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.

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.

Lipid composition of aminopterin-resistant and sensitive strains of Streptococcus pneumoniae. Effect of aminopterin inhibition.

The polar lipids of Streptococcus pneumoniae wild type and aminopterin-resistant strains were analysed. The membrane contained only two acid phospholipids, phosphatidylglycerol and cardiolipin, and a large amount of two glycolipids, glucosyldiglyceride and galactosylglucosyldiglyceride. The unsaturated acyl chains ranged from 58 to 87% of total fatty acids, depending on the strain and on growth conditions. No relation could be established between aminopterin resistance and polar lipid or fatty acid compositions. However, in the presence of bacteriostatic concentrations of aminopterin, the wild type and the resistant mutant did not have the same behavior. The resistant strain maintained its fatty acid composition and a normal [32P]phosphate distribution among phospholipids while the wild type shifted to a higher content in unsaturated fatty acids and to a high relative cardiolipin labelling. Such a differencein [32P] distribution was not observed when bacteriostatic concentrations of chloramphenicol were used, or when growth was stopped after amino acid deprivation induced by high concentrations of isoleucine. The biochemical basis of the aminopterin resistant character of the amiA mutants are not yet well understood but the present study establishes that the mutation confers a certain insensitivity of the lipid metabolism to aminopterin.

Certain properties of isoniazid inhibition of mycolic acid synthesis in cell-free systems of M. aurum and M. avium.

In Mycobacterium tuberculosis isoniazid (INH)-susceptibility and the presence of a thermolabile catalase-peroxidase (T-catalase) are nearly always associated. It is shown in this study that an INH-susceptible strain of M. aurum had a T-catalase activity while its resistant mutants did not, but an in vitro susceptible strain of M. avium had a strong catalase activity without any detectable peroxidase properties. Synthesis of mycolic acids is a genus-specific target for INH and there is an excellent parallelism between INH-susceptibility of intact cells and that of a cell-free system synthesizing mycolic acids. We investigated whether the INH-inhibition of mycolic acid cell-free synthesis was dependent on a T-catalase activity in M. aurum and M. avium: no catalase activity was detectable in any of the cell-free systems tested, and addition of T-catalase from susceptible M. aurum strain to an INH-resistant system did not render it sensitive. So INH can inhibit mycolic acid synthesis independently of the presence of a T-catalase. An INH-susceptible cell-free system prepared from INH-treated (at the MIC) cells was progressively and irreversibly inhibited, while incubation of the same susceptible system in the presence of INH did not result in a significant irreversible inhibition. The possible participation of T-catalase in the irreversible effect of INH is discussed.

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.

Monoglycosyldiacylphenol-phthiocerol of Mycobacterium tuberculosis and Mycobacterium bovis.

The structure of a minor glycolipid of M. tuberculosis (strain Canetti) is shown to be 2-O-methyl-alpha-L-rhamnosyldiacylphenol-phthiocerol. A similar compound with non-methylated rhamnose as sugar moiety was also detected. In the course of this work, the structure of mycoside B from Mycobacterium bovis was reexamined, and was shown to be identical to that of the 2-O-methylrhamnosyldiacylphenol-phthiocerol of the Canetti strain, while it was described as a 2-O-methyl-beta-D-rhamnosyl derivative in the literature. This result is in agreement with the known close relationship between M. tuberculosis and M. bovis. Careful examination of chromatographic fractions containing the above mentioned lipids showed that the occurrence of mycoloyl residues in some phenol-phthiocerol glycolipids, postulated in the literature, was likely to be due to the presence of glycerol monomycolate contaminants.

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.

Mycobacterial glycopeptidolipid interactions with membranes: a monolayer study.

Mycobacterial glycopeptidolipid (GPL) interactions with membranes were analysed with monolayer experiment, using GPLs bearing 3, 1, or 0 carbohydrate residues (GPL3, GPL1, GPL0). Compression isotherms and surface potential determinations suggested that the glycopeptidic moiety of GPL3 permanently dipped in water, while those of GPL1 and GPL0 can lay in the interface. Insertion of GPL molecules into a preformed phospholipid monolayer was observed using GPL3 or GPL1 dispersions, but not from GPL0. It is postulated that the activity of GPL0 is low due to its failure to become inserted into membranes, as is that of GPL3 owing to its insertion only by its acyl chain. GPL1 is likely to disturb membranes by inserting its glycopeptidic moiety into the interface.

A study on the receptor for a mycobacteriophage : phage phlei.

From Mycobacterium phlei, glycolipid fractions have been isolated which inactivate phage Phlei. On the basis of the characteristics of the inactivation (specificity, kinetics, requirement for Ca++) typical of the phage-host cell system, it was concluded that these fractions contain the receptor sites for phage Phlei ; this conclusion was supported by electron microscopic studies. All the active fractions contain four kinds of components : fatty acids, glycerol, sugars (D-lyxose, 6-0-methyl-D-glucose, and low amounts of glucose and mannose), and water-soluble acids. These acids are isolated by degradation of the receptor fractions as oxalic and pyruvic acids. Variations of the ratio oxalic acid/pyruvic acid according to the mode of degradation and the absence of the peak characteristic of the protons of a pyruvic acid residue in the NMR spectrum, suggest that these acids might arise from the splitting of oxaloacetic acid. A tentative structure of the receptor is proposed, in many monoglycerides are linked through keto-acid to a polysaccharide core.

Mycobacterial lipids: a historical perspective.

Mycobacterial lipids have been studied for more than 70 years, due to the fascinating diversity of their structures and biological activities. A historical perspective, and the present status on the structure and activity of major lipids of the outer envelope of mycobacterial cells are presented : mycolic acids, which are main constituents of the cell wall, and glycolipids known for toxic or immunological properties (cord factor, SL, DAT, PGL, GPL, LOS, LAM). As far as possible, it was tried to distinguish between experimentally established knowledge and currently accepted speculations.

