Altered expression profile of mycobacterial surface glycopeptidolipids following treatment with the antifungal azole inhibitors econazole and clotrimazole

Adeline Burguière1, Paul G. Hitchen2, Lynn G. Dover1, Anne Dell2 and Gurdyal S. Besra1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium aviumintracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis, produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis. In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis, suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N-acyl chain in GPL biosynthesis. 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.


Abbreviations: CAD-ES-MS/MS, collision-activated dissociation electrospray mass spectrometry–mass spectrometry; dGPL, de-O-acylated GPL; 6-dTal, 6-deoxytalose; EMB, ethambutol; ETH, ethionamide; GPL, glycopeptidolipid; INH, isoniazid; LOS, lipooligosaccharide; MAC, Mycobacterium aviumintracellulare complex; nsGPL, non-serovar-specific-GPL; PGL, phenolic glycolipid; Rha, rhamnose; ssGPL, serovar-specific-GPL


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The genus Mycobacterium includes the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, which are responsible for tuberculosis and leprosy respectively. Pathogenic mycobacteria produce an array of unusual lipophilic compounds that are required for growth and survival of the organism in the human host (Karakousis et al., 2004). The use of refined analytical techniques coupled with structural characterization has continued to provide detailed structural knowledge on the species- and serotype-specific glycolipids of the genus Mycobacterium (Khoo et al., 1996a; Mederos et al., 1998; Nishiuchi et al., 2004). These highly diverse glycosylated surface antigens have been broadly classified into three major families: the phenolic glycolipids (PGLs), the trehalose-containing lipooligosaccharides (LOSs) and the glycopeptidolipids (GPLs) (Brennan & Nikaido, 1995; Vergne & Daffé, 1998).

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 C26–34 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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Fig. 1. Generic structure of nsGPL. R1=H or CH3; R2=3,4-di-O-CH3-Rha or 2,3,4-tri-O-CH3-Rha; Ac, acetyl.

 
These complex surface glycolipids and other waxes, such as phthiocerol dimycocerosates, are found associated with and interspersed within the outer leaflet of the mycobacterial cell wall permeability barrier. The mycolic acids present in the inner leaflet of this permeability barrier are covalently tethered via their esterification to the arabinan portion of the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex that represents the essential core structure of the mycobacterial cell wall. Several anti-mycobacterial drugs, such as isoniazid, ethionamide and ethambutol, have been shown to inhibit the biosynthesis of mAGP (Banerjee et al., 1994; Khoo et al., 1996b). In the case of ethambutol, inhibition of arabinogalactan biosynthesis leads to an accumulation of trehalose mono- and dimycolate. In addition, analysis of MAC clinical isolates revealed altered GPL profiles following EMB treatment (Khoo et al., 1999).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
M. smegmatis mc2155 strain was grown in either Sauton's medium containing 0·05 % tyloxapol or LB medium containing 0·05 % Tween 80 at 37 °C with agitation at 180 r.p.m. in an orbital incubator.

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 0·7–0·8). MIC values were measured by serial dilution of M. smegmatis cultures onto LB agar plates containing the drugs in a concentration range of 5–30 µg ml–1 for ETH and 0·05–10 µg ml–1 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 5–20 µg ml–1 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 ml–1) or clotrimazole (10 µg ml–1) was then added and the cultures reincubated with gentle agitation for 1 h. Sodium [1,2-14C]acetate (1 µCi ml; 37 kBq ml–1) 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. 40–60 °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 10–30 nl min–1. The drying gas used was nitrogen and the collision gas was argon, with the collision gas pressure maintained at 10–4 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 µl–1 solution of [Glu1]-fibrinopeptide B in acetonitrile/5 % aqueous acetic acid (1 : 3, v/v).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Effect of azole antifungal drugs on M. smegmatis growth
Azole antifungal drugs such as econazole and clotrimazole belong to a class of azole derivatives that inhibit the cytochrome P450 14{alpha}-lanosterol demethylase, also termed CYP51. They are competitive inhibitors that bind reversibly to the haem iron moiety of CYP51 via a specific nitrogen atom in the azole ring (Hitchcock et al., 1990; Ji et al., 2000). The cytochromes P450 (P450s) are haem-thiolate enzymes that are able to oxygenate a large variety of substrates (Lewis, 1996). These haem-containing mono-oxygenases, which are involved in drug metabolism/detoxification and steroid synthesis in mammals (Guengerich, 2001; Imai et al., 1993), are found in virtually all life forms. The recent determination of the genome sequence of M. tuberculosis revealed 19 genes encoding P450s, the highest number found in any bacterium to date, pointing to important physiological roles (Cole et al., 1998). A CYP51 orthologue has recently been characterized in M. smegmatis and its role in sterol biosynthesis degradation confirmed by demonstration of its 14{alpha}-lanosterol demethylase activity (Jackson et al., 2003). Previously, this type of P450 was considered to be found exclusively in eukaryotes (Nes, 1974).

