Paul W Bowyer

Ruwani S Gunaratne

Munira Grainger

Chrislaine Withers-Martinez

Sasala R Wickramsinghe

Edward W Tate

Robin J Leatherbarrow

Katherine A Brown

Anthony A Holder
email: aholder@nimr.mrc.ac.uk

Deborah F Smith

Ruwani S Gunaratne §

Anthony A Holder §

Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

Keywords: Plasmodium falciparum, N-myristoylation, codon optimisation, SPA, inhibition, benzothiazole NMT, myristoyl CoA:protein N-myristoyltransferase; SPA, scintillation proximity assay

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INTRODUCTION

Malaria is a devastating disease with an estimated 300-500 million cases and >1 million deaths per year [1]. Resistance to established anti-malarial drugs is increasing the need for the development of new compounds to combat the disease. Here we describe investigations into the N-myristoyltransferase of Plasmodium falciparum as a potential target for development of novel chemotherapeutics.

Myristoyl-CoA protein N-myristoyltransferase (NMT; EC 2.1.3.97) is an enzyme that catalyses the co-translational transfer [START_REF] Wilcox | Acylation of proteins with myristic acid occurs cotranslationally[END_REF] of the fatty acid, myristate (C 14:0 ), from myristoyl-CoA to the N-terminal glycine of target eukaryotic proteins, as well as to viral and bacterial proteins myristoylated within the host cell [START_REF] Resh | Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins[END_REF][START_REF] Maurer-Stroh | N-terminal N-myristoylation of proteins: refinement of the sequence motif and its taxon-specific differences[END_REF][START_REF] Maurer-Stroh | Myristoylation of viral and bacterial proteins[END_REF]. The reaction proceeds via an ordered Bi-Bi mechanism [START_REF] Rudnick | Kinetic and structural evidence for a sequential ordered Bi Bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-CoA:protein Nmyristoyltransferase[END_REF] in which myristoyl-CoA initially binds, followed by a putative structural rearrangement and binding of the N-terminus of the protein substrate. Myristate transfer to the N-terminal glycine of the protein substrate and step-wise dissociation of CoA followed by the myristoylated protein completes the reaction [START_REF] Farazi | Structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoylCoA and peptide provide insights about substrate recognition and catalysis[END_REF][START_REF] Sogabe | Crystal Structures of Candida albicans N-Myristoyltransferase with Two Distinct Inhibitors[END_REF].

NMT has been extensively investigated as a drug target against pathogenic fungi (reviewed in [START_REF] Sikorski | Selective peptidic and peptidomimetic inhibitors of Candida albicans myristoylCoA: protein N-myristoyltransferase: a new approach to antifungal therapy[END_REF]10]) and also identified as a potential target in kinetoplastid parasites [START_REF] Gelb | Protein farnesyl and Nmyristoyl transferases: piggy-back medicinal chemistry targets for the development of antitrypanosomatid and antimalarial therapeutics[END_REF] as well as a novel anti-cancer agent [12][START_REF] Ducker | Two Nmyristoyltransferase isozymes play unique roles in protein myristoylation, proliferation, and apoptosis[END_REF][START_REF] Selvakumar | Potential role of N-myristoyltransferase in cancer[END_REF][START_REF] Selvakumar | Nmyristoyltransferase 2 expression in human colon cancer: cross-talk between the calpain and caspase system[END_REF]. Genetic analyses of NMT have shown recessive lethality in Saccharomyces cerevisiae, while NMT is an essential gene in Candida albicans, Cryptococcus neoformans [START_REF] Lodge | Targeted gene replacement demonstrates that myristoyl-CoA: protein Nmyristoyltransferase is essential for viability of Cryptococcus neoformans[END_REF][START_REF] Weinberg | Genetic studies reveal that myristoylCoA:protein N-myristoyltransferase is an essential enzyme in Candida albicans[END_REF], Trypanosoma brucei and Leishmania major [START_REF] Price | Myristoyl-CoA:protein N-myristoyltransferase, an essential enzyme and potential drug target in kinetoplastid parasites[END_REF].

Comparative analyses of human and fungal NMTs have shown that the peptide pocket is less well conserved than the myristoyl-CoA binding site [START_REF] Kishore | Comparison of the acyl chain specificities of human myristoyl-CoA synthetase and human myristoyl-CoA:protein N-myristoyltransferase[END_REF]. Although myristoyl-CoA analogues have been shown to have anti-viral activity [START_REF] Cordo | Myristic acid analogs are inhibitors of Junin virus replication[END_REF], selective inhibition can be best achieved by targeting the peptide-binding pocket. For example, inhibitors of the NMT peptide-binding pocket in pathogenic fungi are capable of inhibiting C. albicans NMT (CaNMT) with IC 50 values in the nanomolar range [10, [START_REF] Devadas | Novel biologically active nonpeptidic inhibitors of myristoylCoA:protein N-myristoyltransferase[END_REF][START_REF] Yamazaki | Synthesis of potent and selective inhibitors of Candida albicans N-myristoyltransferase based on the benzothiazole structure[END_REF][START_REF] Ebiike | Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 2[END_REF] and show >1000-fold selectivity over human NMTs. There are two NMT genes in humans, (Homo sapiens N-myristoyltransferase 1 and 2 [24,[START_REF] Giang | A second mammalian N-myristoyltransferase[END_REF]). Their protein products, HsNMT1 and HsNMT2, show 73% identity to each other and 40% -50% identity with the NMTs of C. albicans, T. brucei and P. falciparum. Investigations into inhibitors of HsNMT1 have identified a family of compounds based around a cyclohexyl-octahydro-pyrrolo[1,2-a] pyrazine scaffold with IC 50 values in the low micromolar range [START_REF] French | Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-based inhibitors of human Nmyristoyltransferase-1[END_REF].

NMT activity has also been studied in kinetoplastid parasites. Establishment of robust expression and purification protocols for recombinant forms of T. brucei NMT (TbNMT) and L. major NMT (LmNMT) in Escherichia coli has enabled compounds to be tested for inhibition of these enzymes, in a piggy-back approach [START_REF] Gelb | Protein farnesyl and Nmyristoyl transferases: piggy-back medicinal chemistry targets for the development of antitrypanosomatid and antimalarial therapeutics[END_REF], leading to identification of inhibitors selective for TbNMT [START_REF] Panethymitaki | Characterisation and selective inhibition of Myristoyl CoA: protein Nmyristoyl transferase from Trypanosoma brucei and Leishmania major[END_REF]. In comparison, only low expression of recombinant P. falciparum NMT (PfNMT) (12 μg L -1 ) has been reported to date [28] and this has limited the initiation of similar studies. However, like TbNMT and LmNMT, PfNMT has considerable potential as an antimalarial drug target. PfNMT is encoded as a single copy gene (PF14_0127) with detectable mRNA in the asexual blood stage forms and the recombinant protein shows differential inhibition profiles compared to HsNMT1 [START_REF] Giang | A second mammalian N-myristoyltransferase[END_REF]. A range of N-myristoylated substrates from P. falciparum (including GAP (PFL1090w) [START_REF] Rees-Channer | Dual acylation of the 45 kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites[END_REF], GRASP, (PF10_0168) [START_REF] Struck | Re-defining the Golgi complex in Plasmodium falciparum using the novel Golgi marker PfGRASP[END_REF] and CDPK (PFB0815w) [START_REF] Moskes | Export of Plasmodium falciparum calcium-dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N-terminal membrane anchor motifs[END_REF]) have been identified biochemically. In addition, more than 40 potential substrates with a high likelihood of being N-myristoylated have been identified by bioinformatic and biochemical predictions; these include ADP Ribosylation Factors, CDPKs and several PfEMP1s [START_REF] Bowyer | Studies on the N-myristoyltransferase of Plasmodium falciparum[END_REF]. The presence of these known and predicted substrates of PfNMT suggests that inhibition of this enzyme would disrupt a range of biochemical pathways, ultimately resulting in loss of parasite viability.

To further study the potential of PfNMT as a drug target, we describe here the improved expression and purification of a recombinant form of the enzyme from E. coli, utilising a synthetic codon-optimised PfNMT gene. Furthermore, we also report the development of a scintillation proximity assay (SPA) amenable to high-throughput screening and demonstrate how this assay has been used for the identification of inhibitors showing activity against both recombinant PfNMT and the cultured asexual stages of P. falciparum.

METHODS

Codon optimisation for PfNMT expression in E. coli

The codon usage in the native PfNMT gene was modified to give a new synthetic gene with codons optimal for expression in E. coli. The synthetic gene was designed with the help of a Perl script program, CODOP, that allows codon optimisation for a given host organism [33]. The program allowed the insertion of desired restriction sites and generated 40-mer oligonucleotides for both strands of the gene with a melting temperature for each of the 20 nucleotide overlaps of around 60 o C. The oligonucleotides were checked for presence of repeats, stem-loops and overlaps using the Genetics Computer Group software package (version 8-Unix). A total of 60 oligonucleotides were obtained; the two end fragments were longer (49 and 61 nucleotides) to include the restriction sites BamHI and EcoRI for subsequent subcloning. The sequence was then assembled and amplified as described [START_REF] Withers-Martinez | PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome[END_REF], achieving a reduction in AT content from 73% to 60%. The amplified fragment was cloned into the vector pTrcHisA (Invitrogen) and the sequence confirmed. The resulting plasmid, pTrcNMT, was transformed into E. coli BL21(DE3)pLysS for expression studies.

