Transketolase in Trypanosoma brucei

Molecular & Biochemical Parasitology 179 (2011) 1–7

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Molecular & Biochemical Parasitology

Transketolase in Trypanosoma brucei Sabine A. Stoffel a , Vincent P. Alibu b , Jane Hubert c , Charles Ebikeme d , Jean-Charles Portais c , Frédéric Bringaud d , M. Ernst Schweingruber e , Michael P. Barrett b,∗ a

Pevion Biotech AG, Worblentalstrasse 32, CH-3063 Ittigen/BE, Switzerland Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom c MetaSys (Functional Analysis of Metabolic Systems, Metabolomics, Fluxomics, Ingénierie des Systèmes Biologiques et des Procédés) UMR5504, UMR792 CNRS, INRA, INSA, ISBP/INSA, 135 Avenue de Rangueil, 31077 Toulouse, France d Centre de Résonance Magnétique des Systèmes Biologiques (RMSB), UMR5536, Université Segalen Bordeaux, CNRS, 146 rue Léo Saignat, 33076 Bordeaux, France e Institute of Cell Biology, University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland b

a r t i c l e

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Article history: Received 7 October 2010 Received in revised form 15 April 2011 Accepted 20 April 2011 Available online 6 May 2011 Keywords: Trypanosoma brucei Transketolase Pentose phosphate pathway Metabolic profiling Metabolism

a b s t r a c t A single copy gene, encoding a protein highly similar to transketolase from other systems, was identified in the Trypanosoma brucei genome. The gene was expressed in E. coli and the purified protein demonstrated transketolase activity with Km values of 0.2 mM and 0.8 mM respectively for xylulose 5-phosphate and ribose 5-phosphate. A peroxisomal targeting signal (PTS-1) present at the C-terminus of the protein suggested a glycosomal localisation. However, subcellular localisation experiments revealed that while the protein was present in glycosomes it was found mainly within the cytosol and thus has a dual localisation. Transketolase activity was absent from the long slender bloodstream form of the parasite and the protein was not detectable in this life cycle stage, with the RNA present only at low abundance, indicating a strong differential regulation, being present predominantly in the procyclic form. The gene was knocked out from procyclic T. brucei and transketolase activity was lost but no growth phenotype was evident in the null mutants. Metabolite profiling to compare wild type and TKT null mutants revealed substantial increases in transketolase substrate metabolites coupled to loss of sedoheptulose 7-phosphate, a principal product of the transketolase reaction. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Transketolase (TKT) is a key enzyme of the non-oxidative branch of the pentose phosphate pathway. This pathway provides ribose 5-phosphate for nucleotide biosynthesis and also NADPH used in reductive biosyntheses and defense against oxidative stress [1,2]. The pathway, in some organisms, also provides other phosphorylated sugars for anabolic purposes. Transketolase has broad substrate specificity transferring 2-carbon units between a range of ketose donors and aldose acceptors [3]. Specialised derivatives that catalyse other reactions have also evolved. These include 1-deoxylulose-5-phosphate synthase (which uses glycoaldehyde from pyruvate to generate 1-deoxyxylulose 5-phosphate) [4] and

Abbreviations: TKT, transketolase; PPP, pentose phosphate pathway; ENO, enolase; ALD, aldolase; 6-PGDH, 6-phosphogluconate dehydrogenase; Xu5P, xylulose 5-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose 7-phosphate. ∗ Corresponding author. Tel.: +44 141 330 6904; fax: +44 141 330 4600. E-mail address: [email protected] (M.P. Barrett). URL: http://www.gla.ac.uk:443/ibls/staff/staff.php?who=PAGP|S (M.P. Barrett). 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.04.006

dihydroxyacetone synthase (which uses formaldehyde as an acceptor substrate and xylulose 5-phosphate as donor to produce glyceraldehyde 3-phosphate and dihydroxyacetone [5]). Transketolase has also been co-opted as a putative crystallin in vertebrate eyes [6] and also a binding partner of the regulatory protein marR in E. coli [7]. The structure of the enzyme has been determined from several systems including yeast [8], E. coli [9], maize [10] and the protozoan parasite Leishmania mexicana [11]. The enzyme is a homodimer and its mechanism of action has been determined using kinetic and structural analysis (reviewed in 8). In common with several other enzymes that catalyze decarboxylations of ␣-ketoacids or the transfer of ketol groups from donor ketoses to acceptor aldoses, transketolase requires thiamine diphosphate (TDP) as a cofactor [12,13]. The interaction between transketolase and thiamine has been thoroughly investigated in Saccharomyces cerevisiae [14–16]. In the pentose phosphate pathway, transketolase is usually depicted as performing two reactions, transferring a 2-carbon unit from xylulose 5-phosphate to ribose 5-phosphate to yield sedoheptulose 7-phosphate and to erythrose 4-phosphate yielding fructose 6-phosphate. Transketolase has also been shown to play a role in

