Alpha-mycolic Acids Research Articles

Free enzyme dynamics of CmaA3 and CmaA2 cyclopropane mycolic acid synthases from Mycobacterium tuberculosis: Insights into residues with potential significance in cyclopropanation

Mycolic acids are long chain alpha-alkyl beta-hydroxy fatty acids that are major constituents of the cell wall of Mycobacterium tuberculosis. M. tuberculosis produces three main types of mycolic acids, alpha mycolic acids and keto and methoxy mycolic acids. Cycloproponated mycolic acids make the cell wall less permeable, contribute to antibiotic resistance and host immunomodulation and protect from injury. Cyclopropanation is catalyzed by enzymes of the Cyclopropane Mycolic Acid Synthase (CMAS) family. In the current study, we addressed two CMAS enzymes, proximal alpha cyclopropane mycolic acid synthase (PcaA/CmaA3) and keto cyclopropane Mycolic acid synthase (CmaA2). All-atom Molecular Dynamics (MD) simulations were performed for these enzymes for a timeframe of 100ns each (in triplicate), using GROMACS. Based on the PDB structures of apo and holo states of related CMAS enzymes, we generated a framework which helped us correlate active or inactive states of the enzymes to different conformations sampled by the enzymes during MD simulations. Dynamics suggested that the free or unbound enzymes have intrinsic memory and sample different states of catalysis even in the absence of the substrate/cofactor. Additionally, we find that F200, P201 and W204 may have functional significance. MD simulation of CmaA2 was performed with the objective of gaining insights into the putative role of a loop insert. Analysis showed that acidic residues of this loop possibly play an important role during the active state by forming salt bridges. The insights gained in this study can potentially be utilized for design of effective inhibitors against CMAS enzymes.

Standardization of natural mycolic acid antigen composition and production for use in biomarker antibody detection to diagnose active tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is characterized by the abundance of species specific, antigenic cell wall lipids called mycolic acids. These wax-like molecules all share an identical, amphiphilic mycolic motif, but have different functional groups in a long hydrophobic hydrocarbon mero-chain that divide them into three main classes: alpha-, keto- and methoxy-mycolic acids. Whereas alpha-mycolic acids constitutively maintain an abundance of around 50%, the ratio of methoxy- to keto-mycolic acid types may vary depending on, among other things, the growth stage of M. tuberculosis. In human patients, antibodies to mycolic acids have shown potential as diagnostic serum biomarkers for active TB. Variations in mycolic acid composition affect the antigenic properties and can potentially compromise the precision of detection of anti-mycolic acids antibodies in patient sera to natural mixtures. We demonstrate this here with combinations of synthetic mycolic acid antigens, tested against TB patient and control sera. Combinations of methoxy- and α-mycolic acids are more antigenic than combinations of keto- and α-mycolic acids, showing the former to give a more sensitive test for TB biomarker antibodies. Natural mixtures of mycolic acids isolated from mature cultures of M. tuberculosis H37Rv give the same sensitivity as that with synthetic methoxy- and α-mycolic acids in combination, in a surface plasmon resonance inhibition biosensor test. To ensure that the antigenic activity of isolates of natural mycolic acids is reproducible, we cultured M. tuberculosis H37Rv on Middlebrook 7H10 solid agar plates to stationary growth phase in a standardized, optimal way. The proportions of mycolic acid classes in various batches of the isolates prepared from these cultures were compared to a commercially available natural mycolic acid isolate. LC-MS/MS and NMR data for quantitation of mycolic acids class compositions show that the variation in batches is small, suggesting that the quality of the results for anti-mycolic acid antibody detection in the TB patients should not be affected by different batches of natural mycolic acid antigens if prepared in a standard way.

Development of Novel Bacteriostatic Agents against Mycobacterium tuberculosis Using In silico Techniques

The cell wall of Mycobacterium tuberculosis is made up of mycolic acids which are found to play important role in pathogenesity by modification of double bonds at specific sites on mycolic acid precursors by the action of cyclopropane mycolic acid synthases (CMASs) that belong to a family of S-adenosyl-methionine-dependent methyl transferases. PcaA is an cyclopropane mycolic acid synthase required for cording, persistence and virulence of Mycobacterium tuberculosis and modifies mycolic acid by cyclopropanation of proximal double bond to cis cyclopropane generating alpha mycolic acid. A molecular docking of selected compounds was performed and the differences in their binding modes were investigated in order to design novel lead compound that can act as better antitubercular agent targeting cyclopropane mycolic acid synthases.

