molecular microbiology first published online 9 february ...beverleylab.wustl.edu/pdfs/184. murta et...

Methylene tetrahydrofolate dehydrogenase/cyclohydrolaseand the synthesis of 10-CHO-THF are essential inLeishmania major

Silvane M. F. Murta,†§ Tim J. Vickers,§

David A. Scott‡ and Stephen M. Beverley*Department of Molecular Microbiology, Box 8230,Washington University School of Medicine,660 S. Euclid Avenue, St. Louis, MO 63110, USA.

Summary

10-Formyl tetrahydrofolate (10-CHO-THF) is a keymetabolite in C1 carbon metabolism, arising throughthe action of formate-tetrahydrofolate ligase (FTL)and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase(DHCH). Leishmania major possesses single DHCH1and FTL genes encoding exclusively cytosolic pro-teins, unlike other organisms where isoforms occurin the mitochondrion as well. Recombinant DHCH1showed typical NADP+-dependent methylene tetrahy-drofolate DH and 5,10-methenyltetrahydrofolate CHactivities, and the DH activity was potently inhibitedby a substrate analogue 5,10-CO-THF (Ki 105 nM),as was Leishmania growth (EC50 1.1 mM). Previousstudies showed null ftl - mutants were normal, raisingthe possibility that loss of the purine syntheticpathway had rendered 10-CHO-THF dispensable inevolution. We were unable to generate dhch1 - nullmutants by gene replacement, despite using a widespectrum of nutritional supplements expected tobypass DHCH function. We applied an improvedmethod for testing essential genes in Leishmania,based on segregational loss of episomal comple-menting genes rather than transfection; analysis of~1400 events without successful loss of DHCH1 againestablished its requirement. Lastly, we employed‘genetic metabolite complementation’ using ectopi-cally expressed FTL as an alternative source of10-CHO-THF; now dhch1 - null parasites were readily

obtained. These data establish a requirement for10-CHO-THF metabolism in L. major, and providegenetic and pharmacological validation of DHCH as atarget for chemotherapy, in this and potentially otherprotozoan parasites.

Introduction

Leishmania are important pathogens infecting millions ofpeople worldwide. The symptoms of these infectionsrange from skin lesions and disfiguring necrosis in theface, to lethal pathology in the liver and spleen, dependingon the Leishmania species involved. Although lastingimmunity is possible, no vaccine is presently available,and the drugs used in chemotherapy suffer from variousdeficiencies, such as toxicity, high cost and emergingresistance (Kedzierski et al., 2006; Mishra et al., 2007).Consequently, the development of alternative therapies isan urgent priority.

Folate derivatives are essential cellular cofactors in thesynthesis of thymidine, purines and the amino acidsglycine and methionine, in the metabolism of histidine andserine (Nzila et al., 2005; Christensen and MacKenzie,2006) and for mitochondrial protein synthesis through theformylation of initiator methionyl-tRNAMet (Kozak, 1983).The metabolism of reduced folate cofactors is of particularinterest in drug discovery, since dihydrofolate reductase(DHFR) can be efficiently targeted in antitumour and anti-microbial chemotherapy (Schweitzer et al., 1990; Then,2004; Nzila et al., 2005). While current antifolates areineffective against Leishmania because DHFR inhibition isbypassed by pteridine reductase 1 (PTR1), which is rela-tively insensitive (Bello et al., 1994), combined inhibitorstrategies may overcome this (Hardy et al., 1997; Cavaz-zuti et al., 2008). In this work we explore a potential newarena for chemotherapy, the enzymes responsible for thesynthesis of 10-formyl tetrahydrofolate (10-CHO-THF).

Three known enzyme activities mediate production of10-CHO-THF (Fig. 1) (Appling, 1991; Christensen andMacKenzie, 2006; 2008). In one pathway formate is addeddirectly onto THF in an ATP-dependent reaction byformate-tetrahydrofolate ligase (FTL, EC 6.3.4.3)(Rabinowitz and Pricer, 1962). Alternatively, 10-CHO-THF

Accepted 12 January, 2009. *For correspondence. E-mail [email protected]; Tel. (+1) 314 747 2632; Fax (+1) 314 747 2634.Present addresses: †Centro de Pesquisas Rene Rachou, FIOCRUZ,Belo Horizonte, Minas Gerais, 30190-002, Brazil; ‡Burnham Institutefor Medical Research, 10901 North Torrey Pines Road, La Jolla, CA92037, USA. §Both authors contributed equally to this work.

Molecular Microbiology (2009) 71(6), 1386–1401 � doi:10.1111/j.1365-2958.2009.06610.xFirst published online 9 February 2009

© 2009 The AuthorsJournal compilation © 2009 Blackwell Publishing Ltd

arises in two steps from 5,10-methylene-tetrahydrofolate(5,10-CH2-THF), beginning with oxidation to 5,10-methenyl-tetrahydrofolate (5,10-CH=THF) by a NADP+ orNAD+-dependent methylene tetrahydrofolate dehydroge-nase (DH, EC 1.5.1.5 or 1.5.1.15 respectively) (Hatefiet al., 1957; Moore et al., 1974). 5,10-CH=THF is thenhydrolysed to 10-CHO-THF by methenyltetrahydrofolatecyclohydrolase (CH, EC 3.5.4.9). These enzymes occurphysically in various combinations, with monofunctionalenzymes such as a human, bacterial and plant FTLs,bifunctional enzymes with dehydrogenase and cyclohy-drolase activities (referred to as DHCH enzymes), or tri-

functional enzymes with all three activities (referred to asthe C1-THF synthases) (Tan et al., 1977; Appling, 1991;Nour and Rabinowitz, 1991; Christensen and MacKenzie,2006; 2008). Adding to this complexity, multiple genesoften encode these activities in a single organism, withhumans expressing both a trifunctional C1-synthase inthe cytoplasm (MTHFD1), and a bifunctional DHCH(MTHFD2) and a monofunctional FTL (MTHFD1L) tar-geted to the mitochondrion (Christensen et al., 2005a).Other eukaryotes express differing combinations of bothcytosolic and organellar DHCH and FTLs (Hanson andRoje, 2001; Christensen and MacKenzie, 2006).

The two products of these enzymes have severalknown functions. 5,10-CH=THF is best known as an inter-mediate leading to 10-CHO-THF (Appling, 1991; Hansonand Roje, 2001; Christensen and MacKenzie, 2006),although it also occurs in some DNA photolyases, andfunctions in mammalian histidine catabolism (Stanger,2002; Weber, 2005). These pathways have not beendescribed in Leishmania and their properties in otherorganisms suggest they are unlikely to be essential.10-CHO-THF acts as a formyl donor in purine biosynthe-sis, and in bacteria and eukaryotic organelles for formy-lation of the initiator methionyl-tRNAMet to produce fMet-tRNAMet (Kozak, 1983; Appling, 1991; Varshney et al.,1993; RajBhandary, 1994; Christensen and MacKenzie,2006). Interestingly, many protozoans including trypano-somes and Leishmania lack the de novo purine pathwayand rely exclusively on salvage (Carter et al., 2008), elimi-nating the need for 10-CHO-THF in this capacity. Theimportance of initiator Met-tRNAMet formylation is thesubject of some controversy. While initiator Met-tRNAMet

formylation is thought originally to be essential in bacteria,recent data suggest that is not always the case (Newtonet al., 1999; RajBhandary, 2000). Mitochondrial initiatorMet-tRNAMet formylation is dispensable in the yeast Sac-charomyces cerevisiae but may be required in humans;even when not essential, the consequences of its lossvary widely (Li et al., 2000; Vial et al., 2003; Spencer andSpremulli, 2004). Studies in the protozoan parasite Try-panosoma brucei, a member of the protozoan subfamilytrypanosomatidae to which Leishmania belongs, showthat Met-tRNA is imported into the mitochondrion and thenformylated, where it is used for mitochondrial protein syn-thesis (Tan et al., 2002; Charriere et al., 2005). RNAiknock-down of the formyl-Met-tRNAMet transferase (FMT)shows little phenotype. However, negative results in RNAiknock-downs sometimes can arise through incompleteinhibition, a particular problem in cofactor metabolismwhere even low levels of THF-dependent metabolites canfulfil normal metabolic needs. Importantly, simultaneousRNAi knock-down of the T. brucei FMT with the mitochon-drial initiation factor IF2 yield significant growth inhibition(Charriere et al., 2005), possibly signifying the importance

Fig. 1. 10-Formyl-THF metabolism in Leishmania. Followingsynthesis through the activity of dihydrofolate reductase (DHFR,encoded by the bifunctional DHFR-TS) or pteridine reductase 1(PTR1), tetrahydrofolate (THF) can enter C1 metabolic pathwaysby direct formylation in an ATP-dependent reaction byformate-tetrahydrofolate ligase (FTL, LmjF30.2600). Alternatively,THF is converted into 5,10-methylene-tetrahydrofolate(5,10-CH2-THF) through the activity of serinehydroxymethyltransferase (SHMT). 10-CHO-THF can be generatedin two steps from 5,10-CH2-THF, through oxidation to5,10-methenyl-tetrahydrofolate (5,10-CH=THF) by aNADP+-dependent methylenetetrahydrofolate dehydrogenase (DH,LmjF26.0320), and hydrolysis to 10-CHO-THF bymethenyltetrahydrofolate cyclohydrolase (CH, LmjF26.0320). InL. major the DH and CH activities are contained in the singlebifunctional protein DHCH1, which like FTL is localized exclusivelyto the cytosol (T.J. Vickers, S.M.F. Murta, M.A. Mandell and S.M.Beverley, submitted). While 10-CHO-THF is used in the de novopurine synthetic pathway in many organisms, this pathway isabsent in Leishmania and many parasitic protozoans (Carter et al.,2008). Current data suggest 10-CHO-THF may be utilized in themitochondrion for methionyl-tRNA formylation, the only uniquefunction thus far uncovered by genomic and metabolic studies inLeishmania.

