Gilbert Lanéelle

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.

Mechanism of isoniazid uptake in Mycobacterium tuberculosis

Initial transport kinetics of isoniazid (INH) and its uptake at the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x 10(-6) M to 10(-2) M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 degrees C) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity at the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.

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.

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.

Ethambutol inhibition of glucose metabolism in mycobacteria: a possible target of the drug.

The addition of D-arabinose, D-galactose, D-glucosamine, or D-mannose to the growth medium of Mycobacterium smegmatis suppressed the inhibitory effects of ethambutol both on acetate labeling of cell wall-linked mycolic acids and on the increase in the delipidated cell dry weight. The addition of D-glucose or D-fructose had no effect. It is proposed that ethambutol inhibits an early step of glucose conversion into the monosaccharides used for the biosynthesis of structurally and biologically important cell wall polysaccharides: arabinogalactan, arabinomannan, and peptidoglycan. Images

Cell-free synthesis of mycolic acids in Mycobacterium aurum: radioactivity distribution in newly synthesized acids and presence of cell wall in the system

Distribution of radiolabelling in different parts of the newly synthesized mycolic acids, by a cell-free system from Mycobacterium aurum previously described, is examined, [1-14C]acetate being the precursor. By oxidation cleavage of mycolic acids and examination of the fragments, it was shown that acetate was not uniformly incorporated into the molecule: the methyl terminal part was not labelled, while the central fragments--between unsaturations or between oxygenated functions (oxo or ester) and unsaturations--presented the major part of radioactivity, suggesting the elongation of a preformed compound that the cell-free extract was unable to synthesize. Moreover, the side-chain R2-CH2-COOH was only weakly labelled compared to the central fragments. Since non-hydroxylated fatty acids were not synthesized by the system, it is suggested that de novo C18 fatty acids may be elongated with C2 units by the cell-free extract into C22 fatty derivative, only a low level of labelling being recorded (two C2 units for all the molecule). A scheme is proposed to summarize the main results. Identification of meso-DAP which is a characteristic amino-acid of the peptidoglycan in Actinomycetes and analysis of the profiles of total fatty esters, demonstrated that the cell-free extract is partly constituted by fragments of the cell wall as has already been noticed by examination of micrographs of the extract.

Ellipticine derivatives interacting with model membranes. Influence of quaternarization of nitrogen-2

Four compounds of the ellipticine family were examined in their interaction with liposomes and with an isolated bacterial membrane. The physicochemical methods used detected only minor differences between the properties of the amphiphilic drugs (ellipticine and 2-methyl-ellipticinium) and the two dipolar drugs (9-hydroxy-ellipticine and 2-methyl 9-hydroxy-ellipticinium). The amphiphilic drugs were able to become associated to anionic liposomes in a 20-30% excess of charge neutralization, and seem to penetrate deeper into the lipid layer than the two dipolar drugs. It was also shown that ellipticine penetrates deeper into liposomes membrane than into natural membrane used. In contrast with what can be postulated from the literature dealing with the behaviour of quaternarized drugs, it seems that ellipticine and its quaternarized analogues present fast diffusion through multilayered vesicles. On the whole, the membrane effects of the ellipticines studied here are not different for quaternarized drugs and for drugs not permanently charged, but are influenced by the existence on the molecules of a second polar function.

Ethylenic mycolic acid biosynthesis: extension of the biosynthetic model using cell-free extracts of Mycobacterium aurum and Mycobacterium smegmatis

The hypothetical schemes proposed for the biosynthesis of unsaturated mycolic acids (R1-CH(OH)-CH(R2)-COOH) of Mycobacteria cell walls were experimentally tested by using cell-free extracts either of Mycobacterium aurum or of Mycobacterium smegmatis which produce two kinds of unsaturated mycolic acids (mono and dialkene), [1-14C]acetate being the precursor. Examination of specific radioactivities, in the presence or in the absence of isoniazid, an antituberculous drug inhibiting mycolic acid synthesis, showed that saturated C22 and C24 acids play a role as precursors of two distinct parts of the mycolic acids. Moreover, determination of labelling distribution into mycolic acid fragments obtained by oxidative and pyrolytic cleavages showed first that the side chain R2 and the methyl end R1 both have these C22 and C24 saturated fatty acids as common precursors. Secondly, it is thought that the fragments located between the methyl end R1 and the side chain R2 mainly result from elongation steps (one or two successive additions of seven or eight C2 units according to the mycolic acid type) and a biosynthetic model is proposed for unsaturated mycolic acids extending the published models and illustrating the missing step in monoalkene formation.

Ellipticine-induced alteration of model and natural membranes

The modification of certain membrane properties by ellipticine derivatives was examined. The amphiphilic 9-methoxyellipticine was the most efficient in disorganizing membrane structure and in inducing leaks of liposomes, haemolysis of intact human erythrocytes. This drug and the dipolar 9-amino- and 9-hydroxyellipticine were equally efficient in decreasing the surface charge of membranes and in inhibiting the respiration of an isolated bacterial membrane.

Adjuvant lipopeptide interaction with model membranes.

The cationic lipohexapeptide Pam3Cys-Ser-(Lys)4 is a synthetic model for the triacylated N-terminal part of bacterial lipoproteins, and it is used as an adjuvant and macrophage activator. The amphiphilic lipopeptide was injected below a phosphatidylserine monolayer at the air-water interface. It interacted with the interface, as seen by a decrease in the surface potential (deltaV), and it was inserted in the monolayer, until surface charge neutralization was reached, as seen by the parallel increases of deltaV and of the surface pressure. No insertion occurred above 29 mN/m. The interaction kinetics was sensitive to ionic strength and to the nature of acidic phospholipids and of their acyl chains, but the final equilibrium was independent of these factors. Addition of the lipopeptide to large unilamellar vesicles (LUVs) induced their aggregation, and an exchange of lipids between fluorophor-labelled and non-labelled LUVs. However, no fusion was observed, just as reported for polylysine. The lipopeptide strongly inhibited calcium-induced fusion of PS LUVs, in contrast to the published effect of polylysine. This was probably due to inhibition of calcium fixation on liposomes, since it was observed that the lipopeptide efficiently displaced 45Ca2+ from a PS monolayer. In addition, a phospholipid segregation was observed in SUVs for a few ten micromolar of the lipopeptide.