Location of the mycolyl ester substituents in the cell walls of mycobacteria.

The question of the precise location of mycolic acids, the single most distinctive cell wall entity of members of the Mycobacterium genus, has now been addressed. The free hydroxyl functions of the arabinogalactan component of the mycobacterial cell wall were O-methylated under conditions in which the mycolyl esters were not cleaved. Subsequent replacement of the mycolyl functions with O-ethyl groups resulted in an acid- and base-stable differentially O-alkylated surrogate polysaccharide, more amenable to analysis. Complete hydrolysis, reduction, acetylation, and gas chromatography/mass spectrometry revealed the unexpected finding that the mycolyl substituents were selectively and equally distributed on the 5-hydroxyl functions of terminal- and 2-linked arabinofuranosyl (Araf) residues. Further analysis of the O-alkylated cell wall through partial acid hydrolysis, NaB[2H]4 reduction, pentadeuterioethylation, and gas chromatography/mass spectrometry demonstrated that the mycolyl units are clustered in groups of four on the previously recognized nonreducing terminal pentaarabinosyl unit [beta-Araf-(1----2)-alpha-Araf)2-3, 5-alpha-Araf. However, only about two-thirds of the available pentasaccharide units are so substituted. Thus, the antigenicity of the arabinan component of mycobacterial cell walls may be explained by the fact that about one-third of the pentaarabinosyl units are not mycolyated and are available for interaction with the immune system. On the other hand, the extreme hydrophobicity and impenetrability of the mycobacterial cell may be explained by the same motif also acting as the fulerum for massive esterified paraffin residues. New fundamental information on the structure of mycobacterial cell walls will aid in our comprehension of its impenetrability to antibiotics and role in immunopathogenesis and persistence of disease.

Distribution of a novel mycolic acid in species of the genus Mycobacterium.

We found that Mycobacterium porcinum ATCC 33776T (T = type strain) contains a new kind of mycolic acid with a methoxy group at the omega-1 position. This mycolic acid was identified by comparing it with the previously described methoxymycolic acids. The patterns of mycolic acid methyl esters from 418 strains belonging to 44 species of mycobacteria were studied by using thin-layer chromatography. In addition to M. procinum ATCC 33776T, representative strains of M. porcinum, Mycobacterium fortuitum, "Mycobacterium peregrinum," Mycobacterium senegalense, and a recently isolated Mycobacterium sp. contained appreciable amounts of the newly described mycolic acid.

[Identification of twenty-eight species mycobacteria with their cellular fatty acids by capillary gas chromatography].

Cellular fatty acid (FA) of 28 species of mycobacteria were analysed by capillary gas chromatography (GC). There were more than sixty volatile FA before C12:0 have no any difference between species both in quality and quantity. The identification scheme of Mycobacterium based on cellular FA was presented here. According to this scheme, all the tested species could be divided into 8 groups by the FA peak height order of C18:1, C18:0 and TBSA which were quite stable; after that, 17 species (60%) might be identified to species level with help of some distinctive FA such as C19:0, C21:0. Most pathogenic bacteria could be separated by the scheme (M. tuberculosis, M. bovis and some complexes). This scheme was also demonstrated to be objective rationality by computer cluster analysis with cellular FA components.

Determination of in vivo and in vitro drug effects of mycobacteria from the mass spectrometric analysis of single organisms.

Laser microprobe mass analysis of single bacterial organisms allows the determination of their intrabacterial ratio of sodium-to-potassium ions and the registration of fragment ions originating from their organic bacterial cell matrices as mass fingerprint spectra. It has been established previously that the intrabacterial cation ratio provides information on the physiological state of an individual bacterial cell. In the present experiments it is also shown, with different cultivable mycobacterial species and strains (drug sensitive and resistant) exposed to various drugs, that data derived from the evaluation of the mass fingerprint spectra reflect changes in the degree of impairment. The analysis of Mycobacterium leprae derived from a limited number of skin biopsies of lepromatous/borderline lepromatous leprosy patients under World Health Organization-recommended multiple-drug therapy (WHO/MDT) showed impairment of the organisms with both of the methods of measurement in proportion to the duration of treatment except in one case. In one M. leprae population from a patient who had been treated for 19 months, the fingerprint evaluation gave the first evidence for an insufficient response to treatment. This was further confirmed by the unusual frequency distribution of the Na+,K+ ratios which revealed the existence of two subpopulations, one impaired and one unimpaired.

A new type of serine-containing glycopeptidolipid from Mycobacterium xenopi.