Overexpression and purification of recombinant PfNMT, HsNMT1 and CaNMT

Expression of N-terminally His-tagged PfNMT from pTrcNMT was induced in E. coli BL21(DE3)pLysS in the presence of 100 µg ml -1 ampicillin. A single bacterial colony was grown for 16 h in L-broth (LB) at 37 o C with shaking at 220 r.p.m. Inoculation (1:20) of fresh LB was followed by growth at 37 °C, 220 r.p.m. to an OD 600 of 0.6. The culture temperature was reduced to 30 °C before induction with 0.1 mM IPTG (isopropyl β-D-thiogalactoside) and growth at 30 °C for 4-5 h. Cells were pelleted by centrifugation (14000 g, 10 min) and resuspended in cell lysis buffer (300 mM NaCl, 50 mM NaH 2 PO 4 , 5 mM DTT, 20 mM imidazole, pH 7.0 supplemented with 2% v/v Triton X-100) in the presence of EDTA-free mini complete protease inhibitors (Roche). Cells were then treated with lysozyme (1 mg ml -1 , 30 min on ice), followed by sonication on ice (three rounds of 2 x 10 s pulses at 15 W). The soluble fraction was isolated by two rounds of centrifugation (40000 g, 4 °C) and recombinant His-tagged protein recovered by immobilized metal affinity (IMAC) resin using Ni-Sepharose (GE Healthcare) according to the manufacturer's protocol, with elution by 500 mM imidazole.

Fractions containing NMT activity were pooled and buffer exchanged into 50 mM NaCl, 100 mM NaH 2 PO 4, 5 mM DTT, pH 7.0 by sequential concentration and dilution (3 rounds on Centricon YM-30 spin columns; Millipore) to a final volume of 2 mL. The sample was filtered (0.44 μM filter) before applying to a Sephacryl S-100 column connected to an AKTA FPLC system (GE Healthcare), maintained at 4 °C, using at a flow rate of 0.2 mL min -1 . Fractions containing NMT activity were pooled, diluted to 50 mL in 50 mM NaH 2 PO 4, 5 mM DTT, pH 6.5 and applied using a 50 mL superloop to a ReSOURCE S strong cation exchange column. Elution was conducted using a 0 -1 M NaCl gradient in the same buffer at a flow rate of 2 mL min -1 .

Products were analysed by SDS-PAGE and the identity of the major band (ca. 50 kDa) confirmed as PfNMT by MALDI-TOF mass spectrometry.

Plasmids encoding HsNMT1 (in pET20b) and CaNMT (in pET11c), were provided by Pfizer (Sandwich, Kent, UK). Recombinant forms of these proteins were expressed in E. coli BL21(DE3)pLysS as above and cells lysed in 50 mM TrisHCl, 2 mM EGTA, pH 8.6 (HsNMT) or 20 mM TrisHCl, 1 mM DTT pH 7.4 (CaNMT). For both soluble lysates, NMT activity was enriched by anion exchange using a 30 mL DEAE Sepharose column, with elution over a 0 -1 M NaCl gradient at a flow rate of 3 mL min -1 . All NMTs were stored in 25% glycerol at -20 ºC.

NMT activity and inhibition assay

Biontinylated peptide substrates based on the N-terminal sequence of P. falciparum ADP ribosylation factor 1 (PfARF1) were prepared by solid phase peptide synthesis (SPPS) using standard Fmoc/ t Bu chemistry [START_REF] Fields | Solid phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids[END_REF]. The peptides GLYVSRLFNRLFQKK(Biotin)-NH 2 and GLYVSRLFNRLFQK(Biotin)-NH 2 (PfARFlong and PfARFshort respectively) were synthesized, purified by reverse phase HPLC using a C18 column (Waters) and freeze dried. Controls in which the N-terminal glycine was replaced by alanine were also prepared. These substrates were used to develop a scintillation proximity assay (SPA) for NMT activity amenable to a 96well plate format. [ 3 H]-myristoyl-CoA (GE Healthcare) was supplemented with unlabelled myristoyl-CoA (Sigma) to achieve required specific activities (generally 8 Ci/mmol). Recombinant enzyme was prepared as described above. 100 μL reactions contained equal volumes of buffer (or inhibitor), recombinant NMT, 125 nM [ 3 H]-myristoyl CoA (8 Ci mmol -1 ) and 125 nM biotinylated peptide substrate. All solutions were prepared using assay buffer (30 mM Tris, 0.5 mM EGTA, 0.5 mM EDTA, 2.5 mM DTT adjusted to pH 7.4 with HCl, 0.1% Triton X-100). DMSO was present at a final concentration of 1% (v/v). The reactions were initiated by addition of peptide substrate after a 5 min preincubation and allowed to proceed for 30 min at 37 ºC before termination with 100 μL of stop solution (50 mg/ml SPA beads in PBS/0.05% sodium azide diluted 50 times with 1:1 0.2 M phosphoric acid buffered to pH 4.0 with NaOH and 1.5 M MgCl 2 ). The plate was then sealed and beads allowed to settle for > 8 h before scintillation counting using a Chameleon plate reader (Hidex, Finland).

K M determinations

The apparent K M for the myristoyl-CoA binding of PfNMT was determined by varying the myristoyl-CoA concentration from 0 -500 nM (8 Ci mmol -1 ) at a constant peptide concentration (500 nM) and enzyme concentration using the scintillation proximity assay. Similarly the apparent K M for the biotinylated peptide substrates was determined over the range 78 -500 nM

Inhibition assay

Inhibitors, prepared as 100 mM stock solutions in DMSO, were diluted as required in assay buffer and used to replace the 25 μL buffer fraction in the standard SPA described above.

Initially, inhibitors were screened at a single concentration (50 μM) to identify the compounds which produced the most effective inhibition of the target NMT. These were then further evaluated by determination of IC 50 values. Inhibitor concentrations were varied from 100 nM -1 mM at a constant myristoyl-CoA concentration (125 nM). The resulting data were plotted using GraFit and IC 50 values determined from a four-parameter fit. Each IC 50 value reported is the mean obtained from triplicate assays.

Parasite culture and inhibitor testing

The asexual erythrocytic stages of P. falciparum 3D7 were cultured using a modification of the procedures of Trager and Jensen [START_REF] Trager | Human malaria parasites in continuous culture[END_REF]. Parasites were cultured in RPMI1640 supplemented with 0.5% w/v albumax I (GibcoBRL), 2 mM L-glutamine, 25 mM HEPES, 24 mM NaHCO 3 , 25 μg ml -1 gentamycin, 16 μg ml -1 hypoxanthine, 1.6 mg ml -1 glucose. Human erythrocytes were obtained from the National Blood Transfusion Service. Cultures were maintained at 37 ºC in a haemocrit of 0.5-1% and a gas mixture of 7% CO 2 , 5% O 2 and 88% N 2 . Tight synchronisation was achieved using the Percoll method [37]. Schizonts, isolated by centrifugation through 70% Percoll (1000 g, 11 min), were added to fresh erythrocytes for 1 h, to allow invasion to occur, before removal of the residual schizonts by lysis with 5% w/v sorbitol in PBS. 70% Percoll was prepared by mixing Percoll 9:1 with 10 x PBS and mixing this 7:3 with RPMI 1640.

Selected compounds were added to the culture medium at a final concentration of 100 μM or 10 μM with 1% (v/v) DMSO. Inhibitor-free controls, both with and without 1% DMSO, were included. Experiments were initiated with three different stages of synchronised parasites at a parasitaemia of approximately 1%. Ring stage (ca. 5 h post invasion), trophozoite stage (ca. 27 h) and schizont stage (ca. 42 h) parasites were all allowed to develop, release and re-invade new red blood cells and progress to approximately 30 h in the next cycle. This resulted in a total

RESULTS and DISCUSSION

Codon optimisation improves the yield of PfNMT

In an attempt to improve heterologous expression of PfNMT in E. coli, codon optimisation of PfNMT was undertaken to remove codons rarely used in E. coli. These changes also resulted in an overall reduction in AT content from 73% to 60% (see supplementary figure). Expression of soluble, active protein was achieved by creating an expression construct in which the optimised PfNMT coding sequence was cloned into the BamH1 and EcoR1 restriction sites of the pTrcHisA expression vector. High levels of PfNMT overexpression were also observed using pET 28a and pGEX 5X-1 expression vectors but in these cases, the proteins remained insoluble (data not shown).

A three-stage protein purification strategy was developed. Purification of PfNMT from E. coli lysates by IMAC using Ni Sepharose was the preferred method for the capture stage of the purification. This resin is tolerant to low concentrations of DTT, which was found to reduce precipitation of PfNMT on storage. Mutation of Cys228 to Ser was also observed to reduce precipitation of purified PfNMT (PWB, unpublished observations). However, as Cys228 is predicted to reside at the bottom of the peptide-binding pocket (based upon comparisons with other known NMT structures), all subsequent work was carried out with purified recombinant wild-type enzyme stabilised with DTT rather than the C228S mutant (which has the potential to alter substrate binding).

Sequential stages in the purification of PfNMT are shown in Figure 1. PfNMT is the major band following capture with Ni-Sepharose IMAC (Lane 1, apparent molecular mass ~50 kDa, consistent with predictions from primary sequence analysis). Concentration of this material and subsequent separation by size exclusion chromatography yielded a fraction containing PfNMT with a retention time consistent with a monomeric form of the enzyme. This fraction also contained some contaminating species with lower molecular masses (lane 2). In the absence of DTT, a second peak with a retention time consistent with a 75 kDa protein complex was also observed; this peak was eliminated in the presence of 5 mM DTT (data not shown). The final stage of the purification used cation-exchange chromatography (see Methods) and resulted in the separation of PfNMT from all but one significant impurity (Lane 3). This was identified, by peptide mass fingerprinting, as an E. coli Cap-DNA recognition protein with a predicted mass of 24 kDa. The protein band observed at 50 kDa was confirmed as PfNMT by peptide mass fingerprinting with > 60% peptide coverage. The overall yield was typically 400 μg L -1 representing a >20-fold improvement on previous methods [28]. Glycerol was added to a final concentration of 25% (v/v) in the PfNMT sample which was then stored at -20 ºC. When stored in this way PfNMT remained stable for > 2 years with negligible loss in activity (as tested in the SPA). Storage at 4 ºC resulted in formation of a precipitate and loss of activity; this precipitation was decreased, but not eliminated, in the C228S variant.