DHCH1 and 10-CHO-THF are essential in L. major 1387

© 2009 The AuthorsJournal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1386–1401

of fMet-tRNAMet for essential mitochondrial proteinsynthesis.

Previously we and others used database mining tosurvey the 10-CHO-THF synthetic pathway in Leishmaniaand other trypanosomatids (Opperdoes and Coombs,2007; T.J. Vickers, S.M.F. Murta, M.A. Mandell and S.M.Beverley, submitted). The Leishmania major genomeencodes an active monofunctional FTL and a predictedbifunctional DHCH encoded by the DHCH1 gene. Bothproteins were localized exclusively in the cytoplasmconsistent with the absence of a predicted N-terminalmitochondrial targeting sequence, as shown by immunob-lotting of cellular fractions as well as GFP fusions toDHCH1 (T.J. Vickers, S.M.F. Murta, M.A. Mandell and S.M.Beverley, submitted). Unexpectedly, ftl- null mutantsshowed normal viability and virulence. As noted above, theabsence of purine synthesis mitigates the need for10-CHO-THF in Leishmania, and potentially this couldreduce or even eliminate this requirement globally.

Here we characterize the role of DHCH1 in L. majormetabolism through enzymatic, genetic knockout andpharmacological tests. First we show that DHCH1encodes a functional protein with NADP-dependent DHand CH activities, with kinetic properties similar to thoseseen in other organisms. We used both traditionaltransfection-based and a new plasmid segregationalapproach to establish that DHCH1 was essential. Notably,overexpression of FTL enabled the recovery of dhch1-

mutants, establishing through ‘genetic metabolite comple-mentation’ that 10-CHO-THF is essential. Lastly weshowed that Leishmania growth and DHCH1 activity canbe inhibited by a specific DHCH inhibitor. In total thesedata provide genetic and pharmacological validation ofDHCH as a target for antiparasitic chemotherapy.

Results

Enzymatic activity of recombinant L. major DHCH1

Previously we used database mining of trypanosomatidgenomes to identify gene LmjF26.0320 as DHCH1 as theonly functional DHCH in L. major (T.J. Vickers, S.M.F.Murta, M.A. Mandell and S.M. Beverley, submitted). Ahexahistidine-tagged DHCH1 was expressed in Escheri-chia coli and the recombinant protein was purified bymetal-affinity chromatography (Fig. S1) (T.J. Vickers,S.M.F. Murta, M.A. Mandell and S.M. Beverley, submitted).Dehydrogenase activity was measured by following theconversion of 5,10-CH2-THF to 5,10-CH=THF, while thecyclohydrolase activity was monitored by following theconsumption of 5,10-CH=THF, yielding 10-CHO-THF (seeExperimental procedures). Purified DHCH1 showed both5,10-CH2-THF dehydrogenase and 5,10-CH=THF cyclo-hydrolase activities, with specific activities of 22 � 2 and

6.3 � 0.8 mmol min-1 mg-1 respectively. In comparison withthe activities of the porcine cytosolic multifunctionalC1-synthase the L. major enzyme has a slightly higherdehydrogenase activity and a lower cyclohydrolase activ-ity, with the porcine enzyme having activities of 7.5 and22 mmol min-1 mg-1 respectively (Tan et al., 1977).

The kinetic properties of the dehydrogenase reactionwere determined and are summarized in Table 1. Thereaction followed typical Michaelis–Menten kinetics andcould be saturated with either substrate. In mammals, thecytoplasmic DH activity of the trifunctional C1-synthase isdependent on NADP+ (Tan et al., 1977), whereas themitochondrial DH is dependent on NAD+ (Mejia andMacKenzie, 1988). The L. major DHCH1 dehydrogenaseactivity was highly dependent on NADP+, with no activitydetected with NAD+ (limit of detection 0.3 mmol min-1 mg-1,about 1% of the activity seen with NADP+). While the DHactivity of human mitochondrial DHCH with NAD+ requiresphosphate and magnesium ions (Christensen et al.,2005b), when tested in the presence of both 5 mM(K+)PO4 and 5 mM MgCl2 L. major DHCH1 still showed noNAD+-dependent activity (data not shown). These dataare consistent with the conservation of residues inLmDHCH1 that interact with the NADP+ cofactor in thehuman cytosolic enzyme, notably Arg173 that interactswith the coenzyme 2′ phosphate group in the humanenzyme’s active site (Allaire et al., 1998) (Fig. S2). Incomparison with the human enzymes, the LeishmaniaDHCH1 dehydrogenase activity appears most similar tothat of the NADP+-dependent cytoplasmic C1-synthase,with a lower Km for the coenzyme and lower turnover thanthe human mitochondrial DHCH MTHFD2 (Pawelek andMacKenzie, 1998).

Attempts to generate DHCH1 knockout by ‘classic’double replacements

Leishmania are predominantly diploid and thus inactiva-tion of most genes requires two rounds of gene replace-ment (Cruz et al., 1991). While some chromosomes showaneuploidy in some wild-type (WT) Leishmania strains,preliminary CGH data suggest that chromosome 26

Table 1. Kinetic constants of recombinant Leishmania majorDHCH1.

SubstrateKm (apparent)(mM) kcat

a (s-1)kcat/Km

(M-1 s-1) ¥ 105

5,10-CH2-THFb 120 � 10 14 � 1 1.2NADP+c 38 � 6 10 � 1 2.6

a. Calculated assuming one active site per polypeptide.b. Measured with 1 mM NADP+ as the fixed substrate.c. Measured with 250 mM 5,10-CH2-THF as the fixed substrate.All values are given as the average +/- standard deviation.

1388 S. M. F. Murta, T. J. Vickers, D. A. Scott and S. M. Beverley �


bearing DHCH1 is disomic in the L. major line studiedhere (E. Kruvand and S. Beverley, in preparation). Thus,we constructed two targeting fragments, with the DHCH1ORF replaced by ones encoding puromycin (PAC) orhygromycin B (HYG) resistance, flanked by ~1 kb of 5′and 3′ sequence (Fig. 2A). Electroporation of either frag-ment separately into WT L. major followed by plating onselective media yielded transfectant colonies efficiently atcontrol frequencies (not shown). Analysis of 20 clonaltransfectant lines showed that all were heterozygotes(DHCH1/Ddhch1::PAC or DHCH1/Ddhch1::HYG, abbrevi-ated hereafter as PAC/+ or HYG/+) as expected (Fig. 2A,Fig. S3, and data not shown). These studies used PCRtests with primer pairs containing one located outside ofthe targeting fragment to either the 5′ or 3′ side, partneredwith a marker-specific primer (Fig. S3, Fig. 2B and C anddata not shown).

Next the HYG/+ or PAC/+ heterozygotes were submit-ted to a second round of electroporation with the remain-ing targeting fragment (PAC or HYG respectively) andplated on semi-solid media containing both puromycinand hygromycin B. PCR tests of 12 doubly resistant clonaltransfectant lines showed that each contained both theplanned HYG and PAC DHCH1 replacements (Fig. 2Band C). Nonetheless, the clonal transfectants retained acopy of DHCH1, as revealed by PCR amplification of anintact DHCH1 ORF (Fig. 2D), and grew normally (data notshown). This finding of successful ‘double replacement’accompanied by retention of the WT gene has been seenpreviously in attempts to target other essential genes inLeishmania, arising through generation of aneuploid ortetraploid parasites with additional chromosomes bearingthe target gene (Cruz et al., 1993; Dumas et al., 1997;Ilgoutz et al., 1999a; Vergnes et al., 2005; Balana-Fouce