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 ml–1 respectively. These values were lower than those for ETH (MIC 20 µg ml–1) and INH (MIC 4 µg ml–1). Clotrimazole was also more effective than EMB (MIC 2 µg ml–1). 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 ml–1 and clotrimazole at 15 µg ml–1, 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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Fig. 2. Inhibitory effect of econazole (a) and clotrimazole (b) on M. smegmatis. Cultures were grown in Sauton's medium supplemented with 0·05 % tyloxapol under the following treatments: 0·2 % DMSO control ({blacklozenge}); 5 µg azole ml–1 ({blacksquare}); 10 µg azole ml–1 ({blacktriangleup}); 15 µg azole ml–1 ({lozenge}); and 20 µg azole ml–1 ({square}). Each experiment was performed three times; representative results are shown. Insets: chemical structures of the azole antifungal drugs used in this study.

 
Effect of azole antifungal drugs on M. smegmatis lipid synthesis
Lipids labelled by the incorporation of [14C]acetate were extracted from the cell envelope of M. smegmatis cultures grown either with or without sub-MIC (10 µg ml–1) econazole or clotrimazole treatment (see Methods). Analysis of cell wall-bound mycolic acids revealed a profile as typically observed for M. smegmatis of {alpha},{alpha}'- and epoxymycolic acids, indicating that econazole and clotrimazole had no inhibitory effect on mycolic acid synthesis (data not shown). Analysis of the extractable apolar and polar lipids by 2D TLC across the breadth of polarities observed in Mycobacterium spp., i.e. from apolar waxes to polar phosphatidylinositol mannosides, revealed a cluster of polar lipids only observed in solvent system D that were inhibited by the addition of either econazole or clotrimazole.

Based on their migration in solvent system D and charring with {alpha}-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. 3a–c). 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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Fig. 3. 2D TLCs of base-treated polar lipids extracted from M. smegmatis: (a) control, (b) econazole-treated (10 µg ml–1), and (c) clotrimazole-treated (10 µg ml–1) cultures. Equivalent amounts of lipid extracts (20 000 c.p.m.) were run in system D, CH3Cl/CH3OH/H2O (100 : 14 : 0·8, by vol.) in the first direction and CH3Cl/acetone/CH3OH/H2O (50 : 60 : 2·5 : 3, by vol.) in the second direction. Kodak O-mat film was exposed for 5 days and developed using a X-OGraph.

 


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Fig. 6. Relative abundance of dGPLs. The relative abundance (percentage of control) was determined for each dGPL from M. smegmatis cultures: control (black bars), treated with 10 µg econazole ml–1 (white bars), and treated with 10 µg clotrimazole ml–1 (grey bars). The inset represents 1D TLC of dGPLs from the base-treated polar lipids extracted from M. smegmatis: lane U, extracts from untreated bacteria; lanes E and C, extracts from econazole- and clotrimazole-treated cultures respectively. Equivalent amounts of lipid extracts (20 000 c.p.m.) were run using CH3Cl/CH3OH/H2O (90 : 10 : 1, by vol.) and quantified by phosphoimaging over 3 days.

