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.     

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.     

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.     

Tat–Dependent Translocation of an F420–Binding Protein of Mycobacterium tuberculosis

F₄₂₀ is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F₄₂₀ has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N–terminal signal sequence and is annotated as an F₄₂₀–dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co–purify with F₄₂₀ during expression experiments in M. smegmatis. We also show that the Rv0132c–F₄₂₀ complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F₄₂₀–binding protein being a substrate for the Tat pathway and of the presence of F₄₂₀ outside of the cytosol in any F₄₂₀–producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F₄₂₀ biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.     

Unexpected Abundance of Coenzyme F420-Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F₄₂₀, which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F₄₂₀ biosynthesis nominated many actinobacterial proteins as candidate F₄₂₀-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5′-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F₄₂₀-producing species. The LLM and PPOX families were observed in F₄₂₀-producing species as well as species lacking F₄₂₀ but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism''s ability to make F₄₂₀ allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F₄₂₀-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate F₄₂₀-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F₄₂₀ biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F₄₂₀ for its redox reactions. This dependence and the cofactor''s rarity may make F₄₂₀-related proteins promising drug targets.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an actinobacterium that presents a number of clinical challenges. For example, due to the high frequency of drug-resistant mutants, TB antibiotic regimens require long courses of treatment and a combination of three or more separate drugs (37). Long courses of combination therapy contribute to noncompliance, which in turn has led to an increase in the occurrence of multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (39). There is a clear need for additional tuberculosis-specific drugs that, in combination with the current pharmacopeia, can shorten the course of treatment and increase its effectiveness.Biological features that are present in mycobacteria but rare or absent in other organisms are useful targets for treating TB. For example, mycobacteria have “mycolic” fatty acids present in their cell walls that distinguish them from all other bacteria. Four major anti-TB drugs (isoniazid, cycloserine, ethambutol, and ethionamide) are known to target enzymes involved in the biosynthesis of the mycobacterial cell wall, and others, such as pyrazinamide, caprazamycin, and caprolactams, may do so as well (34, 43).Similarly, the enzyme cofactor F₄₂₀ (Fig. ​(Fig.1),1), a deazaflavin analog of flavin mononucleotide (FMN), is absent from humans but distributed sporadically and sparsely among prokaryotes and observed universally in the mycobacteria (including being encoded by the reduced genome of Mycobacterium leprae). It has been suggested that the reduced F₄₂₀ (F₄₂₀H₂) produced by the action of the F₄₂₀-dependent glucose-6-phosphate dehydrogenase (4) under aerobic conditions may protect mycobacterial cells from macrophage-generated NO₂ (31). Moreover, Rv3547 from M. tuberculosis uses reduced F₄₂₀ in the activation of the NO₂-containing antitubercular drug candidate PA-824 (40). Overall, F₄₂₀ may confer an advantage to mycobacteria in anaerobic environments because it has a lower redox potential than NADP (5).Open in a separate windowFIG. 1.Flavonoid cofactor structures. (A) FMN. (B) Coenzyme F₄₂₀. Note that coenzyme F₄₂₀ typically contains 5 to 7 side chain glutamate residues in mycobacterial species (3).The sporadic phylogenetic distribution of F₄₂₀ provides an opportunity for the application of comparative genomic methods. We introduced partial phylogenetic profiling (PPP) to efficiently discover protein families codistributed with such patterns of biological traits (21). Unlike earlier profiling methods, PPP does not require the prior accurate determination of protein families for success. This method is well suited to the identification of F₄₂₀-dependent enzyme families, which may have distributions only partially spanning the entire profile. PPP analysis is further augmented by SIMBAL (sites inferred by metabolic background assertion labeling) (36). This technique can pinpoint sites discriminating F₄₂₀ binding from FMN binding and subsequently identify additional correlated genes that are undetectable by PPP.Here we demonstrate how comparative genomics, namely, profiling, can strongly associate sets of genes in a particular genome of interest with a biologically important trait, generating numerous experimentally testable hypotheses. This analysis has indicated a pervasive and presumably important feature of M. tuberculosis and its lifestyle. The lack of F₄₂₀-based reactions in humans or their associated gut flora and their prevalence in M. tuberculosis may provide another drug target.     

