Proc. Natl. Acad. Sci. USA Vol. 91, pp. 7149-7153, July 1994 Medical Sciences Primary structure and expression of the dihydropteroate synthetase of Plasmodium falciparum (nadaria/slfadoxie/drug ressance/folate) TONY TRIGLIA AND ALAN F. COWMAN The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia Communicated by J. F. A. P. Miller, March 14, 1994 (receivedfor review November 8, 1993)

ABSTRACT The dihydropteroate synthetase lates has identified sequence differences, and the role ofthese (DHPS) from Plasmodiwnfakipqrum is involved in the mech- differences in sulfonamide/sulfone resistance is discussed. anism ofaction ofthe sulfone/sulfonamide group ofdrugs. We describe the cloning and sequencing ofthe gene encoding theP. MATERIALS AND METHODS falcqparum DHPS enzyme and show that it is a bifunctional Parasites and DNA. The origin ofP. falciparum clones and enzyme that includes dihydro-6-hydroXymethylpterin pyro- strains are as described (14, 15). Parasites were maintained in phosphokinase (PPPK) at the N terminus of DHPS. The gene asynchronous cultures (16). encodes a putative protein of83 kDa that contains two domains DNA Cloning. Two degenerate oligonucleotides, DHPS1 that are homologous with the DHPS and PPPK of (5'-CCWGATWCTTTTWCTGAW) and DHPS2 (5'-WC- other organisms. The PPPK-DHPS gene is encoded on chro- CWGGTCTWGTTGWTTCWCCWCCWATATC; W = A or mosome 8 and has two introns. An antibody raised to the PPPK T) were used in a PCR with NF7 genomic DNA to obtain a region of the protein was found to recognize a 68-kDa protein 130-bp fiagment. Genomic DNA was digested with EcoRI that is expressed throughout the asexual life cycle of the and Mun I, and a library was made in AgtlO. The library was parasite. We have determined the sequence of the DHPS screened with the cloned 130-bp fragment, a 3.7-kb fragment portion of the gene from sulfadoxine-sensitive and -it P. was obtained, and the regions were sequenced. A 4.7-kb fakcparum clones and identified sequence differences that may fragment was obtained from an EcoRI library, and portions have a role in sulfone/sulfonamde resistance. were sequenced to produce the full gene sequence. In ex- periments designed to determine the presence ofan intron in The sulfones and sulfonamides are used extensively as pro- genomic DNA, mRNA was converted to single-stranded phylactic agents against human malaria caused by Plasmo- cDNA ready for PCR amplification. diumfalciparum. Sulfadoxine is the most commonly used of Pulsed-Field Gradient Gel Eectrophoresis. this group of compounds, and it is known to inhibit 7,8- were fractionated in a contour-clamped homogeneous elec- dihydropteroate synthetase (DHPS) (1), which catalyzes the tric field apparatus (17) for 48 hr, with a 150-sec pulse at 160 following reaction in the folate biosynthetic pathway: p-ami- V in a 1.0%6 agarose gel. nobenzoic acid + 6-hydroxymethyldihydropterin pyrophos- Peptide and Antibody Methods. A 15-residue peptide, cor- phate-* dihydropteroic acid. Resistance to sulfadoxine alone responding to aa 353-366 (Fig. 1) with a terminal cysteine and to the combination of sulfadoxine and pyrimethamine residue was made and conjugated to diphtheria toxoid (Chi- have become widespread, which has reduced the efficacy of ron Mimotopes, Melbourne, Australia). The nonconjugated these drugs (2). peptide was covalently attached to a 6%6 beaded agarose Resistance to sulfadoxine in P. falciparum could occur by support via its terminal cysteine residue (Pierce). a number of mechanisms, and there is evidence both for an Expresion Clone Constrution. A clone expressing aa. altered DHPS and for the presence of alternative pathways 301-668 was obtained by PCR and cloned into pGEX2 (18) to (3). It has been shown that conversion of sulfadoxine to the produce a fusion protein with glutathione-S-transferase toxic analogue ofdihydropteroate was reduced in resistant P. (GST). This clone is called E. coli-PfDHPS. falciparum, suggesting that mutations in the DHPS enzyme Affinity-purified antibodies to the DHPS peptide were used may alter affinities of substrate binding. It has also been at a dilution of 1:800, whereas antibodies against the P. shown that increases in both synthesis of p-aminobenzoic falciparum homologue of hsp70 (Pfhsp7o) (19) were used at acid and salvage of folates (4) may also be involved in the a 1:4000 dilution. Bound antibody was detected with horse- mechanism of resistance to these compounds. radish peroxidase-coupled goat anti-rabbit IgG and devel- It has been known for many years that Plasmodia have both oped by ECL, a luminescence-based method (Amersham). DHPS and 7,8-dihydro-6-hydroxymethylpterin pyrophos- phokinase (PPPK) activity (5, 6) and that these two enzyme RESULTS activities seem to copurify, suggesting that they exist as a Coning and Sequence of the PPPK-DHPS Gene. As a first bifunctional enzyme. PPPK catalyzes the reaction in the folic step toward understanding the molecular basis of sulfona- acid synthetic pathway before DHPS. Recently the se- mide/sulfone resistance we chose to isolate the gene for quences of several PPPK (7-10) and DHPS (11-13) DHPS, which is known to be involved in the mechanism of have been determined from various bacterial sources and action ofthis group ofdrugs. Oligonucleotides corresponding Pneumocystis carinji. In this paper we describe the cloning of to two conserved regions ofthe DHPS-encoding gene from a the gene and characterization ofthe protein encoding DHPS from P. falciparum.* Sequence analysis of the DHPS- Abbreviations: DHFR, dihydrofolate reductase; DHPS, 7.8- encoding gene from sulfadoxine-resistant and -sensitive iso- dihydropteroate synthetase; GST, glutathione-S-transferase; PfDHPS, Plasmodium falciparum DHPS; PPPK, 7,8-dihydro-6- hydroxymethylptenn pyrophosphokinase; 1fhsp70, Plasmodium The publication costs ofthis article were defrayed in part by page charge falciparum hsp70; PfPPPK, Plasmodium falciparum PPPK. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U07706). 7149 Downloaded by guest on September 25, 2021 7150 Medical Sciences: Triglia and Cowman Proc. Natl. Acad. Sci. USA 91 (1994)

