The mmaA2 gene of Mycobacterium tuberculosis encodes the distal cyclopropane synthase of the alpha-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.     

A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis

Colonial morphology of pathogenic bacteria is often associated with virulence. For M. tuberculosis, the causative agent of tuberculosis (TB), virulence is correlated with the formation of serpentine cords, a morphology that was first noted by Koch. We identified a mycobacterial gene, pcaA, that we show is required for cording and mycolic acid cyclopropane ring synthesis in the cell wall of both BCG and M. tuberculosis. Furthermore, we show that mutants of pcaA fail to persist within and kill infected mice despite normal initial replication. These results indicate that a novel member of a family of cyclopropane synthetases is necessary for lethal chronic persistent M. tuberculosis infection and define a role for cyclopropanated lipids in bacterial pathogenesis.     

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria

Background

Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/Principle Findings

We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the α- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/Significance

This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.     

Redundant Function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis Cyclopropanation of Oxygenated Mycolates

The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2, mmaA2 and cmaA1, or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.Mycobacterium tuberculosis infection is an ongoing global health crisis. Alleviation of this crisis will require a multidisciplinary approach that must include new antibiotics active against M. tuberculosis. A growing body of literature implicates cell envelope lipids in the pathogenesis of M. tuberculosis infection (5-10, 14-15, 20). The enzymatic pathways that synthesize M. tuberculosis cell envelope lipids are the target of presently available antituberculosis antimicrobials and may be candidates for future antibiotic development.The mycolic acids of M. tuberculosis are alpha-alkyl, beta-hydroxy fatty acids which are 75 to 85 carbons in length (3). There are three classes of major mycolic acids: alpha-, methoxy-, and ketomycolates (Fig. ​(Fig.1).1). Whereas all mycobacteria synthesize mycolic acids, only pathogenic mycobacteria (for example, M. tuberculosis, M. leprae, M. avium, and M. bovis) produce significant quantities of mycolic acids with cyclopropane rings, three-member carbon rings which are added to the meromycolate chain (3). Alpha-mycolates have two cis cyclopropane rings, while methoxy- and ketomycolates have either a cis or trans cyclopropane ring at the proximal position, the latter with a distal methyl branch (Fig. ​(Fig.1).1). In contrast to the case with Escherichia coli, which encodes a single cyclopropane fatty acid synthase (CFAS) (16-17), the M. tuberculosis genome encodes a family of S-adenosyl methionine-dependent methyltransferases that modify cell envelope mycolic acids with methyl branches and cyclopropane rings. Despite substantial amino acid identity, systematic characterization of M. tuberculosis null mutants in each of these methyltransferases has revealed highly specific functions which were not revealed when the enzymes were overexpressed in M. smegmatis (12, 26, 28). Deletion of pcaA greatly reduces synthesis of the proximal cyclopropane ring of the alpha-mycolates (15), whereas deletion of mmaA2 greatly reduces the distal cyclopropane of the same lipid (13). Loss of mmaA2 also causes a mild impairment of methoxymycolate, but not ketomycolate, cis cyclopropanation (13). Similar genetic approaches established cmaA2 as the only trans cyclopropane synthase of oxygenated mycolates (14), while loss of mmaA3 abolishes methoxymycolates, a spontaneous mutation found in many M. bovis BCG strains (4, 11). Finally, deletion of mmaA4 abolishes synthesis of both methoxy- and ketomycolates (10). Recent chemical-genetic analysis of this enzyme family indicates that combined inhibition of their function is lethal to M. tuberculosis, strongly supporting an approach targeting this enzyme family for antimicrobial development (2, 8-19, 25).Open in a separate windowFIG. 1.Chemical structures of the major mycolic acids of M. tuberculosis. Cyclopropane rings and methyl branches are shown and annotated with the methyltransferase responsible for their synthesis.In addition to this essential role in combination, recent evidence implicates individual cyclopropane modifications as important determinants of M. tuberculosis host-pathogen interactions. Inactivation of pcaA causes attenuation of M. tuberculosis in the mouse model of infection while stimulating less-severe granulomatous pathology (15, 23). In contrast, deletion of cmaA2 has no effect on bacterial loads during mouse infection but causes hypervirulence while inducing more-severe granulomatous pathology (24). Inactivation of mmaA4, which leads to an absence of methoxy- and ketomycolates, causes a severe growth defect during the first 3 weeks of infection (10). All of these studies implicate the fine structure of mycolic acids in the pathogenesis of M. tuberculosis infection. One mechanism by which cyclopropanation mediates pathogenesis is through altered inflammatory activity of trehalose dimycolate (TDM), an inflammatory glycolipid. The cyclopropane content of TDM is a major determinant of its inflammatory activity, and this altered TDM is responsible for the virulence phenotypes of cyclopropane-deficient M. tuberculosis strains (9, 23-24).Despite major advances in our understanding of the biosynthesis and pathogenetic function of cyclopropanated mycolic acids through genetic approaches, the methyltransferase(s) that synthesizes the cis cyclopropane ring on the methoxy- and ketomycolates is unknown. In addition, the function of the MmaA1 methyltransferase has not been explored through construction of a null mutant. Prior experiments found that overexpression of mmaA1 in M. tuberculosis resulted in accumulation of trans-unsaturated and -cyclopropanated oxygenated mycolates (27). These data suggested that MmaA1 acts in the biosynthesis of trans-cyclopropanated oxygenated mycolates either by adding the methyl branch distal to the cyclopropane ring or as a cis-trans isomerase or both. In addition, although there was a defect in cis cyclopropanation of methoxymycolates in the ΔmmaA2 strain, this defect was mild, suggesting redundancy with another unidentified enzyme. In this article, we define novel functions for three cyclopropane synthases using a new selectable marker to construct M. tuberculosis strains deficient in multiple mycolic acid methyltransferases. Through this approach, we show that CmaA2 and MmaA2 are redundant for cis cyclopropanation of the proximal position of the methoxymycolates and ketomycolates and that MmaA1 is upstream of CmaA2 in trans cyclopropanation.     

