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School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Correspondence
Gurdyal S. Besra
g.besra{at}bham.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). TB control has been made difficult in recent years by the apparent synergy between the causative agent, Mycobacterium tuberculosis, and HIV (Paolo & Nosanchuk, 2004). In addition, the length and complexity of current TB treatment regimens results in poor patient compliance, a major contributing factor in the emergence of multi-drug-resistant TB (MDR-TB) (Kaye & Frieden, 1996) and extensively drug-resistant (XDR)-TB (Centers for Disease Control and Prevention, 2006). Given this backdrop, the effective control of TB requires the identification of new drug targets and the discovery of novel drugs.
Mycolic acids are one of the most distinctive features of the mycobacterial cell wall. These unusual 
-alkyl, β-hydroxy fatty acids are essential for bacterial survival and, when esterified to the non-reducing termini of the arabinogalactan-peptidoglycan cell wall core, form the inner leaflet of a formidable cell wall permeability barrier (Brennan & Nikaido, 1995; Dover et al., 2004). Mycobacteria are unusual in that they possess both a mammalian-type fatty acid synthase I (FAS-I), which carries all of the necessary enzymic activities and carrier functions on a single polypeptide (Smith et al., 2003), and a bacterial type fatty acid synthase II (FAS-II), in which disassociable enzymes interact with an acyl carrier protein (ACP) AcpM that tethers the growing fatty acyl chain between their active sites (Kremer et al., 2001). M. tuberculosis FAS-I conducts de novo synthesis of intermediate length (principally C16 and C24) fatty acids. FAS-II, however, is incapable of de novo fatty acid synthesis and accepts short-chain (C16) acyl-CoA primers from FAS-I via a condensation reaction carried out by β-ketoacyl-ACP synthase III (mtFabH) (Brown et al., 2005). The newly formed β-ketoacyl-ACP is reduced by a β-ketoacyl-ACP reductase (MabA) (Banerjee et al., 1998) to form a β-hydroxyl-acyl-ACP intermediate. This product is then dehydrated by an unknown β-hydroxyacyl-ACP dehydratase, followed by further reduction by an enoyl-ACP reductase (InhA) to complete the FAS-II cycle (Kikuchi & Kusaka, 1984; Banerjee et al., 1994). Subsequent FAS-II cycles are initiated by the acyl-ACP primed β-ketoacyl-ACP synthases KasA and KasB, respectively (Kremer et al., 2000; Mdluli et al., 1998; Schaeffer et al., 2001) to afford a meromycolic acid (C56), which is then condensed with a C26 fatty acid (Gande et al., 2004; Portevin et al., 2005; Takayama et al., 2005). The oxo-mycolic acid intermediate is then reduced to form the mature mycolic acid (Lea-Smith et al., 2007).
A number of flavonoids have been shown to inhibit both fungal and human FAS-I (Li & Tian, 2004; Li et al., 2002; Wang et al., 2003). Flavonoids comprise a large group of polyphenolic secondary metabolites that are widespread throughout the plant kingdom (Koes et al., 1994). They are all based on a flavan skeleton, consisting of two aromatic rings interconnected by a three carbon atom heterocyclic ring (Tasdemir et al., 2006). More than 6400 flavonoids have been shown to have various interesting properties, including antibacterial, antiprotozoal, anti-inflammatory, dietary antioxidant, vascular and oestrogenic activities, mainly mediated through inhibition of oxidases and NADH usage (Cos et al., 1998; Harborne & Williams, 2000).
Zhang & Rock (2004) have recently shown that epigallocatechin gallate and related flavonoids are potent inhibitors of Escherichia coli β-hydroxyacyl-ACP reductase and enoyl-ACP reductase (Table 1). More recently, Tasdemir et al. (2006) have shown that a number of flavonoids inhibit β-hydroxyacyl-ACP dehydratase of Plasmodium falciparum (Table 1).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Liquid cultures of M. bovis BCG were grown at 37 °C in Sauton's medium containing 25 µg kanamycin ml–1 and 50 µg hygromycin ml–1 (Sauton, 1912). The MIC required to inhibit the growth of 99 % of tested cultures, MIC99, was calculated to indicate the antimycobacterial potency of each flavonoid. MIC99 values of flavonoids against M. bovis BCG, M. bovis BCG pVV16 and M. bovis BCG pVV16-Rv0636 were determined by Alamar Blue according to the manufacturer's protocol (Celltiter-Blue; Promega) followed by MIC99 calculations over the concentration range 0–200 µg ml–1 (Franzblau et al., 1998).