TTA 1 aatatttctoaatataactattaaatgttttastcagraattoaaaatatatatattatqtatgqttattagaqagccaqa 1874 ATA AAT ATT AM GAA AAT AAT AAA AGG ATA TATGTA TTA AAA CAT AGA ATT TCT TAT 81 aaataatttctgtcatatattttaatttttaagttctatantcgataccaaqtattaaatgaacacctatatatgtatat 366 I N I K E N N K R I Y V LK D R I S Y L 161 atatatatatatgtatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatgtatqt 1934 AAA CAA AM ACA AAT ATT ITT CIA ATA TTA AAT ITT AAT TAT CAT TCT TTT TCA GAT CGA 241 ataatatatatatttacaacttaaactataaaagtgaataaaaaaaaaaaaaaaaaaaotattaaaaaaaaotgaaacattt V Y S S D C 321 acaqtaataatantaqtagaaaataaaacaaactatttagtaaaaotaaatoaaagtagcagttaaaaaaaaottctaaaaaa 386 K E K T N I C I LN V N D F 401 atatttgcgccaaacttttaaattaqtatctatatctaactaaaa8aaataaaaatatatatatatatatataacatat ATA AAT GM 481 1994 GCT ATT TTT ITT GAA CCT MA CIT GCT ITT CM AGA ATG TTT GM ATG GCT atataataaaagtatttaattttantaataatoaaattttqcgtaaaganaetctatatatattttacacatatatttatt 406 G I F V E P K R A V Q R M F E M I N E I 561 tatttatgqccatatatatatnat ATC CAA ACT ATA CAA GAA CTA ATA CTT TCT CAG CAA HAT AAA 1 M E T I Q E L I L S E E NK 2054GCT ACT ITT ATA CAT ATA CIT CGA CAA TCC TCT GCT CCT TTT ITT ATA CCT MT CCA AAA 426 A S V I D I G G E S S A P F V I P N P K 627 ACT AAT ATT CCC GTA TTA AAC TTA CCA ACA AAT CAT AGA AGA AAC GCT GTG TTC ATT CTA W2_1 F I 15 T N I A V L NL C T N D R R N A V L I L Kl,3D7,7GS G TTA C 2114 ATT ACT GM AGA CAT TTAGTA GTA CCTGTA TTA CAA TTA TTT CAA AAA GAA TGG MT CAT 687 CAA ACT GCT CTC CAC CTT CTC CAA AAA TAT qtagaaaaaangtataataaataaatoaataaat V Q Q 35 E T A L H L V E K Y L G 446 I S E R D L V V P L L F K E W N D AAC 755 acacaaacaaag-aatatatatatatatatatatatatatatatatatatatatatatatatatqtgtatatacatact.t 2174 ATA AAA MT MA ATT ITT AAA TGT CAT GCG AMA CCA ATT ATA ACT ATT GAT ACA ATT 835 ttatatacatacctttatatacatacttttatatacatatcactctttgattctatatatttttatttttcactttgtag 466 I K N K I V K C D A K P I I S I D T I N 915 GA AAA ATT ATT AAT ACG TCC TAC TTG TAT GM ACC GTT CCA GAA TAC ATT ITA TTA CAT 2234 TAT AAT ITT TTT AAA GAA TGT GTT CAT AAT GAT TTA ITT CAT ATA TTA AAT CAT ATT ACT 47 K I I NT S Y L Y E T V P E Y I V L D 486 Y N V F K E C V D N D L V D I LN D I S 974 AAA AAC GAA ACT TIC CAA AAA ATA AAC MG CAT TGT CGT ATA TAT GAT ITT AAT TAT ATA 2294 GCT TGT ACA MT AAT CCA CAA ATT ATA AAA TTA TTA AAA AAA A AAC AM TTC TAT ACT 66 K K E S C E K I N K D C R I Y D V N Y I 506 A C T N N P E I I KLLK K K N K F Y S CM AAT TTA CAA CAA TCT AAA TAT GAA GAG AAT MA GM TTA ATT CAT 2354 GTA ITT CTA ATG CAT MA AGA CIA AAT CCA CAT ACA ATG CAT AAA CTA ACA AAT TAT CAT 1034 AAC GAA TTC ATG D 86 N E L M 0 N L E E S K Y E E N K E L I D 526 V V L M H K R C N P H T M D K L T N Y 1094 AAA TGT CAA CAA TAT CM ACA TTT TTC MA MT GCA AAA ITT GAT AAT ACT ATT CTA AAG 2414 AAT CTA ITT TAT GAT ATA MM AAT TAT TTA CM CM AGA TTA AAT TTT CTT GTA TTA MT 106 K C E E YE T F L KN G K V DN S I L K 546 N L V Y D I K N Y L E Q R L N F L V L N 1154 GAA CTA AAT GTA CAA AAT TAT TTA TTA CAA TGT MT AAT ATA ATA GTA AAG AAT GAC GAA 2474 GGA ATA CCT CGT TAT AGG ATA CTA TTT CAT ATT CIA TTA GGA TTT GCG AAM AAA CAT CAT 126 E V N V E N Y L L E CN N I I V K N D E 566 1 I P R Y R I L F D I I L G F A K K H D Kl C 1214 ATA ATG AAA AAT AAT TTA AGC AAA TAT AAA CAT AM TAT TAT ACT AGC TAC TTT TAT AAT 146 I M K N N L S K Y K D K Y Y T S Y F Y N 2534 CAA TCT ATT AAA CTC TTA CM AAT ATA CAT GTA TAT GAT GAG TAT CCA CTT TTT ATT CGA 586 0 S I K L L Q N I H V Y D E Y P L F I G 1274 TTC ACA GTT GTA ITT AAA ACT TTT GTA AAT CAT CCT CTT ACT ATG TTG GTA GTT ATA AAA 166 L T V V V K T F V N D P L S M L V V I K 2594 TAT TCA AGA MA AGA TTT ATT CCC CAT TIC AT AAMT CAT CM MT ITT CTA ATA AMT ACA 606 Y S R K R F I A H C M N 0 Q N VV I N T 1334 TAT ATT GAA CM TTA ATG AAA AGG CM MT GTA AAA GAG MA GAA AAA TTT CM AAT CGT W2md S 186 Y I E E L H K R E N V K E K E K F E N R 2654 CM CAA AM TTA CAT CAT CM CM CM MT CAA MT MM AAT ATT GTG GAC AAA TCA CAC 1394 ATA ATA CAT ATA CAT ATT CTA TTT TTT MT CAT TTT ACA ATC TTT ATG AAA AAC ATA AAA 626 0 Q K L H D E Q Q N E N K N I V D K S H 206 I I D I D I L F F N D F T I F M K N I K 2714 AAT TG1 ATG TTT CAG ATG AAT TAC ATG AGG MA GAC MC GAT CAA CTT TTA TAT CM AAA Q 1454 TTG CAA AAA AAT ATG ATT TAT MA ATA CTC TCA MA TAT ATT CAT TTG GM AGA GAT ATA 6461N 1 M F Q M N Y M R K D K D L LY 0 K 226 L E K N M I Y K I L S K Y I H L E R D I 2774 MT ATA TGT I gtqtgtttaaaaa-aaaaaaaattcaaatqaqtatacaaaaqtaacaattstatatatgttacat 1514 AAA AAT CIA MT GAC AAT ATG TCT MMA TA MT ATG CAT MA GAT ATA MT CTT AAT MT 666 N I C I 246 K N G N D N M S K V N M D K D I N L N N 2850 ataaaatataaataatatatattcatgtatatqtatttatgtatttctttcag IT GGA TTA GCA ATT GCT 1574 MC AAT MT ATA MA AAA AAA MT AAT MT GAT ATT CAT TGT GAT TGT GTG CAT CAG MC 670 1 L A I A 266 N N N I K K K N NN D I D C D C V D Q K 2921 TCC TAC ACC TAT TAT MA MI GTA GAT CTA ATA AGA ITT CAT CAC GTT TTA CAA ACA AM 1634 ATG AAT AAT CAT GTI AAT AAT AAA AAT TAT ATA MT TCT TTT AGA GAT CCA CAA CM ATA 675 S Y S Y Y K K V D L I R V H D V L E T K 286 M N N