1 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
2 Department of Biological Sciences, Imperial College, London, SW7 2AZ, UK
Correspondence
Gurdyal S. Besra
g.besra{at}bham.ac.uk
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ABSTRACT |
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INTRODUCTION |
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PGLs were discovered some years ago in pathogenic mycobacteria, such as Mycobacterium bovis (Chatterjee et al., 1989; Daffé et al., 1987
), Mycobacterium kansasii (Fournie et al., 1987
; Riviere et al., 1987
) and Mycobacterium marinum (Navalkar et al., 1965
; Sarda & Gastambide-Odier, 1967
). PGLs all possess similar aglycon structures, mainly formed from polymethyl-branched fatty (mycocersoic) acids esterified to a diol (phenolphthiocerol A) unit. However, their oligosaccharide units, and hence their inherent antigenicity, are species-specific (Brennan, 1988
; Daffé, 1989
; Dobson et al., 1990
; Minnikin, 1982
). The identification of a PGL as a species-specific antigen of the leprosy bacillus renewed interest in this molecule as a potential antigen for serodiagnosis (Hunter et al., 1982
). LOSs were first described by Ballou and colleagues as pyruvylated forms of glycosylated acyltrehaloses in mycobacteria (Kamisango et al., 1985
). More recently, the full extent of glycosylation, ubiquity, antigenicity, and species-specificity of this class of glycolipids have been demonstrated by several research groups (Besra et al., 1993a
, 1994
; Daffé et al., 1991
; Gilleron et al., 1993
).
The third class of glycolipids, the GPLs, constitute a major outer-layer glycolipid of several non-tuberculous mycobacteria (Aspinall et al., 1995; Brennan & Nikaido, 1995
). For instance, they are the major glycolipids found within disseminated Mycobacterium aviumintracellulare complex (MAC) infections, the most common bacterial infection in AIDS patients (McNeil et al., 1987
). The GPLs not only represent surrogate markers for mycobacterial species, such as Mycobacterium xenopi (Besra et al., 1993b
; Riviere & Puzo, 1991
), but also provide the basis for MAC serovariation (Krzywinska et al., 2004
). GPLS are also found in saprophytic mycobacteria, such as Mycobacterium smegmatis, and in animal pathogens, such as M. avium paratuberculosis, Mycobacterium porcinum and Mycobacterium senegalense (Camphausen et al., 1988
).
The GPLs contain a common tripeptide-amino alcohol core, D-phe-D-allo-thr-D-ala-L-alaninol, modified on the amino-terminal D-Phe with a mixture of amide-linked 3-hydroxy and 3-methoxy C2634 fatty acids (Belisle et al., 1993b; Brennan & Goren, 1979
; Daffé et al., 1983
). The lipopeptide core is invariably substituted with 6-deoxytalose (6-dTal), which is linked to the allo-Thr residue and may be variably O-acetylated. Another invariant substitution involves linking of rhamnose, which may be variably O-methylated, to the terminal alaninol residue (Patterson et al., 2000
) (Fig. 1
). These apolar GPLs are termed the non-serovar-specific GPLs (nsGPLs) and are found in all members of the MAC and M. smegmatis (Aspinall et al., 1995
; Belisle & Brennan, 1989
; Brennan & Goren, 1979
). The nsGPLs can be further extended with haptenic oligosaccharides linked to the 6-dTal to produce the highly antigenic serovar-specific GPLs (ssGPLs), which provide the basis for MAC serovariation (Krzywinska et al., 2004
). Although the biosynthesis of the GPLs has not been fully elucidated, models for the synthesis of the lipopeptide core (Belisle et al., 1993a
, b
), its glycosylation (Billman-Jacobe et al., 1999
) and its methylation (Eckstein et al., 2003
) have been proposed. Briefly, an N-3-hydroxy-acyl-Phe acceptor is modified by the sequential addition of Thr, Ala and alaninol residues by the peptide synthetase product of mps (Billman-Jacobe et al., 1999
). Subsequently glycosyltransferases, GtfB and GtfA, catalyse the glycosylation of the allo-threonine and alaninol residues with rhamnose and 6-deoxytalose respectively (Eckstein et al., 2003
). A series of methyltransferases methylate the rhamnosyl residue of GPLs in situ to form nsGPLs (Jeevarajah et al., 2002
; Patterson et al., 2000
). MtfD and MtfC have been proposed to methylate the Rha residue at positions 3 and 4 respectively in M. avium (Eckstein et al., 2003
). Methylation at position 2 also occurs in Mycobacterium butyricum and M. smegmatis (Jeevarajah et al., 2004
; Khoo et al., 1995
) and is presumably carried out by an unidentified third methyltransferase.
