Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria.

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl ß-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Mycolic acids: structures, biosynthesis, and beyond.

Mycolic acids are major and specific lipid components of the mycobacterial cell envelope and are essential for the survival of members of the genus Mycobacterium that contains the causative agents of both tuberculosis and leprosy. In the alarming context of the emergence of multidrug-resistant, extremely drug-resistant, and totally drug-resistant tuberculosis, understanding the biosynthesis of these critical determinants of the mycobacterial physiology is an important goal to achieve, because it may open an avenue for the development of novel antimycobacterial agents. This review focuses on the chemistry, structures, and known inhibitors of mycolic acids and describes progress in deciphering the mycolic acid biosynthetic pathway. The functional and key biological roles of these molecules are also discussed, providing a historical perspective in this dynamic area.

Mycobacterium leprae phenolglycolipid-1 expressed by engineered M. bovis BCG modulates early interaction with human phagocytes.

The species-specific phenolic glycolipid 1 (PGL-1) is suspected to play a critical role in the pathogenesis of leprosy, a chronic disease of the skin and peripheral nerves caused by Mycobacterium leprae. Based on studies using the purified compound, PGL-1 was proposed to mediate the tropism of M. leprae for the nervous system and to modulate host immune responses. However, deciphering the biological function of this glycolipid has been hampered by the inability to grow M. leprae in vitro and to genetically engineer this bacterium. Here, we identified the M. leprae genes required for the biosynthesis of the species-specific saccharidic domain of PGL-1 and reprogrammed seven enzymatic steps in M. bovis BCG to make it synthesize and display PGL-1 in the context of an M. leprae-like cell envelope. This recombinant strain provides us with a unique tool to address the key questions of the contribution of PGL-1 in the infection process and to study the underlying molecular mechanisms. We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses. PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation. Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells.

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

Deciphering the genetic bases of the structural diversity of phenolic glycolipids in strains of the Mycobacterium tuberculosis complex.

Phenolic glycolipids (PGL) play a major role in the virulence of mycobacteria, notably in strains of the Mycobacterium tuberculosis complex and in Mycobacterium leprae. The structure of the carbohydrate domain of these compounds is highly variable, and the genetic bases for these variations remain unknown. We demonstrated that the monoglycosylated PGL formed by Mycobacterium bovis differs from the triglycosylated PGL synthesized by M. tuberculosis (PGL-tb) because of the following two genetic defects: a frameshift mutation within the gene Rv2958c, encoding a glycosyltransferase involved in the transfer of the second rhamnosyl residue of the PGL-tb, and a deletion of a region that encompasses two genes, which encode a GDP-D-mannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/reductase, required for the formation of activated L-fucose. Expression of these three genes in M. bovis BCG allowed synthesis of PGL-tb in this recombinant strain. Additionally, we showed that all M. bovis, Mycobacterium microti, Mycobacterium pinnipedii, and some Mycobacterium africanum strains harbor the same frameshift mutation in their Rv2958c orthologs. Consistently, the structure of PGLs purified from M. africanum (harboring the Rv2958c mutation) and M. pinnipedii strains revealed that these compounds are monoglycosylated PGL. These findings explain the specificity of PGL-tb production by some strains of the M. tuberculosis complex and have important implications for our understanding of the evolution of this complex.

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.

Breaking down the wall: fractionation of mycobacteria.

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.

The acyl-AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-CoA carboxylase components.

Mycolic acids are major and specific long-chain fatty acids of the cell envelope of several important human pathogens such as Mycobacterium tuberculosis, M. leprae, and Corynebacterium diphtheriae. Their biosynthesis is essential for mycobacterial growth and represents an attractive target for developing new antituberculous drugs. We have previously shown that the pks13 gene encodes condensase, the enzyme that performs the final condensation step of mycolic acid biosynthesis and is flanked by two genes, fadD32 and accD4. To determine the functions of the gene products we generated two mutants of C. glutamicum with an insertion/deletion within either fadD32 or accD4. The two mutant strains were deficient in mycolic acid production and exhibited the colony morphology that typifies the mycolate-less mutants of corynebacteria. Application of multiple analytical approaches to the analysis of the mutants demonstrated the accumulation of a tetradecylmalonic acid in the DeltafadD32::km mutant and its absence from the DeltaaccD4::km strain. The parental corynebacterial phenotype was restored upon the transfer of the wild-type fadD32 and accD4 genes in the mutants. These data demonstrated that both FadD32 and AccD4-containing acyl-CoA carboxylase are required for the production of mycolic acids. They also prove that the proteins catalyze, respectively, the activation of one fatty acid substrate and the carboxylation of the other substrate, solving the long-debated question of the mechanism involved in the condensation reaction. We used comparative genomics and applied a combination of molecular biology and proteomic technologies to the analysis of proteins that co-immunoprecipitated with AccD4. This resulted in the identification of AccA3 and AccD5 as subunits of the acyl-CoA carboxylase. Finally, we used conditionally replicative plasmids to show that both the fadD32 and accD4 genes are essential for the survival of M. smegmatis. Thus, in addition to Pks13, FadD32 and AccD4 are promising targets for the development of new antimicrobial drugs against pathogenic species of mycobacteria and related microorganisms.

Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex.

Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans, produce highly specific long chain beta-diols, the dimycocerosates of phthiocerol, and structurally related phenolic glycolipid (PGL) antigens, which are important virulence factors. In addition, M. tuberculosis also secretes glycosylated p-hydroxybenzoic acid methyl esters (p-HBAD) that contain the same carbohydrate moiety as the species-specific PGL of M. tuberculosis (PGL-tb). The genes involved in the biosynthesis of these compounds in M. tuberculosis are grouped on a 70-kilobase chromosomal fragment containing three genes encoding putative glycosyltransferases: Rv2957, Rv2958c, and Rv2962c. To determine the functions of these genes, three recombinant M. tuberculosis strains, in which these genes were individually inactivated, were constructed and biochemically characterized. Our results demonstrated that (i) the biosynthesis of PGL-tb and p-HBAD involves common enzymatic steps, (ii) the Rv2957, Rv2958c, and Rv2962c genes are involved in the formation of the glycosyl moiety of the two classes of molecules, and (iii) the product of Rv2962c catalyzes the transfer of a rhamnosyl residue onto p-hydroxybenzoic acid ethyl ester or phenolphthiocerol dimycocerosates, whereas the products of Rv2958c and Rv2957 add a second rhamnosyl unit and a fucosyl residue to form the species-specific triglycosyl appendage of PGL-tb and p-HBAD. The recombinant strains produced provide the tools to study the role of the carbohydrate domain of PGL-tb and p-HBAD in M. tuberculosis pathogenesis.

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains.

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.

A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.

Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.

Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene.

Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of the pks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.