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protection against oxidative stress in yeast [17,18] and in mammals [19–21] given its role in redirecting sugar phosphates towards the oxidative branch which generates NADPH. Glycolysis has been studied in considerable detail in trypanosomatid parasites such as Trypanosoma brucei [22–24], the causative agent of sleeping sickness, Trypanosoma cruzi the causative agent of Chagas’ disease and the Leishmania species responsible for leishmaniasis. The pentose phosphate pathway has, however, only received attention relatively recently in these organisms [25–30]. Transketolase has previously been characterized in L. mexicana, where it was shown to have a dual localisation between cytosolic and glycosomal compartments [11]. It was reported that in T. brucei, transketolase activity could be detected in the procyclic forms representative of the parasites found in the tsetse fly midgut but not bloodstream form organisms [25] and a recent proteomic analysis of the glycosomal preparations in trypanosomes [31] also found this enzyme in glycosomes of procyclic but not bloodstream form organisms. Bioinformatic analysis, based on the presence of predicted peroxisomal targeting sequences [32], also placed transketolase in glycosomes. To date, however, a detailed analysis of the enzyme in T. brucei has been lacking hence we set out to use genetic modification to determine roles of this enzyme in T. brucei and coupled this to metabolic profiling of parasites from which the gene was deleted.

Procyclic forms of the strain 427 were stably transformed as described [38]. Aliquots of 1 mL were transferred to each well of the 24-well plates. After an 18-h incubation to allow recovery, 0.5 mL of fresh medium containing selective antibiotic was added to each well (75 ␮g mL−1 hygromycin or 45 ␮g mL−1 neomycin). Independent antibiotic-resistant clonal cell lines were obtained after 10–12 days. 2.3. Cloning and heterologous expression of the T. brucei TKT gene T. brucei transketolase (TbTKT, accession number Tb927.8.6170) was PCR amplified from genomic DNA of the Lister 427 strain using oligonucleotides oMB44 (5 -CAA GGA TCC ATG TCG TTT AAT GAT AAC C-3 ) and oMB45 (5 -CCA AAG CTT TCA CAA ATG GGA CCG CTT C-3 ). The PCR fragment was sub-cloned into pGEM-T Easy (Promega) and then into pET28a(+) (Novagen) using BamHI and HindIII (underlined primer sequences) to encode an N-terminal His-tagged protein. The resultant plasmid, named pMB-G27, was transformed into E. coli BL21 (DE3). Overexpression was induced with 1 mM IPTG for 16 h at 16 ◦ C and purified using Ni2+ resin chromatography on a BioCAD system. The purity of the preparation was assessed by SDS PAGE electrophoresis and Coomassie blue staining. 2.4. TbTKT activity assays