H V N N K N Y I N S F R D P Q E I 2981 TCC ITT TTG GAT ITT TTA ACA MA ATA CAC CAA GTG TM tttacaaaa. aagt-gcaacatqtqa 1694 ATA AAC AAT ATC GTA CAT AAT ATT CM TTT TTA TCC ATT CCT CAT GTG TAT ACA ACA CAC 695 S V L D V L T K I D Q V 306 I N N M V D N I E F L S I P R V Y T T H 304 8 t taaacat at atata tatatatat atatat 1754 AGA TAT AGC ATA CTT TTA TGC TTA AAT GAT ATG ATA CCC GAA TAT AAC CAT AAT GTT TTA 326 R Y S I L L C LN D M I P E Y K H N V L 1814 AAT AAT ACC ATC AGA TGT TTA TAT MC AAA TAT GTI ACT All ATG AAA CAA CAA TAT L7T 346 N N T I R C L Y N K Y V S R M K E Q Y N FIG. 1. DNA sequence and deduced protein sequence of the PPPK-DHPS gene of P. falciparum. The complete nucleotide sequences of NF7 and FC27 isolates, both of which are identical, are shown together with the sequences of the DHPS-encoding gene (from aa 358) for four other isolates and clones-3D7, K1, 7G8, and W2mef. Only amino acid differences are shown. The arrows indicate the boundaries of the oligonucleotides used in the PCR to amplify the DHPS gene for sequencing. The sequences to which degenerate oligonucleotides were made for PCR amplification of PfDHPS are overlined. number of organisms (7-9, 12, 13) were used in a PCR to finned that Met-585 was the start of the coding region of this amplify the homologous gene from genomic DNA ofthe NF7 gene. A similar PCR experiment was done with oligonucle- isolate of P. falciparum. A PCR product of 130 bp was otides-spanning the putative stop codon at position 3017 to sequenced and found to show significant homology to other confirm the 3' end of the gene (data not shown). DHPS genes. This was used as a probe to screen NF7 Struure of PPPK-DHPS Protein. Comparison of the genomic DNA libraries and resulted in clones spanning the C-terminal region of the reading frame identified shows that entire DHPS-encoding gene. the region from aa 379-706 encodes the DHPS enzyme from A region of3077 bp was fully sequenced that had three open P.falciparum, as it has strong homology ofbetween 561% and reading frames from 585-720, 915-2783, and 2904-3016 bp 64% with DHPS enzymes from other organisms (7-9,12, 13). (Fig. 1). The region from nt 721-914 and 2784-2903 contained The DHPS domain (aa 379-706) has an overall length of 328 stop codons in all three reading frames (Fig. 1) and was more aa, which resembles all other DHPS enzymes (Figs. 2 and 3) A+T-rich than the surrounding DNA, suggesting that introns and 25 amino acids that are absolutely conserved throughout may interrupt the coding sequence. Oligonucleotides sp evolution (Fig. 2), suggesting a role for these residues in these regions were used in PCR reactions with cDNA synthe- substrate binding and catalysis. sized from poly(A)+ RNA purified from the NF7 isolate ofP. Comparison of the region N-terminal of the PfDHPS falciparum (data not shown). The resulting fiagments were protein with other organisms shows homology with the PPPK sequenced and shown to have nt 721-914 and 2784-2903 enzyme, suggesting that the amino-terminal 378 aa encode removed, confirming the presence of a 194-bp and a 120-bp the PPPK enzyme to produce a bifunctional protein that intron, respectively. These introns contain conserved intron would contain the activity for two steps in the folate biosyn- splice acceptor and donor sites typical ofother malarial genes thetic pathway. The P. falciparum PPPK (PfPPPK) domain (20). Translation of the processed NF7 sequence beginning (aa 1-378) shows homology of between 45% and 57% with with the presumptive initiatng methionine at nt 585 gave an other PPPK enzymes. The PfPPPK domain has two inser- open reading frame of2118 bp encoding 706 aa with a deduced tions of 92 aa (residues 35-126 and 220-311) with respect to molecular mass of 83 kDa (Fig. 1). all other PPPK enzymes. These regions are not introns, as The flanking DNA sequences both upstream and down- oligonucleotides flanking the insertions amplify cDNA that stream of the proposed open reading frame are highly A+T- for the second insertion is identical in sequence to genomic rich (Fig. 1) and have many stop codons consistent with these DNA (data not shown). The PfPPPK and PfDHPS enzyme being noncoding regions. To confirm this, oligonucleotides domains are separated by a hinge region that shows no were used in PCR reactions with cDNA from the NF7 isolate homology to the same region of the only other joined PPPK- that spanned the region upstream ofthe putative methionine DHPS enzyme, that of P. carinji (Fig. 2). initiation codon and the downstream intron. This procedure PPPK-DHPS Is a Single-Copy Gene on 8. To showed unequivocally that the region upstream of the me- determine if the PPPK-DHPS gene is a single copy in the P. thionine was present within the spliced transcript and con- falciparum genome, NF7 genomic DNA was digested with Downloaded by guest on September 25, 2021 Medical Sciences: Triglia and Cowman Proc. Natl. Acad. Sci. USA 91 (1994) 7151