Codon optimisation and the creation of synthetic P. falciparum genes have been shown to be an effective strategy for improving expression efficiency in heterologous expression systems such as P. pastoris [START_REF] Kocken | High-level expression of the malaria blood-stage vaccine candidate Plasmodium falciparum apical membrane antigen 1 and induction of antibodies that inhibit erythrocyte invasion[END_REF], baculovirus [START_REF] Withers-Martinez | PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome[END_REF], murine cells [START_REF] Narum | Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice[END_REF] and E. coli [START_REF] Dutta | Purification, characterization, and immunogenicity of the refolded ectodomain of the Plasmodium falciparum apical membrane antigen 1 expressed in Escherichia coli[END_REF]. However, in a recent extensive study investigating ~1000 P. falciparum open reading frames in an E. coli expression system [START_REF] Mehlin | Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes[END_REF], only a fraction of the selected sequences were successfully expressed (337, of which only 63 provided significant levels of soluble protein). Further investigation of sequences with little or no expression was conducted using alternative expression approaches and revealed little advantage to codon optimisation (3 out of 12 gave insoluble protein) and insect cell expression systems (1 out of 17 proteins previously obtained in the insoluble fraction gave soluble protein). Although some success was obtained with each of these alternatives, no ideal system for expression of P. falciparum proteins could be determined. Most recently Vedadi et al. [START_REF] Vedadi | Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms[END_REF] have reported the effective use of an E. coli expression platform to obtain structural information for apicomplexan proteins by attempting expression of ~400 P. falciparum genes along with selected orthologues. The differing expression profiles of orthologous proteins in the E. coli system resulted in successful structural determinations for significantly increased numbers of the target proteins or their orthologues. Expression studies have not been conducted on other apicomplexan NMTs to date. In this study, we have concentrated on the P. falciparum NMT and demonstrated the benefits of codon optimisation for soluble protein expression from this particular gene.

Development of a scintillation proximity assay for NMT activity

The N-myristoylation reaction transfers myristate from myristoyl-CoA to the N-terminus of a peptide/protein substrate. The majority of the assays developed for monitoring NMT activity have used radiolabel transfer to achieve high sensitivity with subsequent separation of product by HPLC or binding to phosphocellulose paper [START_REF] Panethymitaki | Characterisation and selective inhibition of Myristoyl CoA: protein Nmyristoyl transferase from Trypanosoma brucei and Leishmania major[END_REF][START_REF] Glover | N-myristoylation of p60src. Identification of a myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues[END_REF][START_REF] King | N-myristoyl transferase assay using phosphocellulose paper binding[END_REF]. Whilst these can be successfully used to monitor inhibition of NMT, we favoured an approach more suited to high throughput screening and later automation. For this purpose, we elected to use a scintillation proximity assay (SPA).

The success of such an assay relies on incorporation of radiolabel into a product that can be selectively bound to the immobilised scintillant. In the case of N-myristoylation, 3 H-myristate is transferred to a biotinylated peptide substrate that is bound to streptavidin-coated scintillant beads after termination of the enzymatic reaction. The presence of the scintillant in the bead eliminates the liquid handling stages required by the most similar assay reported [START_REF] French | Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-based inhibitors of human Nmyristoyltransferase-1[END_REF] which uses streptavidin-coated 96-well plates to isolate the 3 H-labelled peptide product before washing, to remove unreacted 3 H-myristate, and addition of a liquid scintillant to facilitate counting.

Choice of a suitable NMT substrate was critical for successful assay development. We have previously shown that recombinant P. falciparum ADP ribosylation factor 1 (PfARF1) is a PfNMT substrate both in vitro and in vivo [28,[START_REF] Rees-Channer | Dual acylation of the 45 kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites[END_REF], while reconstituted N-myristoylation assays in E. coli have demonstrated that PfARF1 is also a substrate for CaNMT and HsNMT1 (data not shown).

On the basis of this information, two biotinylated peptides (PfARFshort and PfARFlong, Table 1) were synthesized (by SPPS) for use as artificial PfNMT substrates. PfARFshort and PfARFlong contain residues 2-15 or 2-16, respectively, of PfARF1 (omitting the N-terminal initiator methionine). Both peptides also contain a biotinylated lysine replacing the naturally occurring lysine as the C-terminal residue. These longer peptides were used in preference to the octapeptides used in many NMT substrate studies [46][START_REF] Rocque | A comparative analysis of the kinetic mechanism and peptide substrate specificity of human and Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase[END_REF][START_REF] Towler | Aminoterminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase[END_REF], to prevent the C-terminal biotinylated lysine from interacting with the peptide binding pocket, thereby minimising potential steric hindrance which could adversely affect the N-myristoylation reaction. Both peptides were confirmed as PfNMT substrates in an HPLC-based activity assay (data not shown). PfARFlong was demonstrated as the better SPA substrate in the same HPLC assay and subsequently in K M determinations using the SPA (K M 1.2 μM ± 0.4 vs 7.1 μM ± 1.3 for PfARFshort).

Development of the SPA was therefore initiated using the PfARFlong substrate (Figure 2). First, the dependence of radioactive incorporation on the presence of both enzyme and peptide substrate was confirmed (Figure 2A). Substituting PfARFlong with AlaARF (in which the Nterminal glycine is replaced by alanine; Table 1) resulted in no detectable signal over background levels. This is consistent with published fungal NMT data, demonstrating that an Nterminal glycine residue is required for enzymatic N-myristoylation [START_REF] Towler | Aminoterminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase[END_REF]. The SPA format also requires discontinuous sampling, taking single time point measurements for each reaction condition and enzyme tested. In order to determine the rate limiting concentrations of NMT required for constant reaction rate over a standard period (allowing initial rate determinations), increasing concentrations of CaNMT, HsNMT and PfNMT were titrated over a 30 min reaction (Figure 2B). A peptide concentration was selected to allow total binding to the scintillant beads, thus eliminating competition for binding sites between unreacted biotinylated substrate and Nmyristoylated product. It is possible to use larger quantities of biotinylated peptide in the reaction (greater than the bead capacity), but the decreased signal resulting from the competitive binding and the additional error dependent on SPA bead concentration made this approach less favourable. The bead capacity for PfARFlong was found to be 50-100 pmol mg -1 .

To further validate the optimised SPA conditions developed above, CaNMT and a known inhibitor of its activity, UK362091 (Pfizer), were used in an IC 50 determination (Figure 2C). The IC 50 value recorded (11.6 ± 1.5 nM) is consistent with that previously determined at Pfizer (19 nM, unpublished data). In addition, we have previously reported the favourable comparison of the SPA with a more laborious phosphocellulose paper binding assay, using TbNMT and a biotinylated substrate based on the T. brucei CAP5.5 protein [START_REF] Panethymitaki | Characterisation and selective inhibition of Myristoyl CoA: protein Nmyristoyl transferase from Trypanosoma brucei and Leishmania major[END_REF]. In view of these collective results, the optimised SPA methods could now be applied to the screening of fungal NMT inhibitors against PfNMT and HsNMT1 in a 96-well plate format.

Inhibitors based around a benzothiazole scaffold can inhibit PfNMT in vitro

Initial screening for inhibition of PfNMT was conducted using five of the fungal NMT inhibitors described previously in studies on TbNMT and LmNMT [START_REF] Panethymitaki | Characterisation and selective inhibition of Myristoyl CoA: protein Nmyristoyl transferase from Trypanosoma brucei and Leishmania major[END_REF]. Compounds CP005240 and CP014553, identified as inhibitors of TbNMT, were also identified as the most potent inhibitors of PfNMT in vitro (IC 50 values of 360 nM and 280 nM respectively, Table 2). However, in contrast to TbNMT, relative inhibition (as compared with HsNMT1) was also achieved with UK370485.

Forty-three compounds (provided by Pfizer), each containing the same benzothiazole scaffold as UK370485, were screened for inhibitory activity at 50 μM against both PfNMT and HsNMT1.

Seven compounds showed >25% inhibitory activity against recombinant PfNMT (data not shown). IC 50 values were generated for these, together with four non-benzothiazoles, previously analysed against LmNMT and TbNMT [START_REF] Panethymitaki | Characterisation and selective inhibition of Myristoyl CoA: protein Nmyristoyl transferase from Trypanosoma brucei and Leishmania major[END_REF] (labelled * in Table 2). Table 2 shows that three benzothiazole-containing compounds (1, 4 and 7) have IC 50 values < 50 μM for PfNMT. All the compounds shown in Table 2 showed some selectivity for PfNMT, with the exception of compound 2 (UK370710) which is more inhibitory to HsNMT than PfNMT.

The structures of these active benzothiazole compounds are shown in Figure 3.

Comparison reveals that the cyclohexyl linker region between the benzothiazole functionality and the aromatic group contains two distinct regiochemistries: 1,4-substituted (compounds 1-3), and (R,S) 1,3-substituted (compounds 4-7) in contrast to the starting compound UK370485, which is trans-1,4. The effect of modifications at the C-6 position of the benzothiazole group can be seen in the reversal of selectivity between PfNMT and HsNMT1 upon change from small fluorine or hydrogen atoms (compounds 3 and 1) to the bulky pyridin-2-yl functionality (compound 2). The majority of compounds that showed inhibition of PfNMT contain the dimethylamide group that is known to form favourable binding interactions in the peptide-binding pocket of CaNMT (Pfizer). Optimisation of both the C-6 substitution and the linker region is currently in progress and presents an opportunity to increase specificity and selectivity of the inhibitors.

Benzothiazole based inhibitors are toxic to cultured asexual stages of P. falciparum

Compounds 1, 3, 4 and 7 (Figure 3 and Table 2) were applied at concentrations of 100 μM and 10 μM to synchronised trophozoite stage P. falciparum 3D7 (approximately 27 h post invasion).

Figure 4 shows the effect of these compounds on total parasitaemia, compared to controls containing 1% DMSO (v/v) only. In all cases, there is a significant reduction in parasitaemia at 100 μM to a level similar to the starting parasitaemia (ca. 1%). In the case of compounds 1 and 3 at 10 μM, there is approximately an 80% reduction in parasitaemia compared to the controls with DMSO only (t-test, P < 0.03). This inhibitory effect is greater than that observed for compounds CP005240 and CP014553, despite the higher IC50s of the benzothiazoles in the SPA (Table 2).