2. Material and methods 2.1. Trypanosome stocks Procyclic T. brucei (strain 427) cells [33] were grown at 27 ◦ C in SDM-79 medium [34] supplemented with 5% heat-inactivated fetal bovine serum. The bloodstream form stock BS 221 (synonymous with MiTat 1.2/221; the bloodstream form of strain 427) was cultured at 37 ◦ C in a humidified atmosphere of 5% CO2 in HMI-9 medium [35] (BioConcept, Allschwil, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (BioConcept). 2.2. Genetic manipulation of the transketolase gene in T. brucei TKT alleles were replaced sequentially with resistance genes for the antibiotics neomycin and hygromycin. Flanking sequences upstream (311 bp) and downstream (355 bp) of the TKT open reading frame were amplified by PCR and cloned into plasmids either side of genes encoding neomycin resistance (NEO) or hygromycin resistance (HYG). The nucleotide sequences used to amplify the 5 UTR were 5TKf: 5 -GGG TAC CTG CGC CAT TTC TTT TTC TCT-3 with 5TKr: 5 -GAA GCT TCG TCT TTG GTC AAT GCT CTG-3 . The sequences of oligonucleotides to amplify the 3 UTR were: 3TKf: 5 -GGG ATC CGG TTT GCT TTT GCC ATG TTT-3 and 3TKr: 5 -GTC TAG ATT TTG TGT GCC TAA CGA ACG-3 . The resulting deletion cassettes were released from the plasmid using restriction enzymes BamHI and HindIII, purified by ethanol precipitation, and resuspended to 0.2 ␮g DNA ␮L−1 in water prior to electroporation into trypanosomes. To over-express TKT in procyclic cells, the open reading frame was PCR-amplified, using the following primers: TKoverexH-f: 5 -AAG CTT ATG TCG TTT AAT GAT AAC C-3 ; TKoverexB-r: 5 CCT AGG TCA CAA ATG GGA CCG CTT C-3 and then cloned into the plasmid pHD1485 (with PARP promoter and an N-terminal Myc-tag), transfected into 427-1313 cells constitutively expressing the tet repressor [36] which were then selected with 25 ␮g mL−1 hygromycin [37]. Induction of overexpression was achieved by adding tetracycline at a concentration of 1 ␮g mL−1 to cell cultures inoculated at a cell density between 0.4 and 1.0 × 106 cells mL−1 . Overexpression was dectected by northern blot analysis and increase in enzymatic activity.

Before use, trypanosomes were washed by suspension and centrifugation in STEN buffer (250 mM sucrose, 25 mM Tris–HCl (pH 7.5), 1 mM EDTA and 150 mM NaCl). The cells were then lysed by incubation in TE buffer (10 mM Tris/HCl, 1 mM EDTA) containing, 0.15% Triton X-100 and complete protease inhibitor cocktail (F. Hoffmann-La Roche Ltd.) and incubated at room temperature for 20 min. The supernatant was separated from cell debris by centrifugation at 13,000 rpm at 4 ◦ C for 30 min. The supernatant was used to determine enzyme activities immediately. Protein concentration was determined by the Bradford method (Biorad). All assays were performed at room temperature; the reaction mixtures were equilibrated for 3–5 min at this temperature, and the reactions started by addition of the cell-free extract (1 × 107 cells assay−1 ) or the purified recombinant TbTKT. The reaction mixture, was as described [11]. Kinetic parameters (Km and Vmax ) for the recombinant protein purified from E. coli were derived for ribose 5-phosphate by keeping xylulose 5-phosphate constant at 2 mM and varying ribose 5-phosphate as doubling dilutions (10 mM – 0.156 mM) or ribose 5-phosphate constant at 10 mM and varying xylulose 5-phosphate as doubling dilutions (2 mM0.03125). Alternatively a reaction where fructose 6-phosphate acts as ketose donor linked to glyceraldehyde 3-phosphate, generating erythrose 4-phosphate was used [39]. 2.5. Southern and northern blot analysis Genomic DNA from procyclic cells was digested with restriction endonuclease BclI. Southern blotting was performed by standard protocols [42]. TKT, neomycin and hygromycin resistance genes were each labeled using the DIG (digoxigenin labelling) system (Roche Diagnostics, Basel, Switzerland) and used to probe blots at high stringency according to standard procedures [40]. The signals were scanned with a Luminescent Image Analyzer LAS-1000plus machine (Fujifilm) with exposure times of 5–15 min. For northern blots total RNA was extracted from 5 × 107 to 1 × 108 cells (from procyclic and bloodstream form trypanosomes) by hot phenol extraction. 11 ␮g of RNA was loaded per lane, and RNA separated by electrophoresis then blotted according to standard procedures [40].