EC MT YIT;UIGTSLASPLEQVN...... Bs MNNIAYIALGSNI GDRETYLR...... Sp KPWAPVHLSLDTCSVTIHRRKQ ARIaALGSNMGIDKQANLK...... PC VEIVRSRSCFSSNNYIKS ENS:IDN E T SLSN L GNRIKFIL ...... P f MMETIOQELILSE.ENKTN4AjVLNLJTDDRRNAVLILETALHLVEKYLGKIINTSYLYETVPEYIVLDKKESCEKINKDCRIYDVNYINE 87

E c ...... A ALK aL GD IPES Hi SV S RPODO ... PDYLNAA GLE TSLA.PEE LLNH Bs ...... K.TSL...L...O0IALVALLHOHAAVTVSSIYETDPSFNRTPPL YE ...AA FLNM EI TSLN.PFELLEL Sp ...... QIAjIDKLRAR.GIHLK ESOCLSDG LSGA AD...GCFANO ETLP.ADLLET PC ...... D AIEKM.SIKGIK TSMLBESKG YFKDO ... P AFYINA aC Ovcv|TSIH.NEO LLFE P f LMONLEESKYEENKELIDKCEEYETFLKNGKVDNSILKEVOVENYLLECNNI VI NNDEIMKNNLSKYK5KYYTS Y Y LT KLTFF PLS 4 VV 18 3 Ec TUR I EiCWK aEE..PW.GRPTLDLDMTL FGN E NJ...... B s T g I E ENLGRBREV. . .R WGPRT.DLDINREN...... Sp LLAIEjSELGRVREV ... HWGPRLIDLD IFIVEDOIL...... PC LIL IE ELj.RXyKVVID. Rj.ERC|DLDOSjFYGRKII...... P f IKYIELMKEEL JENVKEKEKFENtI D NDFT FMKNIKLEKNMIYKILSKYIHLERDIKNGNDNMSKVNMDKDINLNNNNNIKKKNNNDIDC 279