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

The ultrastructural locations of the coenzyme F₄₂₀-reducing formate dehydrogenase and coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F₄₂₀-dependent formate dehydrogenase activity or F₄₂₀-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.Abbreviation PBST Phosphate-buffered saline containing 0.1% (v/v) Triton X-100     

Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1

Cofactor F₄₂₀, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F₄₂₀-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F₄₂₀-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F₄₂₀ + glucose-6-phosphate → F₄₂₀H₂ + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F₄₂₀ cofactor required for numerous F₄₂₀H₂-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F₄₂₀-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F₄₂₀-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F₄₂₀H₂-dependent reductases in industry and bioremediation.

     

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.     

High-yield production of coenzyme F420 in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space

Coenzyme F₄₂₀ is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F₄₂₀-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F₄₂₀ levels are produced in microorganisms, F₄₂₀ availability is a serious bottleneck for research and application. Recent advances in our understanding of the F₄₂₀ biosynthesis enabled heterologous overproduction of F₄₂₀ in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F₄₂₀ biosynthesis in E. coli. However, it still led to the production of low amounts of F₄₂₀ and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module (“core”) addressed parts of the riboflavin biosynthesis pathway and F_O synthase for the conversion of GTP to the stable F₄₂₀ intermediate F_O. The enzymes of the second module (“decoration”) were chosen to turn F_O into F₄₂₀. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F₄₂₀-derived fluorescence. This approach yielded the highest titer of coenzyme F₄₂₀ produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F₄₂₀-dependent bioprocesses as well as applications of F₄₂₀-dependent enzymes in biocatalysis.     

Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications.     

Function of H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum in coenzyme F420 reduction with H2

Summary In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F₄₂₀ (F₄₂₀) with H₂ are present: (1) the F₄₂₀-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N ⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H₂-forming methylenetetrahydromethanopterin dehydrogenase and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F₄₂₀-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H₂-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F₄₂₀ with H₂ is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system. Received: 9 September 1997 / Accepted: 30 October 1997     

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.     

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced. Received: 12 May 1997 / Accepted: 1 July 1997     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.     

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.     

Interpreting Expression Data with Metabolic Flux Models: Predicting Mycobacterium tuberculosis Mycolic Acid Production

Metabolism is central to cell physiology, and metabolic disturbances play a role in numerous disease states. Despite its importance, the ability to study metabolism at a global scale using genomic technologies is limited. In principle, complete genome sequences describe the range of metabolic reactions that are possible for an organism, but cannot quantitatively describe the behaviour of these reactions. We present a novel method for modeling metabolic states using whole cell measurements of gene expression. Our method, which we call E-Flux (as a combination of flux and expression), extends the technique of Flux Balance Analysis by modeling maximum flux constraints as a function of measured gene expression. In contrast to previous methods for metabolically interpreting gene expression data, E-Flux utilizes a model of the underlying metabolic network to directly predict changes in metabolic flux capacity. We applied E-Flux to Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB). Key components of mycobacterial cell walls are mycolic acids which are targets for several first-line TB drugs. We used E-Flux to predict the impact of 75 different drugs, drug combinations, and nutrient conditions on mycolic acid biosynthesis capacity in M. tuberculosis, using a public compendium of over 400 expression arrays. We tested our method using a model of mycolic acid biosynthesis as well as on a genome-scale model of M. tuberculosis metabolism. Our method correctly predicts seven of the eight known fatty acid inhibitors in this compendium and makes accurate predictions regarding the specificity of these compounds for fatty acid biosynthesis. Our method also predicts a number of additional potential modulators of TB mycolic acid biosynthesis. E-Flux thus provides a promising new approach for algorithmically predicting metabolic state from gene expression data.     

Homologous npdGI Genes in 2,4-Dinitrophenol- and 4-Nitrophenol-Degrading Rhodococcus spp.

Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F₄₂₀ reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F₄₂₀. Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F₄₂₀-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F₄₂₀-dependent glucose-6-phosphate dehydrogenases and F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F₄₂₀-dependent enzymes involved in catabolism.     

Carbon Source-Induced Modifications in the Mycolic Acid Content and Cell Wall Permeability of Rhodococcus erythropolis E1

The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.     

A Common Mechanism of Inhibition of the Mycobacterium tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C₁₈, C₂₀, and C₂₂ fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.