Mutations in Dihydropteroate Synthase Are Responsible for Sulfone and Sulfonamide Resistance in Plasmodium Falciparum (Malaria͞sulfadoxine͞drug Resistance)
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Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13944–13949, December 1997 Medical Sciences Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum (malariaysulfadoxineydrug resistance) TONY TRIGLIA*†,JOHN G. T. MENTING*†,CRAIG WILSON‡, AND ALAN F. COWMAN*§ *The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia 3050; and ‡Geographic Medicine, University of Alabama at Birmingham, Birmingham, AL 35294 Communicated by Gustav J. V. Nossal, University of Melbourne, Victoria, Australia, September 18, 1997 (received for review May 16, 1997) ABSTRACT Plasmodium falciparum causes the most se- become less useful as the parasite develops resistance (6); vere form of malaria in humans. An important class of drugs however, despite this they are still used extensively in Africa in malaria treatment is the sulfoneysulfonamide group, of where resistance is not yet widespread. which sulfadoxine is the most commonly used. The target of The DHPS gene has been cloned from P. falciparum and sulfadoxine is the enzyme dihydropteroate synthase (DHPS), encodes a putative bifunctional enzyme with 7,8-dihydro-6- and sequencing of the DHPS gene has identified amino acid hydroxymethylpterin pyrophosphokinase (PPPK), the enzyme differences that may be involved in the mechanism of resis- before DHPS in the folate pathway (7, 8). All the protozoan tance to this drug. In this study we have sequenced the DHPS DHPS enzymes sequenced so far have been either bifunctional gene in 10 isolates from Thailand and identified a new allele with both PPPK and DHPS domains, as in P. falciparum (7, 8), of DHPS that has a previously unidentified amino acid or trifunctional as in Pneumocystis carinii with another enzyme difference. We have expressed eight alleles of P. falciparum from the folate pathway dihydroneopterin aldolase (DHNA), PPPK-DHPS in Escherichia coli and purified the functional PPPK, and DHPS activities (9). The Toxoplasma gondii DHPS enzymes to homogeneity. Strikingly, the Ki for sulfadoxine enzyme is also likely to be bifunctional (10). varies by almost three orders of magnitude from 0.14 mM for Identification of amino acid differences in DHPS between the DHPS allele from sensitive isolates to 112 mM for an sulfadoxine-sensitive and -resistant isolates of P. falciparum enzyme expressed in a highly resistant isolate. Comparison of has suggested that mutations of this enzyme may be involved the Ki of different sulfonamides and the sulfone dapsone has in the mechanism of resistance to this antimalarial drug (7, 8); suggested that the amino acid differences in DHPS would however, no direct evidence for this has yet been obtained. A confer cross-resistance to these compounds. These results point mutation in a highly conserved region of the DHPS gene show that the amino acid differences in the DHPS enzyme of of E. coli confers sulfonamide resistance (11), and a 2-aa sulfadoxine-resistant isolates of P. falciparum are central to insertion in DHPS of both Neisseria meningitidis and Strepto- the mechanism of resistance to sulfones and sulfonamides. coccus pneumoniae has also been shown to confer sulfonamide resistance (12, 13). However, in both N. meningitidis and The sulfonamideysulfone group of compounds is used exten- Streptococcus pyogenes, sulfonamide resistance may be a result sively in the treatment of bacterial diseases including infections of multiple mutations in DHPS genes, which suggests they have caused by Streptococcus spp., Neisseria spp., and Mycobacte- arisen not by mutation of the sensitive gene but by intraspecies rium spp as well as many parasitic infections involving Pneu- or interspecies transfer of genetic material (13, 14). mocystis, Toxoplasma, Cryptosporidium, and Plasmodium. To test whether the amino acid differences found in DHPS Members of this group of chemotherapeutic compounds, such between sulfadoxine-resistant and -sensitive isolates of P. as sulfadoxine and dapsone, inhibit the enzyme dihydrop- falciparum are important in the drug resistance phenotype, we teroate synthase (DHPS) (1), a component of the folate have expressed eight different PPPK-DHPS alleles in E. coli, biosynthetic pathway. The enzyme DHPS catalyzes the con- purified the active enzymes, and determined their kinetic densation of p-aminobenzoic acid (pABA) with 6-hydroxy- parameters. We show that mutations within the P. falciparum methyldihydropterin pyrophosphate to yield 7,8-dihydrop- DHPS gene are responsible for the mechanism of sulfadoxine teroate (1). Plasmodia generate the vast majority of their resistance in P. falciparum. folates de novo, and hence inhibition of DHPS by these drugs leads to depletion of dTTP and decreased DNA synthesis (2). MATERIALS AND METHODS However, there is also some evidence for folate salvage by the Parasites and DNA. The origin of P. falciparum