Structure–function relationships of the antigenicity of mycolic acids in tuberculosis patients

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

Temperature-dependent Regulation of Mycolic Acid Cyclopropanation in Saprophytic Mycobacteria: ROLE OF THE MYCOBACTERIUM SMEGMATIS 1351 GENE (MSMEG_1351) IN CIS-CYCLOPROPANATION OF α-MYCOLATES

The cell envelope is a crucial determinant of virulence and drug resistance in Mycobacterium tuberculosis. Several features of pathogenesis and immunomodulation of host responses are attributable to the structural diversity in cell wall lipids, particularly in the mycolic acids. Structural modification of the alpha-mycolic acid by introduction of cyclopropane rings as catalyzed by the methyltransferase, PcaA, is essential for a lethal, persistent infection and the cording phenotype in M. tuberculosis. Here, we demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Interestingly, alpha-mycolic acid cyclopropanation was inducible in cultures grown at 25 degrees C. The growth temperature modulation of the cyclopropanating activity was determined by high resolution magic angle spinning NMR analyses on whole cells. In parallel, quantitative reverse transcription-PCR analysis showed that MSMEG_1351 gene expression is up-regulated at 25 degrees C compared with 37 degrees C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of alpha-mycolates. The functional equivalence of PcaA and MSMEG_1351 was established by cross-complementation in the MSMEG_1351 knock-out mutant and also in a DeltapcaA strain of Mycobacterium bovis BCG. Overexpression of MSMEG_1351 restored the wild-type mycolic acid profile and the cording phenotype in BCG. Although the biological significance of mycolic acid cyclopropanation in nonpathogenic mycobacteria remains unclear, it likely represents a mechanism of adaptation of cell wall structure and composition to cope with environmental factors.

Temperature dependence of the Langmuir monolayer packing of mycolic acids from Mycobacterium tuberculosis

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (α-MA), cyclopropyl methoxy mycolic acid (MeO-MA), and cyclopropyl ketomycolic acids (Keto-MA) from Mycobacterium tuberculosis were obtained by thermodynamic analysis of the surface pressure ( π) vs. average molecular area ( A) isotherms at temperatures in the range of 10–46 °C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature ( T) and the π values. In the Langmuir monolayer of Keto-MA, the carbonyl group in the meromycolate chain apparently touches the water surface to give the molecule a W-shape in all the temperatures and surface pressures studied. Keto-MA formed a rigid solid condensed film, with four hydrocarbon chains packing together, not observed in the others. In contrast, the monolayer films of α-and MeO-MAs having no such highly hydrophilic intra-chain groups in the meromycolate chain were mostly in liquid condensed phase. This novel insight into the packing of mycolic acids opens up new avenues for the study of the role of mycolic acids in the mycobacterial cell envelopes and pathogenic processes.

The synthesis of a single enantiomer of a major alpha-mycolic acid of Mycobacterium tuberculosis.

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

Accurate Molecular Mass Determination of Mycolic Acids by MALDI-TOF Mass Spectrometry

Mycolic acids, major and specific long-chain fatty (C70-C90) acid components of the mycobacterial cell envelope, were analyzed for the first time using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry operating in a reflectron mode. The various types of purified mycolates from representative mycobacterial species were analyzed using 2,5-DHB as matrix, because less than 10 pmol of mycolates was sufficient to obtain well-resolved mass spectra composed exclusively of pseudomolecular [M + Na]+ ions consistent with the structures deduced from the chemical analytical techniques applied to these molecules. Examination of the MALDI mass spectra demonstrated that the chain lengths of the various mycolates correlated with the growth rate of mycobacterial strains. Although slow growers, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, produced a series of odd carbon numbers (C74-C82) of alpha-mycolic acids, rapid growers synthesized both odd and even carbon numbers. In addition, the main chain of oxygenated mycolic acids from slow growers were four to six carbon atoms longer than the corresponding alpha-mycolic acids, whereas rapid growers elaborated oxygenated homologues possessing the same chain lengths as their alpha-mycolic acids. Furthermore, a comparative analysis of the crude fatty acid mixtures from a wild-type strain of M. tuberculosis and its isogenic mutant effected in the synthesis of oxygenated mycolates by MALDI mass spectrometry revealed structural differences between the alpha-mycolates from the two strains. Thus, this technique appeared to be a rapid and highly sensitive technique for the analysis of mycolic acids, not only by providing accurate molecular masses and new structural information, but also by both reducing sample consumption and saving time.