Changes in parasite morphology were also monitored following the application of different compound concentrations at three different start-points (5 h, 27 h and 42 h post invasion), followed by continuous culture until the mid-trophozoite stage (for controls) in the next cycle (data not shown). In all samples treated with potent compounds, a short survival time was indicated by the presence of dead or dying parasites (as assessed by abnormal morphology) that progressed only a short distance through the life-cycle. However, these dead or dying parasites were infrequent; instead, the large reduction in parasitaemia between experimental samples and controls resulted from the complete absence of detectable Giemsa-stained parasites, suggesting that these cells degenerate quickly and are incapable of invasion and development. Some parasites that did not die in the strongly-inhibited samples were characterised by considerably slower development, with parasites still at the ring stage when control cultures were at the trophozoite stage (> 10 h delay over a 48 h time course; data not shown). In the cultures treated with10 μM of compounds 1 and 3, the effect on both reduction in parasitaemia and delay in development was reduced, although not significantly. Parasites grown in the presence of 10 μM of compounds 4 and 7 were indistinguishable from the controls.

The reasons for the striking differences in morphology observed between compounds 1, 3 and 4, 7 are not known but may be due to different levels of uptake and accumulation by the cultured cells. Additional off-target effects of these compounds, distinct from their inhibitory role against PfNMT, cannot be discounted at this stage.

Outlook

Previous investigations into parasitic protozoan NMT inhibitors have focused on a "piggy-back" approach, using compounds originally identified as NMT inhibitors for the fungal pathogens, C. albicans and Aspergillus fumigatus [START_REF] Gelb | Protein farnesyl and Nmyristoyl transferases: piggy-back medicinal chemistry targets for the development of antitrypanosomatid and antimalarial therapeutics[END_REF][START_REF] Panethymitaki | Characterisation and selective inhibition of Myristoyl CoA: protein Nmyristoyl transferase from Trypanosoma brucei and Leishmania major[END_REF]. We have also reported a small-scale study using peptide aptamers for inhibition of PfNMT [START_REF] Tate | Peptide-based inhibitors of N-myristoyl transferase generated from a lipid/combinatorial peptide chimera library[END_REF]. The piggy-back screening approaches for PfNMT reported here have identified a benzothiazole-based compound that shows anti-parasitic activity in culture, prompting testing of a range of structurally related compounds. This has led to the identification of several structurally-related compounds with IC 50 values < 50 μM, that show some selectivity over HsNMT1 and anti-parasitic activity against cultured P. falciparum. These results compare favourably with those obtained for the highest affinity inhibitors identified in screens of HsNMT1 by French et al. [START_REF] French | Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-based inhibitors of human Nmyristoyltransferase-1[END_REF], using a library of 14,000 compounds. Notably, one compound, containing the benzothiazole scaffold, was also found to have an IC 50 < 50 μM against HsNMT1, demonstrating the value of this piggy-back experimental approach in rapidly identifying drug-like molecules with a potential for further development to provide inhibitors of both HsNMT and PfNMT.

The compounds reported here represent the first evidence of a related family of molecules capable of inhibiting PfNMT. All these compounds were originally synthesised as part of an investigation into inhibitors of fungal NMTs and the discovery of compounds with high HsNMT1 (open circles). A close approximation to a linear fit demonstrates that enzyme concentrations can be selected so that the amount of enzyme is rate limiting under the experimental conditions selected. A.u., arbitrary units.

C. An IC 50 value for inhibitor UK 362091 with CaNMT was determined using the SPA.

Inhibitor concentrations were varied from 1 μM -42 pM in otherwise identical reactions (125 nM PfARFlong peptide, 125 nM myristoyl-CoA, 3 ng CaNMT). A plot of cpm, after 30 min incubation at 37 ºC, against inhibitor concentration allows a four-parameter fit to derive the point at which the signal is half maximal, the IC 50 . The error bars are the standard deviation around the mean from a triplicate repeat. The IC 50 value obtained is 11.6 nM (see inset), consistent with a previous evaluation of 19 nM (Pfizer, unpublished data).

Table 1 Peptides evaluated as substrates of PfNMT

The peptides listed were synthesised by standard Fmoc t Bu SPPS from Rink-Amide MBHA resin pre-loaded with biotinylated lysine, giving a C-terminal amide and glycine or alanine as the Nterminal residue. ARF peptides are based on the N-terminal sequence of PfARF1. K M values for the substrate peptides demonstrate that ARFlong is a better substrate than ARFshort. Peptides with Ala replacing Gly are not substrates for PfNMT.

Peptide

  at a constant myristoyl-CoA concentration of 250 nM. The data obtained were used to generate 6 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20070692 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070692 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society initial rates of reaction from which K M values were obtained using the GraFit software package (version 5.0.13, Erithacus Software, UK: www.erithacus.com/grafit/index.htm). The results quoted are the mean from triplicate assays.

  inhibitor exposure time of 68, 53 and 31 h for the ring, trophozoite and schizont stages, respectively. At the end of each experiment, parasite morphology was examined and the numbers of infected RBCs were determined by Giemsa staining and light microscopy, counting > 2000 red blood cells per sample. The results shown are the mean of a triplicate repeat. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20070692 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070692 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1

 1 Figure 1 Purification of recombinant PfNMT Recombinant PfNMT was enriched in three stages from E. coli soluble lysate, prepared following protein expression in a 50 L fermenter. Products after pooling of fractions showing NMT activity from each purification stage were separated by SDS-PAGE as shown. Lane 1 -Capture stage using Ni-Sepharose IMAC. Lane 2 -Intermediate purification on Sephacryl S-100 size exclusion column. Lane 3 -Final stage using ReSourceS cation exchange (pooled active NMT fractions shown). M, molecular mass markers. In Lane 3, the band migrating at approximately 50 kDa (+) was identified as PfNMT by peptide mass fingerprinting. The major impurity (*) was characterized by peptide mass fingerprinting and is most likely to be an E. coli Cap-DNA Recognition protein (gi:2098303), consistent with the observed molecular mass of 24 kDa.

Figure 2

 2 Figure 2 Development of SPA for NMT activity

Figure 3

 3 Figure 3 Structures of PfNMT inhibitors based around a benzothiazole core structure

Figure 4

 4 Figure 4 Inhibitors of PfNMT in vitro reduce P. falciparum parasitaemia in culture

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20070692 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070692 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20070692 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070692 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 23 Aug 2007 as manuscript BJ20070692 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070692 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 .

 1 Figure 1. Purification of recombinant PfNMT

Figure 2 .

 2 Figure 2. Development of SPA for NMT activity

Figure 3 .

 3 Figure 3. Structures of PfNMT inhibitors based around a benzothiazole core structure

Figure 4 .

 4 Figure 4. Inhibitors of PfNMT in vitro reduce P. falciparum parasitaemia in culture

Table 2 IC 50 values for benzothiazole inhibitors showing activity at 50 μM

 2 IC 50 values were determined using the SPA as described for PfNMT and HsNMT1. Compounds previously evaluated as inhibitors of TbNMT and LmNMT (*;[START_REF] Panethymitaki | Characterisation and selective inhibition of Myristoyl CoA: protein Nmyristoyl transferase from Trypanosoma brucei and Leishmania major[END_REF]) were investigated together

	with benzothiazole-based inhibitors (structures 1-7 in Figure 3) that show inhibitory activity at 50
	μM.
	Inhibitor	IC 50 value determined in SPA (μM)
		PfNMT	HsNMT
	CP 005240 (*)	0.36 +/-0.04	1.8 +/-0.3
	CP 014553 (*)	0.28 +/-0.12	1.6 +/-0.5
	CP 144568 (*)	300 +/-78	>1000
	UK 370485 (*)	232 +/-46	>1000
	UK 145974 (*)	> 1000	>1000
	UK370309 (4)	17 ± 1	331 ± 29
	UK370509 (1)	30 ± 3	286 ± 93
	UK370624 (5)	53 ± 5	656 ± 210
	UK370709 (6)	111 ± 7	711 ± 133
	UK370710 (2)	115 ± 9	28 ± 9
	UK362799 (3)	68 ± 4	309 ± 58
	UK370713 (7)	37 ± 4	329 ± 84

We thank Tanya Parkinson, Andy Bell and colleagues at Pfizer for technical advice and helpful discussions. This work was funded by the Wellcome Trust (061343, programme grant to DFS; 065514, studentship to PWB) and the U.K. Medical Research Council.


Karen Côtes

Rabeb Dhouib

Isabelle Douchet

Henri Chahinian

Alain De Caro

Frédéric Carrière

Stéphane Canaan
email: canaan@ibsm.cnrs-mrs.fr

Rabeb Dhouib #

Characterization of an exported monoglyceride lipase from Mycobacterium

Keywords: Mycobacterium tuberculosis, M. tuberculosis, Terrific Broth, TB, Triacylglycerol, TAG, Diacylglycerol, DAG, Monoacylglycerol, MAG, β-cyclodextrin, β- lipase, Rv0183, expression, monoglyceride, mycobacterium, exported, immunolocalization

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Synopsis

The Rv0183 gene of the M. tuberculosis H37Rv strain, which has been annotated as a probable lysophospholipase, was cloned and expressed in E. coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolyzed monoacylglycerol substrates with a specific activity of 290 U.mg -1 at 37°C. Rv0183 hydrolyzes both long chain di-and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with monoacylglycerol.