An unknown immunogenic glycopeptidolipid, named GPL X-1, was isolated from Mycobacterium xenopi, which is a nontuberculous mycobacterium responsible for pulmonary and disseminated infectious diseases mainly occurring in immunocompromised patients. The glycopeptidolipid was purified until homogeneity, in the native form, by direct phase high performance liquid chromatography. A new route is proposed for the structural elucidation of its unusual lipopeptidic core. The presence of allothreonine (aThr), phenylalanine, and serine in the molecular ratio 1:1:2, respectively, was established by reverse phase high performance liquid chromatography analysis of the phenylthiocarbamyl amino acid derivatives. From the molecular mass (1828 Da) of the native glycopeptidolipid, determined by cesium ion liquid secondary ion mass spectrometry using the amphipathic triethylene glycol monobutyl ether matrix, it was deduced that the tetrapeptide was amidified by a dodecanoic acid. The complete structure, C12-Ser-Ser-Phe-aThr-OCH3, of the lipopeptidic core was established by pyrolysis electron impact-mass spectrometry of the native glycopeptidolipid. To date, this is the first example of a mycobacterial glycopeptidolipid with a C12-tetrapeptidic core containing serine. A novel approach, based on two dimensional 1H,1H correlated spectroscopy analysis of the native and peracetylated GPL X-1, was developed, allowing the structural determination of the monosaccharidic residues with their alkali-labile groups "in situ" on the whole complex molecule. 2-O-Acyl-alpha-L-Rhap, alpha-L-Rhap, 2,4-di-O-acyl-6-deoxy-alpha-L-Glcp, 2,3,4-tri-O-Me-alpha-L-Rhap, and 3-O-Me-6-deoxy-alpha-L-Talp were identified, where Me, Rhap, and Talp are methyl, rhamnopyranosyl, and talopyranosyl, respectively. The latter two were localized at the carbohydrate non-reducing ends, and the C-3's of the remaining monosaccharide residues were found involved in the interglycosidic linkage. The alpha anomeric configurations were inferred from the JC-1,H-1 heteronuclear coupling constants, and the L absolute configurations for all the monosaccharide residues were established by gas chromatography analysis of the trimethylsilyl (+/-)-2-butyl glycosides. Finally, by pyrolysis electron impact mass spectrometry of peracetylated GPL X-1, the following tetrasaccharide appendage structure was proposed: 2,3,4-tri-O-Me-L-Rhap(alpha 1----3)-2-O-lauryl-L-Rhap(alpha 1----3)-L-Rhap- (alpha 1----3)-2,4-di-O-(acetyl,lauryl)-6-deoxy-alpha-L-Glcp. Compared to the oligosaccharidic glycopeptidolipid structures, the particular features of the GPL X-1 tetrasaccharide structure arise from the presence of monosaccharide residues esterified by C12 fatty acids and from the absence of the basal disaccharide core, L-Rhap-(alpha 1----2)-6-deoxy-alpha-L-Talp.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Further stereochemical studies of phthiocerol and phenol phthiocerol in mycobacteria.

Mycobacterium tuberculosis, M. marinum and some other pathogenic species elaborate waxes A, based on a long-chain beta-diol (phthiocerol and companion compounds) and polymethyl-branched fatty acids. The stereochemical studies conducted on waxes A showed that those of M. tuberculosis, M. leprae and M. kansasii differ from waxes A isolated from M. marinum and M. ulcerans by the absolute configuration of the methyl-branched chiral centres occurring in both the long-chain beta-diols and the fatty acyls. Furthermore, the two mycobacterial groups also differ in the stereochemistry of the beta-diol chiral centres.

Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis.

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.

Chemotypes of Mycobacterium malmoense based on glycolipid profiles.

Thin-layer chromatographic analysis of 72 Finnish clinical mycobacterial isolates presumptively identified as Mycobacterium malmoense revealed four major glycolipid profiles with two minor variations. An additional glycolipid profile was found in three British M. malmoense-like strains. No clear distinction between the strains could be made by means of gas chromatography of cellular fatty acids. The two M. malmoense-specific constituents, 2-methyleicosanoate and 2,4,6-trimethyltetracosanoate, were detected in all strains. The frequency of chemotypes other than that of the type strain was 8% among the Finnish isolates. This variation should be recognized when confirmative identification of mycobacteria is based on thin-layer chromatography of glycolipid extracts.