Plasmids and DNA manipulation.
The E. coli mycobacterial shuttle vector pVV16 (a gift from Varalakshmi Vissa, CSU, CO, USA) containing the hsp60 promoter and encoding a 6-histidine C-terminal tag was used for overexpression of M. tuberculosis Rv0636. All DNA manipulations were performed using standard protocols, as described by Sambrook & Russell (2001). PCR amplification was performed using the upstream primer 5'-GATCGATCCATATGGCGCTGCGTGAGTTC-3' and the downstream primer 5'-GATCGATCAAGCTTCGCTTACTTCGCCGAG-3', which contain NdeI and HindIII restriction sites, respectively (underlined). The 430 bp PCR product was then digested with NdeI and HindIII and ligated with similarly digested pVV16, giving rise to pVV16-Rv0636. The coding sequence of the recombinant gene was verified by DNA sequencing.
Determination of the in vivo effects of flavonoids on fatty acid and mycolic acid synthesis.
M. bovis BCG cultures were grown to an OD600 of 0.4 in the presence of 0.25 % Tween 80. The flavonoids were added at various concentrations followed by incubation at 37 °C for 8 h, and then 1 µCi 1,2-[14C]acetate ml–1 (57 mCi mmol–1; GE Healthcare, Amersham Bioscience) was added to the cultures, followed by further incubation at 37 °C for 16 h. The 14C-labelled cells were harvested by centrifugation at 2000 g followed by washing with PBS. The 14C-labelled control and flavonoid-treated cells were then subjected to alkaline hydrolysis using 5 % aqueous tetrabutylammonium hydroxide (TBAH) at 100 °C overnight, followed by the addition of 4 ml CH2Cl2, 500 µl CH3I and 2 ml water, followed by mixing for 30 min. The upper aqueous phase was discarded following centrifugation and the lower organic phase washed thrice with water and evaporated to dryness. The resulting fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) were dissolved in diethyl ether, insoluble residues were removed by centrifugation and the ether solution was evaporated to dryness and redissolved in 200 µl of CH2Cl2. An equivalent aliquot (20 µl) of the resulting solution of FAMEs and MAMEs was subjected to thin-layer chromatography (TLC) using silica gel plates (5735 silica gel 60F254; Merck), developed in petroleum ether/acetone (95 : 5). Autoradiograms were produced by overnight exposure of Kodak X-Omat AR film to the plates to reveal 14C-labelled FAMEs and MAMEs.
Preparation of cytosolic fractions, FAS-I and FAS-II assays.