The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using monoolein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser 110, Asp 226 and His 256, as confirmed by the results of site directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologs of Rv0183 can be identified in other mycobacterial genomes such as M. bovis, M. smegmatis, and even M. leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and the culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

Introduction

Although much research has been focused on Tuberculosis during the last century, it is still the most deadly infectious disease worldwide. It has been estimated that one third of the World's population has been contaminated by, Mycobacterium tuberculosis (M. tuberculosis), the etiologic agent responsible for this disease. Despite the existence of effective drug treatments, 2.5 to 3 million people die of this disease every year (http://www.who.int/mediacentre/factsheets/fs104/en/print.html). The rise of Tuberculosis is directly linked to the spread of AIDS and the development of new multi drug resistance (MDR) strains. M. tuberculosis is a highly complex microorganism, and a better understanding of the biochemistry and the physiological role of the enzymes involved in the various metabolic pathways taken by the infectious process could lead to designing new therapeutic agents to fight the bacillus.

Among the various metabolic pathways involved, lipid metabolism is thought to be one of the main pathways taken by mycobacteria, and this pathway therefore needs to be studied in detail. Studies on the lipids present in mycobacteria have been going on for the last 40 years, and considerable attention has focused on the lipids involved in the cell wall. Many unusual lipids, including phthiocerol dimycocerosates, mycolic acid, glycolipids, polyketides, and glycans, have been found to be involved in the virulence and pathogenicity of M. tuberculosis [1][START_REF] Minnikin | Lipids: complex lipids, their chemistry, biosynthesis and roles[END_REF][3][4].

An intracellular lipid storage process was recently described in M. tuberculosis, forming organites known as lipid bodies. These organites are composed of triacylglycerols (TAG) and other lipids, such as wax esters, polyhydroxyalkanoic acids (PHA) and monomeromycolyldiacylglycerol [4][5][6][7][8][9]. It has been proposed that these lipids probably provide a carbon source promoting the growth of the bacteria during the chronic infection phase [7,10]. It might therefore store the energy required during long periods of bacterial, dormancy [11] and serve as storage form of fatty acids (FA) required for membrane lipid formation [4]. The storage of these lipids in vivo may therefore be crucial to the survival of the bacteria and allow them to adapt to the environment provided by the host cells.

It has been suggested by in vitro studies that the accumulation or the presence of FA in the culture medium may stimulate the dormancy phase of the bacteria: the growth rate of most mycobacteria can be enhanced by adding oleic or palmitic acid to the culture medium [12][13][14].

FA can be generated in vivo by the hydrolysis of host cell lipids [7,10]. During the formation of granulomas, mycobacteria are mixed with phospholipids originating from dead cells (host cells and bacteria), which may be hydrolyzed by phospholipase A and C (PLA and PLC, respectively), leading to the release of lysophospholipids, fatty acids and diacylglycerols (DAG) [15]. The resulting DAG may then be hydrolyzed by a secreted or exported lipase and the microorganism will absorb FA released. A synergy of this kind between lipases and phospholipases has been previously described in the case of another pathogen, Pseudomonas aeruginosa, which degrades lung surfactants (phospholipids) via the joint action of a PLC and a lipase [16]. Excreted PLA and PLC have been previously described in M. tuberculosis [17,18], but no extracellular lipase has been identified so far in this strain. Two intracellular lipolytic enzymes which may participate in the degradation of intracellular TAG have been identified and purified so far in mycobacteria. The first of these is a native enzyme which was purified from M. phlei [19] and characterized. The second lipolytic enzyme was the expression product of the Rv3097c gene from the strain M. tuberculosis H37Rv in Escherichia coli [20].

Using the proteins deduced from the complete genome sequence of M. tuberculosis [21], it H37Rv (accession number Rv0183). This enzyme shows 34% to 36% amino acid sequence identity with monoglyceride lipases from human and mouse adipocytes, respectively [22,23].

Contrary to what was suggested by genome annotation, which identified Rv0183 as a lysophospholipase, this enzyme does not hydrolyze lysophospholipids but hydrolyzes neutral lipids, showing a clear-cut preference for monoacylglycerols (MAG). Polyclonal antibodies specific to Rv0183 were prepared and used to determine the location of the enzyme in M.

tuberculosis. Based on all our biochemical data, it seems possible that Rv0183 may contribute to the growth of M. tuberculosis bacilli and be involved in the hydrolysis of host cell lipids during the infection process.

Materials and methods

Materials

Pfx DNA polymerase, pDonR221, and pDest14 plasmids were purchased from Invitrogen.

Rosetta pLysS E. coli cells were purchased from Novagen. Ni 2+ -Agarose gel was obtained from Amersham Biosciences. All vinyl esters, triacylglycerols, cholesterol oleate, phenylmethylsulfonide fluoride (PMSF), sodium taurodeoxylcholate (NaTDC), sodium deoxylcholate (DOC), gum Arabic (GA), Triton X-100, diethyl p-nitrophenyl phosphate (E600) and goat anti rabbit immunoglobulins conjugated to peroxydase were obtained from Sigma-Aldrich. Tripropionin was purchased from Acros Organics, and pure mono-and diolein were purified from a commercial low grade DL-α-monoolein from Fluka. Tetrahydrolipstatin (THL) was a gift from Hoffmann la Roche. isopropyl β-D-thiogalactoside (IPTG) was from Sigma. Phthiocerol dimycocerosates, glycerol monomycolates and phosphatidyl-inositol dimannoside were provided by Dr. Martine Gilleron from IPBS-UMRS 5089 (Toulouse, France).

Methods

Cloning:

The DNA fragment of the Rv0183 open reading frame (ORF) was amplified by PCR from the bacmid BAC-Rv165 provided by the Pasteur Institute [21,24]. The full Rv0183 sequence proposed by Camus et al. [25] was analyzed using the software program the SignalP 3.0 Server, which identified a putative signal sequence. A truncated form of Rv0183 without the signal sequence (starting from Thr 46) was therefore produced. The primers used, which contained the attB1, Shine Dalgarno, kozak, 6 His-Tag and a Tobacco Etch Virus (TEV) NIa (TEV) site sequences at the 5' end and the attB2 recombination site at 3' end, are presented in Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. manufacturer's instructions (Gateway, Invitrogen). The DNA sequence of the Rv0183 ORF was confirmed by performing DNA sequencing (MilleGen Biotechnologies, France). The recombinant Rv0183 (rRv0183) protein without the 6 His tail and the TEV site is composed of 279 residues and has a calculated molecular mass of 30187 Da, a theoretical isoelectric point of 5.8 and a theoretical extinction molecular coefficient of 0.858 M -1 .cm -1 at 280 nm.

Mutagenesis:

Three Rv0183 mutants in which the putative catalytic residues Ser 110, Asp 226 and His 256 were mutated into Ala were obtained using the Quickchange ® site-directed mutagenesis system (Stratagene, La Jolla, CA, USA). A fourth mutant built in with Asp 226 was replaced by Asn. The oligonucleotides used for PCR mutagenesis are specified in Table 1.

Expression and purification of recombinant and Rv0183 mutants:

E. coli Rosetta pLysS cells were transformed with the pDest14-His-Rv0183 plasmid and grown overnight in LB medium with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37 °C. On the following day, the culture medium was diluted 20 times with Terrific Broth (TB) medium containing antibiotics and grown at 37 °C. Protein expression was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) when the OD 600 had reached the 0.6-0.8 range.

Temperature was then decreased to 25 °C and cells were grown overnight. After 16 hours, cells were harvested at 4 °C by centrifugation at 6,500 g for 20 min. The pellet was resuspended in ice-cold lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.25 mg/mL lysozyme; 30 mL per initial culture liter) and stored overnight at -80 °C. The cell suspension was thawed on ice for 1 hour and 10 µg/mL DNAse I and 20 mM MgSO 4 (final concentration) were then added. The mixture was incubated for 30 min. The supernatant containing the recombinant protein was separated from the cell debris by performing a centrifugation step at 17,000 g for 30 min. This supernatant was loaded (3 mL/min) using a FPLC chromatography system (Amersham Biosciences) onto a Ni 2+ Agarose column (1 mL resin per 5 mg of recombinant protein) previously equilibrated with buffer A (10 mM Tris-HCl pH 8.5, 150 mM NaCl) containing 10 mM imidazole. The column was then washed using 5 column volumes of 5% and 10% of buffer B (buffer A+500 mM imidazole). rRv0183 was eluted with 100% buffer B. After analysis by SDS-PAGE [26], the eluted fractions containing the enzyme were pooled at a concentration of around 4 mg/mL and loaded onto a size exclusion chromatography (Superdex 200) in 10 mM Tris-HCl pH 8.5 buffer containing 150 mM NaCl. The 6 His-tag in the N-terminal position of rRv0183 was removed by TEV digestion and purified by exclusion from a Ni 2+ -Agarose column.

Proteolytic digestion of rRv0183 by TEV was performed overnight at room temperature using a TEV/rRv0183 ratio of 1/10. During the purification process, the amounts of rRv0183 were monitored by measuring the enzyme activity. After the purification process, the protein concentration was calculated from the OD value measured at 280 nm using the extinction molar coefficient (0.858 M -1 .cm -1 ). The enzyme was then concentrated to 12 mg/mL and stored at -80 °C.

pH-stat kinetic assays:

Enzymatic hydrolysis of solutions and emulsions of various esters was monitored potentiometrically for at least 5 min at various temperatures using a pH-stat (TTT 80, Radiometer, Copenhagen, Denmark). Assays were performed in 15 mL of 2.5 mM of Tris-HCl buffer at pH 7.5, containing 150 mM NaCl and 3 mM NaTDC. Fatty acid release was automatically titrated with 0.1 N NaOH. The specific activity of the Rv0183 enzyme was expressed in units per milligram of protein (U.mg -1 ), where one unit corresponds to the release of one micromole of fatty acid per minute.

Analysis of lipolysis products by thin layer chromatography (TLC):

In order to check whether rRv0183 was able to hydrolyze α-2-monoacylglycerol (2-MAG), this compound was generated from triolein using Human Pancreatic Lipase (HPL). HPL is known to specifically hydrolyze the sn-1 and sn-3 ester bonds of triacylglycerol and to specifically release 2-MAG.