clones and parasite (3). The human host lacks most of this biosynthetic strains is shown in Table 1. The PR145, 336, 338, 342, 345, 347, pathway, and consequently many effective chemotherapeutic 327, 343, 352, and 346 isolates were kindly provided by Sodsri agents have been developed that target some of the enzymes Thaithong of the WHO Collaborating Centre on the Biological involved in the synthesis of pyrimidines. Characterization of Malaria Parasites, Chulalongkorn Univer- In the case of Plasmodium falciparum, the causative agent of sity, Bangkok, Thailand. Parasites were maintained in asyn- the most lethal form of human malaria, sulfadoxine has been chronous cultures as described (15). used extensively for prophylaxis. Usually, this has been in Expression Constructs. The full-length PPPK-DHPS gene combination with the antifolate drug, pyrimethamine, which from P. falciparum was amplified from D10 cDNA in two inhibits the enzyme dihydrofolate reductase (DHFR) (4), halves using a unique AccI restriction site at position 963 in the because the combination of these drugs shows a marked synergism in their antimalarial effect (5). These drugs have Abbreviations: DHFR, dihydrofolate reductase; DHNA, dihydrone- opterin aldolase; DHPS, 7,8-dihydropteroate synthetase; pABA, p- The publication costs of this article were defrayed in part by page charge aminobenzoic acid; PPPK, 7,8-dihydro-6-hydroxymethylpterin pyro- phosphokinase. payment. This article must therefore be hereby marked ‘‘advertisement’’ in †T.T. and J.G.T.M. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed at: Walter and Eliza © 1997 by The National Academy of Sciences 0027-8424y97y9413944-6$2.00y0 Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, PNAS is available online at http:yywww.pnas.org. Melbourne, Australia 3050. e-mail: [email protected]. 13944 Downloaded by guest on September 29, 2021 Medical Sciences: Triglia et al. Proc. Natl. Acad. Sci. USA 94 (1997) 13945 Table 1. Genotypes and sulfadoxine sensitivity of P. falciparum lines and genotypes of DHPS constructs Amino acid Isolate Origin Sulfadoxine 436 437 540 581 613 Construct Reference D10, HB3, ItG2, M24, NF7 PNG, Africa, Brazil S S A K A A D10-C (7, 8) 3D7 Africa R S G K A A 3D7-C (7, 22) Tak9y96 SE Asia R A G K A A Tak9y96-C (22) K1 SE Asia R S G K G A K1-C (7) W2mef, Dd2 SE Asia R F G K A S W2mef-C (7) PR145, 336, 338, 342, 345, 347 Thailand ND A G E A A PR145-C This publication PR327, 343, 352 Thailand ND S G E G A This publication PR346 Thailand ND A G E G A This publication S A K G A D10-CyG581 S G E A A 3D7-CyE540 ND, not determined. cDNA sequence where the A of the start codon is position 1. exchange buffer (10 mM TriszHCly5mMDTTy0.1 mg/ml The four oligonucleotides were: P1 (59-ATATATATACCA- PMSFy1 mM leupeptiny1 mM pepstatin, pH 8.6) before load- TGGAAACTATACAAGAA) with the introduced NcoI site ing onto a Mono Q HR 5y5 column (Pharmacia). Proteins underlined, P2 (59-CTTTTAATACATATATCCTTTTAT), were eluted with a gradient of 0–0.35 M NaCl in anion- P3 (59-TTAGAGATCCACAAGAAATAATAAAC), and P4 exchange buffer. At this pH the addition of DTT was essential. (59-GTTGAATTCCATGTTTGCACTTTCC) with the intro- The purified protein was stored in gel filtration buffer by y duced EcoRI site underlined. The two halves of the PPPK- ultrafiltration buffer exchange as described above and finally DHPS gene were cloned into the NcoI and EcoRI sites of the concentrated to 0.3–0.5 ml. PPPK-DHPS was detected in fractions by activity assay, probing Western blotted protein pTrc expression vector (Invitrogen) to express the native y PPPK-DHPS enzyme. A region of instability, encompassing a with affinity-purified anti-DHPS antibody, or on SDS PAGE stretch of 12 nucleotides at position 1551–1562, was mutated stained for protein. to the sequence AAAGAAGAAGAA by PCR mutagenesis. For purification of the D10-C enzyme, the final recovery was 11.1%, which represented 0.04% of the initial protein. Rou- This did not change the encoded amino acids but stabilized the tinely, around 0.2 mg of pure enzyme was obtained from 400 sequence in E. coli. The PPPK-DHPS D10 construct (D10-C) mg of starting protein. was then used as the starting plasmid for all subsequent Enzyme Assays. The PPPK-DHPS enzymes were assayed constructs (Table 1). The 3D7 (3D7-C) and W2 mef (W2 using a modification of a published procedure (16). The assay mef-C) alleles of PPPK-DHPS were constructed by amplifying was dependent on the presence of both PPPK and DHPS DNA fragments, which contained the required mutations, activities, but the DHPS activity component was isolated by from genomic DNA and subcloning into the D10 construct by measurement of the conversion of [3H]pABA to [3H]dihy- using appropriate restriction enzymes. Similarly, the K1 (K1- dropteroate. Enzyme activity was found to be dependent on y y C), Tak 9 96 (Tak 9 96-C), and PR145 (PR145-C) PPPK- protein concentration; hence, BSA was included in assay DHPS alleles were made by substituting amplified fragments reaction mixtures.