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Recently, we have shown that azole antifungal agents are potent inhibitors of mycobacterial growth (McLean et al., 2002b). It is well established that azole antifungal agents are also potent inhibitors of cytochrome P450-monooxygenases (Lamb et al., 1999
), several of which act on a variety of lipid substrates (Lewis, 1996
). Given the complexity of the mycobacterial envelope in terms of lipophilic compounds, in this study we examined the inhibitory properties of the antifungal agents econazole and clotrimazole in relation to the synthesis of complex lipids in M. smegmatis.
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METHODS |
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Determination of MIC.
M. smegmatis mc2155 was used to test the efficacy of two azole antifungal drugs, econazole and clotrimazole, and three anti-mycobacterial drugs, ethionamide (ETH), ethambutol (EMB) and isoniazid (INH). M. smegmatis was grown in LB medium at 37 °C with agitation at 180 r.p.m. until exponential phase was reached (OD600 0·70·8). MIC values were measured by serial dilution of M. smegmatis cultures onto LB agar plates containing the drugs in a concentration range of 530 µg ml1 for ETH and 0·0510 µg ml1 for the other drugs. All assays were performed in triplicate and the experiment was repeated twice.
Effect of azoles on the growth of M. smegmatis.
M. smegmatis mc2155 was grown overnight in LB medium at 37 °C with agitation at 180 r.p.m. An aliquot (3 ml) of the culture was transferred into a flask containing fresh Sauton's medium (50 ml) supplemented with 0·05 % tyloxapol and returned to the orbital incubator and agitated at 180 r.p.m. When the OD600 reached 0·1, econazole or clotrimazole was added in a concentration range of 520 µg ml1 in a final concentration of 0·2 % DMSO. DMSO (0·2 %) was also added to control cultures. Growth of the cultures was followed by regular OD600 measurements over 30 h.
Labelling of lipids and mycolic acids from M. smegmatis.
Radioactive labelling of lipids was performed to determine the effect of the azole antifungal drugs on the M. smegmatis cell envelope. M. smegmatis mc2155 was grown in Sauton's medium (5 ml) supplemented with 0·05 % tyloxapol at 37 °C in an orbital incubator at a speed of 180 r.p.m. to an OD600 of 0·3. Econazole (10 µg ml1) or clotrimazole (10 µg ml1) was then added and the cultures reincubated with gentle agitation for 1 h. Sodium [1,2-14C]acetate (1 µCi ml; 37 kBq ml1) was added and the cultures reincubated for a further 6 h. The cells were then harvested; the pellet was washed and freeze-dried.
Extraction and analysis of lipids by TLC.
Labelled lipids were extracted according to the procedures of Dobson et al. (1985). Initially, non-polar lipids were extracted from cells by mixing with 2 ml CH3OH/0·3 % NaCl (100 : 10, v/v) and 1 ml petroleum ether (b.p. 4060 °C) on a mixing rotator for 15 min. The mixture was then centrifuged (3 min at 3000 r.p.m.), and the upper layer removed and stored. Petroleum ether (1 ml) was then added to the lower fraction, and the solution was mixed and recentrifuged as previously described. The combined upper layers containing the apolar lipids were dried under a gentle stream of nitrogen. The apolar lipids were resuspended in CHCl3/CH3OH (2 : 1, v/v) (200 µl); aliquots (20 µl) were dried in a scintillation vial and then mixed with 10 ml scintillation fluid and their radioactivity measured. The polar lipids were further extracted by adding 2·3 ml CHCl3/CH3OH/0·3 % NaCl (90 : 100 : 30, by vol.) to the lower aqueous methanol layer. The solution was mixed for 1 h, centrifuged and the supernatant removed and stored. The above step was repeated twice by the addition of 0·75 ml CHCl3/CH3OH/0·3 % NaCl (50 : 100 : 40, by vol.) and mixing for 30 min. Defatted cells were kept for further analysis (see below). A mixture of CHCl3/0·3 % NaCl (1 : 1, v/v; 2·6 ml) was finally added to the pooled supernatants. The mixture was mixed for 5 min, centrifuged and the lower layer recovered and dried under a gentle stream of nitrogen. The polar lipids were resuspended in CHCl3/CH3OH (2 : 1, v/v; 200 µl); an aliquot (20 µl) was dried in a scintillation vial and then mixed with 10 ml scintillation fluid and its radioactivity measured. Equivalent amounts (20 000 c.p.m.) of each sample were separated by two-dimensional (2D) analytical TLC for both apolar (systems A, B, C and D) and polar lipids (systems D and E) as described by Dobson et al. (1985)
and visualized by exposing to Kodak X-Omat film for 5 days.