 
Characterization of dGPLs
In order to conduct structural analyses, the four tentatively assigned de-O-acylated GPLs (dGPLs) from M. smegmatis were purified by preparative TLC and termed (from bottom to top) dGPL-I, -II, -III and -IV (Fig. 4). Analysis of the underivatized dGPLs by electrospray-mass spectrometry (ES-MS) gave a major signal in each spectrum corresponding to the [M+Na+] molecular ion. The molecular ions of dGPL-I, -II, -III and -IV were observed at m/z 1159, 1173, 1173 and 1187 respectively (data not shown) and are consistent with the reported GPLs from M. butyricum (Khoo et al., 1995). Further dGPL sequence information was afforded by collision-activated dissociation (CAD) ES-MS/MS of per-O-deuteriomethylated derivatives. All four dGPLs yielded similar spectra with characteristic fragment ions indicative of the location of the native O-methyl groups. The CAD-ES-MS/MS mass spectra for dGPL-I, -II, -III and -IV are shown in Fig. 5 with the assignment of key fragment ions shown schematically. The spectrum of per-O-deuteriomethylated dGPL-I (Fig. 5a) shows fragment ions consistent with the presence of both an O-deuteriomethylated Rha attached to ‘C-terminal’ alaninol and an O-deuteriomethylated N-acyl chain, which result from derivatization and are therefore indicative of dGPL-I lacking any of the additional O-methyl groups associated with the other dGPLs. The spectrum of per-O-deuteriomethylated dGPL-II (Fig. 5b) shows fragment ions at m/z 903, 709, 494 and 394, consistent with an additional O-methyl group located on the N-acyl chain. In contrast, whilst dGPL-III affords the same molecular ion at m/z 1309, the fragment ions at m/z 906, 706, 491 and 391 generated during CAD analysis are consistent with the incorporation of a third O-methyl group into the 2,3,4-tri-O-methyl-Rha attached to ‘C-terminal’ alaninol (Fig. 5c). Previous sugar analyses of dGPLs suggest that this final methylation should be of the hydroxyl group occupying the 2 position. Finally, the fragment ions resulting from analysis of the dGPL-IV molecular ion are consistent with the presence of both of these O-methyl groups (Fig. 5d).



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Fig. 4. dGPLs purified after preparative TLC. GPLs were purified by preparative TLC and revealed by {alpha}-naphthol staining following development with CH3Cl/CH3OH/H2O (90 : 10 : 1, by vol.).

 


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Fig. 5. CAD-ES-MS/MS spectra of per-O-deuteriomethylated dGPLs from M. smegmatis: (a) dGLP-I; (b) dGPL-II; (c) dGPL-III; (d) dGPL-IV. Fragment ion assignments are illustrated schematically in each panel.

 
Quantification of radiolabelled dGPLs by phosphoimaging revealed that in untreated M. smegmatis dGPL-III is highly expressed, representing 51·9 % of total dGPL, with lesser amounts of dGPL-I (25·0 %), dGPL-IV (14·3 %) and dGPL-II (8·8 %) (see inset Fig. 6). It is clear that GPL biosynthesis is inhibited by the azole antifungal drugs at sub-MIC concentrations; the proportion of the different dGPLs changed significantly upon treatment with econazole (Figs 3b and 6), but with clotrimazole (Figs 3c and 6) the production of all dGPLs was decreased strikingly. The abundance of each dGPL was carefully quantified following treatment with either econazole or clotrimazole and is represented in Fig. 6. When M. smegmatis was treated with 10 µg econazole ml–1, the overall synthesis of total dGPLs remained the same in comparison to the control cultures. However, the relative abundance of dGPL-I and dGPL–II decreased by 65 and 23 % respectively, whereas the amounts of dGPL-III and dGPL-IV increased by 28 and 78 % respectively. The effect of clotrimazole (10 µg ml–1) was more dramatic (Figs 3c and 6), with the biosynthesis of all four dGPLs being strongly inhibited (16 % residual dGPL). Individually, dGPL-I was inhibited by 99 %, dGPL-II by 82 %, dGPL-III by 83 % and dGPL-IV by 62 % compared with untreated M. smegmatis.

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.


   ACKNOWLEDGEMENTS
 
This work was supported by the Darwin Trust of Edinburgh. G. S. B. acknowledges support as a Lister Institute-Jenner Research Fellow and from the Medical Research Council (UK).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 2 February 2005; accepted 21 February 2005.



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