Fig. 2. Attempts to generate dhch1 - null mutants by ‘classic’ double replacements.A. Planned deletion of the DHCH1 ORF by homologous gene replacement using targeting fragments with PAC or HYG resistance markers. Inthe first round, parasites were transfected with either construct separately; in this diagram the round starting with PAC is shown. Heterozygousreplacement lines were obtained with high efficiency. In the second round, the PAC/+ heterozygote (formally, Ddhch1::PAC/DHCH) wastransfected with the HYG targeting fragment, with the expectation of yielding homozygous dhch1 - null mutants. Instead, only aneuploidparasites showing both the two planned replacements but retaining WT DHCH1 were recovered (formally,Ddhch1::PAC/DHCH/Ddhch1::SAT/Ddhch1::PAC/DHCH1).B. PCR tests of planned PAC replacements at DHCH1. Homologous replacement was visualized by PCR amplification using a primer directedagainst sequences located within the PAC ORF, and a primer located on the DHCH1 chromosome outside the targeting fragment (indicated byheavy line) on the 5′ (white arrows) or 3′ (black arrow) side (SMB2731/2558 and SMB2771/2557, Table S1). The sizes of the predicted PCRproducts are shown. WT, wild-type DNA; NC, negative control (no DNA template), #1–8, eight independent lines with PAC replacements atDHCH1.C. PCR tests of planned HYG replacements at DHCH1. This experiment is similar to that described in (B), except that primers were directedagainst sequences located in the HYG targeting fragment (SMB2731/2562 and SMB2771/2561, Table S1). #1–6, six independent lines withHYG replacements at DHCH1.D. PCR tests showing retention of DHCH1 in second-round transfectants. Amplifications were performed using primers directed againstsequences within the L. major DHCH1 ORF (SMB3125/3126, Table S1). Lines #1–4 represent double replacement with PAC/HYG and lines#5–8 with HYG/PAC.



et al., 2008). Analysis of the DNA content of the doublytargeted lines above by flow cytometry showed WT DNAcontents (data not shown), suggested that these werelikely to be aneuploids, bearing at least one ‘WT’ chromo-some along with the planned HYG and PAC replacements(Fig. 2A).

Attempts to generate DHCH1 knockout by doublereplacement using different nutritional supplements

We attempted to bypass the consequences of DHCH1ablation through metabolic complementation. In thesestudies, the second-round transfections above wererepeated, but with parasites grown and plated on mediacontaining various supplements potentially able to bypassthe DHCH1 requirement. First we included metabolitesknown to participate and/or arise in C1 folate metabolism,including glycine, serine, formate, tetrahydrofolate, thymi-dine and folinic acid. Second, parasites were grown withthe supplements above plus the C1 folates 5,10-CH=THFand 10-formylfolic acid (10-CHOFA), the first being anintermediate metabolite formed through the DH activity ofDHCH1 and the second constituting a potential precursorto 10-formyl-THF. While the ideal supplement would havebeen 10-CHO-THF, this metabolite is very unstable;however, under the mildly alkaline pH of Leishmaniaculture media (7.4), spontaneous isomerization of 5,10-CH=THF to 10-CHO-THF occurs over time (Rabinowitz,1963; Brouwer et al., 2007). Lastly, since the expectedrole of 10-CHO-THF in Leishmania arises through its rolein mitochondrial protein synthesis, we attempted tobypass this potential requirement by performing transfec-tions in the presence of pyruvate and uridine, two metabo-lites that have been proposed to bypass the need formitochondrial DNA and presumably the proteins encodedtherein (Sen et al., 2007).

In total, 65 independent clonal lines were analysed, all ofwhich yielded the presumptive aneuploidy phenotype bythe PCR assay described above (data not shown), i.e.parasites bearing both the planned HYG and PAC replace-ments while retaining at least one copy of the DHCH1 ORF,without the recovery of any dhch1- null mutants.

Deletion of chromosomal DHCH1 in the presence ofectopically expressed DHCH1

An important control was whether successful targetingcould be achieved in the presence of ectopic DHCH1expression, as this would render unlikely the possibilitythat molecular events neither anticipated nor tested inour strategy above had occurred. First, DHCH1 wasexpressed from the multicopy episomal pXNG4 vector,which bears a nourseothricin (SAT) resistance marker(D. Scott and S. Beverley, in preparation). DHCH assaysof WT protein extracts showed a specific activity of

1 nmol min-1 mg-1, a value not significantly different fromthe assay background in controls lacking NADP+, whereasWT/pXNG4-DHCH1 transfectants showed a specific activ-ity of 31 nmol min-1 mg-1, at least 30-fold greater. pXNG4-DHCH1 was then introduced into the HYG/+ or PAC/+heterozygotes described previously, and the second chro-mosomal allele in these lines targeted by transfection asbefore (Fig. 3A). To visualize the chromosomal DHCH1allele, PCR using a forward primer located 5′ and outside ofthe DHCH1 targeting fragment and a reverse primerlocated within the DHCH1 ORF was used (Fig. 3B).

In contrast to the results above with parasites lackingectopic DHCH1 expression, the chromosomal DHCH1was now readily lost in all 12 independent clonal trans-

Fig. 3. Deletion of chromosomal DHCH1 in the presence ofectopic DHCH1.A. Scheme for the deletion of chromosomal DHCH1 in thepresence of ectopically expressed DHCH1. First, a pXNG4-DHCH1episomal expression construct was transfected into aDHCH1/Ddhch1::PAC heterozygote described in Fig. 2A and 10/10transfectants analysed were confirmed to have the predictedgenotype depicted. These DHCH1/Ddhch1::PAC [pXNG4-DHCH1]lines were then transfected with the HYG DHCH targeting fragmentand selected for resistance to all three antibiotics; of the 12 clonallines tested, all showed the planned replacement of thechromosomal DHCH1 locus (formally, Ddhch1::HYGDHCH1/Ddhch1::PAC [pXNG4-DHCH1]).B. PCR tests showing loss of the chromosomal DHCH1. Todistinguish the ectopic DHCH1 from the chromosomal locus, DNAfrom candidate lines was subjected to PCR tests using a primerdirected against sequences located within the DHCH1 ORF, and aprimer located on the DHCH1 chromosome outside of the targetingfragment on the 5′ side (SMB2731/3126, Table S1). WT, wild-typeDNA control; NC, negative control lacking any DNA template. Thearrow marks the size of the predicted fragment found in WT DNA,which is absent in the chromosomal dhch1 - knockouts.



fectants tested (Fig. 3B). PCR tests showed that theselines contained both the HYG and PACDdhch1- replace-ments, while retaining the DHCH1 ORF borne on pXNG4-DHCH1 (data not shown). Thus, in the presence ofectopically expressed DHCH1, both chromosomalDHCH1 alleles could be successfully eliminated, yieldingparasites that were genetically dhch1-/+pXNG4-DHCH1.These methodological controls further support the conclu-sion that under the conditions tested, DHCH1 is essential.

Genetic metabolite complementation permits therecovery of DHCH1 replacement

A requirement for DHCH1 was somewhat surprising,since Leishmania possess a second cytoplasmic pathwayfor 10-CHO-THF synthesis through FTL, whose enzy-matic activity has been verified (T.J. Vickers, S.M.F.Murta, M.A. Mandell and S.M. Beverley, submitted)(Fig. 1). This could indicate that DHCH1 has an unantici-pated function, or alternatively, that the activity of the FTLpathway was insufficient for metabolic needs in theabsence of DHCH1. Indeed, evidence that the FTL

pathway is of secondary importance comes from the factthat ftl- mutants could be readily generated without anyapparent effect on growth in vitro or virulence in animalinfections (T.J. Vickers, S.M.F. Murta, M.A. Mandell andS.M. Beverley, submitted).

Thus we asked whether provision of 10-CHO-THF byoverexpression of FTL using the multicopy episomalvector pXNG4-FTL could bypass the requirement forDHCH1 (Fig. 4A). pXNG4-FTL was introduced into theheterozygous PAC/+ or HYG/+ lines, and FTL overexpres-sion was confirmed by Western blot analysis (Fig. S4).These parasites were then submitted to a second round oftargeting as before. Analysis of 12 independent transfec-tant lines showed that all now lacked the DHCH1 ORFentirely, and Western blot analysis with a polyclonal anti-DHCH1 antiserum confirmed a complete loss of DHCH1protein (Fig. 4B and C). The ability of elevated FTLexpression to enable parasites to readily dispense withDHCH1 is strong evidence of genetic metabolite comple-mentation, presumably through the product of FTL activ-ity, 10-CHO-THF, and in turn argues strongly that theessential role of DHCH1 is the provision of this metabolite.

Fig. 4. Deletion of chromosomal DHCH1 in the presence of ectopic FTL overexpression.A. Scheme for the deletion of chromosomal DHCH1 in the presence of ectopically expressed FTL. A pXNG4-FTL episomal expressionconstruct was transfected into a DHCH1/PAC (DHCH1/Ddhch1::PAC) heterozygote described in Fig. 2A and 10/10 transfectants analysed hadthe predicted genotype depicted. These DHCH1/PAC/+FTL lines (DHCH1/Ddhch1::PAC [pXNG4-FTL]) were then transfected with the HYGDHCH1 targeting fragment as described in the legend to Fig. 3A; of the 12 clonal lines tested, all showed the planned replacement of thechromosomal DHCH1 locus and were thus dhch1 -/+FTL (Ddhch1::HYG/Ddhch1::PAC [pXNG4-FTL]).B. PCR tests showing loss of the chromosomal DHCH1. This was performed as described in the legend to Fig. 3B. NC, negative controllacking DNA; WT, WT DNA; pXNG-DHCH1, WT transfected with pXNG-DHCH1. Pac4, Pac5 and Hyg7 are three different lines showing anabsence of the predicted chromosomal DHCH1 PCR product (shown by arrow).C. Western blot showing loss of DHCH1 expression in dhch1 -/+FTL transfectants. The lines are as described in (B), extracts from which wereprobed with polyclonal rabbit antisera against DHCH1 (1:3000) (T.J. Vickers, S.M.F. Murta, M.A. Mandell and S.M. Beverley, submitted).