The Mycobacterium tuberculosis cmaA2 Gene Encodes a Mycolic Acid trans-Cyclopropane Synthetase

Infection with Mycobacterium tuberculosis remains a major global health emergency. Although detailed understanding of the molecular events of M. tuberculosis pathogenesis is still limited, recent genetic analyses have implicated specific lipids of the cell envelope as important effectors in M. tuberculosis pathogenesis. We have shown that pcaA, a novel member of a family of M. tuberculosis S-adenosyl methionine (SAM)-dependent methyl transferases, is required for alpha-mycolic acid cyclopropanation and lethal chronic persistent M. tuberculosis infection. To examine the apparent redundancy between pcaA and cmaA2, another cyclopropane synthetase of M. tuberculosis thought to be involved in alpha-mycolate synthesis, we have disrupted the cmaA2 gene in virulent M. tuberculosis by specialized transduction. Inactivation of cmaA2 causes accumulation of unsaturated derivatives of both the methoxy- and ketomycolates. Analysis by proton NMR indicates that the mycolic acids of the cmaA2 mutant lack trans-cyclopropane rings but are otherwise intact with respect to cyclopropane and methyl branch content. Thus, cmaA2 is required for the synthesis of the trans cyclopropane rings of both the methoxymycolates and ketomycolates. These results define cmaA2 as a trans-cyclopropane synthetase and expand our knowledge of the substrate specificity of a large family of highly homologous mycolic acid methyl transferases recently shown to be critical to M. tuberculosis pathogenesis.

Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice.

Members of the Mycobacterium tuberculosis group synthesize a family of long-chain fatty acids, mycolic acids, which are located in the cell envelope. These include the non-oxygenated alpha-mycolic acid and the oxygenated keto- and methoxymycolic acids. The function in bacterial virulence, if any, of these various types of mycolic acids is unknown. We have constructed a mutant strain of M. tuberculosis with an inactivated hma (cmaA, mma4) gene; this mutant strain no longer synthesizes oxygenated mycolic acids, has profound alterations in its envelope permeability and is attenuated in mice.

Production and partial characterization of antibody to cord factor (trehalose 6,6'-dimycolate) in mice.

Antibody production against the trehalose 6,6'-dimycolate (TDM, cord factor) of Rhodococcus ruber, a non-pathogenic species of the Actinomycetales group, was investigated in mice by repeated intraperitoneal injection of TDM in water-in-oil-in-water micelles without carrier protein. The antigenic TDM was isolated and purified chromatographically from the chloroform-methanol extractable lipids of R. ruber. The hydrophobic moiety of this TDM was composed of two molecules of monoenoic or dienoic alpha-mycolic acids with a carbon chain length ranging from C44 to C48 centering at C46. To detect the antibody, an enzyme-linked immunosorbent assay (ELISA) system was employed using plastic plates coated with TDM. The antibody reacted against the TDM of R. ruber. The antibody was reactive in similar fashion against glycosyl monomycolates differing in the carbohydrate moiety, such as that of glucose mycolate (GM) and mannose mycolate (MM), obtained from R. ruber. Moreover, the antibody reacted against mycolic acid methyl ester itself when it was used as the antigen in ELISA, and trehalose did not absorb the antibody to TDM or inhibit the reaction. These results indicate that the epitope of TDM recognized by the antibody is mycolic acid, an extremely hydrophobic part of the molecule. Next, we prepared monoclonal anti-TDM antibody (moAb) in mice myeloma cells to examine its biological activities and the role of humoral immunity in mycobacterial infection. MoAb reacted against the TDM, glycosyl mycolate, and mycolic acid methyl ester in ELISA in the same manner as our polyclonal antibody did. The administration of moAb suppressed granuloma formation in the lungs, spleen, and liver induced by TDM and inhibited the production of interleukin-1 (IL-1) and chemotactic factor, which is reported to precede granuloma formation.

A new rapidly growing mycobacterial species, Mycobacterium murale sp. nov., isolated from the indoor walls of a children's day care centre.