The 14C-labelled mycolic acid methyl esters (MAMEs) were then extracted from the defatted cells as described previously (Kremer et al., 2000). Briefly, mycolic acids were released from the defatted cells by treatment with 1 ml 5 % tetrabutyl ammonium hydroxide at 100 °C, overnight. The samples were allowed to cool and CH2Cl2 (4 ml), water (3 ml) and CH3I (100 µl) were added and the entire contents mixed for 30 min. The upper aqueous layer was discarded, and the lower organic layer washed twice with water (4 ml). The lower organic layer containing the MAMEs was dried under a gentle stream of nitrogen, resuspended in CH2Cl2 (200 µl) and an aliquot (20 µl) dried in a scintillation vial and then mixed with 10 ml of scintillation fluid and its radioactivity measured. Equivalent amounts (20 000 c.p.m.) were separated by TLC developed in petroleum ether/acetone (95 : 5, v/v) and exposed to Kodak X-Omat film for 5 days.
In order to remove acetyl/acyl substituents and simplify the TLC patterns of the base-stable GPLs, an alkali treatment was performed. After the addition of 4 ml CHCl3/CH3OH/0·8 M NaOH (10 : 10 : 3, by vol.) to the dried polar lipid fractions, the solution was incubated at 55 °C for 15 min, allowed to cool at room temperature, then CHCl3 (1·75 ml) and H2O (0·25 ml) were added. The solution was mixed on a rotator for 10 min and centrifuged at 4000 r.p.m. for 5 min. The upper layer was removed and the lower organic layer washed twice with 2 ml CHCl3/CH3OH/H2O (3 : 47 : 48, by vol.). The lower organic layer containing the base-stable de-O-acylated GPLs (dGPLs) was dried under a gentle stream of nitrogen and resuspended in CHCl3/CH3OH (2 : 1; 200 µl); an aliquot (20 µl) was dried in a scintillation vial and then mixed with 10 ml scintillation fluid and its radioactivity measured. Equal counts (20 000 c.p.m.) were subjected to 2D TLC in system D or 1D TLC in CHCl3/CH3OH/H2O (90 : 10 : 1, by vol.). The 2D TLCs were visualized by exposing to Kodak X-Omat film for 5 days and 1D TLCs were quantified by phosphoimaging over 3 days.
Purification of GPLs from M. smegmatis.
Apolar lipids were extracted from 20 g dried cells by stirring in 440 ml methanolic saline (40 ml 0·3 % NaCl and 400 ml CH3OH) and 440 ml petroleum ether for 2 h. The cells were centrifuged at 3000 r.p.m. for 5 min. The resulting biphasic solution was separated and the upper layer containing the apolar lipids was recovered. An additional 440 ml petroleum ether was added, followed by mixing and harvest as described above. The two upper petroleum ether fractions were combined and dried under reduced pressure.
To isolate the polar lipids, 520 ml CHCl3/CH3OH/0·3 % NaCl (9 : 10 : 3, by vol.) was added to the lower layer and the solution stirred for 4 h. The mixture was filtered and the filter cake re-extracted twice with 170 ml CHCl3/CH3OH/0·3 % NaCl (5 : 10 : 4, by vol.). To the combined filtrates CHCl3 (290 ml) and 0·3 % NaCl (290 ml) were added. The mixture was stirred for 1 h, allowed to settle, and the lower layer containing the polar lipids dried under reduced pressure. An alkali treatment was then performed on the dried polar lipids as described above. The base-treated polar lipids were then separated on 10 cmx20 cm plastic-backed TLC plates of silica gel 60 F254 (Merck), run in CHCl3/CH3OH/H2O (90 : 10 : 1, by vol.). The plates were then sprayed with 0·01 % 1,6-diphenyl-1,3,5-hexatriene dissolved in petroleum ether/acetone (9 : 1, v/v) and the dGPLs visualized under UV light. Following detection the plates were redeveloped in toluene; the corresponding dGPL bands were scraped from the plates and extracted from the silica gel using CHCl3/CH3OH (2 : 1, v/v) and subjected to mass spectroscopy analyses (see below).