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.     

Phosphorylation of mycobacterial PcaA inhibits mycolic acid cyclopropanation: consequences for intracellular survival and for phagosome maturation block

Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG ΔpcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the ΔpcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the ΔpcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.     

Mycobacterium bovis BCG genes involved in the biosynthesis of cyclopropyl keto- and hydroxy-mycolic acids

The resurgence of tuberculosis and the emergence of multidrug-resistant mycobacteria necessitate the development of new antituberculosis drugs. The biosynthesis of mycolic acids, essential elements of the mycobacterial envelope, is a good target for chemotherapy. Species of the Mycobacterium tuberculosis complex synthesize oxygenated mycolic acids with keto and methoxy functions. In contrast, the fast-growing Mycobacterium smegmatis synthesizes oxygenated mycolic acids with an epoxy function. We describe the isolation and sequencing of a cluster of four genes from Mycobacterium bovis bacillus Calmette–Guérin (BCG), coding for methyl transferases, and which, when transferred into M. smegmatis , allow the synthesis of ketomycolic acid, in addition to an as yet undescribed mycolic acid, hydroxymycolic acid. These oxygenated mycolic acids, unlike the regular mycolic acids of M. smegmatis , and similar to the mycolic acids of M. bovis , are highly cyclopropanated. Furthermore, there is a perfect match between the structures of the keto- and the hydroxy-mycolic acids. We propose a biosynthetic model in which there is a direct relationship between these two types of mycolic acid.     

Crystal structures of mycolic acid cyclopropane synthases from Mycobacterium tuberculosis.

Mycolic acids are major components of the cell wall of Mycobacterium tuberculosis. Several studies indicate that functional groups in the acyl chain of mycolic acids are important for pathogenesis and persistence. There are at least three mycolic acid cyclopropane synthases (PcaA, CmaA1, and CmaA2) that are responsible for these site-specific modifications of mycolic acids. To derive information on the specificity and enzyme mechanism of the family of proteins, the crystal structures of CmaA1, CmaA2, and PcaA were solved to 2-, 2-, and 2.65-A resolution, respectively. All three enzymes have a seven-stranded alpha/beta fold similar to other methyltransferases with the location and interactions with the cofactor S-adenosyl-l-methionine conserved. The structures of the ternary complexes demonstrate the position of the mycolic acid substrate binding site. Close examination of the active site reveals electron density that we believe represents a bicarbonate ion. The structures support the hypothesis that these enzymes catalyze methyl transfer via a carbocation mechanism in which the bicarbonate ion acts as a general base. In addition, comparison of the enzyme structures reveals a possible mechanism for substrate specificity. These structures provide a foundation for rational-drug design, which may lead to the development of new inhibitors effective against persistent bacteria.     

坑酸分枝杆菌和相关菌全细胞枝菌酸甲基酯的薄层分析

Mycolic acid methanolysates of whole-cell in Mycobacterium and related bacteria were analysed by thin-layer chromatography. The experimental results show that five of twenty-two species, M. tuberculosis, M. bovis, M. kansasii, M. marinum and M. gastri have similar pattern of mycolates, composed of alpha-mycolates, methoxymycolates, ketomycolates and two unknown components. M. gilvum, M. phleri, M. avium, M. intracellulare, M. xenopi and M. nonchromogenicum contain alpha-mycolates, ketomycolates and wax-ester. The patterns of TLC for other tested species were different from each other. Nocardia, Rhodococcus and Corynebacterium show a relatively simple pattern which principally contain alpha-mycolates. The four genus can be differentiated. Spots of mycolic acids of nine strains Mycobacterium sp. isolated from patients in this hospital were similar to M. tuberculosis. These strains were also identified to the same result as above by traditional methods. The method is of value in the classification and identification of Mycobacterium.     