Scotochromogenic mycobacterial isolates from water-damaged parts of indoor building materials of a children's day care centre represented a phenetically and genetically distinct group of strains. A 16S rDNA dendrogram (1243 bp) showed that the closest species to the new strain MA112/96T was Mycobacterium abscessus. Phylogenetic and phenetic analyses (100 characteristics) grouped the new isolates with M. abscessus, Mycobacterium vaccae, Mycobacterium aurum and Mycobacterium austroafricanum. Ribotyping with Pvull restriction distinguished the 5 isolates from the other 12 most closely related species by the major bands at 6.5-7 kb and 13-15 kb. The cell morphology of the new isolates was typical of mycobacteria, electron microscopy revealed a triple-layered cell wall with an irregular electron-dense outer layer. They grew at 10-37 degrees C, with no growth at 45 degrees C in 5 d. The gene encoding the secreted 32 kDa protein, specific to mycobacteria, was detected by PCR. The main whole-cell fatty acids were characterized by high tuberculostearic acid 10Me-C18:0 (17% at 28 degrees C), which increased with increasing growth temperature (22% at 37 degrees C). The other main fatty acids were C18:1 cis9 and C16:0 (21-20% each), followed by, C17:1 cis9 (14%), C16:1 cis10 (8%) and also a high amount of C20 alcohol (9%). alpha-Mycolic acids, keto-mycolates and wax esters were present (C60-C90), MK-9(H2) (90%) and MK-8(H2) were the main menaquinones. The cellular phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidyl inositolmannosides and diphosphatidylglycerol. Polyamine content was low. G+C content was 72.9 mol%. The new isolates are proposed as a new species, Mycobacterium murale sp. nov. The type strain is MA112/96T (= DSM 44340T).

Mycobacterium alvei sp. nov.

A new species of rapidly growing, nonphotochromogenic mycobacteria, Mycobacterium alvei, is described. The inclusion of this organism in the genus Mycobacterium is based on its acid fastness, its mycolate pattern, and its G + C content. A study of six strains showed that they form a homogeneous group with an internal phenotypic similarity value of 97 +/- 2.22%. DNA relatedness studies showed that the six M. alvei strains which we studied form a single DNA hybridization group which is less than 49% related to 14 other species of the genus Mycobacterium; the deltaTm values determined for the strains which exhibited higher levels of DNA homology were all greater than 7.9 degrees C. A lipid analysis showed that tuberculostearic acid was present. Docosanoic and tetracosanoic acid methyl esters were detected as mycolic acid cleavage products. All six isolates which we tested contained alpha-mycolic acids and relatively large amounts of a new kind of mycolic acid containing a methoxy group of omega-1 position, a characteristic that has not been described previously in mycobacteria. Strain CR-21 is the type strain; a culture of this strain has been deposited in the Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur, Paris, France, as strain CIP 103464.

Thermally adaptive changes of mycolic acids in Mycobacterium smegmatis.

The effect of growth temperature on mycolic acid composition in eight strains of Mycobacterium smegmatis was investigated by gas chromatography/mass spectrometry. A change in growth temperature from 45 to 20 degrees C caused a shift in the subclass and molecular species composition of mycolic acids. The relative amount of alpha'-mycolic acids to alpha-mycolic acids decreased, and that of hydroxy mycolic acids increased at lower temperatures. Moreover, the proportion of shorter-chain species of alpha-mycolic acids increased, and those of longer-chain species of alpha-mycolic and hydroxy mycolic acids decreased. This observation seems to be due to the changes of the chain length of meromycolates because the alpha-alkyl chain unit of mycolic acids was not affected. The ratio of odd to even carbon-numbered alpha-mycolates decreased as the growth temperature was lowered. In contrast, the molecular species composition of alpha'-mycolic acid was not influenced by the growth temperature.

Structure and Molecular Species Composition of Three Homologous Series of -Mycolic Acids from Mycobacterium spp.

Three homologous series of alpha-mycolic acids (dicyclopropanoyl acids, monocyclopropanoyl monoenoic acids and dienoic acids) from 16 rapidly growing and 14 slowly growing mycobacteria were separated by argentation thin-layer chromatography and analysed by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives. Mycobacterial species were separated into five groups. Strains of group A contained similar amounts of even and odd carbon-numbered dienoic acids, with a methyl branch on the odd acids and a C24-alpha-unit, as typified by Mycobacterium fortuitum and M. chitae. Group B strains possessed similar amounts of even carbon-numbered dicyclopropanoyl alpha-mycolic acids and odd carbon-numbered unsaturated acids with C22- and C24-alpha-units, as found in M. phlei and M. diernhoferi. Group C strains contained mainly even carbon-numbered dicyclopropanoyl acids with C22- and C24-alpha-units, as shown by M. vaccae and M. aurum. Group D strains possessed mainly odd carbon-numbered dienoic acids with a methyl branch and a C24-alpha-unit, as seen in M. triviale and M. nonchromogenicum. Group E strains had mainly even carbon-numbered dicyclopropanoyl acids with C24- or C26-alpha-units, as found in M. avium and M. tuberculosis. Many rapidly growing mycobacteria also produced alpha'-mycolic acids which were shorter in the length of the main carbon chain but whose alpha-units were the same as those in alpha-mycolic acids from the same species. These alpha'-mycolic acids had either one or two double bonds and showed variations in both their unsaturation and overall size, which may be useful in taxonomic studies.