Forcing loss of pXNG4-DHCH1 by negative selection

As a final test probing the essentiality of DHCH1 in WTLeishmania, we applied a method building on ‘plasmidshuffling’ approaches common in yeast genetics. Plasmidsegregation-based approaches have the advantage ofallowing the separation of tests of gene function fromtransfection, which is relatively inefficient (D. Scott andS.M. Beverley, in preparation). In our studies this utilizedtwo features of the episomal pXNG4 vector, namely thepresence of both a negative selective marker (a modifiedHSV thymidine kinase gene) and a positive GFP fluores-cence marker (Fig. 5A). The starting point was the dhch1-/pXNG4-DHCH1 parasite described earlier, where thechromosomal DHCH1 was ablated and parasite growthmaintained by the episomal pXNG4-DHCH1 (Figs 3and 5A).

dhch1-/pXNG4-DHCH1 parasites were grown briefly(24 h, or ~3 doublings) in the absence of nourseothricin(selective for the SAT marker of episomal pXNG4) but inthe presence of ganciclovir, which through the action ofHSV TK selects against parasites bearing pXNG4. Over

this period little effect on parasite growth was seen. Then,parasites were subjected to flow cytometry, to assessplasmid copy number by following expression of the GFPmarker. Two populations of cells were evident followingthis treatment: first, ‘bright’ cells showing strong fluores-cence (> 200 FU; M2 gate in Fig. 5B) and presumablyhigh copy numbers of pXNG4-DHCH1; and ‘dim’ cells,showing background fluorescence (2–20 FU; M1 gate inFig. 5B), presumably either lacking pXNG4-DHCH1, orbearing just a few copies. Controls showed that in theabsence of ganciclovir the dhch1-/+pXNG4-DHCH1 para-sites retained high GFP fluorescence, while WT cellsshowed background fluorescence only (Fig. 5C and D).As our previous results indicated that DHCH1 was essen-tial, we expected that the ‘bright’ cells retaining pXNG4-DHCH1 would be viable, while the ‘dim’ cells would suffera growth disadvantage and perhaps perish.

We used flow cytometry to select single cells into indi-vidual wells of a 96-well microtitre plate, focusing on the‘dim’ (M1) or ‘bright’ (M2) parasites from the M1 popula-tion shown in Fig. 5B. We inoculated parasites into platescontaining M199 media containing puromycin and hygro-

Fig. 5. Plasmid segregational tests of DHCH1 essentiality.A. Starting dhch1-/+pXNG4-DHCH1 transfectant. The structure of one of the lines generated as described in Fig. 3A is shown, additionallyshowing the TK (ganciclovir sensitivity), GFP and SAT resistance markers encoded on the pXNG4-DHCH1 vector.B. Forced loss of pXNG4-DHCH1 by negative selection and sorting. The line depicted in (A) was grown for 24 h in the presence of ganciclovirbut in the absence of nourseothricin. Fluorescent (bright) and non-fluorescent (dim) parasites were assigned using the M1 and M2 gatesshown, and single cells were sorted into 96-well plates containing M199 medium and/or various supplements (see Experimental procedures).With ‘bright’ cells, 478/672 (71%) of the wells yielded growth. In contrast, only 48/2016 of wells inoculated with ‘dim’ cells (2.4%) showedgrowth; all of these continued to grow in the presence of nourseothricin (SAT resistance) establishing retention of the pXNG-DHCH1 episome.C. Retention of pXNG4-DHCH1 in the absence of ganciclovir selection. The line shown in (A) was grown 24 h without ganciclovir prior to GFPflow cytometry.D. WT L. major subjected to GFP flow cytometry.



mycin B (to guarantee retention of dhch1- cells) butlacking ganciclovir or nourseothricin (pXNG4 markers).Several experiments were performed with media supple-mented additionally with the various metabolitesdescribed earlier.

In these studies, 478/672 of the wells inoculated witha single ‘bright’ parasite grew out (71%), a value similarto that seen with controls probing the effects of sorting,recovery and growth of single cells in the cells (50–80%). In contrast, in wells receiving a single ‘dim’ para-site, growth was seen in only 48/2016 (2.4%) (Fig. 5B).Importantly, all 48 ‘dim’ parasite lines retained thepXNG4-DHCH1 plasmid, as judged by their ability togrow in the presence of nourseothricin (SATR) and/orDHCH1 PCR tests. We presume these cells arose eitherby imperfect sorting, and/or from cells originally express-ing low levels of GFP due to the presence of only one ora few copies of pXNG4-DHCH1. After correcting for theintrinsic plating efficiency, we estimate that in totalapproximately 1430 cells (71% of 2016) had beenscored for their ability to grow in the absence of DHCH1.Thus plasmid shuffling enabled us to extend the strin-gency of our selection from a total of 65 events scoredby the ‘classic’ transfection-based approach, to a valuemore than 20-fold higher.

Interaction of L. major DHCH1 with novel antifolates

To corroborate the conclusion that DHCH activity wasessential for Leishmania growth, we turned to pharmaco-logical tests. The substrate analogue 5,10-CO-THF ((2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H-imidazol[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioicacid) was previously shown to be a potent inhibitor of theDHCH activity of the human cytosolic C1-synthase(Temple et al., 1982; Tonkinson et al., 1998). We predictedthat this compound should also be a potent inhibitor of theLeishmania DHCH1, based on the overall sequence con-servation, including the residues shown to interact with thisinhibitor in structural studies of the human C1-synthaseDHCH domain (Schmidt et al., 2000) (Fig. S2). Indeed,5,10-CO-THF was a potent inhibitor of the DH activity ofrecombinant Leishmania DHCH1, with the data fitting to asimple competitive model with respect to 5,10-CH2-THF,giving a Ki of 105 � 2 nM (Fig. 6). This was about 10-foldhigher than the Ki of 18 � 5 nM seen for the DH activity ofthe human cytosolic C1-synthase (Schmidt et al., 2000).While 5,10-CO-THF was shown previously to be a weakinhibitor of the human DHFR (Ki > 100 mM) (Temple et al.,1982), 100 mM 5,10-CO-THF did not inhibit the activity ofLeishmania DHFR-thymidylate synthase (DHFR-TS) (datanot shown) nor FTL (T.J. Vickers, S.M.F. Murta, M.A.Mandell and S.M. Beverley, submitted). L. major DHCH1was also not inhibited by pteridine analogues shown pre-

viously to inhibit L. major DHFR or PTR1, including meth-otrexate or hydrophobic diamino-quinazoline, -pyrimidineor -pteridine analogues [compound 34: 2,4-diamino-6-(3,4-dichlorophenoxy)-quinazoline; compound 70:2,4-diamino-6-benzyl-5-(3-phenylpropyl)-pyrimidine; andcompound 25: 2,4-diamino-6,7-diisopropylpteridine] (Belloet al., 1994; Hardy et al., 1997) (tested at 50 mM; data notshown).

Toxicity of folate analogues to cell lines

5,10-CO-THF inhibited L. major growth with an EC50 of1.1 � 0.04 mM (Table 2). Parasites overexpressingDHCH1 (WT/pXNG4-DHCH1 or dhch1-/+pXNG4-DHCH1) showed four- to fivefold resistance (P < 0.0001and P < 0.0003 respectively; Table 2), consistent with thestudies of purified DHCH above. Parasites overexpress-ing FTL (WT/pXNG4-FTL or dhch1-/pXNG4-FTL) alsoshowed threefold resistance (P < 0.0001 and P < 0.0002respectively; Table 2), implying that FTL activity could actas a metabolic bypass for DHCH inhibition. Consistentwith the enzyme inhibition data, DHCH1 or FTL overex-pressors did not show resistance to methotrexate or twoof the hydrophobic antifolates tested above (compounds34 and 70; Table 2). Interestingly, even in the absence ofa DHCH target (dhch1-/pXNG4-FTL line), growth wasinhibited with an EC50 in the mM range (Table 2), suggest-ing 5,10-CO-THF has other targets in Leishmania. FTLcan be excluded as it is not sensitive to 5,10-CO-THF, andwhile the bridged structure of the inhibitor does not closely

Fig. 6. Inhibition of L. major DHCH1 by 5,10-CO-THF. Thedehydrogenase activity of purified recombinant DHCH1 (1.3 mg)was assayed in the presence of 1 mM NADP+ and varyingconcentrations of 5,10-CH2-THF substrate (12.5–250 mM) in thepresence of no (�), 1.5 mM (�), 3 mM (�) or 5 mM (�) inhibitor.The lines show a global fit of these data to a simple competitivemode of inhibition.



resemble that of 10-CHO-THF, it seems probable that thetarget could be other folate-dependent enzymes or trans-porters within the cell.