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2.6. Western blot analysis Rabbit polyclonal antibodies against Leishmania major transketolase (LmTKT) [11] were purified through columns packed with TbTKT-coupled Affigel 10 (Bio-Rad) according to the manufacturer’s instructions. Elution of the specific antibodies was performed with 0.1 M glycine pH 2.5. To avoid protein denaturation due to the extreme pH, samples were collected in Eppendorf tubes containing 2 M Tris–HCl pH 8.3. 5 × 106 trypanosomes were centrifuged at 2500 rpm for 5 min in a microfuge. The pellets were resuspended in 1 mL of PBS and again centrifuged. The supernatant was aspirated, the cells resuspended in Laemmli buffer and denatured on a heating block at 95 ◦ C for 5 min. Cell lysates were resolved by SDS-PAGE in 10% polyacrylamide gels, and transferred by electroblotting to nitrocellulose membranes (Hybond ECL, GE Healthcare). The blots were probed overnight with the T. brucei TKT-epiptope enriched anti-LmTKT antibodies (1/10), washed and then incubated with anti-rabbit horseradish peroxidase conjugate (1/2,000, Santa Cruz Inc.) for 1 h. Bound conjugate was detected by the ECL Chemiluminescent system. Where necessary, protein loading was verified by probing for Tb6PGDH.

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mixture was briefly vortexed (≈2 s). The extracts were immediately filtered (0.2 ␮m) and chilled with liquid nitrogen. After lyophilisation, the dried extracts were resuspended in 200 ␮L Milli-Q water prior to analysis. Three replicates were taken from each culture media, sampled and analysed separately. Metabolites involved in different parts of the metabolic network of T. brucei procyclic cells were determined by liquid ion chromatography coupled to tandem mass spectrometry as recently described [43] with measured concentrations of metabolites expressed as a total cellular concentration assuming a volume of 108 cells being equal to 5.8 ␮l. The metabolites under examination included glucose 6phosphate (G6P), fructose 6-phosphate (F6P), fructose 1,6biphosphate (FBP), mannose 6-phosphate (M6P), 2 and 3phosphoglycerate (2/3-PG), phosphoenolpyruvate (PEP) and glycerol 3-phosphate (Gly-3P), fumarate (FUM), malate (MAL), succinate (SUC), citrate (CIT), ␣-ketoglutarate (␣-KG), cis-aconitate (cis-ACO) and orotate (ORO), 6-phosphogluconate (6-PG), ribose 1-phosphate (R1P), ribulose 1-phosphate (Ru1P) (considered together as pentose 1-phosphate, (Pen1P)), ribose 5-phosphate (R5P), ribulose 5-phosphate (Ru5P), xylulose 5-phosphate (Xu5P) (considered together as pentose 5-phosphate (Pen5P)) and sedoheptulose 7-phosphate (S7P).

2.7. Subcellular localisation of TbTKT Digitonin fractionation of trypanosomes was performed by modification of a method previously described [41]. Specifically, 5 × 107 procyclic parasites were washed with STEN buffer and resuspended in 300 ␮L of STEN supplemented with a complete protease inhibitor cocktail (F. Hoffmann-La Roche Ltd.). The cells were differentially permeabilized with 40 ␮g digitonin per 100 ␮g cellular protein (2.5 × 107 cells) for 2 h on ice and centrifuged at 13,000 rpm for two minutes. After TCA precipitation, equal amounts (equivalent of 4 × 106 cells) of the supernatant (S) and pellet (P) were analyzed by SDS-PAGE and blotted with the T. brucei TKT enriched rabbit anti-LmTKT [11]. For comparison, enolase (ENO) and aldolase (ALD) (as cytosolic and glycosomal markers, respectively) were also probed with antibodies kindly provided by Professor P. Michels (University of Louvain). 2.8. Metabolite analysis Wild type 427 and TKT knockout mutants of procyclic T. brucei grown in SDM-79 medium were sampled by a fast filtration method [42] derived from that recently reported [43]. Total sampling time was below 8 s. The extraction of intracellular metabolites was carried out by transferring the filters containing the pellets into 5 mL of boiling water for 30 s. 200 ␮L of a uniformly 13 C-labeled E. coli cell extract was added as quantification internal standard [44], and the