Cytosolic extracts, enriched for FAS-I and FAS-II using ammonium sulfate precipitation, of M. smegmatis mc2155 pVV16 and M. smegmatis mc2155 pVV16-Rv0636 (approx. 10 g) were prepared as described by Kremer et al. (2002). The final extract containing the FAS-I and FAS-II activities, was dissolved in 5 ml buffer (50 mM MOPS, pH 7.9, 5 mM β-mercaptoethanol, 10 mM MgCl2). Protein concentrations were determined using the BCA protein assay reagent kit (Pierce). FAS-I and FAS-II experiments were conducted as described by Slayden et al. (1996) using the 40–80 % ammonium sulfate fraction (Kremer et al., 2002). Here, for FAS-II reactions, 0.1 mg protein, 10 µM palmitoyl-CoA and 45 nCi [14C]malonyl-CoA (52 mCi mmol–1; GE Healthcare) were used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Tasdemir et al., 2006; Zhang & Rock, 2004). Butein, a product of Rhus verniciflua, quercetin from Flaveria bidentis and fisetin from Rhus cotinus target the enoyl-ACP reductases of both P. falciparum and E. coli at relatively low concentrations (Sharma et al., 2007; Tasdemir et al., 2006; Zhang & Rock, 2004). Isoliquirtigenin and 2,2',4'-trihydroxychalcone, related products of Dalbergia odorifera inhibit both the enoyl-ACP and β-ketoacyl-ACP reductases of E. coli (Zhang & Rock, 2004). Interestingly, Tasdemir et al. (2006) also demonstrated that not only do these flavonoids target FAS-II reductases, but they also target the β-hydroxyacyl-ACP dehydratase (Table 1
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The five flavonoids as shown in Table 1 were initially tested for in vivo inhibitory properties against M. bovis BCG, a recognized surrogate for M. tuberculosis in drug susceptibility testing. The MIC99 was calculated to indicate the antimycobacterial potency for each flavonoid. Four of the five tested flavonoids inhibited M. bovis BCG growth. Butein was the most potent of the flavonoids with an MIC99 value of 43 µg ml–1 (157 µM); potency decreased in the series butein>isoliquirtigenin (195 µM, 50 µg ml–1)>2,2',4'-trihydroxychalcone (214 µM, 55 µg ml–1)>fisetin (220 µM, 63 µg ml–1). As quercetin did not affect bacterial growth even at high concentrations (>750 µM), it was not considered further.
Effect of flavonoids on M. bovis BCG fatty and mycolic acid biosyntheses
The biosynthesis of both fatty and mycolic acids in M. bovis BCG exposed to each of the four active flavonoids was followed by labelling with 1,2-[14C]acetate. Analyses of FAMEs and MAMEs extracted from the labelled cells by TLC (Fig. 1) revealed that exposure to all compounds coincided with decreases in the incorporation of label into both FAMEs and MAMEs (Fig. 1). Mycobacteria are unusual in that they produce a broad distribution of FAMEs in terms of hydrocarbon chain length, long-chain (LC-) FAMEs (>C24), and short-chain (SC-) FAMEs (C16–C18). Two bands are often resolved in this TLC system. Essentially, the LC-FAMEs migrate faster, whilst SC-FAMEs are slightly retarded. These data suggest that the flavonoids tested inhibit de novo fatty acid biosynthesis in mycobacteria as well as mycolic acid biosynthesis. Interestingly, the populations of SC-FAMEs and LC-FAMEs, which both derive from FAS-I but with the latter being extended by FAS-II, appeared to be differentially affected by these compounds.
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). Unlike its counterparts that were also active in vivo against M. bovis BCG pVV16 (Table 2), 2,2',4'-trihydroxychalcone inhibited both FAS-I and FAS-II in vitro at low concentrations (Table 3), suggesting that its inhibition of fatty and mycolic acid biosynthesis is mediated by a common mechanism. Butein and fisetin inhibited FAS-II only, whereas isoliquirtigenin inhibited both FAS-I and FAS-II (Table 3), but the latter most acutely. These patterns of inhibition against FAS-I and FAS-II in vitro are consistent with the in vivo effects on fatty acid and mycolic acid biosynthesis, and support the findings that the flavonoids inhibit fatty acid and mycolic acid biosynthesis. The profiles of inhibition gained using butein, fisetin and isoliquirtigenin highlighted these as potential probes of FAS-II function.
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), no genes were strong candidates to encode the β-hydroxyacyl-ACP dehydratase activity required in FAS-II. BLASTP and TBLASTX analyses of the M. tuberculosis genome using FabZ and FabA from E. coli as query sequences failed to identify a specific mycobacterial dehydratase. Castell et al. (2005) identified seven possible (R)-specific hydratases in M. tuberculosis using a specific hydratase 2 motif [G-D-X-N-P-(LIV)-H-X5-A] (Qin et al., 2000) which contains a histidine residue implicated in catalysis. These included protein products of fas, Rv3538, Rv3389c, Rv0130, Rv0249, Rv0241 and Rv0636. The gene encoding FAS-I, fas, could be immediately discounted as encoding the unidentified FAS-II dehydratase. In addition, several of these candidates have been proposed to be non-essential through the existence of viable disrupted mutants generated by transposon mutagenesis (Sassetti et al., 2003). Using this logic, the potential of Rv3538 and Rv0636 as candidates encoding the FAS-II β-hydroxyacyl-ACP dehydratase was affirmed. Examination of the Mycobacterium leprae genome highlighted a high degree of ‘genetic decay’ with many genes being annotated as non-functional pseudogenes, and thus M. leprae has been thought of as maintaining a minimal gene set for mycobacterial pathogenesis (Eiglmeier et al., 2001). Despite this massive genetic decay, M. leprae possesses an intact cell wall and represents a useful reference when considering candidate genes for essential cell-wall-related enzymes (Vissa & Brennan, 2001). Taking this into consideration and the expected conservation of the β-hydroxyacyl-ACP dehydratase in M. leprae, we could exclude Rv3538 as no orthologue appears to be present in the M. leprae genome. Thus the product of Rv0636 remains as the most promising candidate for this essential enzyme.