Utilizaçäo do eletroforese em gel de poliacrilamida-dodecil sulfato de sódio (SDS-PAGE) na identificaçäo de bactérias do gênero Mycobacterium / Utilization of sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) in the identification of bacteria of the geners Mycobacterium

Estudou-se a utilizaçäo da técnica de eletroforese em gel de poliacrilamida-dodecil sulfato de sódio (SDS-PAGE), na identificaçäo do gênero Mycobacterium. Treze espécies diferentes, foram utilizadas para preparar extratos de células íntegras, rompidas por ultra-som, pérolas de vidro e solubilizadas em fenol-ácido acético-água. O método do ultrassom forneceui as melhores separaçöes eletroforéticas. A extraçäo fenol-acéticafalhou em fornecer perfis adequados. A desintegraçäo mecânica por pérolas de vidro, mostrou-se exeqüível na produçäo de extratos e os perfis obtidos foram bastante distintos. As bandas de polipeptideos obtidas por SDS-PAGE foram discriminatórias e possibilitaram a identificaçäo das espécies estudadas. O M. tuberculosis e o M. bovis exibiram perfis bastante semelhantes, ao contrário das espécies M. avium, M. intracellulare e M. scrofulaceum cujos perfis foram diferentes, principalmente, desta última espécie. O M. fortuitum, M. smegmatis e M. phlei também mostraram perfis distintos. O número de polipeptídeos das espécies estudadas variou entre 20 e 46. A SDS-PAGE é um procedimento alternativo que pode ser utilizado na identificaçäo de espécies do gênero Mycobacterium.

High-performance liquid chromatography patterns of mycolic acids as criteria for identification of Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium smegmatis.

Rapidly growing mycobacteria of clinical significance were identified by mycolic acids detected with high-performance liquid chromatography. Mycolic acids from whole cells were extracted, derivatized, and detected by a modified high-performance liquid chromatography procedure in less than 3 h. Use of an internal standard allowed differentiation of Mycobacterium chelonae and Mycobacterium fortuitum by comparison of relative retention times. Peak height ratios were used for subidentification of M. chelonae strains; however, M. fortuitum and Mycobacterium smegmatis could not be separated by this system.

A novel mycolic acid in a Mycobacterium sp. from the environment.

A fast-growing, non-photochromogenic mycobacterium isolated from the environment exhibited, on thin-layer chromatograms, a characteristic pattern of mycolates composed of unsaturated mycolates and also an unknown more polar component. Spectroscopic analysis and chemical degradation showed that this latter component was a novel mycolic acid containing a methoxy group at the omega-1 position (instead of omega-17 and omega-18 in known methoxymycolates), and two double bonds in the long mero aldehyde chain (instead of one as in known mycolates with additional oxygenated groups).

Isolation and analysis by the reductive-cleavage method of linkage positions and ring forms in the Mycobacterium smegmatis cell-wall arabinogalactan.

The Mycobacterium smegmatis arabinogalactan polysaccharide has been isolated from the cell wall by saponification and extraction to remove lipids and subsequent solubilization by treatment with lysozyme. Analysis for neutral sugars demonstrated the presence of D-arabinose and D-galactose in a ratio of 3:1, respectively. Reductive cleavage of the fully methylated polysaccharide in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate and subsequent acetylation in situ gave six partially methylated 1,4-anhydroalditol acetates as the major products and three partially methylated 1,5-anhydroalditol acetates as minor products. Partially methylated 1,5-anhydroalditol acetates were not formed when reductive cleavage was accomplished with triethylsilane and a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate as the catalyst, demonstrating that the polysaccharide is exclusively comprised of furanosyl residues. The partially methylated anhydroalditols so produced were identified by comparison to authentic standards. Their identifies are consistent with the presence in the M. smegmatis arabinogalactan of an octasaccharide repeating unit comprised of a nonreducing terminal D-arabinofuranosyl group, a 2-O-linked D-arabinofuranosyl residue, three 5-O-linked D-arabinofuranosyl residues, a 3,5-di-O-linked D-arabinofuranosyl residue, a 5-O-linked D-galactofuranosyl residue, and a 6-O-linked D-galactofuranosyl residue.

Primary structure of a 7Fe ferredoxin from Streptomyces griseus.

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12,291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the Fe-S clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two Fe-S clusters are conserved.

The carbohydrate- and lipid-containing cell wall of mycobacteria, phenolic glycolipids: structure and immunological properties.

Phenolic glycolipids were first discovered as cell-wall constituents of M. bovis, M. bovis BCG, M. marinum, and M. kansasii. Recently, such compounds were also isolated from M. leprae and have been shown to be specific-species serological markers. Moreover, they seem to be involved, in the case of lepromatous leprosy, in the stimulation of the suppressor T-cells. The functional activities of these phenolic glycolipids over the immune cells stimulation emphasized the role played by these molecules in the mycobacteria pathogenicity. Phenolic glycolipids have also been found in M. gastri and M. tuberculosis strain Canetti. From a structural point of view, these glycolipids contain the same aglycon moiety mainly assigned to phenolphthiocerol diester while the sugar part structure confers to some of these glycolipids their antigenic specificity. The search of immunoreactive glycolipids and their function analysis remain a challenge for chemists and immunologists for the understanding of the mycobacteria pathogenicity.

