Mutations in Dihydropteroate Synthase Are Responsible for Sulfone and Sulfonamide Resistance in Plasmodium Falciparum (Malaria͞sulfadoxine͞drug Resistance)

Home , Dapsone, Pyrimethamine, Sulfadoxine

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 (malaria͞sulfadoxine͞drug 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 sulfone͞sulfonamide 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 ␮M for Identification of amino acid differences in DHPS between the DHPS allele from sensitive isolates to 112 ␮M 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 sulfonamide͞sulfone 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, dihydroneopterin 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 pyrophosphokinase. 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 0027-8424͞97͞9413944-6$2.00͞0 Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, PNAS is available online at http:͞͞www.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) Tak9͞96 SE Asia R A G K A A Tak9͞96-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-C͞G581 S G E A A 3D7-C͞E540 ND, not determined.

cDNA sequence where the A of the start codon is position 1. exchange buffer (10 mM Tris⅐HCl͞5mMDTT͞0.1 mg/ml The four oligonucleotides were: P1 (5Ј-ATATATATACCA- PMSF͞1 ␮M leupeptin͞1 ␮M pepstatin, pH 8.6) before load- TGGAAACTATACAAGAA) with the introduced NcoI site ing onto a Mono Q HR 5͞5 column (Pharmacia). Proteins underlined, P2 (5Ј-CTTTTAATACATATATCCTTTTAT), were eluted with a gradient of 0–0.35 M NaCl in anion- P3 (5Ј-TTAGAGATCCACAAGAAATAATAAAC), and P4 exchange buffer. At this pH the addition of DTT was essential. (5Ј-GTTGAATTCCATGTTTGCACTTTCC) with the intro- The purified protein was stored in gel filtration buffer by ͞ 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 ͞ 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 ͞ ͞ 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. All assays were done at 37°C, and kinetic ͞ into the 3D7 construct. The constructs D10-C G581 and assays were done in triplicate. For the estimations of Ki and Km, 3D7-C͞E540 (Table 1) were obtained as intermediate steps reactions (80 ␮l containing 1 mg͞ml enzyme, 100 mM Tris⅐HCl toward the final K1-C and PR145-C constructs. (pH 8.5), 10 mM MgCl2, 20 mM 2-mercaptoethanol, 80 mM Peptide Synthesis and Antibody Production. A 15-residue 6-hydroxymethylpterin, 5 mM ATP, 1 mg͞ml BSA, pH 8.5) peptide (Chiron) corresponding to amino acids 631–644 of the were commenced by the addition of various amounts of 4 DHPS domain was conjugated to diphtheria toxoid to allow Ci͞mmol [3H]pABA (American Radiolabeled Chemicals, St. antibody production in rabbits. Louis) and either dimethyl sulfoxide (DMSO) or sulfadoxine Purification of PPPK-DHPS. Cultures of each recombinant (Hoffmann–LaRoche) in DMSO. Reactions were sampled at clone were grown at 37°C and induced with 0.5 mM isopropyl appropriate time points, and [3H]dihydropteroate was sepa- 3 ␤-D-galactopyranoside (IPTG) at 30°C. Cells were pelleted, rated from [ H]pABA on PEI-cellulose (Merck) with 0.2 M 3 resuspended in sonication buffer (50 mM phosphate, pH LiCl. The origins were excised and [ H]dihydropteroate was 8.0͞300 mM NaCl͞1 mM EDTA͞2mMDTT͞0.5 mM phe- counted by liquid scintillation. Initial rates of reaction were nylmethylsulfonyl fluoride (PMSF)͞1 ␮g/ml leupeptin͞1 estimated with the kinetics program ENZFITTER, by using linear ␮g/ml pepstatin A͞10 mg/ml DNaseI͞0.1 mg/ml RNase A͞0.1 regression and first-order nonlinear regression analysis, as mg/ml lysozyme), and stirred. Cells were sonicated and cen- appropriate. Ki and Km values were calculated from the vo trifuged at 6,000 ϫ g for 10 min. The supernatant pH was values using the program EZ-FIT (DuPont). Ki values for dapsone, sulfamethoxazole, and sulfathiazole were calculated adjusted to 6.2 with solid Mes, dialyzed against 10 mM ϭ ϩ ϩ from the IC50 data using the equation v Vmax[s]([s] (1 Mes-NaOH, pH 6.2 (cation exchange buffer), centrifuged (10 Ϫ1 Ϫ ϭ ϭ I.Ki )) 1, where v reaction velocity, Vmax maximum min, 18,000 ϫ g), and the supernatant was separated on a ϭ ϭ reaction rate, [s] substrate concentration, Km Michaelis SP-Sepharose HPLC chromatography column (Pharmacia). ϭ ϭ constant, I inhibitor concentration, and Ki inhibitory Proteins were eluted with a gradient of 0–0.45 M NaCl in constant. One unit of enzyme activity is equal to the formation cation-exchange buffer. Fractions containing PPPK-DHPS of 1 ␮mol [3H]dihydropteroate per minute. were combined, and the buffer was exchanged with an Amicon Western Blotting. Protein samples were analyzed by SDS͞ stirred-cell nitrogen-pressure ultrafiltration unit (YM10 mem- PAGE and electrophoretically transferred to nitrocellulose as ͞ brane) with gel filtration buffer (5 mM Mes-NaOH 0.5 M described (17). Bound antibody was detected with horseradish ͞ ͞ ͞ ͞ NaCl 1mMDTT 0.1 mM Na2EDTA 0.2 mM leupeptin 0.2 peroxidase-coupled goat anti-rabbit IgG and developed by mM pepstatin A͞10 mg/ml PMSF, pH 6.2), and proteins were ECL, a luminescence-based method (Amersham). concentrated to Ͻ3 ml. Proteins were separated on a Sephacryl S-300 HR column (Pharmacia) equilibrated with gel filtration RESULTS buffer. Fractions containing PPPK-DHPS were combined, Amino Acid Differences in DHPS. Sequence analysis of the concentrated by ultrafiltration, and diluted 1:7 with anion- DHPS gene from sulfadoxine-resistant and -sensitive isolates Downloaded by guest on September 29, 2021 13946 Medical Sciences: Triglia et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 1. Expression of eight alleles of P. falciparum PPPK-DHPS in E. coli. Eight different alleles of PPPK-DHPS were cloned into the pTrc vector and transformed into E. coli. Extracted proteins were separated by SDS͞PAGE and stained with Coomassie blue (A) or transferred to nitrocellulose and probed with affinity-purified anti-DHPS antibodies (B). The order of the tracks in each panel from left to right is pTrc, D10-C, 3D7-C, Tak9͞96-C, K1-C, W2 mef-C, PR145, D10-C͞G581, and 3D7-C͞E540.