Chemical derivatization for CAD-ES-MS/MS analysis.
Per-O-deuteriomethylation was performed using the NaOH procedure as described previously (Dell et al., 1993). Briefly, NaOH pellets were crushed with DMSO to form a slurry. An aliquot of this slurry was added to dried dGPLs along with 1 ml deuteriomethyl iodide. The reaction was terminated by the addition of water, and per-O-deuteriomethylated dGPLs recovered by chloroform extraction. The chloroform layer was washed several times with water to remove any impurities. After derivatization the reaction products were purified on Sep-Pak C18 (Waters) as previously described (Dell et al., 1994
).
CAD-ES-MS/MS analysis.
CAD-ES-MS/MS spectra were acquired using a Q-TOF (Micromass) instrument. The per-O-methylated dGPLs were dissolved in methanol before loading into a nano-capillary (Proxeon). A potential of 1·5 kV was applied to a nanoflow tip to produce a flow rate of 1030 nl min1. The drying gas used was nitrogen and the collision gas was argon, with the collision gas pressure maintained at 104 mbar. Collision energies used were typically between 50 and 80 eV. Data were acquired and processed using Masslynx software (Micromass). The instrument was pre-calibrated using a 1 pmol µl1 solution of [Glu1]-fibrinopeptide B in acetonitrile/5 % aqueous acetic acid (1 : 3, v/v).
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RESULTS AND DISCUSSION |
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By definition, the MIC is the minimal concentration of the drug necessary to inhibit the growth of 99 % of the bacilli. The MIC values for econazole and clotrimazole as well as three established anti-mycobacterial drugs, ETH, INH and EMB, were determined for M. smegmatis. The results clearly showed that M. smegmatis is sensitive to econazole and especially clotrimazole, with MIC values of 2 and 0·5 µg ml1 respectively. These values were lower than those for ETH (MIC 20 µg ml1) and INH (MIC 4 µg ml1). Clotrimazole was also more effective than EMB (MIC 2 µg ml1). When M. smegmatis was cultured in Sauton's medium, the MIC values were higher than those determined on agar plates, with econazole at 20 µg ml1 and clotrimazole at 15 µg ml1, respectively, resulting in total inhibition of M. smegmatis growth (Fig. 2). DMSO, used as a carrier for the drugs in these experiments, did not affect the growth of M. smegmatis, nor did the use of a different liquid medium (LB broth, data not shown).
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Based on their migration in solvent system D and charring with -naphthol, which is specific for glycolipids, these lipids were tentatively assigned as belonging to the glycopeptidolipid (GPL) family of mycobacterial glycolipids. In order to confirm that they belonged to the alkali-stable family of GPLs and to simplify their profiles, a mild base treatment was performed on the polar lipid extracts to remove the acetyl substituents, and 2D TLC patterns were again produced using solvent system D. Equivalent amounts (20 000 c.p.m.) of control, econazole-treated and clotrimazole-treated samples were applied to 2D TLC plates and developed in direction 1, CHCl3/CH3OH/H2O (100 : 14 : 0·8, by vol.), and direction 2, CHCl3/acetone/CH3OH/H2O (50 : 60 : 2·5 : 3, by vol.) (Fig. 3ac
). To allow further resolution and easier comparison, the same samples (20 000 c.p.m.) were developed in a 1D TLC solvent system, CH3Cl/CH3OH/H2O (90 : 10 : 1, by vol.), and analysed by phosphoimaging (see inset of Fig. 6
).