Assays were performed at 37 °C using HPL (140 µg) and colipase (molar ratio 1:2) in 15 mL of 1 mM Tris-HCl buffer, pH 7.5 containing 2 mM CaCl 2 , 3 mM NaTDC, 150 mM NaCl , and triolein (460 µmol) emulsified with gum arabic (GA) (3.1%, w/v). Five hundred microliters of the reaction mixture were collected at various times: before adding HPL (T0), after 1 hour of incubation with HPL alone and 10, 20 and 120 min after adding rRv0183 (780 µg). The reaction products were then extracted with 5 mL of a chloroform/methanol (2:1, v/v) mixture. The organic phase was separated from the water phase and dried over magnesium sulphate, and the neutral lipids were separated by performing TLC using silica gel plates from Merck and n-heptane/diethyl ether/acetic acid (55:45:1, v/v/v) as the eluent. After spraying the plates with a cupric acetate-phosphoric acid solution, the neutral lipid bands were revealed by heating the plates at 180 °C for 10-15 min.

Monolayer assays:

Lipase activities were determined using a zero order trough, as described by Verger and de Haas [27] using 1,2 dicaprin (DiC10), monoolein, diolein, purified soybean oil and phospholipids (DLPE, DLPG, DLPC and DPPC) as substrates. The aqueous subphase was composed of 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 21 mM CaCl 2 and 1 mM EDTA. In addition, 13 mM β-cyclodextrin (β-CD) were added when long chain glycerides were used as substrates. The long chain FA released upon substrate hydrolysis were solubilised by β-CD to prevent any interference with the surface pressure. The reactions were performed at 25 °C.

The subphase in the reaction compartment was continuously agitated with a 2.0 cm magnetic stirrer set at 250 rpm. The enzyme solution (final concentration 5 nM) was injected through the film over the stirrer. The reaction compartment had a surface area of 100 cm 2 and a volume of 130 mL. The reservoir compartment was 21.9 cm long and 14.8 cm wide.

Effects of pH, temperature and NaTDC on enzyme stability and activity:

The pH stability profile of rRv0183 was determined after incubating the enzyme for 90 min in 1 mL of buffers containing 100 mM NaCl set at various pH values: 100 mM sodium acetate at pH 4.0 and 5.0; 100 mM MES at pH 6.0 and 6.5; 100 mM Hepes at pH 7.0 and 7.5; 100 mM Tris-HCl at pH 8.0 and 100 mM glycine at pH 9.0. The residual activity was determined potentiometrically at 37 °C at pH 7.5 using a monoolein substrate (see pH-stat assays) in the presence of 3 mM NaTDC (final concentration). To draw up the pH and temperature dependence profile, buffer and substrate were pre-equilibrated at selected pH levels. The NaTDC dependence was assessed potentiometrically at 37 °C in 2.5 mM Tris-HCl buffer pH 7.5, 150 mM NaCl containing NaTDC at concentrations ranging from 0 to 6 mM.

Inhibition assay:

Purified rRv0183 (0.7 nmole) in 5 mM Hepes buffer (pH 7.5) containing 500 mM NaCl was pre-incubated at 25 °C with several inhibitors (THL, E600 and PMSF) at a lipase-inhibitor molar ratio of 1/200 in all cases. Each inhibitor was previously solubilized in anhydrous ethanol (10 mM of a stock solution). The reaction was performed in a final volume of 70 µL in the absence or presence of 3 mM NaTDC. The remaining activity was measured potentiometrically as a function of time using a monoolein substrate.

Titration of the sulfhydryl group:

Reaction mixtures were prepared as follows in the presence and absence of 2% SDS: 190 µL of protein solution (0.4 mg/mL) were mixed with 810 µL of 5 mM Hepes pH 7.5, 150 mM NaCl containing 0.35 mM 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB) at 25 °C. A 5 mM stock solution of DTNB was initially prepared in methanol. The reactions were carried out in 1 cm pathlength cells with stirring. The changes in absorbance occurring at 412 nm, corresponding to the formation of the TNB product (ε 412 = 13.9 mM 1 .cm 1 [28]), were monitored on a Kontron spectrophotometer (model SFM 25).

S t a g e 2 ( a ) P O S T -P R I N T Production and purification of anti-rRv0183 polyclonal antibodies:

Antiserum against rRv0183 was prepared in rabbits (Bourgogne Fawn) using 4 mg of purified antigen. Rabbits were injected subcutaneously every 2 weeks with 0.5 mg antigen emulsified with complete Freund's adjuvant. Rabbits were bled by ear vein puncture before the beginning of the immunization procedure (pre-immune sera), then 1 week after the second booster injection and weekly injections were administered thereafter. The antibody response was assessed by performing a direct binding ELISA using rRv0183 antigen. This immunization procedure yielded an anti-Rv0183 serum with a titer of 1/40,000. Polyclonal antibodies were purified from the serum by performing affinity chromatography using a column of immobilized rRv0183 prepared as follows: Eleven milligrams of immobilized rRv0183 were coupled to 3 mL of AffiGel-10 (Bio-Rad) with a high efficiency of up to 90% and packed into an IBF column (diameter 1 cm). Serum was loaded onto the AffiGel-10-rRv0183 column and incubated overnight at 4 °C. After washing the column with 10 mM Tris-HCI pH 7.0 and eluting pure immunoglobulins at an acidic pH level (2.4), the solution was immediately neutralized to pH 7.0 with a 2 M Tris-HCI pH 9.0. Purified antibodies (20 mg) were concentrated at 2 mg/mL and stored at -20 °C.

Western blotting analysis of Rv0183 in cellular fractions from M. tuberculosis:

Samples of M. tuberculosis were kindly provided by the "TB Vaccine Testing and Research

Materials" Contract funded by the NIH and NIAID (National Institutes of Health). Proteins were separated on 12% SDS-PAGE before being transferred onto a nitrocellulose membrane [29]. The purified polyclonal antibodies directed against rRv0183 (final dilution 1:1,000) were incubated for 1 hour with the nitrocellulose membrane previously saturated with 3% dry milk powder (Régilait ® ). After 3 washes, the membrane was incubated for 1 hour with horseradish peroxydase-conjugated anti-rabbit IgG (at a dilution of 1:4,000). The immunoreactive proteins were revealed after covering the membrane with enhanced

Results

Selection of Rv0183:

BLAST searches against all available microbial genomes showed that homologs of Rv0183 gene (without its putative signal peptide sequence) are to be found in many mycobacterial species and other microorganisms such as Burkholderia and Streptomyces sp. Sequence identity between the mycobacteria is 100% in the case of M. bovis, 79% in that of M. leprae and 68% in that of M. smegmatis, corresponding to e-values ranging from 0 to 10 -111 (Figure 1).

To our knowledge, none of the homologs identified among the microbial genomes have been purified and characterized so far. Despite their low sequence identity with lysophospholipases (14 to 18%), these proteins have been annotated as putative lysophospholipases. However, a blast search against human and mouse genomes identified two hits with e-values ranging from 10 -35 to 10 -37 corresponding to a sequence identity ranging from 34% to 36%. Both hits correspond to monoglyceride lipases (MGL) involved in fat mobilization processes in adipose tissues [22,30]. In Rv0183 and all the mycobacterial homologs, two lipase motifs including the GXSXG pentapeptide containing the catalytic Ser and a His-Gly dipeptide are conserved.

In addition, the His and Met residues present in the pentapeptide containing the active serine (G-H-S-M-G) are strictly conserved in Rv0183 and in human and mouse MGL. Secondary structure predictions suggested that Rv0183 may belong to the α/β hydrolase fold family and that it is composed of 8 β-strands and 9 α-helices (Figure 1), but no significant hits have been obtained from the PDB data base with which to build a dependable model.

Cloning, expression and purification of rRv0183:

Under the conditions described in the Methods section, 14 to 15 mg of pure rRv0183 was purified from the 23 mg of protein produced per culture liter (giving a yield of between 60 and 65%). The migration of purified rRv0183 observed upon performing SDS-PAGE Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. corresponds to a molecular mass (of approximately 30 kDa) which is in agreement with the theoretical mass deduced from the DNA sequence (Figure 2, lane 2). The 6 His-Tag in the N-terminal position was removed by performing TEV cleavage with a yield of 70%. The difference between the molecular masses of rRv0183 with and without the 6 His-Tag can be seen in Figure 2. Masses determined by MALDI-TOF mass spectrometry were 31940 Da and 30223 Da, respectively. About 10 mg of purified rRv0183 without the His tail were obtained from 1 liter of culture medium. All biochemical data (see below) were obtained with both forms of the rRv0183 and it was noted that the presence of 6 His-Tag did not affect the biochemical behavior of the enzyme. Further characterization using circular dichroism and dynamic light scattering methods showed that rRv0183 is a monodisperse α/β protein in solution (data not shown).

Biochemical properties:

Substrate specificity

To investigate the potential role of Rv0183, activity measurements using various substrates were performed using the pH-stat, TLC and monomolecular film techniques (Table 2 and Figures 3 and4).

The rRv0183 activity measured using the pH-stat technique showed that this enzyme was not able to hydrolyze emulsified triacylglycerols, whatever the chain length of the substrates tested (Table 2). Only diacylglycerols with short and medium fatty acid chains were hydrolyzed, and specific activities of 27 and 46 U.mg -1 were measured with DiC6 and DiC10 emulsified with gum Arabic. No activity was recorded with the diacylglycerols having the longest chain fatty acids, even in the presence of gum Arabic and detergents (NaTDC, CHAPS or Tween). rRv0183 was found to be active, however, on synthetic monoacylglycerols with short, medium and long chain fatty acids (MC4, 1-monobutyroyl rac glycerol; MC8, 1-monooctanoyl rac glycerol; MC10, 1-monodecanoyl rac glycerol; MC12, 1- 2). The maximum specific activity recorded using monoolein as substrate was obtained at 37 °C and reached 290 U.mg -1 . 1-monopalmitoyl rac glycerol (MC16) was also tested, but under our assay conditions, MC16 was found to be insoluble due to the absence of double bonds, and no lipase activity was recorded.