3. Results 3.1. Transketolase from T. brucei A single copy TKT gene from T. brucei is predicted to encode a protein of 669 amino acids with a molecular mass of 72.6 kDa (www.genedb.org) including a conserved thiamine diphosphate binding site and a “TKT motif”, which is common to all TKT proteins [8]. The protein possesses all features previously proposed as essential for transketolase and it is 63% identical (75% similarity) to the same protein previously characterized in L. mexicana [11]. We expressed a His-tagged version of the T. brucei TKT gene in E. coli and purified the protein using Ni2+ -NTA chromatography. The purified protein was shown to have a Km value of 0.2 ± 0.05 mM for Xu5P with a Vmax value measured at 2.3 ␮mol min−1 mg protein−1 using that substrate (Fig. 1A) and a Km value of 0.8 ± 0.11 mM for R5P and a Vmax value of 1.8 ␮mol min−1 mg protein−1 using that substrate (Fig. 1B). The C-terminus of the protein contains a peroxisome-targeting signal type 1 (PTS-1) sequence (SHL) as previously reported [32], and the protein has previously been identified in highly purified glycosomes isolated from procyclic trypanosomes [31]. A similar situation exists for L. mexicana, although in this parasite while some of the protein could be found in glycosomes it was predominantly cytosolic in promastigote forms of the parasite [11]. We there-

Fig. 1. Kinetic characterisation of TbTKT, (A) Michaelis–Menten plot determining kinetic parameters assaying xyluose 5-phosphate as donor with ribose 5-phosphate concentration fixed (Lineweaver-Burke plot is inset) (B) Michaelis–Menten plot determining kinetic parameters using ribose 5-phosphate as acceptor with xylulose 5phosphate concentration fixed (Lineweaver-Burke plot is inset).

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Fig. 2. TKT in T. brucei. (A) Subcellular localisation of TbTKT. Extracts from procyclic trypanosomes supernatant (S) or pellet (P) from digitonin lysis experiments were analysed by SDS-PAGE and blotted with affinity purified rabbit anti-LmTKT. For comparison, enolase (ENO) and aldolase (ALD) (as cytosolic and glycosomal markers respectively) are shown. (B) Specific activities of TKT from procylic (P) and bloodstream from (B) T. brucei. (C) Western blot using affinity purified anti-LmTKT antibody. Lanes: 1, recombinant LmTKT; 2, recombinant TbTKT; 3, procyclic and 4, bloodstream form T. brucei. (D) Northern blot with 11 mg of total RNA loaded per lane and probed with digoxigenin-labeled TKT. Actin was used as a loading control.

fore studied the subcellular localisation of the T. brucei enzyme using digitonin fractionation (Fig. 2A). While the cytosolic marker enzyme enolase and glycosomal marker aldolase were found associated exclusively with the expected fractions, transketolase was present in both the cytosol, and to a lesser extent, the same fraction as aldolase that we take to be the glycosomes although specific markers for other organelles e.g. the mitochondrion were not used. Densitiometry indicated more than 89% of the protein is cytosolic. Therefore, as in Leishmania, the T. brucei enzyme has a dual localisation. It was previously reported that transketolase activity was absent from bloodstream form T. brucei [25]. TKT assays in procyclic form and bloodstream form organisms showed detectable activity only in procyclic forms (45 mU mg of protein−1 ) (Fig. 2B). Western blot analysis using the anti-LmTKT antibody [11] also recognized a 72 kDa protein band in procyclic but not in bloodstream form organisms (Fig. 2C). Finally, northern blot analysis revealed the gene’s transcript to be substantially more abundant in procyclic form organisms than in bloodstream forms (Fig. 2D). 3.2. Growth of T. brucei procyclic TKT-null mutants and overexpressors To assess possible biological roles of TKT in procyclic forms, a null mutant was constructed. Both alleles of the T. brucei TKT gene were replaced by sequential transformation of 427 procyclic forms with constructs containing the neomycin and hygromycin resistance markers. To verify the correct integration of the cassettes and the replacement of the original gene by resistance markers, genomic DNA from knock-out (tkt−/− ) and wild type trypanosomes was analyzed by Southern blot (data not shown). The null mutants were also tested for TKT activity. We measured a specific activity of 45 mU/mg of protein in wild type cells while activity was not measurable in the tkt−/− trypanosomes (Fig. 3A). The 72 kDa protein recognized by the anti-LmTKT antibody was also recognized in wild-type but not detectable in the tkt−/− knockout line (Fig. 3B), indicating that the TKT gene had been successfully deleted and that the TKT activity in trypanosomes was attributable to the product of that gene.