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Glycopeptidolipids are major species- or type-specific cell surface lipids of several atypical mycobacteria including the pathogenic members of the MAC and the fast-growing saprophytic organism M. smegmatis (Aspinall et al., 1995). Belisle and co-workers have identified several genes encoding glycosyltransferases and methyltransferases involved in the biosynthesis of haptenic oligosaccharides of ssGPLs of M. avium serovar 2. It is believed that nsGPLs are intermediate precursors of ssGPLs in M. avium (Belisle et al., 1991
). However, major questions remain concerning the assembly of the nsGPL lipopeptide core. GPLs have been demonstrated to have a very important role in maintaining the surface properties of the mycobacterial cell envelope (Vergne & Desbat, 2000
). The use of M. smegmatis, which produces only nsGPLs, allowed the identification of a gene, designated mps, encoding a putative peptide synthetase containing four modules, each containing domains for cofactor binding and for amino acid recognition and adenylation involved in the biosynthesis of the common lipopeptide core (Billman-Jacobe et al., 1999
). Recently, the presence of an O-methyltransferase which catalyses the conversion of the fatty acid from 3-hydroxy to the 3-methoxy form was reported (Jeevarajah et al., 2002
).
In this study, we provide evidence that the biosynthesis of nsGPLs from M. smegmatis is inhibited by the azole antifungal drugs econazole and clotrimazole. M. smegmatis possesses four types of nsGPLs varying from one to another by the presence or absence of O-methyl groups on the fatty acyl chain and/or the rhamnose. Based on these structures, two biosynthetic pathways are possible. The most polar dGPL-I, which contains neither of these O-methyl groups, could be a precursor to dGPL-II, which is methylated on the N-acyl chain, and dGPL-III, which is methylated on the terminal 2,3,4-tri-O-methyl-Rha. Both dGPL-II and -III could give rise to dGPL-IV by further methylation. Both routes are plausible, as one methylation event need not affect the other. It appears likely that econazole and clotrimazole inhibit an enzyme involved in the early steps of GPL biosynthesis, possibly at the stage of lipopeptide synthesis. In the case of econazole the inhibition is weaker, possibly reflecting a weaker binding to its target. In addition, this would allow any dGPL-I and -II present prior to inhibition to be converted into dGPL-III and -IV, as we observed in our analyses. However, treatment with clotrimazole results in strong inhibition of dGPL synthesis. It has been recently demonstrated that azole antifungal drugs bind tightly to M. tuberculosis CYP121 (McLean et al., 2002a) and CYP51 (Bellamine et al., 1999
; Guardiola-Diaz et al., 2001
) and M. smegmatis CYP51 (Jackson et al., 2003
). Although the present study does not allow us to identify an intracellular target, it would appear likely that the drugs inhibit P450(s) in vivo. Moreover, the synthesis of the amide-linked long-chain fatty acid of nsGPLs represents a likely target as it is hydroxylated and it represents a precursor common to all GPLs. Given the roles of characterized P450s for instance CYP4C1 catalyses fatty acid hydroxylation in the cockroach (Lewis, 1996
) and CYP107 from Saccharopolyspora erythraea catalyses 6-deoxyerythronilide hydroxylation (Nelson et al., 1993
) we speculate that a cytochrome P450 might be involved in fatty acid hydroxylation in the biosynthetic pathway of GPLs. This is believed to be the first report that a P450, whose action is inhibited by econazole and clotrimazole, may be involved in GPL biosynthesis.
Conclusion
These results have an important implication in understanding mycobacterial cell envelope synthesis and potentially cytochrome P450 function. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum, M. peregrinum, M. chelonae and M. abscessus. As a result we are currently seeking to identify the cellular target of econazole and clotrimazole which results in the inhibition of GPL biosynthesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Banerjee, A., Dubnau, E., Quemard, A. & 7 other authors (1994). inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263, 227230.[Medline]
Belisle, J. T. & Brennan, P. J. (1989). Chemical basis of rough and smooth variation in mycobacteria. J Bacteriol 171, 34653470.[Medline]
Belisle, J. T., Pascopella, L., Inamine, J. M., Brennan, P. J. & Jacobs, W. R., Jr (1991). Isolation and expression of a gene cluster responsible for biosynthesis of the glycopeptidolipid antigens of Mycobacterium avium. J Bacteriol 173, 69916997.[Medline]
Belisle, J. T., Klaczkiewicz, K., Brennan, P. J., Jacobs, W. R., Jr & Inamine, J. M. (1993a). Rough morphological variants of Mycobacterium avium. Characterization of genomic deletions resulting in the loss of glycopeptidolipid expression. J Biol Chem 268, 1051710523.
Belisle, J. T., McNeil, M. R., Chatterjee, D., Inamine, J. M. & Brennan, P. J. (1993b). Expression of the core lipopeptide of the glycopeptidolipid surface antigens in rough mutants of Mycobacterium avium. J Biol Chem 268, 1051010516.