Accordingly, CLUSTAL W alignment of the characterized (R)-enoyl-CoA hydratase of Aeromonas caviae (Fukui & Doi, 1997) and the β-hydroxyacyl dehydratase domain of the essential C. glutamicum FasA (Radmacher et al., 2005) with M. tuberculosis Rv0636 and its supposed orthologue M. bovis Mb0655 showed that both mycobacterial polypeptides contain numerous similarities with these bona fide dehydratases. Particularly strong conservation is apparent in the immediate vicinity of the Asp31 and His36 catalytic dyad (numbering from PDB entry 1IQ6) (Hisano et al., 2003) (Fig. 2).
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; Kremer et al., 2000; Larsen et al., 2002). To investigate whether the putative M. tuberculosis dehydratase Rv0636 is a target for flavonoids, we overexpressed M. tuberculosis Rv0636 from plasmid pVV16-Rv0636 in M. bovis BCG. This recombinant plasmid was constructed by cloning Rv0636 downstream of the hsp60 promoter of the low-copy-number E. coli–Mycobacterium shuttle vector pVV16. The use of this relatively strong promoter ensures constitutive expression of cloned genes in mycobacteria. In analyses of M. bovis BCG pVV16-Rv0636 we observed MIC99 values of 59 µg ml–1 (216 µM) and 64 µg ml–1 (250 µM) for butein and isoliquirtigenin, respectively. In comparison to M. bovis BCG pVV16, which we used as a control, the overproduction of Rv0636 conferred some resistance to these agents (Table 3). A high level of resistance was not observed, indicating that the flavonoids might target several FAS-II enzymes. This was not surprising since flavonoids inhibit several FAS-II enzymes as shown in inhibition studies using E. coli and P. falciparum (Sharma et al., 2007; Tasdemir et al., 2006; Zhang & Rock, 2004) (Table 1). The overexpression of Rv0636 also had little or no effect on both 2,2',4'-trihydroxychalcone and fisetin activity in vivo against M. bovis BCG (Table 3). Together, these data suggest that fisetin might inhibit FAS-I, but in a manner distinct from isoliquirtigenin and butein.
The resistance observed upon overexpression of Rv0636 against butein activity was further investigated in liquid culture (Fig. 3a). When butein was introduced at 100 µg ml–1 to exponentially growing shaken liquid cultures, growth was immediately depressed. In an identical experiment, however, the growth of M. bovis BCG pVV16-Rv0636 was not inhibited by butein (Fig. 3a). Furthermore, on overexpression of Rv0636 the incorporation of radiolabel into SC-/LC-FAMEs and MAMEs (Fig. 3b) was unaffected by exposure to butein up to 100 µg ml–1.