Ec ...... ER TVr DMKN GF PLFFOfiAELVFIDGEMLROILHTRAFDKLNKW* Bs ...... ETFOVLVPLRMYE|I|QLQLAEICOOVEKE EATSAETDQEGVRVWKQKSGVDEF* MAQ Sp.. DLILPHPYIAERLFVLES L0EI AIaHFIN ILKOPHRNLYDALKK* PC ...... ISEIIPNRVLERSL LLDISLILVDVTGLS ASYFEKIVDHDIK.....PVLPFLYK Pf DCVDQKMNNHVNNKNYINSFRDPYEIINNMVDNINFI E YTTH YSIULC NDMIOEYKVLNNTIRCLYNKYVSRMK..... QYNINIKE 370

Nm MARHVWOAGRFEIGLDIP DGGVYSQNAOTA AHNEQLLKEGAD DIGGEST SGADYVSP(EEEAWR EPVLAEVAGW EC MKLFAOGTSLDLSHPH MGOLNVTPDSFSDGGTH.NSLIDA KHH|A|NLMNAGA TIIDI[GGESTRPGIAAEVIS vEEELORVIPVVEAIAOR Bs HTIDOTOVIHTKPSALSYREKTL MGILNVTPDSFSDGGKY.DSLDKIALHLNIAIKEMIDDGAHNIIDIGGESTRPGIAEC|VtSEDEEMSoRVIPVIERITKE Sp MSSTANNATIItI]NVTPDSFSDGGIOFF.ALEOA 0IARKLIIAEGAIS MDIGGESTRPGSSYVEIEEEIIQRVPVIKAORKE PC NKSIDF ...... SFRSYKATINILN kTDSFFDGGIIH..SDS IDVEKFINA|GTIIDIG GLSTRPGSYIIP EEEIFVj|I[PAIKYLOKTE Pf NNKRIYVL KDRISYLKE N IFFEP.KRDVORMFEMUNEUPSV ESSAPFVIPNPRSRDLVLQLFOKE 462 *DHPS

Nom ..G7P ...... SLDTRRTVIME K LAL GID I i AALNDEGAVELLAROAD..TGICLMHOM GLPK ...... TMQI Ec F.E|VW|I.S|V||SVDTSKPE IRESA VG . I|IND I|RSLSEPGALEAAA.ETG. .LPVCLMNMOGNPK .H|M.Q.G.N.P. TMOE Bs L.G|VP ...... ISVDTYKASVADEVK AG A.SINDIWGAKHDPKMASVAAEHN. .VPIVLMNRP ......

SpPc ...... PC S.DVLSPKILI ASODTFRSEAEQ VKLAAGA.DLVNDITGLMGDEKMPHVVAEAR.GPSLVMNDISGGRYDPKMFNTVARLK..VPICI PMRGN...... FLTMEN WN ...... SIDTNKSOVAEA V D YPf KDAIKLI|I N I S|I|DJT|FNRSNEV|AKECQVDKND .SL|[LN I|SAGGCTYDNN KMPENTIIARKLKLKKKNKFYSV...... KRGNPH...... VP..CIM.|MR..N...... AQVVIMFNPVMARPOOPSSLIFPNFGFGOAFTEEE..MD..K.F54NM0

Nm NPKYQD V. VGEVARYLKAR SAEC I AAG I AP QRDOILOP G FGLFKPLHN IALMRHLPTELM AE ... GFPL LGSR ST ELT G.. Ec APKYDDV.FAEVNRYFIEQ. IARCEQAOGAKEKLLLDPGF..GFG|K|NLSH|N|YS ARL A||F. H.H F NLPLLVIMSRKSMIGRLL ...... Os ERNYNDL .LPDMLSDLMES .VKIAVEAGVDERNOIOLDPGI. .GFPIKITYHDINLAVMNKL..I.FSG .. LGYP VILLATSRRRFOURVL...... PC LTDYGTD MLEOSTDEL.EKLLINSAEK| GEPRNIHLDPGL..GFSR.TLOLNAEL... GP RTFTG...... LADFETLPIEELMEAFFERALARAAEAGOAPENLLDPGIGFGLTIK..KEINLLLLRDLDKL.HQRRFNKaLKSVNCFNGLPjWJK .RGYPIFLG SRRRFVINILEENGFEVNPE PLTDGTDPf IEQLTNYDNLVYD..FPIE|EKNYLME|RLNFLVLNGIPRYRLDPINS KSG |PRW |IIDPGL..GAKKO| ...... GS||TLH3TTGIIEL|RRFLP|[L|||SRS.NDKSCF LL ...... KEYPLF GYSRKRF|IACMNIONvVLNTN 626