Activity of rRv0183 was also checked on 2-MAG, the physiological MAG usually produced by 1, 3-regioselective lipase. Triolein was first converted into 2-MAG and free oleic acid by adding HPL prior to rRv0183 (Figure 3, lane 2). Ten minutes after rRv0183 was added to the reaction mixture, 2-MAG decreased rapidly, and it was completely hydrolyzed within 20 min (Figure 3, lane 3 and 4). These data shown that rRv0183 is able to hydrolyze both 2-MAG and 1-rac MAG. Since we also established that rRv0183 is not able to hydrolyze dioleylglycerols (Table 2), the disappearance of 1,2-dioleyl rac glycerol observed after adding rRv0183 (Figure 3, lanes 3-5) can be attributed to the HPL activity also present in the reaction mixture. Overall, the concomitant action of HPL and rR0183 results in the complete conversion of triolein into free oleic acid (Figure 3, lane 5). These data support the idea that rRv0183 is a monoglyceride lipase, and that this enzyme is responsible for completing the process of TAG lipolysis initiated by other lipases.

Using the monolayer technique, we also observed that rRv0183 shows considerable rates of activity on 1-rac monoolein and 1,2 dicaprin, as well as being able to hydrolyse diolein and purified soybean oil monomolecular films (Figure 4). The activities measured using a 1,2 dicaprin substrate were comparable to those recorded with dog gastric lipase (DGL) under similar conditions [31]. When 1-rac monoolein was used as the substrate, the lipase activity increased with the pressure up to the collapse value (30 mN.m -1 ) without reaching a maximum. With 1,2 dicaprin, diolein and purified soybean oil substrates, the activities reached a maximum of 11, 8, 3 mmole.cm -2 .min -1 .M -1 at 15, 20 and 7 mN.m -1 , respectively, and decreased at higher surface pressure values. The activity recorded with diolein and purified soybean oil was found to be almost zero before the collapse of the monomolecular film. When 1,2-dicaprin was used as the substrate, the hydrolytic activity decreased more slowly as a function of the surface pressure without reaching zero, even near the collapse value. This result might explain why the rRv0183 activity was recorded with emulsified dicaprin using the pH-stat technique (46 U.mg -1 ), whereas no activity was found to occur on diolein, triolein or purified soybean oil under these assay conditions. Although the monomolecular film technique does not make it possible to determine specific activities with each of substrates, these data clearly indicate that rRv0183 is able to hydrolyze TAG, DAG and MAG and that these compounds can therefore be accommodated in the active site of the enzyme. However, the absence of activity observed on long chain DAG and TAG emulsions under bulk conditions may possibly be due to the fact that the physical state of the substrate induces denaturation of the enzyme at the oil/water interface, or else it may show that rRv0183 has a poor affinity for oil-water interfaces.

The fact that rRv0183 showed high rates of activity on monoglycerides under bulk conditions probably results from rRv0183 having a better affinity for micellar substrates rather than emulsified substrates. This is an important finding for understanding the role of Rv0183 in vivo.

Since Rv0183 has been annotated in the genome as a lysophopholipase, we checked its activity using lyso-PC as substrate (data not shown). Despite many attempts to induce activity in this case, we did not observe any hydrolysis of this substrate. Actually, this result was not very surprising, since the homologous human and mouse monoglyceride lipases do not hydrolyze lysophospholipids [22,23,30]. In order to investigate another potential substrate, trials were performed with several phospholipids using the monomolecular film technique. 2). We also checked the activity of rRv0183 on lipids constituting the cell wall such as phthiocerol dimycocerosates, glycerol monomycolates and phosphatidylinositol dimannoside. No activity was detected on these various subtrates (data not shown).

Chain length dependence

rRv0183 was also found to hydrolyse synthetic vinyl esters with various acyl chain lengths.

To determine the acyl chain length specificity, the specific activity of rRv0183 was measured on these vinyl esters and monoacylglycerols having 4 to 18 carbon atoms, and the highest values were those recorded with monoacylglycerol substrates having between 8 and 18 carbon atoms (data not shown). Similar specific activities were recorded with vinyl laurate (VC 12 ), vinyl butyrate (VC 4 ) and vinyl propionate (VC 3 ) (64, 45 and 57 U.mg -1 , respectively;

Table 2). These data show that the enzyme activity does not depend significantly on the chain length, and that the active site can therefore accommodate substrates with short, medium and long acyl chains.

pH stability and pH dependent activity

The pH stability and the pH dependent activity of rRv0183 were investigated using a monoolein substrate (Figure 5A and5B). The results obtained show that rRv0183 is stable at alkaline pH values ranging from 7.5 to 9 and that 100% residual activity occurs at pH 9 after 1 hour of incubation at room temperature (Figure 5A). At lower pH values, the residual activity dropped rapidly to 50% at pH 6.5, and it was completely abolished after 1 hour of incubation at pH levels of 6.0 and below. The curve describing the activity of rRv0183 as a function of the pH (Figure 5B) is superimposable on the pH stability curve (Figure 5A). Since the purified rRv0183 was found to be unstable at pH values below pH 6.5, it was impossible to determine the real effects of the pH on the rate of enzyme turnover at lower pH levels. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Temperature dependence

The temperature dependence profile obtained using the monoolein substrate (Figure 5C)

shows that the specific activity of rRv0183 increased linearly 3-fold from 25 °C to 50 °C before decreasing slowly at higher temperatures. Although rRv0183 showed a high rate of activity at 55 °C, it cannot be classified as a thermostable enzyme, since no residual activity was detected after 1 h of incubation at 55 °C, which suggests that rRv0183 is completely denatured at this temperature. Based on these data, 37 °C was used in all the present biochemical studies. At this temperature, it was possible to test volatile vinyl substrates as well as triglycerides and the activity of the enzyme remained linear during several minutes on all the substrates tested.

NaTDC dependence

Lipase activity on MAG and DAG was enhanced by adding various detergents, such as tetradecyltrimethyl-ammonium bromide (a cationic detergent), Triton X-100 (a non-ionic detergent), CHAPS (a zwitterionic detergent), DOC (an anionic detergent) (data not shown)

and NaTDC (an anionic detergent) (Figure 5D). In the absence of detergent, no activity was recorded with monoolein, DiC 6 , DiC 10 and VC 12 substrates. When monoolein was used as the substrate, the lipolytic activity increased with the NaTDC concentration, peaked at 1 mM NaTDC, and then remained stable up to 5 mM NaTDC (Figure 5D).

The fact that the activity profiles recorded with other detergents under the same assay conditions were similar to those obtained with NaTDC, shows that the charge of the detergent does not affect the activity of rRv0183. These data suggest that the binding of rRv0183 to substrate micelles, which is a prerequisite for lipolytic activity to occur, is probably not mediated by electric charges but only by hydrophobic interactions. NaTDC, which is commonly used in lipase assays, was used here to measure the activity of Rv0183. The

Absence of the disulfide bridge

The amino acid sequence of rRv0183 possesses two cystein residues. Under native conditions, no free sulfhydryl groups were detected during the incubation of rRv0183 with the DTNB reagent. However, after adding 2% SDS to the rRv0183 protein solution in the absence of reducing agent, two moles of sulfhydryl group were titrated per mole of enzyme. These results show that two free cysteines are present but that they are not involved in a disulfide bridge and not accessible to the solvent in the native Rv0183.

Identification of the catalytic triad

The high level of sequence identity observed between Rv0183 and the human and mouse monoglyceride lipases, in which a catalytic triad has been identified [22,30], led us to study the possible involvement of Ser 110, Asp 226 and His 256 in the catalytic triad of Rv0183

(Figure 1). The corresponding Ala mutants were expressed and purified and correct folding was checked using the circular dichroism technique. The enzymatic activity on vinyl esters and monoglycerides was found to be completely abolished when the following residues were mutated: Ser 110 into Ala, Asp 226 into Ala or Asn, and His 256 into Ala.

Effects of serine esterase inhibitors on rRv0183 activity

After preincubating pure rRv0183 with E600, PMSF or THL at a molar excess of 200 in the absence of NaTDC, the lipolytic activity observed on monoolein was inhibited (Figure 6A): after a 180-min incubation step, the inhibition rate of rRv0183 was 100% with PMSF, 80% with E600 and only 22% with THL. In the presence of 3 mM NaTDC, the inhibitory process was accelerated. The activity was completely inhibited after a 25-min incubation step with PMSF and a 103-min incubation step with E600. After a 150-min incubation step with THL, the rate of rRv0183 inhibition was 62% (Figure 6B). These results differ from those obtained with classical lipases such as HPL, DGL and TLL, the inhibition of which requires the presence of a detergent such as NaTDC: these enzymes are known to have a lid domain covering the active site, and only in the presence of NaTDC the lid adopt an open conformation giving inhibitors access to the active site [32,33]. The kinetic behavior of rRv0183 in the presence of an inhibitor supports the idea that the catalytic site of this enzyme is probably not covered by a lid.

Immunolocalization of rRv0183 in cell fractions:

Anti-Rv0183 polyclonal antibodies (pAbs) were used to determine the cellular and subcellular distribution of Rv0183 in M. tuberculosis. As shown in Figure 7, pAbs recognized the pure rRv0183 (lane 1) as well as the native Rv0183 present in the M. tuberculosis culture medium (lane 3) and the cell wall (lane 4). As can be seen from Figure 7, Rv0183 was not detected in the culture medium filtrate passed through a 0.22 µm filter (lane 2), which suggests that Rv0183 bound to large particles. No trace of enzyme was observed in either the membrane or the cytosol fractions (lane 5, 6, 7). The fact that native Rv0183 was detected in the culture medium is in agreement with data obtained in studies in which the M. tuberculosis proteome was analyzed [34,35].