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). The over-representation of the Rv0636 protein afforded protection to FAS-II activity against both butein and isoliquirtigenin in ratios that were almost identical to the changes observed in MIC99 values on Rv0636 overexpression in M. bovis BCG, which points towards the FAS-II lesion observed here bearing significance in vivo. In accordance with our perception that these two agents exert their toxicity via a different route, we observed no protection of FAS-II activity by Rv0636 over-representation in vitro when using 2,2',4'-trihydroxychalcone and fisetin. Considering our in vivo and in vitro observations collectively, Rv0636 is an apparent cellular target of butein and isoliquirtigenin and its function seems intimately linked to the function of FAS-II, i.e. meromycolyl extension. Therefore, it represents an extremely strong candidate for the unidentified β-hydroxyacyl-ACP dehydratase of mycobacterial FAS-II.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), but recently the emergence of MDR-TB (Kaye & Frieden, 1996) and XDR-TB (Centers for Disease Control and Prevention, 2006) has come to the fore; an untreatable epidemic of XDR-TB cannot be excluded. In this worrying climate, the need for the discovery and characterization of key enzymes involved in essential mycobacterial biosynthetic pathways towards the definition of novel targets for chemical intervention and the testing of novel classes of potential inhibitors is apparent.
Previously, flavonoid compounds have been shown to be active against E. coli and P. falciparum in vivo and have been shown to inhibit FAS-II components (Sharma et al., 2007; Tasdemir et al., 2006; Zhang & Rock, 2004). Five flavonoids were evaluated for their potential as mycobacterial FAS-II inhibitors in vivo. Our results show that four of the five tested flavonoids compounds were active against M. bovis BCG. MICs for these compounds ranged between 150 to 220 µM, with butein being the most effective against M. bovis BCG at a concentration of 157 µM (43 µg ml–1).
Analysis of the effects that exposure to these compounds exerted upon the biosyntheses of fatty and mycolic acids demonstrated that all four active compounds affected both systems with the inhibition of mycolate biosynthesis appearing more acute. Although the fatty acyl products of FAS-I provide primers for extension to meromycolate precursors of mycolic acids, the effects on FAS-II appear to be more complex than a simple deprivation of primer supply brought about via FAS-I inhibition. This is illustrated by the disparity between FAS-I and FAS-II inhibition caused by butein and isoliquirtigenin (Table 3). Although the effects of both inhibitors on mycolate biosynthesis are very similar, isoliquirtigenin appears to be a more potent inhibitor of fatty acid biosynthesis than butein. In general, the biosynthesis of all subtypes of mycolates appeared to be equally affected, and thus the inhibition of a common meromycolate precursor is likely, which is consistent with a lesion in FAS-II.
Confirmation of the activity of these flavonoids in vitro implied that they could be used as potential antitubercular agents, but also as a tool for the identification of the unidentified FAS-II dehydratase. In terms of target definition, a key resource was provided by the work of Sassetti et al. (2003) who applied high density transposon mutagenesis and mapping of insertion sites to identify non-essential genes for M. tuberculosis growth. The availability of several mycobacterial genome sequences, especially that of the obligate intracellular pathogen M. leprae, allows us to make useful, biologically informed comparisons across Mycobacterium. Historically, M. tuberculosis FAS-II has proven as a clinically relevant target (Banerjee et al., 1994; Dover et al., 2007; Kremer et al., 2000). Previous analyses by Castell et al. (2005) first suggested that Rv0636 of M. tuberculosis H37Rv is a good candidate for the unidentified β-hydroxyacyl-ACP dehydratase of FAS-II. The gene encoding Rv0636 is not only predicted to be essential (Sassetti et al., 2003), but is highly conserved over numerous mycobacterial species such as M. bovis, M. smegmatis and importantly M. leprae. We have shown that the specific hydratase 2 motif (Qin et al., 2000) which contains an Asp-His catalytic dyad is present in Rv0636. Furthermore, our attempts at homology modelling of Rv0636 using Aeromonas caviae (R)-enoyl CoA hydratase as a template suggest that Rv0636 might also adopt a comparable hot-dog fold like those of other FAS-II dehydratases E. coli FabA and P. falciparum FabZ (A. K. Brown, unpublished results). Taken together, these observations suggest that Rv0636 represents an excellent candidate for the FAS-II dehydratase.