Nm .EANAAE[r VHGS ...... V AAALASVAR GAQIVRVHDVKATADALKVWEALGINL* Ec .NVGPSEIRILSIG S ...... L AICAVIAAMOGAHIIRVHDVKETVEAMRVVEATLSAKENKRYE* Bs .DLPPEEIRIAE GT ...... G..A TVCLGIOKGCD VRVHDVKQIARMAKMMDAMLNKGGVHHG* Sp TELGFRN R DTAS ...... AHVTSIAARQGVEVVRVHDVASHRMAVEIASAIRLADEAENLDLKQYK* V I N V Pc .DNM PKDDRIW T ...... V AVVASIS GD CD RVH YEMYKR ISKMSDAIWKEIY* Pf OKLHDEQONENKNIVDKSHNWMFOMNYMRKDKDQLLYQKNICGGL IASYSYYKKVLOIRVHDVILETKSVLDVLTKIDQV* 7 06 FIG. 2. Comparison ofthe predicted PfPPPK and PfDHPS amino acid sequences. Protein sequence data are from the following references: Ec, E. coIl (10); Nm, Neisseria meningitidis (14), Bs, Bacillus subtilis (8), Sp, Streptococcus pneumoniae (7, 11), and Pc, P. carinji (9). Pf stands for PJDHPS amino acid sequence from the NF7 isolate. Boxed areas indicate amino acid identity shared by >50%6 of sequences, dots indicate spaces inserted into the sequence to provide optimal homology, asterisks indicate stop codons, and numbers indicate amino acid residues of the P. falciparum protein, corresponding to those in Fig. 1. The # symbol represents amino acids where differences occur between the DHPS sequence of different P. falciparum isolates. Note that the first amino acid of the P. carinii sequence is position 270 of the Fas gene. The approximate position where the PPPK and DHPS domains begin is indicated. four different enzymes, blotted, and probed with radiolabeled electrophoresis was used to determine the chromosomal DNA encompassing the DHPS-encoding gene (Fig. 4A). A location of the PPPK-DHPS gene. A probe encompassing the single hybridizing band was obtained with each enzyme, DHPS gene hybridized to a single chromosomal band in all P. suggesting that the PPPK-DHPS gene is present within the P. falciparum isolates (Fig. 4B), and this was unambiguously falciparum genome as a single copy. Pulsed-field gradient gel identified as chromosome 8. To test if the mechanism of sulfadoxine-resistance in P. Intran itron falciparum involved PPPK-DHPS gene amplification, we \/I 378 \/06 used Southern blot analysis to probe genomic DNA of PPPK DHPS P. falciparum resistant and sensitive isolates (Fig. 5). Probes spanning both Insertion Insertion Insertion the DHPS gene and the single-copy DHFR gene (16) were hybridized to BstYI-digested DNA from known sulfadoxine- resistant clones K1 and W2mef (3), as well as other isolates. The DHPS fragment hybridized to the expected 1260-bp 1 160 290 462 740 fragment, and DHFR hybridized to a 1000-bp fragment. In all FasA DHNA PPPK DHPS P. carinii isolates the intensity of hybridization to DHPS is equal to that seen with DHFR, indicating that PPPK-DHPS is present as FiG. 3. Organization of dihydroneopterin aldolase (DHNA), single PPPK, and DHPS enzymes. Proteins are aligned to indicate the a copy in all isolates tested (Fig. 5). regions of predicted amino acid sequence that show significant The PPPK-DHPS Enzyme Is Expressed Throughout the homology. Length of each protein is shown at right of each box. Asexual Life Cycle. Antibodies were raised to a peptide Location of introns and insertions within the P. falciparum PPPK- corresponding to aa 353-366 ofthe PPPK-DHPS enzyme and DHPS gene are shown. affinity-purified on a peptide-agarose support. These anti- Downloaded by guest on September 25, 2021 7152 Medical Sciences: Triglia and Cowman Proc. Nad. Acad. Sci. USA 91 (1994)

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- 2300 S - 68 -

43 - -1352

4- CHROM. 8 - 603 Antibody:, PtDHPS PtDHPS, PiDH PS PiDHPS Peptide Peptide FIG. 6. The PPPK-DHPS enzyme is a 68-kDa protein. (A) -31 0 Uninfected erythrocytes (RBC; 5 x 105) and synchronized tropho- zoites (TROPHS; 5 x 105) were separated in each well by SDS/ PAGE. The proteins were immunoblotted and probed with affinity- purified anti-DHPS antibodies (eft lanes) or the same antibody FIG. 4. The P. falciparum PPPK-DHPS gene is a single-copy preabsorbed with the peptide used to generate the antibodies (right gene located on chromosome (CHROM.) 8. (A) Genomic DNA firom lanes). (B) E. coli proteins or proteins from E. coli expressing the NF7 parasite was digested with Ssp I (lane S), Rsa I (lane R), Dra PfDHPS fused with GST (E. coli-PfDHPS) were separated by I (lane D), and Apo I (lane A), fractionated, and hybridized with a SDS/PAGE and immunoblotted. Blots were probed with the same 130-bp PfDHPS fiagment bounded by the degenerate oligonucleo- antibodies as in A. tides DHPS 1 and DHPS 2. (B) Chromosomes from various P. falciparum clones were separated by pulsed-field gradient gel elec- cycle and sampled aliquots at 8-hr intervals throughout the trophoresis and hybridized with the same probe as in A. 48-hr asexual cycle. Equal numbers of parasites were sepa- rated by SDS/PAGE, and an immunoblot of the transferred bodies recognize a protein of an apparent molecular mass of proteins was probed with the anti-PPPK-DHPS antibodies. 68 kDa in purified trophozoites (Fig. 6A). To prove that the A single band of 68 kDa was observed predominantly in the antibodies were specific to the P. falciparum PPPK-DHPS trophozoite stage, but some protein was evident in all of the they were preabsorbed with the same peptide used to raise time points sampled (Fig. 7). An immunoblot of the same the immune response, and the reactivity to the 68-kDa samples when probed with antibodies to Pfhsp7O, as ex- protein could be removed. To further demonstrate the spec- pected, showed a band of 70 kDa that increased in intensity ificity ofthe antipeptide antibodies a GST fusion protein was as the parasite matured. made in Escherichia coli that included 39 kDa of the PPPK- Sequence Differences in the DHPS-Encodlng Gene. Previ- DHPS protein (aa 301-678) and 26 kDa of the GST protein. ously it has been shown that metabolism ofsulfadoxine to the The antipeptide antibodies recognize a band of -65 kDa and toxic analog is reduced in resistant P.falciparum, suggesting another at 45 kDa in E. coli transformed with a plasmid mutations in the DHPS enzyme that alter substrate binding. expressing the PPPK-DHPS fusion protein but show no To analyze this possibility two oligonucleotides encompass- reactivity in untransformed E. coli (Fig. 6B). The 45-kDa ing the entire DHPS gene (indicated as arrows in Fig. 1) were band is presumably a proteolytic breakdown product from used to amplify and sequence DNA from five other P. the expected 65-kDa protein. Reactivity with both the 65-kDa falciparum clones and isolates (FC27, W2mef, K1, 3D7, and and 45-kDa bands was abolished by preabsorbing on the 7G8). W2mef and K1 have previously been shown to be peptide to which the antibodies were raised. Taken together, sulfadoxine-resistant (3), FC27 was isolated from Papua, these results prove that the antipeptide antibodies are specific New Guinea before any resistance was described, and the for the P. falciparum PPPK-DHPS protein. other three clones are of unknown sulfadoxine sensitivity. To determine the stage specificity ofPPPK-DHPS expres- Among the six P. falciparum clones sequence differences sion we synchronized parasites in the ring stage of the life within DHPS occurred at only four positions (Fig. 1), with a single nucleotide change resulting in a changed amino acid in

