thickness direction. The observation of topography and cross section of the monolayers proposed a series of schematic pictures for the lateral arrangement of the alkyl chains of α-MAMAI in the monolayer at different surface pressures. The proposed molecular conformations were consistent with the surface pressure−area isotherm. The results suggested that the α-MAMAI molecules partially took a flat-on stance in the gas-state monolayer and that the full extension of the longer alkyl chain of the molecule in the monolayer was possible only when the monolayer was highly compressed to the solid state.

Methanol-utilizing Mycobacterium strains isolated from soil.

Gram-positive facultative methylotrophs were isolated by enrichment cultures with methanol as the sole source of carbon and energy. These bacteria were acid-fast, non-sporeforming, nonmotile rods, and grew on methanol and methylamine, but not on methane. The deoxyribonucleic acid base composition was 66 to 69mol% guanine plus cytosine. The cellular fatty acids consisted of a large amount of straight-chain saturated C16:0 acid, straight-chain unsaturated C16:1 and C18:1 acids, and 10-methyl C19:0 acid. The major quinone was menaquinone MK-9(H2). The principal amino acids in the cell wall was meso-diaminopimelic acid. There was a large amount of α-mycolic acid, a small amount of α′- ycolic acid and dihydroxy mycolic acid. The facultative methylotrophs appeared to be included in the genus Mycobacterium.

Analysis of fatty acids by negative ion gas chromatography/tandem mass spectrometry: structural correlations between alpha-mycolic acid chains and delta-5-monounsaturated fatty acids from Mycobacterium phlei.

The analysis of a complex mixture of monounsaturated fatty acids from mycobacterium phlei is achieved by capillary gas chromatographic separation of their pentafluorobenzyl esters, formation of gas-phase carboxylate anions by electron capture ionization and decomposition of these anions by collision activation. This method allows the structural determination of fatty acid isomers by examination of their collisionally activated dissociation mass-analysed ion kinetic energy spectra. A good correlation is observed between the structure of the unsaturated fatty acids ranging from C24 to C27 and the methyl terminal part of the main chain of diunsaturated mycolic acids.

Separation of Fatty Acids as Theirp-Bromophenacyl Esters on a C30-Bonded Silica Column by High Performance Liquid Chromatography

Abstract A new C30-bonded silica column was developed for high performance liquid chromatography. This column was tested for the fractionation of fatty acids as their p-bromophenacyl esters by the reverse-phase mode. Certain pairs of fatty acid esters that are very difficult to separate on a C18-bonded silica column, i. e., arachidonic (C20:4)-palmitoleic (C16:1); elaidic (trans C18:1)-vaccinic (cis C18:1); behenic (C22:0)-nervonic (C24:1); and arachidonic (C20:0)-erucic (C22:1) esters, were completely resolved on the C30-bonded column using solvent gradients of acetonitrile: water and acetonitrile: p-dioxane. A solvent system of methylene chloride: acetonitrile (2:1, v/v) was developed for this column to achieve good separation of a homologous series of extremely nonpolar C76 to C82 α-mycolic acid esters from Mycobacterium tuberculosis H37Ra.

Separation of structural studies of the molecular species of monomycolates and dimycolates of alpha-D-trehalose present in Mycobacterium phlei (author's transl)

Mixtures of dimycolates of alpha-D-trehalose (cord factor) and monomycolates have been isolated from Mycobacterium phlei and separated as trimethylsilyl derivatives according to the polarity of the fatty acid residues. The free glycolipids can be recovered by mild hydrolysis. Silylation and disilylation reactions did not induce any isomerisation. The structure of these trehalose esters has been determined by using a series of reactions elaborated on synthetic acyl-sugar models. Free hydroxyl groups are transformed into tetrahydropyran ethers, deacylated by dimsyl sodium, methylated and the sugar derivatives are hydrolyzed. The O-methyl-sugars obtained contain a methyl ether group located at the position where the acyl group was present initially. Identification by gas chromatography of the O-methyl-sugars thus allows the location of the fatty acid residues in the glycolipid. It has been demonstrated that no migration occurs. Two types of 6-monomycoloyl-alpha-D-trehalose have been isolated, differing by the nature of the mycolic acid residues. One of them, called MA, contains "alpha-phlei mycolic acid". The other one, called MB, contains "gamma-phlei mycolic acid" which is the ester of 2-eicosanol with the omega-carboxyl function of a dicarboxylic mycolic acid. Three types of 6,6'-dimycoloyl-alpha-D-trehalose (cord factor) have been obtained. Two of them are symmetrical diesters, containing either two residues of alpha-mycolic acid, or two residues of gamma-mycolic acid. The last one is an unsymmetrical diester, the hydrolysis of which gives one mole of alpha-mycolic acid and one mole of gamma-mycolic acid. The ratio of the different diesters in the cord factor fraction might be explained by some equilibrium between the different 6-ester groups or by a transformation in situ of one species into another one by biochemical modification of the mycolic acid residues.

Sur les acides mycoliques de P6

An α-mycolic acid was isolated from P6, an unclassified mycobacterium, scotochromogen, and its structure was determined. This acid has a chain of 80 carbons and two rings of cyclopropane. It was found that the entire structure of this α-mycolic acid was similar to that from M. smegmatis and M. phlei.

Studies on Mycolic Acid and Related Compounds. III. Synthesis of α-Mycolic Acid found in Brevannes Strain of Human-type Tubercle Bacilli (1).

Condensation of 2-hexacosyl-3-acetoxytriacontanoic acid chloride and ethyl benzyl tetracosylmalonate gave ethyl 2-tetracosyl-3-oxo-4-hexacosyl-5-acetoxydotriacontanoate whose reduction with sodium borohydride finally afforded the objective 2-tetracosyl-3, 5-dihydroxy-4-hexacosyldotriacontanoic acid, C82H164O4.

Studies on Mycolic Acid and Related Compounds. IV. Synthesis of α-Mycolic Acid found in Brevannes Strain of Human-type Tubercle Bacilli. (2).

Studies on Mycolic Acid and Related Compounds. IV. Synthesis of α-Mycolic Acid found in Brevannes Strain of Human-type Tubercle Bacilli. (2).