Consistent with its candidature as the unidentified FAS-II dehydratase, the overexpression of M. tuberculosis Rv0636 in M. bovis BCG conferred resistance to butein and isoliquirtigenin, probably via flavonoid binding. Analysis of fatty acid and mycolic acid metabolism in vivo revealed the partial reversion to untreated FAME and MAME profiles in treated cells. These finding were echoed in our in vitro assays with over-representation of Rv0636 providing partial protection of FAS-II activity. When considered collectively, these observations suggest that elevated Rv0636 concentrations protect FAS-II from the action of these two agents that have previously been shown to inhibit β-hydroxyacyl-ACP dehydratases. As the other components of FAS-II are known, we can assert that Rv0636 represents the dehydratase of FAS-II. This however, requires formal proof; our ongoing studies involve purification of an active recombinant enzyme and the development of functional assays using relevant long-chain β-hydroxyacyl thioester substrates.
Interestingly, in consideration of the structure of anti-FAS-II flavonoids described here, a common structural feature became apparent. If one considers that the ketone group borne by each of these flavonoids emulates the carbonyl group of a substrate fatty acid, they all possess a 2,3 double bond consistent with a product mimic. Furthermore, the most potent inhibitors of FAS-II do not bear the oxygen-containing cycle possessed by fisetin and quercetin.
The activity of these compounds against M. bovis BCG identifies a new area for antitubercular drug development and the implication of Rv0636 as the unidentified β-hydroxyacyl-ACP dehydratase of FAS-II provides impetus towards characterization of this enigmatic enzyme, which surely represents an important potential target for future drug development studies.
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ACKNOWLEDGEMENTS |
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Edited by: W. Bitter
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Banerjee, A., Sugantino, M., Sacchettini, J. C. & Jacobs, W. R., Jr (1998). The mabA gene from the inhA operon of Mycobacterium tuberculosis encodes a 3-ketoacyl reductase that fails to confer isoniazid resistance. Microbiology 144, 2697–2704.
Belanger, A. E., Besra, G. S., Ford, M. E., Mikusova, K., Belisle, J. T., Brennan, P. J. & Inamine, J. M. (1996). The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc Natl Acad Sci U S A 93, 11919–11924.
Brennan, P. J. & Nikaido, H. (1995). The envelope of mycobacteria. Annu Rev Biochem 64, 29–63.[CrossRef][Medline]
Brown, A. K., Sridharan, S., Kremer, L., Lindenberg, S., Dover, L. G., Sacchettini, J. C. & Besra, G. S. (2005). Probing the mechanism of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III mtFabH: factors influencing catalysis and substrate specificity. J Biol Chem 280, 32539–32547.
Castell, A., Johansson, P., Unge, T., Jones, T. A. & Backbro, K. (2005). Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial survival during infection, has a double hotdog fold. Protein Sci 14, 1850–1862.[CrossRef][Medline]
Centers for Disease Control and Prevention (2006). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs – worldwide, 2000–2004. MMWR Morb Mortal Wkly Rep 55, 301–305.[Medline]
Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S. & other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef][Medline]
Cos, P., Ying, L., Calomme, M., Hu, J. P., Cimanga, K., Van Poel, B., Pieters, L., Vlietinck, A. J. & Vanden Berghe, D. (1998). Structure–activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. J Nat Prod 61, 71–76.[CrossRef][Medline]
Dover, L. G., Cerdeno-Tarraga, A. M., Pallen, M. J., Parkhill, J. & Besra, G. S. (2004). Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae. FEMS Microbiol Rev 28, 225–250.[CrossRef][Medline]
Dover, L. G., Alahari, A., Gratraud, P., Gomes, J. M., Bhowruth, V., Reynolds, R. C., Besra, G. S. & Kremer, L. (2007). EthA, a common activator of thiocarbamide-containing drugs acting on different mycobacterial targets. Antimicrob Agents Chemother 51, 1055–1063.
Dye, C. (2006). Global epidemiology of tuberculosis. Lancet 367, 938–940.[CrossRef][Medline]
Eiglmeier, K., Parkhill, J., Honore, N., Garnier, T., Tekaia, F., Telenti, A., Klatser, P., James, K. D., Thomson, N. R. & other authors (2001). The decaying genome of Mycobacterium leprae. Lepr Rev 72, 387–398.[Medline]
Franzblau, S. G., Witzig, R. S., McLaughlin, J. C., Torres, P., Madico, G., Hernandez, A., Degnan, M. T., Cook, M. B., Quenzer, V. K. & other authors (1998). Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol 36, 362–366.