0 r- r-r- N1 Go) E RINGS TROPHS S, S P RINGS TROPHS S S R LL C u y ( cm u 7 rn-1 Z cn o r B --- e.r- 3. A an o CO UD IT N ° 7OtD o C-zo- bp

--O 68Kd - -- 70Kd _ 'A - 1266 1 u .1 - DHPS

1000 _M' - DHFR

Antibody anti- PIDHPS antil Pfhsp7O

FIG. 5. The DHPS-encoding gene is not amplified in sulfadoxine- FIG. 7. The PPPK-DHPS protein is expressed throughout the P. resistant P. falciparum parasites. Genomic DNA from FC27 (sulfa- falciparum asexual cycle. NF7-synchronized parasites were sampled doxine-sensitive), K1, and W2mef (sulfadoxine-resistant) (3) and at 8-hr intervals over 40 hr. Proteins from 5 x 106 purified parasites three other clones were digested with BstYI, fractionated, and or 5 x 106 uninfected erythrocytes (RBC) were loaded in each track, hybridized both with a probe spanning the DHPS gene and with a separated by SDS/PAGE and immunoblotted. Filters were probed 500-bp probe from the coding region of the P. falciparum dihydro- with affinity-purified anti-DHPS antibodies (A) or anti-Pfhsp7O folate reductase (DHFR) gene. Calculated sizes of the hybridizing antibodies (B) and developed as described. TROPHS, trophozoites; BstYI fragments are shown in bp. S, schizonts; S/R, schizonts and rings; Kd, kDa. Downloaded by guest on September 25, 2021 Medical Sciences: Triglia and Cowman Proc. Natl. Acad. Sci. USA 91 (1994) 7153