Grown in standard SDM79 medium, the tkt−/− trypanosomes did not exhibit any differences in growth rate nor morphology compared to the parental 427 strain nor was the nucleus-kinetoplast ratio abnormal (data not shown). Thus, TKT is not essential for cell survival and cell division under standard laboratory conditions. To detect possible phenotypes of trypanosomes overexpressing TKT we cloned the gene into the pHD1485 vector [32], which contains a tetracycline inducible PARP promoter in addition to a hygromycin resistance marker and appropriate trypanosome RNA processing signals. After transfection, the procyclic trypanosomes were selected for resistance to hygromycin and subsequently induced with tetracycline for 48 h. Total RNA from induced and notinduced cells was extracted and TKT RNA levels were determined by northern blot analysis. The tet-induced strain exhibits enhanced TKT activity (Fig. 4A) increased mRNA levels (Fig. 4B) when compared with the uninduced cells. We found that overexpression of TKT has no effect on growth rate with cells in the induced and not-induced state behaving similarly in terms of doubling time, morphology and also in the comparison of nucleus-kinetoplast ratio. 3.3. Intracellular metabolite profile of tkt−/− mutant versus wild type procyclic cells In spite of there being no discernable phenotype in in vitro growth medium, we went on to profile changes to the parasite’s metabolic profile. The determination of intracellular metabolite levels was carried out by using a rapid procedure to quench the metabolic activity, separate the cells from the culture medium and extract the intracellular metabolites. A highly sensitive IC-MS/MS analytical method was then applied to quantify some key intracellular metabolites involved in different part of the metabolic network of procyclic T. brucei [43]. The concentrations of a variety of metabolites are given in Table 1 and those of selected metabolites in Fig. 5. By adding the U-13 C-labeled E. coli extract into both the unlabeled calibration standard mixture and the T. brucei cell extracts, absolute concentrations were obtained. The standard molecule for S7P was unavailable commercially so intracellular levels of this compound

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Fig. 3. Knockout of TbTKT. (A) Specific TbTKT activity (U mg protein−1 ) of wild type procyclic forms (WT) and tkt−/− null mutants (KO). The data represent the mean of three independent measurements. Error bars are standard errors around the mean. (B) Western blot showing expression of TbTKT in Wild type (WT) and tkt−/− mutant(KO) cells. Tb6-PGDH was used as a loading control.

Fig. 4. Overexpression of TbTKT, (A) Specific activities of TbTKT overexpressing (+tet) or not (−tet) cells, expressed as percent of parental cell line 427-1313 (WT). (B) Northern blot of TbTKT overexpression. 11 ␮g total RNA was loaded per lane. The upper panel shows the blot probed with TKT, the lower band being the transcript from the overexpressed gene. The lower panel shows ribosomal RNAs indicating similar loading in each lane.

are expressed as analytical peak area ratio (12 C/13 C) and compared between tkt−/− and wild type procyclic cells. Erythrose 4-phosphate exhibited poor stability in out analytical conditions and thus was not included in analyses. In the cell extracts of procyclic T. brucei, most of the targeted metabolites were well-quantified and their concentrations ranged from around 860 nM to 1.9 mM. Metabolites of the pentose phosphate pathway were strongly influenced by the deletion of transketolase. We observed a slight but significant increase of the level of 6-PG in the tkt−/− mutant compared to the wild type procyclic cells, and a strong increase in the levels of pentose 5-phosphates (R5P, Ru5P, Xu5P) in the tkt−/− cells. Crucially, the level of S7P was 63 fold lower in the tkt−/− mutant than in the wild type cells. The accumulation of the transketolase substrates (R5P, Ru5P, Xu5P) and a loss of the key product, S7P, clearly points to transketolase playing a role anticipated for the classical pathway of transferring a 2 carbon unit between Xu5P and R5P to create the seven carbon derivative.

Among glycolytic intermediates and other sugar phosphates, the intracellular levels of 2/3-PG, PEP and F6P as well as gly-3P, and M6P were somewhat reduced in the tkt−/− mutant compared to the wild type procyclic cells. This indicates that the deletion of transketolase

Table 1 Relative abundance of metabolites. Metabolites

Concentration in wild type (␮M)

Concentration in tkt−/− (␮M)

Fumarate Succinate Malate ␣-KG Orotic acid PEP gly-3P cis-Aconitate 2/3-PG Citrate Pen1P Pen5P G6P F6P M6P 6-PG FBP