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.

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.

Alteration of delta psi-dependent amino acid transports in Streptococcus pneumoniae by the antitumoral drug SOAz.

Interactions of the antitumoral drug SOAz with natural and model membranes are described. Biological studies were carried out with the bacterium Streptococcus pneumoniae taken as a model system. They reveal that SOAz is able to reduce delta psi and the delta psi-dependent amino acid transports without being cytotoxic for the bacteria. With respect to model membranes, leakage studies carried out with Na+ and K+ loaded lipid vesicles demonstrated that SOAz exhibits no ionophore activity. In contrast, the drug is shown to decrease the surface potential of monolayers of acidic phospholipids but without penetrating within the film. The possibility that SOAz might alter the delta psi part of the proton motive force by decreasing the outside surface potential of the bacterial membrane is discussed.

Mise en evidence d'une conformation stable d'un peptidolipide.

The Peptidolipide J (CH(3)-(CH(2))(n)-CO-D Phe-L Ile-L Ile-L Phe-L Ala-O-CH(3)) is obtained also in acidic form by a chromatographic artefact. In chloroformic solutions the ester and the acid behave in a very different way: the acid has a stable conformation, as shown by the infra-red spectra and the optical rotary dispersion; this conformation is stabilized by hydrogen bonding between two peptide chains associated as a carboxylic dimer.

Mycolic acid synthesis: a target for ethionamide in mycobacteria?

Striking structural analogies exist between the two specific antimycobacterial drugs ethionamide (ETH) and isoniazid (INH), and they share several inhibitory properties in susceptible species of mycobacteria. The effect of ETH on mycolic acid synthesis was studied in whole cells and in cell extracts of various species, since this synthesis is one direct target for INH, as we recently demonstrated in cell extracts of Mycobacterium aurum. It was shown in the present study that there is not a direct relationship between ETH susceptibility and mycolic acid inhibition. This observation could explain the lack of cross-resistance between the two drugs. The presence of ETH disturbed mycolic acid synthesis in both resistant and susceptible mycobacteria. Synthesis of oxygenated species of mycolic acid was inhibited, while that of diunsaturated acids was either slightly altered or even increased. In contrast, INH inhibited the synthesis of all kinds of mycolic acids in the same way in all susceptible strains and had no effect on mycolic acid synthesis in resistant strains. In the presence of ETH, the unsaturated mycolic acid molecules presented a methyl end different from the usual one. These data strongly suggest that the normal unsaturated mycolic acid species are not the precursors of the oxygenated types. Moreover, they show that ETH probably acts early in the pathway leading to oxygenated mycolic acid.

Isoniazid inhibition of mycolic acid synthesis by cell extracts of sensitive and resistant strains of Mycobacterium aurum.

Isonicotinic acid hydrazide (isoniazid; INH) inhibition of mycolic acid synthesis was studied by using cell extracts from both INH-sensitive and -resistant strains of Mycobacterium aurum. The cell extract of the INH-sensitive strain was inhibited by INH, while the preparation from the INH-resistant strain was not. This showed that the INH resistance of mycolic acid synthesis was not due to a difference in drug uptake or the level of peroxidase activity (similar in both extracts). As INH did not induce accumulation of any labeled intermediates, it is postulated that the drug acts either on production of labeled chain elongation precursors of mycolic acids or an early step of this elongation. The level of inhibition was not changed by addition of NAD or nicotinamide; thus, INH does not act on mycolic acid synthesis as an NAD antimetabolite. Benzoic or acetic acid hydrazides and known or postulated metabolites of INH (i.e., the corresponding acid, aldehyde, or alcohol) were not inhibitors of cell-free mycolic acid synthesis; the complete structure of INH was required, as already known for inhibition of mycobacterial culture growth. Extracts prepared from INH-treated cells showed reduced mycolic acid synthesis, and the inhibition level was not modified by either extensive dialysis or pyridoxal phosphate. This latter molecule efficiently antagonized INH action by reacting rapidly with INH, as shown by differential spectroscopy. Moreover, pyridoxal phosphate did not release inhibition of INH-treated extracts. It is proposed that INH may covalently react with an essential component of the mycolic acid synthesis system.

Phagosomal pH determination by dual fluorescence flow cytometry.

Several methods have been developed to measure the pH of phagosomes, using fluorescein derivatives as reporter of pH, and spectrofluorimetry, fluorescence microscopy, or flow cytometry as quantification technique. All have major disadvantages, including either a slow or inaccurate response. In the present study, pH determination was achieved on J774-cell phagosomes containing dual-labeled zymozan particles using dual fluorescence flow cytometry with an argonion laser excitation wavelength at 488 nm. This allowed zymozan-containing macrophages to be distinguished from other cells and their fluorescence to be measured rapidly. The use of a new probe, namely Oregon Green 488 which has a pKa lower than carboxyfluorescein with the same maximum excitation and emission wavelengths, allowed investigation of pH value below 5. The dual labeling with Oregon Green 488 and carboxytetramethylrhodamine as pH-sensitive and pH-insensitive probes, respectively, overcame the absence of an isobestic point in the Oregon Green 488 spectrum. The phagosomal pH was determined using a calibration curve of phagosomal pH established by adding ionophores in phagocyte suspension and measuring the fluorescence intensity ratio (535 nm/585 nm) for different pHs. A phagosomal pH of 4.5 +/- 0.1 can be accurately determined. This method permits pH measurements down to 4, even in the presence of nonengulfed reporter particles.