Three types of mycolic acid from Mycobacterium tuberculosis Brévanne: implications for structure-function relationships in pathogenesis.

Saponification of the chloroform-soluble wax from Mycobacterium tuberculosis Brévanne led to the isolation of three classes of mycolic acid containing characteristic functional groups along the methylene backbone: type alpha (two cyclopropane rings); type beta (methoxyl, methyl, and cyclopropane); and type gamma (ketone, methyl, and cyclopropane). The structures of these acids were elucidated principally by mass spectrometry. The high mass region of the keto mycolate is presented showing the meromycolal and molecular ion regions. This is first time a molecular peak for this mycolic acid has been reported. The structure of the keto mycolate was further substantiated by study of the mass spectral fragmentation of its dithioketal derivative. Within each type of acid, a series of homologs was encountered, varying according to the number of methylene units in the backbone chain. Chromatographic and infrared spectrophotometric evidence is presented for the alkali-induced isomerization of the three types of mycolates.     

The specificity of methyl transferases involved in trans mycolic acid biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.     

Quantitative comparison of the mycolic and fatty acid compositions of Mycobacterium leprae and Mycobacterium gordonae

The mycolic and fatty acids of three samples each of Mycobacterium leprae and Mycobacterium gordonae were compared. Acids released by whole-organism alkaline hydrolysis were converted to 4-nitrobenzyl esters and mycolic acids were further derivatized to t-butyldimethylsilyl ethers. Thin-layer chromatography of the derivatized long-chain extracts showed that all three M. leprae preparations contained so-called alpha-mycolates and ketomycolates but that the M. gordonae samples had a methoxymycolate in addition to the above types. Silica gel normal-phase high-performance liquid chromatography of the total mycolic acid derivatives confirmed the lack of detectable amounts of methoxymycolates in M. leprae and reverse-phase chromatography of the individual mycolate types demonstrated the homogeneity of the chain lengths of the mycolic acids in each species. Non-hydroxylated fatty acid 4-nitrobenzyl esters were transformed to methyl esters and examined by gas chromatography. Tuberculostearic (10-methyloctadecanoic) acid was a major component of the lipids of all three M. leprae preparations but it was absent in one M. gordonae strain and a very minor component in the other representatives of this latter species. On the basis of fatty and mycolic acid compositions, therefore, a previously suggested close relationship between M. leprae and M. gordonae was not supported.     

Mycobacterium tuberculosis SigM positively regulates Esx secreted protein and nonribosomal peptide synthetase genes and down regulates virulence-associated surface lipid synthesis

The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions.     

Production and partial characterization of anti-cord factor (trehalose-6,6'-dimycolate) IgG antibody in rabbits recognizing mycolic acid subclasses of Mycobacterium tuberculosis or Mycobacterium avium

An ELISA with cord factor (trehalose-6,6'-dimycolate) is useful for the serodiagnosis of tuberculosis. To clarify the exact antigenic epitope in cord factor, recognized by a rabbit anti-cord factor IgG antibody, and to ascertain the most sensitive and specific diagnostic test antigen, rabbits were immunized with two kinds of cord factors isolated from Mycobacterium tuberculosis or Mycobacterium avium and the reactivities of the sera were tested against cord factors or the component mycolic acid methyl esters by ELISA. The serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against M. tuberculosis cord factor, but less reactive against M. avium cord factor. In contrast, the serum from rabbits immunized with M. avium cord factor was highly reactive against M. avium cord factor but less reactive against M. tuberculosis cord factor. Moreover, the serum from rabbits immunized with M. tuberculosis cord factor reacted against mycolic acid methyl esters, especially methoxy mycolic acid methyl ester. On the other hand, the serum from rabbits immunized with M. tuberculosis cord factor was less reactive against trehalose-6-monomycolate and not reactive against sulfolipid (2,3,6,6'-tetraacyl trehalose 2'-sulfate). From these results, it was concluded that the anti-cord factor IgG antibody, produced experimentally in rabbits, recognized the differences in the cord factor structures, i.e. the hydrophobic moiety rather than the carbohydrate moiety. It was also noted that the serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against methoxy mycolic acid as an epitope. This paper is the first to describe how the anti-cord factor IgG antibody can recognize the mycolic acid subclasses, which differ according to the species of mycobacteria.     

Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.     

Rv3389C from Mycobacterium tuberculosis, a member of the (R)-specific hydratase/dehydratase family

The (R)-specific 3-hydroxyacyl dehydratases/trans-enoyl hydratases are key proteins in the biosynthesis of fatty acids. In mycobacteria, such enzymes remain unknown, although they are involved in the biosynthesis of major and essential lipids like mycolic acids. First bioinformatic analyses allowed to identify a single candidate protein, namely Rv3389c, that belongs to the hydratases 2 family and is most likely made of a distinctive asymmetric double hot dog fold. The purified recombinant Rv3389c protein was shown to efficiently catalyze the hydration of (C(8)-C(16)) enoyl-CoA substrates. Furthermore, it catalyzed the dehydration of a 3-hydroxyacyl-CoA in coupled reactions with both reductases (MabA and InhA) of the acyl carrier protein (ACP)-dependent M. tuberculosis fatty acid synthase type II involved in mycolic acid biosynthesis. Yet, the facts that Rv3389c activity decreased in the presence of ACP, versus CoA, derivative and that Rv3389c knockout mutant had no visible variation of its fatty acid content suggested the occurrence of additional hydratase/dehydratase candidates. Accordingly, further and detailed bioinformatic analyses led to the identification of other members of the hydratases 2 family in M. tuberculosis.     

Synthesis and properties of methyl 5-(1'R,2'S)-(2-octadecylcycloprop-1-yl)pentanoate and other omega-19 chiral cyclopropane fatty acids and esters related to mycobacterial mycolic acids

A 23-26-carbon chain length range of omega-19 (1'R,2'S) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been prepared. The key cyclopropyl intermediate, (1'R,2'S)-(Z)-1-formyl-2-octadecylcyclopropane, underwent Wittig chemistry with various reagents to provide vinylic precursors, which were selectively reduced to the corresponding saturated omega-19 cyclopropane fatty acids or esters. The 24-carbon omega-19 cyclopropane ester was made by chain elongation of the 23-carbon ester. Saturated and unsaturated chiral cyclopropane acids and esters were assayed, using wall extracts of Mycobacterium smegmatis; the incorporation of 14C-acetate was used to measure inhibition or stimulation of mycolic acid synthesis. Minor inhibition (2-3%) was shown by the 23- and 24-carbon saturated esters; all the other compounds were stimulants. The most effective (38-55%) stimulators of mycolate synthesis were the unsaturated esters with 23- and 26-carbons and the saturated and unsaturated 25-carbon acids.     

Anti-cord factor (trehalose 6,6'dimycolate) IgG antibody in tuberculosis patients recognizes mycolic acid subclasses.

The detection of anti-cord factor (trehalose 6,6'-dimycolate) IgG antibody in active (smear-and/or culture-positive) and inactive (smear-and culture-negative) tuberculosis patients is a useful serodiagnostic tool that can be used for early clinical diagnosis of the disease. We estimated the titers of anticord factor IgG antibody in the sera of tuberculosis patients, and compared them with those of Mycobacterium avium-infected patients. Most of the serum samples obtained from the tuberculosis patients were highly reactive against M. tuberculosis (MTB) cord factor isolated from M. tuberculosis H37Rv, a human-type mycobacterial strain, whereas they were less reactive against M. avium (MAC) cord factor. Similarly, most of the serum samples of the MAC-infected patients were highly reactive against MAC cord factor and less reactive against MTB cord factor. These results suggest that anti-cord factor IgG antibody recognizes the mycolic acid subclasses as an epitope which comprises cord factor, since MTB and MAC cord factor differ in mycolic acid subclasses and molecular species composition. To clarify the exact antigenic epitope in cord factor and to find out a more sensitive and specific diagnostic test antigen, we examined the reactivity of patients' sera to glycolipids containing trehalose (cord factor and sulfolipid) obtained from various mycobacterial species. Furthermore, the reactivity of human antisera to various mycolic acid subclasses (alpha-, methoxy and keto mycolic acids) of MTB cord factor was compared. We found that anti-cord factor IgG antibody in the sera of human tuberculosis patients most strikingly recognized methoxy mycolic acid in the cord factor of M. tuberculosis, whereas it recognized alpha- and keto mycolic acids weakly. Pre-absorption studies of antibody with MTB cord factor or methoxy mycolic acid methyl ester showed that anti-cord factor antibody was absorbed partially, but consistently. This is the first report describing that the specific subclass of mycolic acid from mycobacteria is antigenic in the humoral immune system of human tuberculosis infection.     

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.     

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105)

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).