Discussion

Understanding the molecular mechanisms involved in lipid metabolism in M. tuberculosis is likely to be of both fundamental and clinical importance. Although many studies have been performed on the synthesis of lipids in M. tuberculosis [11], little is known so far about their degradation [20]. No secreted or exported lipases have been identified up to now in M.

tuberculosis, although lipids play an important role in this microorganism: (phospho)-lipids in the host cells may be hydrolyzed into fatty acids and glycerol and absorbed by the mycobacteria. These fatty acids are therefore used to synthesize the lipids (TAG) occurring in the M. tuberculosis lipidic inclusion bodies, which are thought to be required for the survival of the bacteria during the dormant and reactivation phases [7]. During these phases, intra-and extracellular lipases may play an important role in lipid metabolism processes. In the M.

tuberculosis genome, which was completed in a recent study, 24 genes have been annotated

as putative lipases (Lip C to Z except K and S) [20,21]. However, among these 24 targets, 9

do not possess the G-X-S-X-G pentapeptide, which is the minimum moiety included in the

larger lipase consensus pattern [LIV]-{KG}-[LIVFY]-[LIVMST]-G-[HYWV]-S-{YAG}-G-

[GSTAC], according to the PROSITE database. Among the 15 remaining targets, only Lip Y (Rv3097c) has been found so far to be a lipolytic enzyme [20], while Lip H (Rv1399c) and Lip F (Rv3487c) were not classified as lipolytic enzymes after being biochemically characterized [36,37]. However, more specific research on the M. tuberculosis genome based on comparisons with genes coding for biochemically characterized enzymes found to be lipolytic has shown that other genes may be lipases (data not shown). One of these potential lipases, the Rv0183 gene, which codes for a putative lysophopholipase [21], was selected because of its high amino acid sequence identity (~34%) with known mammalian monoglyceride lipases. After having cloned, expressed and biochemically characterized the recombinant Rv0183, this protein was found to be a lipolytic enzyme hydrolyzing long chain Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

monoacylglycerols, which does not show any lysophospholipase activity. This enzyme is the first exported lipase to be identified in M. tuberculosis. The optimum conditions of expression obtained in Rosetta pLysS E. coli cells at 37 °C using Terrific Broth as growth medium yielded around 10.5 mg of purified soluble recombinant protein per liter of culture medium.

The optimum assay conditions were then established using the pH-stat technique, which is a useful means of performing kinetic studies. The biochemical properties of rRv0183 are very similar to these of human and mouse MGL [22,30,38,39]: amino acid sequence alignment (Figure 1) and mutagenesis experiments showed that the catalytic triad is composed of residues Ser 110, Asp 226 and His 256. The results of inhibition studies performed in the absence and presence of NaTDC support the idea that unlike many other lipases, rRv0183

does not possess a lid covering its active site. This is actually not very surprising, since many bacterial lipases have been found to be devoid of a lid domain [40].

The biochemical characteristics of rRv0183 determined using the monomolecular film technique clearly show that this enzyme is also potentially active on long chain TAG and DAG. Therefore, rRv0183 possesses an active site accommodating various glycerides. The absence of rRv0183 activity on TAG and DAG in the pH-stat assay probably resulted from the occurrence of poor interactions between the enzyme and the lipid-water interface, whereas the monomolecular film technique made it possible to screen a larger range of interfacial conditions.

The high levels of expression of rRv0183 obtained here will hopefully lead to further crystallization studies. Information about the rRv0183 3-D structure will help to explain the substrate specificity of the enzyme, which it is difficult to interpret both at the molecular level and in terms of the putative physiological role of Rv0183.

The cellular location of Rv0183 means that native enzyme can be exported through the cell wall and may play a role both in the cell wall and in the culture medium: Rv0183 may act does not play the same physiological role in vivo as the first intracellular lipase identified [20]. Several hypotheses can be put forward, as follows: i) Rv0183 may be able to hydrolyze extracellular TAG, DAG and MAG with different rates, leading to the uptake of free fatty acids and their further storage in lipid inclusion bodies when M. tuberculosis infects adipocytes [10], ii) Rv0183 may hydrolyze diglycerides released by PLC and then the resulting monoglycerides, thus triggering the release of glycerol and absorbable fatty acids, iii) Rv0183 may mainly hydrolyse the monoglycerides resulting from the action of another, as yet unknown exported lipase. The latter scheme is known to occur in human and mouse adipocytes, where the homologous MGL catalyzes the hydrolysis of monoglycerides after the action of adipose triglyceride lipase (ATGL) and hormono-sensitive lipase (HSL) on the TAG and DAG, respectively [41,42].

The absence of Rv0183 in the culture medium passed through a 0.22 µm filter suggests that Rv0183 may aggregate in large vesicles after being exported, or that Rv0183 may be exported from the membrane by these extracellular vesicles. Vesicular trafficking from phagosomes containing bacteria has been found to occur in previous studies [43,44]. Mycobacterial lipids were found to be present in extracellular vesicles isolated from the culture medium. Via the release of these vesicles, the effects of the bacteria may be exerted on more distal cells or tissues rather than being restricted to the nearest cells. In this context, the possibility can not be ruled out that Rv0183 may be involved in trafficking processes of this kind during the infection stage. Although Rv0183 was detected in the culture medium, the processes underlying its up-regulation are not yet known. Its physiological role will now have to be Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

investigated using specific antibodies. This powerful tool combined with electronic microscopy will make it possible to detect the presence of Rv0183 during the process of phagocytosis of mycobacteria by macrophages.

Although Rv0183 is not thought to be one of the main targets involved in the infection process [45,46], this does not rule out the possibility that this enzyme may contribute to the reactivation of the bacteria and help the mycobacteria to survive in the mature granulomas during the dormant phase. This hypothesis is in agreement with previous studies showing that bacteria grown in vitro had a preference for carbohydrates, whereas bacteria grown in vivo preferred fatty acids as their carbon source [47,48].

Likewise, the presence of a homologous enzyme in M. leprae (ML2603), which is known to have only the enzymes necessary for its replication in the host cells, suggests that this enzyme may play an important role in the lifecycle of the microorganism. Studies on the behavior of Rv0183 might also provide indirect information about the lipid metabolism in M. leprae.

In conclusion, the results obtained in the present study show the existence of an exported lipolytic enzyme, which preferentially hydrolyses monoacylglycerols and is closely related to human and mouse monoglyceride lipases. Based on the present biochemical findings, this exported enzyme may be involved in the hydrolysis of host cell lipids during the infection process, but its exact physiological contribution to the process of lipid metabolism still remains to be elucidated. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 1: Oligonucleotides used in this study

  has now become possible to identify proteins which might be involved in the lipid metabolism by comparing their amino acid sequences with those of other known lipases. Here we report on the cloning and biochemical characterization of a lipase exported by M. tuberculosis S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

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  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745

  -S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society chemiluminescence (ECL) reagent (Amersham Biosciences, ref. RPN 2109), after exposing Kodak Biomax MS film to the membrane. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society monolauroyl rac glycerol; MC14, 1-monomyristoyl rac glycerol; MC18:1, 1-monooleyl rac glycerol) (Table

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  rRv0183 was not found to hydrolyze cationic (dilauroylphosphatidylethanolamine, DiC 12 PE), S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

S t a g e 2

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  standard activity assay developed for use with rRv0183 was performed in 2.5 mM Tris-HCl, S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society 150 mM NaCl, 3 mM NaTDC at pH 7.5 at 37 °C using 9.3 mM of monoolein (50 µl) as substrate.

S t a g e 2

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S t a g e 2

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S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society outside the bacilli, and be directly involved in the host lipid degradation process initiated by another partner. Many studies have shown that protein fractions originating from the culture medium are potential candidates for the production of vaccines, and Rv0183 might therefore be one of them. Since no Rv0183 can be detected inside the cytoplasm, Rv0183 obviously

S t a g e 2

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  The underlined bases indicate the site of the mutation. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 :

 1 Figure 1: Amino-acid sequence alignment between Rv0183, without its putative signal

Figure 2 :

 2 Figure 2: SDS-PAGE for purification and TEV cleavage of rRv0183. Protein samples were

Figure 3 :

 3 Figure 3: TLC analysis of triolein degradation by HPL and rRv0183. Reaction was

Figure 4 :

 4 Figure 4: Lipolytic activity of rRv0183 on 1,2 dicaprin, 1 rac monoolein, diolein and purified

Figure 5 :

 5 Figure 5: Kinetic assays on rRv0183 using a monoolein substrate. (A) pH stability, (B) pH

Figure 6 :

 6 Figure 6: Inhibitory effects of E600 (○), THL (◊) and PMSF (□) on rRv0183 activity as a

Figure 7 :

 7 Figure 7: Location of Rv0183 in M. tuberculosis cellular compartments. Aliquots (15 µg of

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Figure 1 S

 1 Figure 1

Figure 2 S

 2 Figure 2

Figure 3 S

 3 Figure 3

Figure 4 S

 4 Figure 4

Figure 5 S

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Figure 6 S

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Figure 7 S

 7 Figure 7

Table

  . The PCR product was cloned into the expression vector pDest14 in line with the

© 2007 The Authors Journal compilation © 2007 Biochemical Society

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Acknowledgements

We thank Dr. Steward Cole and Dr. Nadine Honoré at the Pasteur Institute, Paris, France, for providing us with the BACs library and Marilyne Blémont, CNRS, UPR 9025, Marseille, and France and Emilie Layre, IPBS, UMR5089, Toulouse, France, for their technical assistance. We are indebted to Dr. Jessica Blanc for revising the English manuscript. This work was supported in part by the CNRS and the GIP ANR 06-JCJC-0067-01. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070745 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070745 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2: Specific activities in the hydrolysis of lipids

Experiments were performed with tri, di and monoacylglycerides at 37 °C in 2.5 mM Tris-HCl pH 7.5 buffer, 150 mM NaCl under stirring. Assays with vinyl esters were performed at 25 °C in the same buffer. The procedure used in the experiments on cholesterol oleate was strictly the same as that described by Ben Ali et al. [49]. Activities on phospholipids were determined on monomolecular film at 25 °C (see Materials and Methods section).

Substrates

Specific activity (U.mg