Granuloma formation and hemopoiesis induced by C36-48-mycolic acid-containing glycolipids from Nocardia rubra.

As previously reported (I. Yano, I. Tomiyasu, S. Kitabatake, and K. Kaneda, Acta Leprologica 2:341-349, 1984), Nocardia rubra, one of the nonpathogenic actinomycetes, possesses three classes of mycolic acid-containing glycolipid, i.e., glucose mycolate, trehalose dimycolate, and trehalose monomycolate. The carbon chain length of their mycolic acids is shorter (C36-48) than that in mycobacteria (longer than C70), and the glycolipid consists of only alpha-mycolic acid. One intravenous administration of 500 micrograms of each purified glycolipid to ICR mice in the form of water-in-oil-in-water emulsion without any protein antigens caused prominent granuloma formation in the lungs, spleen, and liver. The lung index in the treated mice was about 3.5 times larger than that in the control mice (given water-in-oil-in-water emulsion only) at 1 week after the injection and then rapidly declined, while spleen and liver indices peaked at 2 weeks after the injection and persisted longer. The granuloma consisted of macrophages, some of which phagocytized glycolipid micelles, lymphocytes, monocytes, and neutrophils. In addition, many small hemopoietic islands were observed in the liver sinusoids, where various immature blood cells were trapped by the prominent cytoplasmic projections of Kupffer cells. The granuloma formation and hemopoiesis observed here are considered to be the most characteristic morphological expression of macrophage activation in these organs. This is the first report to show that such histological changes can be induced by chemically defined and homogeneous mycolic acid-containing glycolipids other than those of mycobacteria.

Regulation of Cell Wall Mycolic Acid Biosynthesis in Acid-Fast Bacteria<subtitle>I. Temperature-Induced Changes in Mycolic Acid Molecular Species and Related Compounds in <italic>Mycobacterium phlei</italic></subtitle>

Molecular species of two major subclasses of mycolic acids from Mycobacterium phlei, alpha-mycolic acids (M1) and dicarboxy mycolic acids (M3), were separated gas-chromatographically and identified mass-spectrometrically. The mycolic acid compositions of extractable and cell wall bound lipids were markedly influenced by growth temperature. Increasing growth temperature from 20 degrees C to 50 degrees C resulted in an increase in longer chain species of both mycolisc acid subclasses with a concomitant decrease in shorter chain homologues. The most abundant molecular species were C76 and C58 of M1 and M3 in the 20 degrees C grown cells, while the 50 degrees C grown cells contained C80 in M1 and C62 in M3, most abundantly. Changes in mycolic acid composition occurred rapidly after growth temperature was raised from 20 degrees C to 50 degrees C with an increase in C62 and a concomitant decrease in C58. Mass fragmentographic analysis revealed that an increase in total carbon numbers of mycolic acids was caused by the elongation of straight chain alkyl unit, without any changes in alpha-branch. Changes in the molecular species composition of secondary alcohols presumably derived from the ester mycolic acids were also observed and an increase in longer species (C20-ol-2) with a concomitant decrease in shorter ones (C18-ol-2) was noted as the temperature rose. An increase in the growth temperature also resulted in a decrease in unsaturated fatty acids in extractable lipids. These observations suggest that mycobacteria alter the molecular species composition of mycolic acid subclasses and phospholipids, in response to growth temperature, to maintain a suitable membrane function.

Characterization of the purified components of a new homologous series of alpha-mycolic acids from Mycobacterium tuberculosis H37Ra

Homologous series of alpha-mycolic acids from Mycobacterium tuberculosis H37Ra were separated according to size as their p-bromophenacyl ester by bonded C18 reverse-phase, high performance liquid chromatography. Mass spectrometry of the prominent components separated by high performance liquid chromatography gave relatively simple spectra and showed a series of components differing by 28 atomic mass units. A total of different structures were identified. The structures were established as follow: (formula see text) where a = 17, 18, 19; b = 10; c = 15, 17, 19, 21; and d = 21, 23. The C76 and C78 acids contained some C24 alpha-branch acid (d - 21), whereas the C80 and C82 acids contained some a = 19 acids. Several new homologous series were revealed.