Characteristic new members of the phthiocerol and phenolphthiocerol families from Mycobacterium ulcerans.

Diacyl phthiodiolone A and phenolphthiodiolone A lipids were isolated from two strains of Mycobacterium ulcerans. The diol units of the phthiodiolone A and phenolphthiodiolone A components were shown to have erythro stereochemistry by infrared spectroscopy and proton nuclear magnetic resonance of an acetal derivative. This stereochemistry is shared only by related diols from M. marinum, the diols from M. bovis, M. kansasii, M. leprae and M. tuberculosis having threo stereochemistry.

Gas chromatographic characterization of the relationship between some Mycobacterium avium and Mycobacterium paratuberculosis strains.

Mycobacterium avium strain P-55 and M. avium strain DENT differ from M. avium strain 16909-338 on the basis of their fatty acid spectra (C14:0, C18:0 and tuberculostearic [TBS] acids) studied by multivariate statistical analyses. Strains P-55 and DENT are closer to M. paratuberculosis strain 5889 than to M. avium strain 16909-338, a finding which is in harmony with earlier immunological observations. The recently isolated M. paratuberculosis strain 385 has proved different from M. paratuberculosis strain 5889.

[In vitro activation of immune cells by a mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate, derived from Gordona aurantiaca].

A mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate (GaGM), derived from Gordona aurantiaca, an acid-fast bacteria closely related taxonomically to Mycobacterium, was investigated for its immune adjuvant activity in vitro. The liposomes containing GaGM showed strong mitogenic effects on murine spleen cells at the doses used (25-100 micrograms/ml), but not on T-cell-depleted spleen cells or macrophage-depleted spleen cells. These results suggest that the mitogenic property of liposomes containing GaGM differs from that of such as lipopolysaccharide, a B-cell mitogen and that its mitogenic effects depend on the presence of macrophages. In addition, liposomes containing GaGM augmented the mixed lymphocyte reaction (MLR) and in vitro induction of cytotoxic T-lymphocytes (CTLs) against allogeneic tumor cells. These results suggest that liposomes containing GaGM have immune adjuvant properties in vitro and the adjuvant activity may be related to such cytokines as interleukin-1 and -2.

Gas chromatography of mycobacterial fatty acids and alcohols: diagnostic applications.

Capillary gas chromatography of cellular fatty acids and alcohols has been used as a routine method for a period of two years in the mycobacterial diagnostic laboratory of Statens institutt for folkehelse, Oslo, Norway. All mycobacteria (165 isolates) other than Mycobacterium tuberculosis (MOTT) and 24 randomly selected M. tuberculosis isolates were studied. Twelve characteristic lipid constituents allowed the construction of a diagnostic scheme. Without exceptions, all 36 examined isolates belonging to the M. tuberculosis-complex were characterized by a relatively high concentration level of hexacosanoic acid (mean: 4%, range: 1-13%), low level of tetracosanoic acid (mean: 1%, range: 0.1-3%), lack of methylbranched acids other than tuberculostearic acid, and lack of fatty alcohols. Members of the MAIS-complex (73 isolates) were all characterized by the general presence of the fatty alcohols 2-octadecanol (mean: 2%, range: 0.1-5%) and 2-eicosanol (mean: 7%, range: 2-21%), relatively high levels of tetracosanoic acid (mean: 5%, range: 1-15%) and lack (or trace) of hexacosanoic acid and methylbranched acids other than tuberculostearic acid. All 16 isolates of M. gordonae were easily recognized by their unique lack of tuberculostearic acid and their content of 2-methyl-tetradecanoic acid (mean: 5%, range: 2-12%), and the M. xenopi isolates were the only examined strains containing the fatty alcohol 2-docosanol (mean: 9%, range: 2-13%). The six M. malmoense strains contained the two unique constituents 2-methyl eicosanoic acid (mean: 3%, range: 1-4%) and 2,4,6-trimethyl tetracosanoic acid (mean: 3%, range: 2-4%). The ten strains of M. kansasii were characterized by 2,4-dimethyl tetradecanoic acid (mean: 5%, range: 1-11%), whereas the seven strains of M. marinum shared 2,4-dimethyl hexadecanoic acid (mean: 4%, range 0.2-12%) as a specific marker.