Fukui, T. & Doi, Y. (1997). Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae. J Bacteriol 179, 4821–4830.
Gande, R., Gibson, K. J., Brown, A. K., Krumbach, K., Dover, L. G., Sahm, H., Shioyama, S., Oikawa, T., Besra, G. S. & Eggeling, L. (2004). Acyl-CoA carboxylases (accD2 and accD3), together with a unique polyketide synthase (Cg-pks), are key to mycolic acid biosynthesis in Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis. J Biol Chem 279, 44847–44857.
Harborne, J. B. & Williams, C. A. (2000). Advances in flavonoid research since 1992. Phytochemistry 55, 481–504.[CrossRef][Medline]
Hisano, T., Tsuge, T., Fukui, T., Iwata, T., Miki, K. & Doi, Y. (2003). Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis. J Biol Chem 278, 617–624.
Kaye, K. & Frieden, T. R. (1996). Tuberculosis control: the relevance of classic principles in an era of acquired immunodeficiency syndrome and multidrug resistance. Epidemiol Rev 18, 52–63.
Kikuchi, S. & Kusaka, T. (1984). Purification of NADPH-dependent enoyl-CoA reductase involved in the malonyl-CoA dependent fatty acid elongation system of Mycobacterium smegmatis. J Biochem (Tokyo) 96, 841–848.
Koes, R. E., Quattrocchio, F. & Mol, J. N. M. (1994). The flavonoid biosynthetic pathway in plants – function and evolution. Bioessays 16, 123–132.[CrossRef]
Kremer, L., Baulard, A., Estaquier, J., Content, J., Capron, A. & Locht, C. (1995). Analysis of the Mycobacterium tuberculosis 85A antigen promoter region. J Bacteriol 177, 642–653.
Kremer, L., Douglas, J. D., Baulard, A. R., Morehouse, C., Guy, M. R., Alland, D., Dover, L. G., Lakey, J. H., Jacobs, W. R. & 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, 16857–16864.
Kremer, L., Nampoothiri, K. M., Lesjean, S., Dover, L. G., Graham, S., Betts, J., Brennan, P. J., Minnikin, D. E., Locht, C., Besra, G. S. & other authors (2001). Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA : AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II. J Biol Chem 276, 27967–27974.
Kremer, L., Dover, L. G., Carrere, S., Nampoothiri, K. M., Lesjean, S., Brown, A. K., Brennan, P. J., Minnikin, D. E., Locht, C., Besra, G. S. & other authors (2002). Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis. Biochem J 364, 423–430.[CrossRef][Medline]
Larsen, M. H., Vilcheze, C., Kremer, L., Besra, G. S., Parsons, L., Salfinger, M., Heifets, L., Hazbon, M. H., Alland, D. & other authors (2002). Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis. Mol Microbiol 46, 453–466.[CrossRef][Medline]
Lea-Smith, D. J., Pyke, J. S., Tull, D., McConville, M. J., Coppel, R. L. & Crellin, P. K. (2007). The reductase that catalyzes mycolic motif synthesis is required for efficient attachment of mycolic acids to arabinogalactan. J Biol Chem 282, 11000–11008.
Li, B. H. & Tian, W. X. (2004). Inhibitory effects of flavonoids on animal fatty acid synthase. J Biochem (Tokyo) 135, 85–91.
Li, X. C., Joshi, A. S., ElSohly, H. N., Khan, S. I., Jacob, M. R., Zhang, Z., Khan, I. A., Ferreira, D., Walker, L. A. & other authors (2002). Fatty acid synthase inhibitors from plants: isolation, structure elucidation, and SAR studies. J Nat Prod 65, 1909–1914.[CrossRef][Medline]
Mdluli, K., Slayden, R. A., Zhu, Y., Ramaswamy, S., Pan, X., Mead, D., Crane, D. D., Musser, J. M. & Barry, C. E., III (1998). Inhibition of a Mycobacterium tuberculosis β-ketoacyl ACP synthase by isoniazid. Science 280, 1607–1610.