Discussion

We show here that L. major expresses an active bifunc-tional NADP+-dependent DHCH encoded by DHCH1(Table 1). Previous studies have localized DHCH1 proteinto the parasite cytosol, consistent with the predicted lackof an N-terminal mitochondrial targeting sequence andthe NADP dependency of the DH activity (T.J. Vickers,S.M.F. Murta, M.A. Mandell and S.M. Beverley,submitted). Attempts to generate dhch1- null mutants byhomologous replacement were unsuccessful, despiteanalysis of a large number of independent events and theuse of metabolic supplements shown in other organismsto rescue DHCH deficiency. Instead, these studies invari-ably yielded parasites bearing the two planned replace-ments, but also bearing extra copies of DHCH1 that mostlikely arose through aneuploidy. The unusual Leishmaniaphenotype of the recovery of planned replacements, butwith retention of the target gene through aneuploidy ortetraploidy, is widely used as an indication that a gene isessential in this organism. As a technical control, chromo-somal dhch1- replacements were readily obtained afterectopic expression of DHCH1. Lastly, we applied aplasmid shuffling approach that allowed us to increase thenumber of events analysed 20-fold: from 65 by classictransfectional approaches, to more than 1400 throughnegative selection and cell sorting. However, we were stillunable to recover a viable dhch1- null mutant (the virtuesand potential of the ‘plasmid shuffle’ methodology inLeishmania are discussed further below). Thus we areconfident in concluding that DHCH1 is an essential genein L. major under these circumstances.

Notably, we were able to completely remove DHCH1following ectopic expression of FTL, which provides analternative metabolic route to 10-CHO-THF synthesis viathe formate tetrahydrofolate ligase activity. This form of

‘genetic metabolic complementation’ suggests that para-site dependence on DHCH1 activity arises through theneed for the ultimate metabolite of both FTL and DHCH1,10-CHO-THF. Hypothetically, the active metabolite couldbe 5,10-CH=THF, as these two metabolites interconvert atsignificant rates under physiological conditions, whichalso precludes measurement of their individual abun-dance in the cell (Brouwer et al., 2007). However, as yetthere is no known essential role in metabolism for 5,10-CH=THF, other than as an intermediate in 10-CHO-THFsynthesis, and we thus favour the conclusion that10-CHO-THF is the relevant metabolite.

Interestingly, the levels of WT FTL activity appear insuf-ficient to rescue the dhch1- parasites, consistent with ourprevious finding that null mutant ftl- parasites were phe-notypically normal when grown in vitro and are fully infec-tive to susceptible mice (T.J. Vickers, S.M.F. Murta, M.A.Mandell and S.M. Beverley, submitted). Hypothetically, itis possible that elevated FTL synthesis or other alter-ations in 10-CHO-THF metabolism could bypass therequirement for DHCH1 under some circumstances, andindeed in other studies we have been able to detectsecond-site mutations including FTL gene amplification inother selections forcing loss of DHCH1 (S. Murta and S.Beverley, unpubl. data).

What is the role of 10-CHO-THF in trypanosomatidmetabolism?

In mammals, known roles of 10-CHO-THF include theprovision of formyl groups in purine biosynthesis, as anintermediate in C1-THF interconversions involved inamino acid metabolism (glycine, methionine, histidine andserine), or the production of fMet-tRNAMet in the mitochon-drion (Appling, 1991; Christensen and MacKenzie, 2006).However, Leishmania are well known to be purine aux-otrophs as their genome lacks the purine biosyntheticenzymes glycinamide ribonucleotide (GAR) and phospho-ribosylaminoimidazolecarboxamide (AICAR) transformy-lases (EC 2.1.1.2 and 2.1.2.3 respectively) (Carter et al.,

Table 2. Effects of 5,10-CO-THF and antifolates compound on growth of FV1 promastigote lines grown in M199 culture medium.

L. major FV1 lines5,10-CO-THFEC50 (mM)

MTX EC50

(nM)Compound 34EC50 (nM)

Compound 70EC50 (nM)

WT 1.1 � 0.04a 36 � 1 680 � 90 440 � 50dhch1 -/pXNG4-DHCH1 4.5 � 0.5 41 � 2 760 � 80 400 � 50dhch1 -/pXNG4-FTL 3.3 � 0.3 40 � 2 540 � 60 420 � 40WT/pXNG4-DHCH1 5.1 � 0.8 n.d. n.d. n.d.WT/pXNG4-FTL 3.7 � 0.5 n.d. n.d. n.d.

a. EC50 for 5,10-CO-THF differs significantly between WT and all other lines tested (P < 0.0001 to P < 0.0003).Compound 5,10-CO-THF is ((2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-4H- imidazo[3,4-f]pteridin-8-yl]benzoyl]amino]pentanedioicacid); MTX: methotrexate; compound 34 is 2,4-diamino-6-(3,4-dichlorophenoxy)-quinazoline; compound 70 is 2,4-diamino-6-benzyl-5-(3-phenylpropyl)-pyrimidine.n.d. not determined.



2008). Similarly, several studies have established thatLeishmania still require the amino acids arising throughthe C1-pathway for normal growth (Scott et al., 2008;Vickers et al., 2006). Thus under the conditions tested,formylation of the initiator Met-tRNAMet currently stands asthe only candidate essential metabolic role. As discussedearlier, the extent to which fMet-tRNAMet synthesis isrequired is a matter of some debate, with differences seenin studies both within and between species in botheukaryotes and prokaryotes. However, the conclusionthat Leishmania require fMet-tRNAMet would be consistentwith the finding that the related trypanosomatid parasiteT. brucei requires formylated initiator methionyl-tRNAMet

for mitochondrial protein synthesis and survival (Tan et al.,2002; Charriere et al., 2005). This would then suggest thatcytoplasmic 10-CHO-THF arising from either FTL orDHCH1 activity in the cytosol must then be transported tothe mitochondrion. While generally it is thought thatmetabolites other than reduced folates are the principalroute of communication for cytosolic-mitochondrialC1-dependent pathways in mammalian cells (Appling,1991), some studies point to trafficking of C1 tetrahydro-folates between the cytosol and mitochondrion in plants(Prabhu et al., 1998; Hanson et al., 2000).

The interpretations above rest on the strength andaccuracy of current genome-based models of trypano-somatid metabolomes. Given the substantial number ofhypothetical proteins of unknown function encoded intrypanosomatid genomes (Berriman et al., 2005;El-Sayed et al., 2005; Ivens et al., 2005; Peacock et al.,2007), one cannot exclude the possibility that Leishma-nia possess novel essential pathways that require10-CHO-THF. Indeed, the L. major genome predicts ahighly degenerate pseudogene (pseDHCH2) andDHCH2-related sequences are completely absent in theLeishmania braziliensis and African trypanosomegenomes (Berriman et al., 2005; El-Sayed et al., 2005;Ivens et al., 2005; Peacock et al., 2007). In contrast,seemingly intact DHCH2s occur in Leishmania infantum,Leishmania mexicana, Crithidia fasciculata and Trypano-soma cruzi, whose predicted proteins show conservationof DHCH active-site residues (T.J. Vickers, S.M.F. Murta,M.A. Mandell and S.M. Beverley, submitted). Interest-ingly, a GFP-tagged LiDHCH2 is localized to the mito-chondrion when expressed in Leishmania donovani (T.J.Vickers, S.M.F. Murta, M.A. Mandell and S.M. Beverley,submitted), although we have not been able to demon-strate DHCH activity associated with overexpression ofWT LiDHCH2 (data not shown). Potentially the occur-rence of a catalytically active DHCH2 could signify anincreased importance of mitochondrial 10-CHO-THFsynthesis in some trypanosomatid species. In the future,direct studies of mitochondrial fMet-tRNAMet synthesismediated by FMT and whether DHCH2 is enzymatically

active in those trypanosomatids that possess it will testthese hypotheses.

Plasmid segregation and shuffling as an improved testof gene function

Currently, the standard approach to determining if a geneis essential in Leishmania is an inability to delete bothchromosomal alleles by homologous gene replacement(Cruz et al., 1991; Barrett et al., 1999). Fortunately, thesestudies are facilitated by the fact that homologous recom-bination occurs far more frequently than non-homologousinsertions in trypanosomatids (Clayton, 1999; Beverley,2003). Ignoring technical failures, the general experienceof most investigators has been that attempts to deleteessential genes in Leishmania yield parasites bearing theplanned allelic replacements but containing additionalgene copies that arise via aneuploidy or polyploidy (Cruzet al., 1993; Dumas et al., 1997; Ilgoutz et al., 1999b;Vergnes et al., 2005; Balana-Fouce et al., 2008). This isoften considered a diagnostic criterion, especially whenaccompanied by successful replacement in the presenceof an ectopic gene.