every case (data not shown). The four amino acid changes sary to express the different alleles in a heterologous expres- observed in the six different P. falciparum isolates all occur sion system. in regions highly conserved among DHPS enzymes from The PPPK-DHPS protein ofP. falciparum is an attractive different organisms (Fig. 2). This observation lends support target for antimalarials, as neither of these two enzyme to the possibility that they may be involved in resistance to activities are present within the human host of the parasite. the sulfone/sulfonamide group of antimalarials. This result suggests that specific highly selective inhibitors against this target protein may be developed and the identi- DISCUSSION fication and analysis ofthis gene represent the first step in this The sulfone/sulfonamides are an important group of antima- process. larial compounds that act either to directly inhibit the P. Note Added in Proof. While this work was in progress, we became falciparum enzyme DHPS or are converted by this enzyme to aware ofsimilar work entitled "Correlation ofsulfadoxine resistance a toxic sulfa analog that inhibits the following enzyme in the with point mutations located within the bifunctional hydroxymeth- folate biosynthetic pathway. To understand the role of the yldihydropterin pyrophosphokinase- gene PfDHPS enzyme both as a target and in the mechanism of of the human malaria parasite Plasmodium falciparum" by D. resistance to these compounds we have cloned the Brooks, P. Wang, M. Read, W. Watkins, P. Sims, and J. Hyde that gene has now been submitted for publication. encoding this enzyme. The gene we have identified has strong homology to DHPS sequences ofother organisms and encodes We thank D. Cappai for technical assistance. This research was a protein of an apparent molecular mass of 83 kDa. However, supported by the Australian National Health and Medical Research the observed size ofthe protein on Council, the United Nations Development Program/World Bank/ SDS/PAGE gels is 68 kDa, World Health Organization Special Program for Research and Train- a situation similar to that seen with the P. carinii DHPS ing in Tropical Diseases. A.F.C. is supported by a Wellcome Trust enzyme, which encodes a protein of 84 kDa but migrates at 69 Senior Research Fellowship and, in part, by an International Re- kDa (21). The PfDHPS protein was found to be joined to a search Scholar's award from the Howard Hughes Medical Institute. region homologous to the PPPK enzyme of other organisms, 1. Zhang, Y. & Meshnick, S. R. (1991) Antimicrob. Agents Che- suggesting that this enzyme in P. falciparum is bifunctional. mother. 35, 267-271. The DHPS protein of P. carinji is, at least, a trifunctional 2. Peters, W. (1987) in Chemotherapy and Drug Resistance in enzyme with a domain of unknown function (Fas A) at the N Malaria (Academic, London). terminus followed by regions encoding dihydroneopterin al- 3. Dieckmann, A. & Jung, A. (1986) Mol. Biochem. Parasitol. 19, dolase (21), PPPK, and also DHPS at the C terminus. The P. 143-147. falciparum PPPK-DHPS protein appears not to bejoined to a 4. Krungkrai, J., Webster, H. K. & Yuthavong, Y. (1989) Mol. region homologous to dihydroneopterin aldolase. Biochem. Parasitol. 32, 25-38. 5. Ferone, R. (1973) J. Protozool. 20, 459-463. The PfPPPK polypeptide has two 92-aa insertions when 6. Ferone, R. (1977) Bull. WHO 55, 291-298. compared with other PPPK enzymes from other organisms. 7. Lopez, P., Greenberg, B. & Lacks, S. A. (1990) J. Bacteriol. Insertions have previously been found within P. falciparum 172, 4766-4774. enzymes compared with the same enzyme from other orga- 8. Slock, J., Stahly, D. P., Han, C.-Y., Six, E. W. & Crawford, nisms. For example, there is a 42-aa insertion within the P. I. P. (1990) J. Bacteriol. 172, 7211-7226. falciparum dihydroorotate dehydrogenase gene homologue 9. Volpe, F., Dyer, M., Scaife, J. G., Darby, G., Stammers, D. K. (22), and there are enlarged variable regions, usually con- & Delves, C. J. (1992) Gene 112, 213-218. 10. Talarico, T. L., Ray, P. H., Dev, I. K., Merrill, B. M. & taining many asparagine residues, within the RNA polymer- Dallas, W. S. (1992) J. Bacteriol. 172, 5971-5977. ase II enzyme of P. falciparum (23). 11. Lopez, P., Espinosa, M., Greenberg, B. & Lacks, S. A. (1987) The observed size of the PPPK-DHPS protein from P. J. Bacteriol. 169, 4320-4326. falciparum in immunoblots is 68 kDa. This size is much 12. Radstrom, P., Fermer, C., Kristiansen, B.-E., Jenkins, A., smaller than that described for the native enzyme from Skold, 0. & Swedberg, G. (1992) J. Bacteriol. 174, 6386-6393. Plasmodia (190-250 kDa. 24, 25), suggesting that the P. 13. Dallas, W. S., Gowen, J. E., Ray, P. H., Cox, M. J. & Dev, falciparum PPPK-DHPS enzyme may exist as a higher I. K. (1992) J. Bacteriol. 174, 5961-5970. multimer. The PPPK-DHPS enzyme is expressed throughout 14. Cowman, A. F., Morry, M. J., Biggs, B. A., Cross, G. A. M. & Foote, S. J. (1988) Proc. Natl. Acad. Sci. USA 85, 9109- the asexual life cycle, suggesting that its functions in folate 9113. metabolism are important in all stages. The protein is more 15. Foote, S. J., Kyle, D. E., Martin, R. K., Oduola, A. M., highly expressed in the trophozoite stage when rapid growth Forsyth, K., Kemp, D. J. & Cowman, A. F. (1990) Nature and DNA replication are occurring, which would allow for an (London) 345, 255-258. increased production of folate precursors when the need 16. Trager, W. & Jensen, J. B. (1978) Nature (London) 273, would be greatest. 621-622. The demonstration that metabolism of sulfadoxine by 17. Chu, G., Vollrath, D. & Davis, R. (1986) Science 234, 1582- DHPS in P. falciparum is reduced in sulfadoxine-resistant 1585. parasites suggested that mutations in this enzyme were 18. Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31-40. involved in the mechanism of resistance to this class of 19. Bianco, A. E., Favaloro, J. M., Burkot, T. R., Culvenor, J. G., Crewther, P. E., Brown, G. V., Anders, R. F., Coppel, antimalarals (3). We have cloned and sequenced the DHPS R. L. & Kemp, D. J. (1986) Proc. Natl. Acad. Sci. USA 83, portion of the gene from a number of different P. falciparum 8713-8717. isolates, some of which are known to be resistant to sulfa- 20. Brown, H. J. & Coppel, R. L. (1991) Mol. Biochem. Parasitol. doxine. Four amino acid differences were identified between 49, 99-110. PfDHPS enzymes that are all present in highly conserved 21. Volpe, F., Ballantine, S. P. & Delves, C. J. (1993) Eur. J. regions, suggesting these differences may be involved in the Biochem. 216, 449-458. mechanism of resistance to sulfones and sulfonamides. Pre- 22. Le Blanc, S. B. & Wilson, C. M. (1993) Mol. Biochem. Para- viously, alterations have been identified in the DHFR en- sitol. 60, 349-352. zyme sequence that mediates pyrimethamine resistance, and 23. Li, W. B., Bzik, D. J., Gu, H. M., Tanaka, M., Fox, B. A. & Inselburg, J. (1989) Nucleic Acids Res. 17, 9621-9636. the only changes found were relevant to the level of resis- 24. McCullough, J. & Maren, T. A. (1974) Mol. Pharmacol. 10, tance to the drug. To prove that the amino acid differences 140 145. that we have identified are important in the mechanism of 25. Walter, R. D. & Konigk, E. (1974) Z. Phys. Chem. 3M. resistance to the sulfones and sulfonamides it will be neces- 431-437. Downloaded by guest on September 25, 2021