Bellamine, A., Mangla, A. T., Nes, W. D. & Waterman, M. R. (1999). Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 96, 89378942.
Besra, G. S., McNeil, M. R., Khoo, K. H., Dell, A., Morris, H. R. & Brennan, P. J. (1993a). Trehalose-containing lipooligosaccharides of Mycobacterium gordonae: presence of a mono-O-methyltetra-O-acyltrehalose "core" and branching in the oligosaccharide backbone. Biochemistry 32, 1270512714.[CrossRef][Medline]
Besra, G. S., McNeil, M. R., Rivoire, B., Khoo, K. H., Morris, H. R., Dell, A. & Brennan, P. J. (1993b). Further structural definition of a new family of glycopeptidolipids from Mycobacterium xenopi. Biochemistry 32, 347355.[CrossRef][Medline]
Besra, G. S., Gurcha, S. S., Khoo, K. H., Morris, H. R., Dell, A., Hamid, M. E., Minnikin, D. E., Goodfellow, M. & Brennan, P. J. (1994). Characterization of the specific antigenicity of representatives of M. senegalense and related bacteria. Zentralbl Bakteriol 281, 415432.[Medline]
Billman-Jacobe, H., McConville, M. J., Haites, R. E., Kovacevic, S. & Coppel, R. L. (1999). Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis. Mol Microbiol 33, 12441253.[CrossRef][Medline]
Brennan, P. J. (1988). In Microbial Lipids, pp. 203298. Edited by C. Ratledge & S. G. Wilkinson. London: Academic Press.
Brennan, P. J. & Goren, M. B. (1979). Structural studies on the type-specific antigens and lipids of the Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum serocomplex. Mycobacterium intracellulare serotype 9. J Biol Chem 254, 42054211.[Medline]
Brennan, P. J. & Nikaido, H. (1995). The envelope of mycobacteria. Annu Rev Biochem 64, 2963.[CrossRef][Medline]
Camphausen, R. T., Jones, R. L. & Brennan, P. J. (1988). Antigenic relationship between Mycobacterium paratuberculosis and Mycobacterium avium. Am J Vet Res 49, 13071310.[Medline]
Chatterjee, D., Bozic, C. M., Knisley, C., Cho, S. N. & Brennan, P. J. (1989). Phenolic glycolipids of Mycobacterium bovis: new structures and synthesis of a corresponding seroreactive neoglycoprotein. Infect Immun 57, 322330.[Medline]
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[CrossRef][Medline]
Daffé, M. (1989). Further specific triglycosyl phenol phthiocerol diester from Mycobacterium tuberculosis. Biochim Biophys Acta 1002, 257260.[Medline]
Daffé, M., Lanéelle, M. A. & Puzo, G. (1983). Structural elucidation by field desorption and electron-impact mass spectrometry of the C-mycosides isolated from Mycobacterium smegmatis. Biochim Biophys Acta 751, 439443.[Medline]
Daffé, M., Lacave, C., Lanéelle, M. A. & Lanéelle, G. (1987). Structure of the major triglycosyl phenol-phthiocerol of Mycobacterium tuberculosis (strain Canetti). Eur J Biochem 167, 155160.[Abstract]
Daffé, M., McNeil, M. & Brennan, P. J. (1991). Novel type-specific lipooligosaccharides from Mycobacterium tuberculosis. Biochemistry 30, 378388.[CrossRef][Medline]
Dell, A., Khoo, K.-H., Panico, M., McDowell, R. A., Etienne, A. T., Reason, A. J. & Morris, H. R. (1993). In Glycobiology: a Practical Approach, pp. 187222. Edited by M. Fukuda & A. Kobata. Oxford: Oxford University Press.