One challenge of the gene replacement-basedapproach is the relative inefficiency of transfection (10-4

or less), limiting the number of events that can bescored. For example, here we analysed 65 events tar-geting the last copy of DHCH1, a number exceeding thattypically reported in tests of Leishmania gene function.Other factors impinging on replacement-based ap-proaches include great variation in transfection effi-ciency, depending on the locus and targeting constructused (Papadopoulou and Dumas, 1997), and that survi-vors can also emerge through mutations unrelated to thetargeted gene (S.M.F. Murta, T.J. Vickers and S.M. Bev-erley, in preparation). In contrast, in the plasmid shufflingapproach, functional tests arise through segregation ofan ectopically expressed gene borne on a plasmidbearing both positive (GFP) and negative (TK) markers,in a line previously engineered to lack the gene of inter-est (Fig. 5). Following a brief period of culture to allowsegregation of the episomal vector, a large number ofcandidate ‘segregants’ can be obtained through appro-priate manipulations of the GFP or TK markers, andrapidly tested. This allowed us to rapidly score ~1400such segregants for DHCH1, at least 20 times thenumber achieved by traditional methods, and if neces-sary many more could have been tested. Thus webelieve that the plasmid segregation method provides asignificant improvement to tests of essential gene func-tion in Leishmania, as first established in the yeastS. cerevisiae (Forsburg, 2001). In the future one canenvisage a number of other applications based on theapproach of ‘plasmid shuffling’.



DHCH1 and 10-CHO-THF metabolism as a target forantiparasitic chemotherapy

Leishmania major folate metabolism shows many differ-ences from that of their human hosts, including a bifunc-tional DHFR-TS (Coderre et al., 1983), a novel pteridinereductase (PTR1) (Bello et al., 1994), a large andcomplex family of folate and biopterin transporters (Ouel-lette et al., 2002), and cytosolic but not mitochondrialsynthesis of 10-CHO-THF (T.J. Vickers, S.M.F. Murta,M.A. Mandell and S.M. Beverley, submitted), amongothers. These factors contribute to profound differences inantifolate sensitivity; for example, the relative insensitivityof PTR1 to many antifolates and its ability to act as ametabolic bypass of DHFR inhibition accounts for therelative lack of efficacy of these drugs against Leishmania(Bello et al., 1994; Nare et al., 1997).

While some folate-metabolizing enzymes are essentialin Leishmania (DHFR-TS, folylpolyglutamyl-synthase)(Cruz and Beverley, 1990; El Fadili et al., 2002), otherstested appear largely or wholly dispensable for growthand virulence, including methylene tetrahydrofolatereductase and the glycine cleavage complex (Vickerset al., 2006; Scott et al., 2008). This may reflect the abilityof the parasite to salvage sufficient quantities of manyC1-THF-dependent metabolites such as amino acids andpurines (Scott et al., 1987; Carter et al., 2008), particularlyin the mammalian stages where the parasites residewithin phagolysosomes that are postulated to be rich infree amino acids (McConville et al., 2007). In contrast, wewere unable to rescue the requirement for DHCH1 withany of a wide array of metabolites known or postulated torescue DHCH deficiency. This could reflect many factorsincluding a deficiency in a non-salvageable metabolite, aconclusion consistent with an essential requirement formitochondrial fMet-tRNAMet.

For a parasite enzyme to be a good drug target it mustbe both essential for parasite survival, and dispensable,absent or substantially different in the host (Fairlamb,2002). In mammals both the cystosolic and mitochondrialforms of DHCH (MTHFD1 and MTHFD2 respectively) areessential for early development (Brody et al., 2002; DiPietro et al., 2002; Parle-McDermott et al., 2005).However, human fibroblasts lacking either enzyme areviable, but are purine or glycine auxotrophs (Patel et al.,2003; Christensen et al., 2005a), and 10-CHO-THFmetabolism is dispensable in the yeast S. cerevisiaewhen grown in rich media (West et al., 1996). This sug-gests that the adult tissues of the human host may be lessdependent on DHCH activity than Leishmania. In con-trast, the 10-CHO-THF pathway is simplified in manyparasitic protozoans, where many species synthesize asingle cytosolic DHCH and FTL (L. major, L. braziliensis),while others synthesize just a cytosolic DHCH (African

trypanosomes) (T.J. Vickers, S.M.F. Murta, M.A. Mandelland S.M. Beverley, submitted). Preliminary genomicanalysis suggests that like African trypanosomes, themalaria parasite P. falciparum also possesses a singleroute to 10-CHO-THF synthesis, via a bifunctional DHCH(Nzila et al., 2005). These data suggest that the relativesimplicity of the 10-CHO-THF pathway in parasitic proto-zoa relative to their human host may render them moresensitive to chemotherapeutic interventions.

While few DHCH inhibitors have been developed,L. major DHCH1 activity and promastigote growth in vitrowere sensitive to the substrate analogue 5,10-CO-THF(Temple et al., 1982). The specificity of this inhibitor wassupported by the findings that DHCH1 overexpressors areless susceptible, and it fails to inhibit DHFR or FTL enzy-matic activity. However, overexpression of FTL did notcompletely overcome 5,10-CO-THF inhibition suggestingit could have other targets beyond DHCH1.

Consistent with the genetic studies above, DHCH inhibi-tors have little activity or toxicity against mammalian cells invitro, or in mouse cancer models, at concentrations below10 mM (Temple et al., 1982; Tonkinson et al., 1998). Inpreliminary studies we found modest activity of 5,10-CO-THF against Leishmania amastigotes in macrophages(EC50 ~ 5 mM), although host cell toxicity was seen above10 mM (S. Hickerson and S. Beverley, unpubl. data). Theseresults are encouraging since 5,10-CO-THF is actuallymore selective for the host than parasite enzymes (Ki = 18versus 105 nM respectively; Schmidt et al., 2000; Fig. 6).While highly conserved, numerous differences betweenLeishmania DHCH1 and the human cytosolic and mito-chondrial DHCHs (Fig. S2) could be exploited to designmore selective and potent inhibitors. DHCH inhibitors havenot received a high priority in cancer chemotherapy pro-grammes due to the poor efficacy for the first ones tested,although a number of antifolates targeting 10-CHO-THFutilizing enzymes such as GAR transformylase have beendeveloped (Zhang et al., 2003). Possibly, similar pharma-cophores may hold promise in targeting 10-CHO-THFmetabolism in Leishmania and other trypanosomatids.

Experimental procedures

Reagents

(6-R,S)-tetrahydrofolate (THF) and 5,10-CH=THF wereobtained from Schircks Laboratories (Jona, Switzerland).5,10-CO-THF was custom synthesized by SAFC Pharma, adivision of Sigma-Aldrich Corporation, according to themethod of Temple et al. (1982); the compound was 96.2%pure by high-performance liquid chromatography. Methotrex-ate and compound 25 were purchased from Sigma, the folateanalogues compound 34 and compound 70 were asdescribed (Hardy et al., 1997). HYG was purchased fromCalbiochem (EMD Biosciences, San Diego, CA) and SATwas from Werner BioAgents (Jena, Germany). All other



chemicals and reagents were of analytical grade and pur-chased from Sigma-Aldrich.

Parasite growth and drug inhibition

All studies used derivatives of L. major clone Friedlin V1promastigotes, grown at 27°C in M199 medium (US Biologi-cals) supplemented with 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) pH 7.4, 50 mMadenosine, 1 mg ml-1 biotin, 5 mg ml-1 haemin, 2 mg ml-1 biop-terin and 10% (v/v) heat-inactivated fetal calf serum. Growthwas determined by seeding parasites at 5 ¥ 105 cells ml-1 atvarious concentrations of drug and counting cells using amodel Z1 Coulter counter when cultures reached late-logphase. The EC50 value is the concentration of drug required todecrease growth by 50%.

Cloning of L. major DHCH1

The DHCH1 coding sequence was amplified with recombi-nant Pfu DNA polymerase (Stratagene) using L. majorgenomic DNA template, prepared by the LiCl mini-prepmethod (Medina-Acosta and Cross, 1993), using primersSMB3528 and SMB3529, which added 5′ NdeI and 3′ BamHIsites respectively. The amplified product was cloned intopCR.blunt (Invitrogen), yielding pCR.blunt.DHCH1 (B6255)and the NdeI–BamHI fragment transferred into the corre-sponding sites of pET15b (Novagen), yielding pET15b-LmjDHCH1 (B6026).

Expression, purification and enzymatic assays ofrecombinant L. major DHCH1

Protein was expressed in E. coli from pET15b-LmjDHCH1 andpurified by metal-affinity chromatography, as described inSupporting information. During the purification and storage ofthis protein the inclusion of 10% glycerol in all buffers wasrequired to prevent loss of activity. In this manner, the enzymecould be flash-frozen with no loss in activity. DHCH activitieswere measured spectrophotometrically, using a temperature-controlled Beckman DU-640 spectrophotometer, essentiallyas described by Tan et al. (1977) following either the formationof 5,10-CH=THF from 5,10-CH2-THF (DH activity) or the con-version of 5,10-CH=THF to 10-CHO-THF (CH activity). 5,10-CH2-THF dehydrogenase assays were carried out in a 0.5 mlvolume at 27°C and contained 25 mM MOPS, pH 7.3, 250 mMTHF, 2.5 mM formaldehyde, 1 mM NADP+ and 30 mM2-mercaptoethanol. Assays were pre-incubated for 5 min toallow formation of 5,10-CH2-THF and reactions initiated by theaddition of enzyme. The reactions were then stopped after5 min by the addition of an equal volume of 1 M HCl and the5,10-CH=THF produced quantified at 350 nm, using a extinc-tion coefficient of 24.9 mM-1 cm-1. DH activity was expressedas mmoles 5,10-CH=THF produced per minute. Formaldehydestocks were freshly prepared from paraformaldehyde, andTHF dissolved just before use in 250 mM triethanolamine-Cl,pH 7, 40 mM 2-mercaptoethanol. Cyclohydrolase activity wasmeasured in 0.5 ml of assays at 27°C containing 25 mMMOPS, pH 7.3, 30 mM 2-mercaptoethanol and 50 mM 5,10-CH=THF. The hydrolysis of 5,10-CH=THF was followed at355 nm, and the non-enzymatic rate subtracted.

