Marie-Antoinette Lanéelle

Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope.

The antigen 85 complex of Mycobacterium tuberculosis consists of three abundantly secreted proteins. The recent characterization of a mycoloyltransferase activity associated in vitro with each of these antigens suggested that they are potentially important for the building of the unusual cell envelope of mycobacteria. To define the physiological role of these proteins, the gene coding for antigen 85C was inactivated by transposon mutagenesis. The resulting mutant was shown to transfer 40% fewer mycolates to the cell wall with no change in the types of mycolates esterifying arabinogalactan or in the composition of non‐covalently linked mycolates. As a consequence, the diffusion of the hydrophobic chenodeoxycholate and the hydrophilic glycerol, but not that of isoniazid, was found to be much faster through the cell envelope of the mutant than that of the parent strain. Taken together, these data demonstrate that: (i) antigen 85C is involved directly or indirectly in the transfer of mycolates onto the cell wall of the whole bacterium; (ii) the enzyme is not specific for a given type of mycolate; and (iii) the cell wall‐linked mycolate layer may represent a barrier for the diffusion of small hydrophobic and hydrophilic molecules.

The biosynthesis of mycolic acids by Mycobacteria: current and alternative hypotheses.

Experimental observations, accumulated during several decades, have allowed an overall scheme for the biosynthesis of the mycolic acids, which are very long chain fatty acids of Mycobacteria to be proposed. But, in almost every step, several hypotheses are compatible with the experimental results, leading to variations of the overall scheme. The aim of this review is to point to some additional possibilities. It is generally assumed that the classical elongation process of fatty acid synthesis produces two long chains, the condensation of which leads to the direct precursors of mycolic acids. But three condensations of four fatty acids, usually synthesized by Mycobacteria, is another hypothesis that could be considered. In the first hypothesis, some methyl or methylene substituents or oxygenated functions are added to the double bonds of an unsaturated precursor, whereas in the second hypothesis, the methylations could help in the building of very long aliphatic chains, and determine the location of double bonds or ramifications. The hypothetical coexistence of two pathways for mycolate biosynthesis is discussed.

Structure and stereochemistry of mycolic acids of Mycobacterium marinum and Mycobacterium ulcerans.

Mycobacterium marinum and M. ulcerans were previously shown to synthesize lipid compounds which are stereochemically different from the corresponding molecules isolated from M. tuberculosis and other species. Stereochemical and biogenetic studies of mycolic acids isolated from M. marinum showed that the absolute configurations of the chiral centres occurring in the mycolates are identical to those of the other mycobacterial species examined so far. Furthermore, all the methyl branches were found to come from methionine whatever the configuration of the centre. The structures of the mycolates synthesized by M. marinum and M. ulcerans were found to be identical, consisting of dicyclopropyl and monocyclopropyl monoenoic mycolates, monoenoic keto- and methoxymycolates, thus reinforcing the taxonomical relationship between the two species.

Molecular dissection of the role of two methyltransferases in the biosynthesis of phenolglycolipids and phthiocerol dimycoserosate in the Mycobacterium tuberculosis complex.

A few mycobacterial species, most of which are pathogenic for humans, produce dimycocerosates of phthiocerol (DIM) and of glycosylated phenolphthiocerol, also called phenolglycolipid (PGL), two groups of molecules shown to be important virulence factors. The biosynthesis of these molecules is a very complex pathway that involves more than 15 enzymatic steps and has just begun to be elucidated. Most of the genes known to be involved in these pathways are clustered on the chromosome of M. tuberculosis. Based on their amino acid sequences, we hypothesized that the proteins encoded by Rv2952 and Rv2959c, two open reading frames of this locus, are involved in the transfer of methyl groups onto various hydroxyl functions during the biosynthesis of DIM, PGL, and related p-hydroxybenzoic acid derivatives (p-HBAD). Using allelic exchange and site-specific recombination, we produced three recombinant strains of Mycobacterium tuberculosis carrying insertions in Rv2952 or Rv2959c. Analysis of these mutants revealed that (i) the protein encoded by Rv2952 is a methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL, respectively, (ii) Rv2959c is part of an operon including the newly characterized Rv2958c gene that encodes a glycosyltransferase also involved in PGL and p-HBAD biosynthesis, and (iii) the enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD produced by M. tuberculosis. These data further extend our understanding of the biosynthesis of important mycobacterial virulence factors and provide additional tools to decipher the molecular mechanisms of action of these molecules during the pathogenesis of tuberculosis.

Structural elucidation by field desorption and electron-impact mass spectrometry of the c-mycosides isolated from Mycoba cterium smegma tis

C-mycosides are superficial type-specific glycopeptidolipids of mycobacterial origin. In the present work, we have shown that field desorption mass spectrometry using the cationization method is a useful method for the molecular weight determination of such compounds. Complementary structural information has been obtained by electron-impact mass spectrometry. Combination of both methods has permitted the elucidation of the structure of the C-mycosides of Mycobacterium smegmatis, ATCC 607. This structure is similar to those described elsewhere, but some minor differences are observed in the lipid portion, mainly in the double bond location.