Dell, A., Reason, A. J., Khoo, K.-H., Panico, M., McDowell, R. A. & Morris, H. R. (1994). Mass spectrometry of carbohydrate-containing biopolymers. Methods Enzymol 230, 108132.[Medline]
Dobson, G., Minnikin, D. E., Minnikin, S. M., Parlett, M., Goodfellow, M., Ridell, M. & Magnusson, M. (1985). Systematic analysis of complex mycobacterial lipids. In Chemical Methods in Bacterial Systematics, pp. 237265. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
Dobson, G., Minnikin, D. E., Besra, G. S., Mallet, A. I. & Magnusson, M. (1990). Characterisation of phenolic glycolipids from Mycobacterium marinum. Biochim Biophys Acta 1042, 176181.[Medline]
Eckstein, T. M., Belisle, J. T. & Inamine, J. M. (2003). Proposed pathway for the biosynthesis of serovar-specific glycopeptidolipids in Mycobacterium avium serovar 2. Microbiology 149, 27972807.[CrossRef][Medline]
Fournie, J. J., Riviere, M. & Puzo, G. (1987). Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. I. Evidence for tetrasaccharide structure of the oligosaccharide moiety. J Biol Chem 262, 31743179.
Gilleron, M., Vercauteren, J. & Puzo, G. (1993). Lipooligosaccharidic antigen containing a novel C4-branched 3,6-dideoxy-alpha-hexopyranose typifies Mycobacterium gastri. J Biol Chem 268, 31683179.
Guardiola-Diaz, H. M., Foster, L. A., Mushrush, D. & Vaz, A. D. (2001). Azole-antifungal binding to a novel cytochrome P450 from Mycobacterium tuberculosis: implications for treatment of tuberculosis. Biochem Pharmacol 61, 14631470.[CrossRef][Medline]
Guengerich, F. P. (2001). Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14, 611650.[Medline]
Hitchcock, C. A., Dickinson, K., Brown, S. B., Evans, E. G. & Adams, D. J. (1990). Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alpha-sterol demethylase purified from Candida albicans. Biochem J 266, 475480.[Medline]
Hunter, S. W., Fujiwara, T. & Brennan, P. J. (1982). Structure and antigenicity of the major specific glycolipid antigen of Mycobacterium leprae. J Biol Chem 257, 1507215078.
Imai, T., Globerman, H., Gertner, J. M., Kagawa, N. & Waterman, M. R. (1993). Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency. J Biol Chem 268, 1968119689.
Jackson, C. J., Lamb, D. C., Marczylo, T. H., Parker, J. E., Manning, N. L., Kelly, D. E. & Kelly, S. L. (2003). Conservation and cloning of CYP51: a sterol 14 alpha-demethylase from Mycobacterium smegmatis. Biochem Biophys Res Commun 301, 558563.[CrossRef][Medline]
Jeevarajah, D., Patterson, J. H., McConville, M. J. & Billman-Jacobe, H. (2002). Modification of glycopeptidolipids by an O-methyltransferase of Mycobacterium smegmatis. Microbiology 148, 30793087.[Medline]
Jeevarajah, D., Patterson, J. H., Taig, E., Sargeant, T., McConville, M. J. & Billman-Jacobe, H. (2004). Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium. J Bacteriol 186, 67926799.
Ji, H., Zhang, W., Zhou, Y., Zhang, M., Zhu, J., Song, Y. & Lu, J. (2000). A three-dimensional model of lanosterol 14alpha-demethylase of Candida albicans and its interaction with azole antifungals. J Med Chem 43, 24932505.[CrossRef][Medline]
Kamisango, K., Saadat, S., Dell, A. & Ballou, C. E. (1985). Pyruvylated glycolipids from Mycobacterium smegmatis. Nature and location of the lipid components. J Biol Chem 260, 41174121.[Abstract]
Karakousis, P. C., Bishai, W. R. & Dorman, S. E. (2004). Mycobacterium tuberculosis cell envelope lipids and the host immune response. Cell Microbiol 6, 105116.[CrossRef][Medline]
Khoo, K. H., Suzuki, R., Morris, H. R., Dell, A., Brennan, P. J. & Besra, G. S. (1995). Structural definition of the glycopeptidolipids and the pyruvylated, glycosylated acyltrehalose from Mycobacterium butyricum. Carbohydr Res 276, 449455.[CrossRef][Medline]
Khoo, K. H., Chatterjee, D., Dell, A., Morris, H. R., Brennan, P. J. & Draper, P. (1996a). Novel O-methylated terminal glucuronic acid characterizes the polar glycopeptidolipids of Mycobacterium habana strain TMC 5135. J Biol Chem 271, 1233312342.
Khoo, K. H., Douglas, E., Azadi, P., Inamine, J. M., Besra, G. S., Mikusova, K., Brennan, P. J. & Chatterjee, D. (1996b). Truncated structural variants of lipoarabinomannan in ethambutol drug-resistant strains of Mycobacterium smegmatis. Inhibition of arabinan biosynthesis by ethambutol. J Biol Chem 271, 2868228690.
