Phenol-phthiocerol glycolipids have been found previously in Mycobacterium leprae, M . kansasii, M. bovis and M . marinum, but not in M . tuberculosis. A search for such glycolipids in this latter species showed that the Canetti strains of M . tuberculosis synthesize a major triglycosyl phenol-phthiocerol, accompanied by minor amounts of other glycolipids with a similar aglycone moiety.The triglycoside moiety has the following structure: 2,3,The aglycone moiety consists in phenol-phthiocerol (two homologs). Its two secondary alcohol functions are esterified by mycocerosic acids (homologs with 26-32 carbon atoms and with 2 -4 methyl branches). The proposed structure differs on several points from the M . leprae glycolipids, but presents some analogy with the major glycolipid of M. kansasii.A minor monoglycosyl phenol-phthiocerol was also studied. Its overall structure is very similar to that of M . bovis, with 2-0-methyl rhamnose as sugar moiety.In the last few years, it has been realized that glycolipids are major surface antigens of pathogenic mycobacteria [l], and important applications are expected from this: the detection and identification of bacteria in patients and the elaboration of semi-synthetic vaccines.Three phenol-phthiocerol trisaccharides isolated from Mycobacterium leprae have attracted much attention, since they were the first antigens to be chemically defined in this important pathogen [l -31.Long ago, phenol-phthiocerol monosaccharides were identified in M . bovis [4], M. marinum [5] and M . kansasii [6]; the structure of the glycolipid of the latter species has been revised recently [7, 81. The four mycobacteria quoted above, as well as M . tuberculosis, are pathogenic for man and have the same kind of mycolic acids in common [9]. However no phenol-phthiocerol glycolipid had been found previously to this paper in the most important pathogen of this genus, M . tuberculosis. We searched for glycolipids in various strains belonging to this species, and a major phenol-phthiocerol trisaccharide, accompanied by minor amounts of a phenol-phthiocerol monosaccharide, was detected in the four Canetti strains of M. tuberculosis tested. The structure of the triglycosyl glycolipid is described here. It differs on several points from the glycolipids of M . leprae, and presents some analogy with the major glycolipid of M. kansasii.

Phthienoic acids constitute a family of dextro-rotary odd-numbered unsaturated fatty acids isolated exclusively from virulent strains of human and bovine tubercle bacilli. In the bacterial cell they are not free and a search for their linked form in complex wall lipids of Mycobacterium tuberculosis (strain Canetti) showed that they esterified trehalose.Structural elucidation of the major phthienoyl trehalose showed the occurrence of five acyl residues located at 2, 2', 3', 4 and 6' positions of trehalose. The acyl substituents were mainly 2,4,6-trimethyl tetracos-2-enoic acid (C27 phthienoic acid) accompanied by its homologs. In addition to these branched fatty acids, straight-chain CI6 and C,* acyls composed about 20% of the substituents. The proposed structure is a new one, both for the mycobacterial-specific glycolipid and for the substituted positions on trehalose.Other minor acyl trehaloses were detected in M . tuberculosis (strain Canetti), differing from the major component by the occurrence of an additional hydroxy fatty acid (3-hydroxy-2,4,6-trimethyl tetracosanoic acid) or by the number of acyl substituents. The major glycolipid presented a weak activity in vitro on mitochondria1 oxidative phosphorylation. These glycolipids and phthienoic acids could serve as virulence indicators.Mycobacterial cell walls are endowed with large amounts of glycolipids belonging to three classes, according to their structure: glycopeptidolipids called mycosides C, phenolic glycolipids, and trehalose-containing glycolipids (for recent reviews see [I -31). While dimycoloyl trehalose (called cord factor) is widespread among mycobacteria 111, the other glycolipids exhibit a more specific distribution in the genus Mycobacterium, allowing identification of species or subspecies by means of the presence of specific antigenic determinants [3]. In the course of establishing such a principle for identification of mycobacterial species, non-tuberculous mycobacteria have been examined, skirting the most serious pathogen Mycobacteriurn tuberculosis [3]. Seeking specific glycolipids of the tubercle bacillus, we recently discovered phenolic glycolipids in the Canetti strains [4]. In a detailed examination of the lipid content of these strains, we found unusual glycolipids and the structure of the major one is described here. This glycolipid is a polyacylated trehalose, where the acyl substituents are mainly phthienoic acids (Fig. 1) isolated so far only from virulent human and bovine strains of the tubercle bacillus [S, 61. MATERIALS AND METHODS Mycohacterial strains and culturesM . tuberculosis (H3,Ra, H37Rv, Canetti) and M . bovis (ATCC 19210 and BCG) were obtained from the Institut Pasteur (Paris). Cultures were grown for 3 weeks on Sauton medium at 37°C and were harvested by filtration during the stationary growth phase. Lipid extractionThe bacteria were kept in CHC13/CH30H (l/l, by vol.) for several days at room temperature to kill them. The cells were extracted twice with CHC13/CH30H (2/1, by vol.) and the crude extracts were po...

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 INHsensitive 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.

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