Tracking the Putative Biosynthetic Precursors of Oxygenated Mycolates of Mycobacterium tuberculosis STRUCTURAL ANALYSIS OF FATTY ACIDS OF A MUTANT STRAIN DEVOID OF METHOXY- AND KETOMYCOLATES

Disruption of the mma4 gene (renamed hma) of Mycobacterium tuberculosis has yielded a mutant strain defective in the synthesis of both keto- and methoxymycolates, with an altered cell-wall permeability to small molecules and a decreased virulence in the mouse model of infection (Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M. A., Yu, K., Quémard, A., Smith, I., and Daffé, M. (2000) Mol. Microbiol. 36, 630–637). Assuming that the mutant would accumulate the putative precursors of the oxygenated mycolates of M. tuberculosis, a detailed structural analysis of mycolates from the hma-inactivated strain was performed using a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, proton NMR spectroscopy, and chemical degradation techniques. These consisted most exclusively of α-mycolates, composed of equal amounts of C76-C82 dicyclopropanated (α1) and of C77-C79 monoethylenic monocyclopropanated (α2) mycolates, the double bond being located at the “distal” position. In addition, small amounts ofcis-epoxymycolates, structurally related to α2-mycolates, was produced by the mutant strain. Complementation of the hma-inactivated mutant with the wild-type gene resulted in the disappearance of the newly identified mycolates and the production of keto- and methoxymycolates of M. tuberculosis. Introduction of the hma gene inMycobacterium smegmatis led to the lowering of diethylenic α mycolates of the recipient strain and the production of keto- and hydroxymycolates. These data indicate that long-chain ethylenic compounds may be the precursors of the oxygenated mycolates of M. tuberculosis. Because the lack of production of several methyltransferases involved in the biosynthesis of mycolates is known to decrease the virulence of the tubercle bacillus, the identification of the substrates of these enzymes should help in the design of inhibitors of the growth of M. tuberculosis.

Acide mycolique epoxydique: un nouveau type d'acide mycolique

Abstract From Mycobacterium fortuitum , a new kind of mycolic acid, containing an epoxy group, has been isolated, for which a structure is proposed, based on chemical degradations and mass spectrometry.

Mycolic acid synthesis by Mycobacterium aurum cell-free extracts.

The first cell-free system capable of synthesizing whole mycolic acids: (R1CH(OH)CH(R2)COOH, with 60 to 90 carbon atoms) from [1-14C]acetate is described and preliminary investigations into some of its requirements and properties are reported. Biosynthetic activity for mycolic acids occurred in an insoluble fraction (40 000 X g pellet) from disrupted cells of Mycobacterium aurum (ATCC 23366-type strain); it produced mycolic acids, but a very small amount of non-hydroxylated fatty acids. The predominant product was unsaturated mycolic acid (type I), while oxo- (type IV) and dicarboxy- (type VI) mycolic acids were synthesized to a lesser extent. When [1-14C]palmitic acid was used as a marker, no labelled mycolic acid was detected. The reaction required a divalent cation (Mg2+ or Mn2+), KHCO3 and O2. Neither CoA, NADH, NADPH nor ATP were necessary, but CoA rather increased the synthesis of non-hydroxylated fatty acids. Glucose or trehalose were not required. Avidin inhibited the biosynthesis of the three types of mycolic acid indicating the presence of a biotin-requiring enzyme in the reaction sequence and therefore a carboxylation step, but citrate had no allosteric effect. Iodoacetamide inhibited the system. These first data are in favor of a complex multienzyme system.

Inteins invading mycobacterial RecA proteins

Five new inteins were discovered in a survey of 39 mycobacterial strains that was undertaken to clarify the role of RecA inteins in mycobacteria. They are all inserted at the RecA‐b site of the recA gene of Mycobacterium chitae, M. fallax, M. gastri, M. shimodei and M. thermoresistibile and belong to the MleRecA allelic family. Sequence analysis showed that although only M. tuberculosis harbours an intein at the RecA‐a site the sequence of the RecA‐b site is well conserved between species. Furthermore, the presence of inteins does not correlate with specific characteristics of the species such as pathogenicity or growth rate.

Diglycosyl phenol phthiocerol diester of Mycobacterium leprae

A diglycosyl phenol phthiocerol diester that had not been previously detected was isolated from M. leprae-infected armadillo tissues. Spectroscopy methods allowed the elucidation of its structure. The diglycoside was a 2,3-di-O-methylrhamnopyranosyl (alpha 1----2)3-O-methylrhamnopyranosyl (alpha 1-linked to the phenolic hydroxyl of phthiocerol dimycocerosates). It differs from the major phenolic glycolipid (PGL I) only by the absence of the terminal 3,6-di-O-methylglucopyranosyl unit. The diglycoside could be an intermediate in the synthesis of the latter antigen or a degradative product in the host detoxification process.