Enzyme inhibition was measured using the dehydrogenaseassay, with stocks of hydrophobic diaminoquinazolines anddiaminopteridines dissolved in DMSO, and 5,10-CO-THFdissolved in 250 mM triethanolamine-Cl, pH 7, 40 mM2-mercaptoethanol, as with THF, and stored at -80°C. 5,10-CO-THF was custom synthesized by Sigma-Aldrich ChemicalPvt. (Bangalore, India); the authenticity of the product wassupported by infrared spectroscopy and proton NMR and itwas estimated to be greater than 96% pure. Data from 5,10-CO-THF inhibition assays were fitted to competitive, non-competitive, uncompetitive and mixed inhibition models bynon-linear regression using Sigmaplot. The DHFR activity ofthe bifunctional L. major DHFR-TS was measured asdescribed (Hardy et al., 1997). Activity in all other drug assayswas expressed as the percentage of a no-drug DMSO control.

DHCH1 replacement constructs

Fusion PCR (Szewczyk et al., 2006) was used to generatethe two deletion constructs, which contained either PAC orHYG drug-resistance cassettes between the 5′ and 3′LmjDHCH1 flanking sequences (Szewczyk et al., 2006).Briefly, the flanking sequences and drug-resistance cassetteswere amplified separately by PCR using primers that produceoverlapping ends. The 5′ and 3′ LmjDHCH1 flankingsequences (743 and 726 bp) were amplified using theprimers SMB2772/2656 and 2657/2773 that added a SphIand BamHI site respectively. PAC and HYG drug-resistancecassettes (600 and 1026 bp) were amplified using theprimers SMB2557/2558 and SMB2561/2562, respectively,that added a linker sequence. Each deletion construct wasthen made by mixing the purified PCR products for the flank-ing sequences and a drug-resistance cassette and usingthese products as templates in a second round of PCR. Thetwo PCR reactions to generate the PAC and HYG constructsboth used the external primers SMB2772 and 2773, andfused the flanking sequences to the correct drug-resistancecassette. These two deletion constructs were then cloned intopGEM-T (Promega), yielding pGEM-T.5′.PAC.3′.LmjDHCH1(B4827) and pGEM-T.5′.HYG.3′.LmjDHCH1 (B4827). Thesequences of these constructs were confirmed by restrictionmapping and sequencing.

DHCH1 replacement and transfections

DHCH1 targeting fragments were digested with SphI andBamHI, and the linear targeting fragments containing PAC(2.1 kb) or HYG (2.5 kb) were transfected separately into WTL. major FV1 promastigotes as described (Robinson and Bev-erley, 2003). Heterozygous clones, DHCH1/Ddhch1::PAC orDHCH1/Ddhch1::HYG (referred as PAC/+ or HYG/+), wereisolated by plating on semi-solid M199 medium containing30 mg ml-1 puromycin or 50 mg ml-1 hygromycin B respectively.Heterozygous PAC/+ or HYG/+ clones were transfected withdeletion constructs containing HYG or PAC cassettes. Theparasites were plated in semi-solid M199 medium containing30 mg ml-1 puromycin and 50 mg ml-1 hygromycin. In attemptsto rescue the growth of DHCH1-/- homozygotes several differ-ent mixtures of different supplements were added to theplating medium. The basic supplements were: glycine



(1 mg ml-1), serine (0.1 mg ml-1), formate (5 mM), THF (5 mM),thymidine (10 mg ml-1) and folinic acid (10 mg ml-1). Additionalsupplements were added to the basic mixture in other plates;these were: 5,10-CH=THF (10 mM), 10-formylfolic acid10-CHOFA (10 mM) or, alternatively, uridine (0.1 mg ml-1) andsodium pyruvate (1 mM). DNA contents of permeabilizedRNase-treated cells were measured by propidium iodidestaining using flow cytometry (Cruz et al., 1993).

PCR primers for assessing chromosomal DHCH1 orPAC or HYG replacements

Generation of the planned PAC or HYG replacements wasconfirmed by PCR, using a primer located 5′ (SMB2731) or 3′(SMB2771) of the DHCH1 ORF targeting fragment and aprimer located within the PAC or HYG genes (SMB2557/2558or SMB2561/2562). The loss of the DHCH1 ORFs was con-firmed using primers specific for the chromosomal DHCH1allele (SMB3125/3126) or using a forward primer located inthe chromosome 5′ (SBM2731) of the DHCH1 ORF targetingfragment and a reverse primer located within the DHCH1gene (SMB3126).

PXNG4 expression constructs

DHCH1 and FTL ORFs were amplified with primers(SMB3125/3126 and SMB3123/3124 respectively) fromL. major genomic DNA, adding flanking BglII sites, anda CCACC sequence preceding the initiation codon.The DHCH1 and FTL ORFs were inserted into pGEM-T(Promega) yielding pGEM-DHCH1 (B6029) and pGEM-FTL(B6030); the DHCH1 and FTL ORFs were then extracted byBglII digestion and inserted into the BglII site of pXNG4(B5840) to create pXNG4-DHCH1 (B6031) and pXNG4-FTL(B6032).

These plasmids were transfected into either WT L. majorFV1, or heterozygote lines (DHCH1/Ddhch1::PAC orDHCH1/D dhch1::HYG) and transformants isolated as before,using plating media containing 100 mg ml-1 nourseothricin andeither 30 mg ml-1 puromycin or 50 mg ml-1 hygromycin. Thepresence of plasmid was confirmed by PCR and FACS analy-sis for the presence of SAT and GFP markers respectively.Three colonies overexpressing FTLor DHCH1 were submittedto second replacement using either the PAC or HYG targetingfragments. The parasites were isolated by plating on semi-solid M199 media containing 100 mg ml-1 nourseothricin,30 mg ml-1 puromycin and 50 mg ml-1 hygromycin.

Single-cell sorting

Prior to flow cytometry, dhch1- /pXNG4-DHCH1 lines wereincubated with 50 mg ml-1 ganciclovir in M199 media for 24 h.Single-cell sorting was then performed based on theirGFP fluorescence as described in Results. Cells werere-suspended in phosphate-buffered saline, and filteredthrough CellTrics 50 mm filters to remove clumps (Partec).Single cells were then recovered using a Dako MoFlo high-speed cell sorter and placed into individual wells of 96-wellplates, each containing 150 ml of M199 medium. Plates wereincubated at 27°C for 1–2 weeks and parasite growth wasscored.

Acknowledgements

We thank A. Hanson and M. Ouellette for discussions. Wethank Sushil Rajan and Sigma-Aldrich Chemical Pvt. forcustom synthesis of 5,10-CO-THF. Single-cell sorting wasperformed with the assistance of the staff of the high-speedsorter core, Alvin J. Siteman Cancer Center, WashingtonUniversity Medical School. The Siteman Cancer Center issupported in part by NCI Cancer Center Support Grant #P30CA91842. This work was supported by National Institutes ofHealth Grant AI21903 (to S.M.B); Brazilian agencies: CNPqand FAPEMIG (to S.M.F.M.); and European MolecularBiology Organization long-term fellowship ALTF 106-2005(to T.J.V.).

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Supplemental information for “Methylene tetrahydrofolate dehydrogenase/cyclohydrolase

and the synthesis of 10-CHO-THF are essential in Leishmania major”, by Silvane M. F.

Murta, Tim J. Vickers, David A. Scott, and Stephen M. Beverley.

Supplemental methods

Expression and purification of recombinant LmjDHCH1

The pET15b-LmjDHCH1 vector was transformed into E. coli strain Rosetta2(DE3)/pLysS. Cells

were grown at 37°C in 800 ml LB media containing 100 μg ml-1 carbenicillin and 12.5 μg ml-1

chloramphenicol to an OD of 0.6. Expression was induced for 4 h with 1 mM isopropyl-β-D-

galactopyranoside and the cells then harvested and frozen.