35.77 ± 6.28 1882.24 ± 201.16 259.29 ± 27.37 86.23 ± 16.95 0.86 ± 0.02 55.59 ± 5.46 178.81 ± 21.01 2.64 ± 0.11 194.98 ± 18.29 306.7 ± 51.14 45.21 ± 4.77 44.79 ± 6.7 120.99 ± 7.67 24.17 ± 1.23 14.85 ± 0.66 20.06 ± 1.57 11.6 ± 0.94

27.03 ± 1.96 1509.42 ± 280.08 65.58 ± 56.86 11 ± 3.82 0.85 ± 0.12 12.28 ± 0.49 55.48 ± 48.23 2.36 ± 0.16 111.66 ± 1.77 358.68 ± 20.89 52.44 ± 6.64 354.18 ± 20.54 114.28 ± 9.61 15.25 ± 1.02 8.63 ± 0.16 35.8 ± 2.87 11.91 ± 0.27

Fig. 5. Analysis of intracellular metabolites by IC-MS/MS. The comparative abundance of selected metabolites is shown in wild type (dark grey bars) and tkt−/− (light grey bars) cells. The full list is given in Table 1. G6P, glucose 6-phosphate; 2/3-PG, 2 phosphoglycerate and 3-phosphogrycerate; PEP, phosphoenolpyruvate; Gly3P, glycerol 3-phosphate; Pen5P, combined ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6, bisphosphate, 6PG, 6-phosphogluconate; S7P, sedoheptulose 7-phosphate.

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affects not only the cellular metabolism around the pentose phosphate pathway itself but also some other enzymes involved in other parts of the central carbon metabolism of procyclic T. brucei. Other glycolytic intermediates such as G6P and FBP were not affected by transketolase deletion. Among organic acids, we observed that the deletion of transketolase does not influence the intracellular concentration of fumarate or succinate, although the intracellular concentration of malate was slightly but significantly reduced in the tkt−/− mutant. Among Krebs cycle intermediates, only ␣-KG, for which an eight fold decrease of concentration was observed in the tkt−/− mutant (from 86.2 ␮M to 11 ␮M) was affected (Table 1).

4. Discussion In parasitic protozoa of the order Kinetoplastida, glucose metabolism has received considerable attention following from the observation that many of the enzymes of the glycolytic pathway are sequestered within a peroxisome-like organelle, the glycosome [22,23]. The pentose phosphate pathway has received less attention, although in Leishmania parasites [27] and in T. cruzi [28–30] a role in defence against oxidative stress has been inferred given that enzymes of the pathway appear to be up-regulated when cells are exposed to oxidative stress. It was previously shown [25] that a full complement of the enzymes of both oxidative and nonoxidative branches of the pathway are found in in vitro cultivated procyclic forms of T. brucei. The form of the parasite that lives in the mammalian bloodstream, however, has only the enzymes of the oxidative branch of the pathway with transketolase and ribulose 5-phosphate epimerase reportedly absent [25]. A TKT gene was identified in the T. brucei genome database. The gene was cloned and expressed in E. coli, and the histidine-tagged protein was purified and assayed for transketolase activity. Km values for R5P and Xu5P were in a similar range to those measured in other organisms. The high Km value compared with far lower measured steady state concentrations of these substrates indicates that the enzyme works far from saturation. This might relate to its having a high control coefficient for flux through the pathway [3] where flux through TKT will be more or less proportional to the abundance of the enzyme. The T. brucei TKT has a typical type 1 peroxisomal targeting sequence (PTS-1) at its carboxy terminus. This has led to the suggestion that the protein is targeted to the trypanosome’s glycosomes [32] and a proteomic analysis of highly purified glycosomes indicated that the protein was found in this organelle in procyclic- but not in bloodstream form organisms [31], although this analysis did not look for the protein in other cellular compartments. To resolve the question of the localisation of transketolase in T. brucei we used antibodies in controlled subcellular fractionation experiments, and found the protein to be principally cytosolic with a smaller fraction found in the cellular fraction containing glycosomes. Thus in procyclic T. brucei, as in L. mexicana, transketolase has a dual localisation in spite of being encoded by a single gene. Based on the crystal structure of the Leishmanial enzyme we previously hypothesized [11] that the C-terminus of the protein, which protrudes through an orifice within the dimer, might alternate between conformations, leaving it either available or not to interact with PEX5 which then carries it into the glycosomes and the same might be speculated for the T. brucei enzyme. The subcellular distribution could then be determined stochastically based upon the relative time that the PTS-1 is exposed or not. Alternatively enzyme binding to ligands could influence conformation and thus localisation. It will be of great interest to investigate the molecular basis of dual localisation in more detail. We confirm the observation that transketolase activity could be detected in procyclic form organisms but not in bloodstream