Khoo, K. H., Jarboe, E., Barker, A., Torrelles, J., Kuo, C. W. & Chatterjee, D. (1999). Altered expression profile of the surface glycopeptidolipids in drug-resistant clinical isolates of Mycobacterium avium complex. J Biol Chem 274, 97789785.
Kremer, L., Douglas, J. D., Baulard, A. R. & 9 other authors (2000). Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. J Biol Chem 275, 1685716864.
Krzywinska, E., Krzywinski, J. & Schorey, J. S. (2004). Phylogeny of Mycobacterium avium strains inferred from glycopeptidolipid biosynthesis pathway genes. Microbiology 150, 16991706.[CrossRef][Medline]
Lamb, D., Kelly, D. & Kelly, S. (1999). Molecular aspects of azole antifungal action and resistance. Drug Resist Updat 2, 390402.[CrossRef][Medline]
Lewis, D. F. V. (1996). In Cytochromes P450: Structure, Function and Mechanism. London: Taylor & Francis.
McLean, K. J., Cheesman, M. R., Rivers, S. L. & 9 other authors (2002a). Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis. J Inorg Biochem 91, 527541.[CrossRef][Medline]
McLean, K. J., Marshall, K. R., Richmond, A., Hunter, I. S., Fowler, K., Kieser, T., Gurcha, S. S., Besra, G. S. & Munro, A. W. (2002b). Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes. Microbiology 148, 29372949.[Medline]
McNeil, M., Tsang, A. Y. & Brennan, P. J. (1987). Structure and antigenicity of the specific oligosaccharide hapten from the glycopeptidolipid antigen of Mycobacterium avium serotype 4, the dominant Mycobacterium isolated from patients with acquired immune deficiency syndrome. J Biol Chem 262, 26302635.
Mederos, L. M., Valdivia, J. A., Sempere, M. A. & Valero-Guillen, P. L. (1998). Analysis of lipids reveals differences between Mycobacterium habana and Mycobacterium simiae. Microbiology 144, 11811188.[Medline]
Minnikin, D. E. (1982). In the Biology of the Mycobacteria, pp. 95104. Edited by C. Ratledge & J. L. Stanford. London: Academic Press.
Navalkar, R. G., Wiegeshaus, E., Kondo, E., Kim, K. & Smith, D. W. (1965). Mycoside G, a specific glycolipid in Mycobacterium marinum (Balnei). J Bacteriol 90, 262265.
Nelson, D. R., Kamataki, T., Waxman, D. J. & 9 other authors (1993). The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol 12, 151.[Medline]
Nes, W. R. (1974). Role of sterols in membranes. Lipids 9, 596612.[Medline]
Nishiuchi, Y., Kitada, S. & Maekura, R. (2004). Liquid chromatography/mass spectrometry analysis of small-scale glycopeptidolipid preparations to identify serovars of Mycobacterium aviumintracellulare complex. J Appl Microbiol 97, 738748.[CrossRef][Medline]
Patterson, J. H., McConville, M. J., Haites, R. E., Coppel, R. L. & Billman-Jacobe, H. (2000). Identification of a methyltransferase from Mycobacterium smegmatis involved in glycopeptidolipid synthesis. J Biol Chem 275, 2490024906.
Riviere, M. & Puzo, G. (1991). A new type of serine-containing glycopeptidolipid from Mycobacterium xenopi. J Biol Chem 266, 90579063.
Riviere, M., Fournie, J. J. & Puzo, G. (1987). A novel mannose containing phenolic glycolipid from Mycobacterium kansasii. J Biol Chem 262, 1487914884.
Sarda, P. & Gastambide-Odier, M. (1967). Structure chimique de l'aglycone du mycoside G de Mycobacterium marinum. Chem Phys Lipids 1, 434444.[CrossRef]
Vergne, I. & Daffé, M. (1998). Interaction of mycobacterial glycolipids with host cells. Front Biosci 3, d865d876.[Medline]
Vergne, I. & Desbat, B. (2000). Influence of the glycopeptidic moiety of mycobacterial glycopeptidolipids on their lateral organization in phospholipid monolayers. Biochim Biophys Acta 1467, 113123.[Medline]
Received 2 February 2005;
accepted 21 February 2005.
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