Frozen cells were thawed on ice and resuspended in 30 ml of lysis buffer (75 mM Na-

phosphate, pH 7.5, 500 mM NaCl, 1 mM benzamidine, 3 μg ml-1 leupeptin, 250 μM 4-(2-amino-

ethyl)benzenesulphonyl fluoride, 10 U ml-1 DNAase I (Sigma), 10,000 U ml-1 lysozyme (Sigma),

1 mM 2-mercaptoethanol, 10% (v/v) glycerol). The cells were then lysed by sonication, (4 x 30 s

bursts), with cooling to <4°C between pulses. An identical extraction procedure was used to

produce extracts of L. major for enzyme assay. After centrifugation (30,000 x g, 1 h, 4°C ),

clarified extract was applied at 2 ml min-1 at 4°C to a nickel-charged 20 ml (1.2 x 11 cm) Ni-

NTA chelating agarose column (Qiagen) equilibrated with binding buffer (50 mM Na-phosphate,

pH 7.5, 200 mM NaCl, 10% (v/v) glycerol). Unbound protein was removed by washing with 50

ml of binding buffer. Retained proteins were eluted with a linear gradient at 1 ml min-1 of 0-

100% of elution buffer containing (50 mM Na-phosphate, pH 7.5, 200 mM NaCl, 10% (v/v)

glycerol, 500 mM imidazole). Fractions containing DHCH1 were pooled.

2

Western blot analysis

Whole cell lysates from L. major lines were prepared from stationary phase parasites in SDS

sample buffer; 5 X 106 cell equivalents/lane. The samples were separated by electrophoresis on a

10% SDS polyacrylamide gel and electrotransferred onto nitrocellulose membranes (Hybond-

ECL; Amersham Biosciences). Antiserum raised against LmjDHCH1 and LmjFTL were used at a

1:3,000 and 1:1,000 dilution, respectively. Secondary antibodies conjugated to peroxidase were

from Amersham Biosciences, and ECL reactions were conducted using PerkinElmer Life

Sciences chemiluminescence kit. In western blot analysis both the anti-FTL and DHCH1

antisera identify a major protein band of the expected molecular weights. The assignment of

these bands to FTL and DHCH1 was confirmed by western blots against null or overexpressor

mutants.

3

Table S1. Oligonucleotide primers used in this work.

Primer number Sequence

SMB 3528 CATATGCCGTCTGCTCAGATCATTGA

SMB 3529 GGATCCCTATGATACGCCCAACGCAGCCT

SMB 2557 ggtaacggtgcgggctgacgCCACCATGACCGAGTACAAGCCC

SMB 2558 cgagatcccacgtaaggtgcTCAGGCACCGGGCTTGCG

SMB 2561 ggtaacggtgcgggctgacgCCACCATGAAAAGCCTGAACTC

SMB 2562 cgagatcccacgtaaggtgcCTATTCCTTTGCCCTCG

SMB 2656 cgtcagcccgcaccgttaccATGATCTGAGCAGACGGCATC

SMB 2657 gcaccttacgtgggatctcgGGCACGTGCGCTCCACTTC

SMB 2731 CGTGTGACACTTTCAAGGTGCGA

SMB 2771 ATTCGACGTTGCTTGCGGT

SMB 2772 GCATGCACGCAAACGGATTGGATGTGT

SMB 2773 GGATCCCACGTGTCTTTGGAACAGAGTG

SMB 3123 AGATCTCCACCATGGCCACCCGGAAGCTGCA

SMB 3124 AGATCTTTACGAAAGACCCACAATCC

SMB 3125 AGATCTCCACCATGCCGTCTGCTCAGATCAT

SMB 3126 AGATCTCTATGATACGCCCAACGCAG

Restriction sites are underlined small type is linker sequence.

4

Figure S1. Purified LmDHCH1.

A 5 μg sample of purified recombinant DHCH1 was separated on a 12% SDS-PAGE gel and

stained with Coomassie Blue.

Figure S2. Alignment of human cytoplasmic and mitochondrial DHCH domains with

Leishmania major DHCH1.

The sequences are L. major DHCH1 and the DHCH domains of the human cytoplasmic C1-

synthase (Swiss-Prot P11586) and mitochondrial DHCH (P13995). Residues identical in all

three proteins are shown boxed and shaded. Structural features have been depicted based upon

the structure of the human C1-synthase DHCH domain (Schmidt et al., 2000). The active site

lysine (K56, ▼) and proximal residues (*) as well as Arg173 implicated in NADP+ binding are

marked (*). Residues implicating in NADP+ binding are shown boxed with a blue outline,

while residues implicated in DHCH inhibitor binding are shown boxed with a gray outline.

Figure S3. PCR confirming heterozygous DHCH1 replacements.

PCR tests of planned HYG (A) or PAC (B) replacements at DHCH1. Homologous replacement

was visualized by PCR amplification using a primer located on the DHCH1 chromosome outside

of the targeting fragment on the 5’ site (SMB2731) and a primer directed against sequences

located with the HYG (SMB2562) or PAC ORF (SMB2558) within the targeting fragment. The

sizes of the predicted PCR products are shown (C). #1-4, four independent heterozygous lines

with HYG replacement +/ HYG and #1-12, twelve independent heterozygous lines with PAC

replacement +/ PAC.

5

Figure S4. Overexpression of FTL in pXNG4-FTL transfectants

Western blot analysis of FTL protein detected with polyclonal rabbit antisera against LmjFTL

(1:1,000) (Vickers et al., 2008) in extracts of the L. major WT, WT+pXNG4-FTL and

heterozygous +/PAC or +/HYG lines transfected with pXNG4-FTL. Protein loading was

assessed with a rabbit anti-L. major histone H2A polyclonal antibody (Iris

Wong and Stephen M. Beverley, manuscript in preparation) at 1: 100,000

dilution.

Fig. S1 Purification of recombinant L. major DHCH1 proteing j p

Fig. S2. Alignment of human DHCH domains and Leishmania major DHCH1

showing homology (shading) and features based on human cDHCH domain structure (Schmidt et al 2000)

---------------------------------MPSAQIIDGKAIAAAIRSELKDKVAALRELYGGRVPG 37LmjDHCH1---------------------------------MAPAEILNGKEISAQIRARLKNQVTQLKEQVPGFTPR 37cMTHFD1MAATSLMSALAARLLQPAHSCSLRLRPFHLAAVRNEAVVISGRKLAQQIKQEVRQEVEEWVAS-GNKRPH 69mMTHFD2

0j▼** * ** *LASIIVGQRMDSKKYVQLKHKAAAEVGMASFNVELPEDISQEVLEVNVEKLNNDPNCHGIIVQLPLP--K 105LmjDHCH1

LAILQVGNRDDSNLYINVKLKAAEEIGIKATHIKLPRTTTESEVMKYITSLNEDSTVHGFLVQLPLDSEN 107cMTHFD1LSVILVGENPASHSYVLNKTRAAAVVGINSETIMKPASISEEELLNLINKLNNDDNVDGLLVQLPLP--E 137mMTHFD2

HLNENRAIEKIHPHKDADALLPVNVGLLHYKGREPPFTPCTAKGVIVLLKRCGIEMAGKRAVVLGRSNIV 175LmjDHCH1+ Arg 173*

HLNENRAIEKIHPHKDADALLPVNVGLLHYKGREPPFTPCTAKGVIVLLKRCGIEMAGKRAVVLGRSNIV 175LmjDHCH1SINTEEVINAIAPEKDVDGLTSINAGRLARGDLNDCFIPCTPKGCLELIKETGVPIAGRHAVVVGRSKIV 177cMTHFD1HIDERRICNAVSPDKDVDGFHVINVGRMCLDQYS--MLPATPWGVWEIIKRTGIPTLGKNVVVAGRSKNV 205mMTHFD2

GAPVAALLMKE--------NATVTIVHSGTSTEDMIDYLRTADIVIAAMGQPGYVKGEWIKEGAAVVDVG 237LmjDHCH1

TTPVPDPSRKDGYRLVGDVCFEEAAARAAWISPVPGGVGPMTIAMLLENTLEAFKAALGVS 298LmjDHCH1

GAPMHDLLLWN--------NATVTTCHSK--TAHLDEEVNKGDILVVATGQPEMVKGEWIKPGAIVIDCG 237cMTHFD1GMPIAMLLHTDGAHERPGGDATVTISHRYTPKEQLKKHTILADIVISAAGIPNLITADMIKEGAAVIDVG 275mMTHFD2

INYVPDDKKPNGRKVVGDVAYDEAKERASFITPVPGGVGPMTVAMLMQSTVESAKRFLEKFKP 300cMTHFD1INRVHDPVTAKP-KLVGDVDFEGVRQKAGYITPVPGGVGPMTVAMLMKNTIIAAKKVLRLEER 337mMTHFD2

* active site Inhibitor bindingNADP+▼ Lys 56 (catalytic site) * active site Inhibitor bindingNADP+▼ Lys 56 (catalytic site)

Fig. S3 PCR confirming DHCH1 heterozygotes

PAC5′ 3′B

HYG

1953 bp

5′ 3′A + / Hyg

+ / PAC1504 bp

+ /HYGbp 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12

+ / PACCbp

+ / PAC

1504

1000 -1650 -2000 -

1953

Fig. S4 Overexpression of FTL in pXNG4-FTL transfectants

TL TL

pxN

G-F

TL

C/+

pxN

G4-

FT

G/+

pxN

G4-

FT

FTL

112

64

KDa

WT

WT+

PAC

HY

G

FTL

Histone H2A

molecular microbiology first published online 9 february ...beverleylab.wustl.edu/pdfs/184. murta et...

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