Paolo, W. F., Jr & Nosanchuk, J. D. (2004). Tuberculosis in New York city: recent lessons and a look ahead. Lancet Infect Dis 4, 287–293.[CrossRef][Medline]
Portevin, D., de Sousa-D'Auria, C., Montrozier, H., Houssin, C., Stella, A., Laneelle, M. A., Bardou, F., Guilhot, C. & Daffe, M. (2005). The acyl-AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-CoA carboxylase components. J Biol Chem 280, 8862–8874.
Qin, Y. M., Haapalainen, A. M., Kilpelainen, S. H., Marttila, M. S., Koski, M. K., Glumoff, T., Novikov, D. K. & Hiltunen, J. K. (2000). Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity. J Biol Chem 275, 4965–4972.
Radmacher, E., Alderwick, L. J., Besra, G. S., Brown, A. K., Gibson, K. J. C., Sahm, H. & Eggeling, L. (2005). Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum. Microbiology 151, 2421–2427.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sassetti, C. M., Boyd, D. H. & Rubin, E. J. (2003). Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48, 77–84.[CrossRef][Medline]
Sauton, B. (1912). Sur la nutrition minérale du bacille tuberculeux. C R Hebd Seances Acad Sci 155, 860–861.
Schaeffer, M. L., Agnihotri, G., Volker, C., Kallender, H., Brennan, P. J. & Lonsdale, J. T. (2001). Purification and biochemical characterization of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthases KasA and KasB. J Biol Chem 276, 47029–47037.
Sharma, S. K., Parasuraman, P., Kumar, G., Surolia, N. & Surolia, A. (2007). Green tea catechins potentiate triclosan binding to enoyl-ACP reductase from Plasmodium falciparum (PfENR). J Med Chem 50, 765–775.[CrossRef][Medline]
Slayden, R. A., Lee, R. E., Armour, J. W., Cooper, A. M., Orme, I. M., Brennan, P. J. & Besra, G. S. (1996). Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis. Antimicrob Agents Chemother 40, 2813–2819.[Abstract]
Smith, S., Witkowski, A. & Joshi, A. K. (2003). Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res 42, 289–317.[CrossRef][Medline]
Takayama, K., Wang, C. & Besra, G. S. (2005). Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev 18, 81–101.
Tasdemir, D., Lack, G., Brun, R., Ruedi, P., Scapozza, L. & Perozzo, R. (2006). Inhibition of Plasmodium falciparum fatty acid biosynthesis: evaluation of FabG, FabZ, and FabI as drug targets for flavonoids. J Med Chem 49, 3345–3353.[CrossRef][Medline]
Vissa, V. D. & Brennan, P. J. (2001). The genome of Mycobacterium leprae: a minimal mycobacterial gene set. Genome Biol 2, 1–7.[Medline]
Wang, X., Song, K. S., Guo, Q. X. & Tian, W. X. (2003). The galloyl moiety of green tea catechins is the critical structural feature to inhibit fatty-acid synthase. Biochem Pharmacol 66, 2039–2047.[CrossRef][Medline]
Zhang, Y. M. & Rock, C. O. (2004). Evaluation of epigallocatechin gallate and related plant polyphenols as inhibitors of the FabG and FabI reductases of bacterial type II fatty-acid synthase. J Biol Chem 279, 30994–31001.
Received 18 May 2007;
revised 22 June 2007;
accepted 2 July 2007.
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2 µg ml–1.
, M. bovis BCG pVV16; 
, M. bovis BCG pVV16+butein; 
, M. bovis BCG pVV16-Rv0636; 
, M. bovis BCG pVV16-Rv0636+butein. (b) M. bovis BCG pVV16 and M. bovis BCG pVV16-Rv0636 were cultured in liquid medium at an OD600 of 0.4 prior to labelling with 1 µCi 1,2-[14C]acetate ml–1 for 8 h. 14C-labelled SC-/LC-FAMEs and MAMEs were extracted and resolved by TLC. An equivalent aliquot (20 µl) of the resulting solution of SC-/LC-FAMEs and MAMEs was subjected to TLC using silica gel plates developed in petroleum ether/acetone (95 : 5). Autoradiograms were produced by overnight exposure to film to reveal 14C-labelled SC-/LC-FAMEs and MAMEs.