forms [25] and western blots indicate that the protein itself is not detectable in bloodstream form organisms while northern blot analysis indicates that the level of stable transcript is greatly reduced. Measurements of RNA abundance using RNASeq also indicated around a five fold increased TKT RNA abundance in procyclic forms [45], as did microarray analysis [46] and a digital gene expression analysis [47] indicated around a three fold increase. Procyclic tkt−/− null mutants were unaffected in their ability to grow in standard laboratory medium. Moreover, no overt changes to morphology of the parasites were visible. However, metabolic profiling revealed clear differences between wild type and tkt−/− mutants. Loss of TbTKT led to increased intracellular concentrations of substrates including pentose phosphates and reduced intracellular concentration of the main unique product S7P. In addition some other metabolites were also affected by loss of transketolase. These changes point to cross-talk between pathways of central carbon metabolism in response to the genetic perturbation around transketolase. Gross phenotypic changes associated with loss of the enzyme in rich medium were not apparent although it is possible that important roles for the enzyme and the pentose phosphate pathway would only become manifest within the environment of the tsetse fly. Acknowledgements This work was supported by grants from the Swiss National Foundation to MES and the Wellcome Trust to MPB. We thank Prof. C. Clayton for the inducible plasmid pHD1485 and the cell line 4271313. CE, JH, FB and J-CP are supported by the Agence Nationale de la Recherche (ANR) program (grant name METABOTRYP of the ANRMIME2007 call). MPB and FB also thank the BBSRC-ANR Systems Biology programme for financial support of their “Systryp” project. We also thank Gordon Campbell for his technical assistance. References [1] Wood T. Physiological functions of the pentose phosphate pathway. Cell Biochem Funct 1986;4:241–7. [2] Barrett MP. The pentose phosphate pathway and parasitic protozoa. Parasitol Today 1997;13:11–6. [3] Schenk G, Layfield R, Candy JM, Duggleby RG, Nixon PF. Molecular evolutionary analysis of the thiamine-diphosphate-dependent enzyme, transketolase. J Mol Evol 1997;44:552–72. [4] Lois LM, Campos N, Putra SR, Danielsen K, Rohmer M, Boronat A. Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc Natl Acad Sci U S A 1998;95:2105–10. [5] Waites MJ, Quayle JR. The interrelation transketolase and dihydroxyacetone synthase activities in the methylotrophic yeast Candida boidinii. J Gen Microbiol 1981;124:309–16. [6] Sax CM, Salamon C, Kays WT, Guo J, Yu FX, Cuthbertson RA, Piatigorsky J. Transketolase is a major protein in the mouse cornea. J Biol Chem 1996;271:33568–74. [7] Domain F, Bina XR, Levy SB, Transketolase A. an enzyme in central metabolism, derepresses the marRAB multiple antibiotic resistance operon of Escherichia coli by interaction with MarR. Mol Microbiol 2007;66:383–94. [8] Schneider G, Lindqvist Y. Crystallography and mutagenesis of transketolase: mechanistic implications for enzymatic thiamine catalysis. Biochim Biophys Acta 1998;1385:387–98. [9] Asztalos P, Parthier C, Golbik R, Kleinschmidt M, Hübner G, Weiss MS, Friedemann R, Wille G, Tittmann K. Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5phosphate. Biochemistry 2007;46:12037–52. [10] Gerhardt S, Echt S, Busch M, Freigang J, Auerbach G, Bader G, Martin WF, Bacher A, Huber R, Fischer M. Structure and properties of an engineered transketolase from maize. Plant Physiol 2003;132:1941–9. [11] Veitch NJ, Maugeri DA, Cazzulo JJ, Lindqvist Y, Barrett MP. Transketolase from Leishmania mexicana has a dual subcellular localisation. Biochem J 2004;382:759–67. [12] Schellenberger A. Sixty years of thiamine diphosphate biochemistry. Biochim Biophys Acta 1998;1385:177–86.

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