of P. falciparum has shown that there are amino acid differ- on SDS͞PAGE throughout the purification of the D10-C and ences at four positions, and it has been suggested that they may K1-C enzymes showed a single protein band after anion- be involved in the mechanism of resistance to sulfadoxine (7, exchange chromatography, suggesting that the enzymes had 8). To determine whether other differences in the DHPS gene been purified essentially to homogeneity (Fig. 2). Active could occur, we sequenced isolates obtained from Thailand, a PPPK-DHPS enzyme was purified from the eight different alleles of PPPK-DHPS using the same procedure. Analysis by geographic area where there has been extensive use of sulfa- ͞ doxine and pyrimethamine. The PPPK-DHPS gene from 10 SDS PAGE and Coomassie blue staining suggested that all Thai isolates (PR145, 336, 338, 342, 345, 346, 347, 327, 343, and were purified to homogeneity (Fig. 3A). The purified proteins 352) was obtained by PCR and sequenced. The DHPS gene have the predicted size from the sequence of the gene for the P. falciparum PPPK-DHPS enzyme (7, 8), contain both PPPK from the 10 isolates encoded a Glu at amino acid 540 (Glu-540) and DHPS enzyme activities, and are recognized by an anti- rather than Lys-540, which has been described for all other DHPS antibody (Fig. 3B). Therefore, we have purified from E. isolates that have so far been sequenced (Table 1), and also a coli the active P. falciparum PPPK-DHPS bifunctional recom- Gly-437 and Ala-613. The DHFR gene from the Thai isolates binant enzyme. was also sequenced across the amino acids responsible for Kinetic Constants of DHPS Activity and Inhibitory Con- pyrimethamine and cycloguanil resistance (18–21). All had the stants for Sulfadoxine. The kinetic parameters of DHPS same sequence for the DHFR gene: Ala-16, Ile-51, Arg-59, Asn-108, and Leu-164, indicating that all had the genotype that would confer resistance to high levels of pyrimethamine (18–21). Expression of Functional P. falciparum PPPK-DHPS in E. coli. To measure the effects of amino acid changes on the DHPS enzyme we expressed the full-length PPPK-DHPS gene in E. coli. Six plasmid constructs were made that express the D10 (D10-C), 3D7 (3D7-C), Tak9͞96 (Tak9͞96-C), KI (KI-C), W2 mef (W2 mef-C), and PR145 (PR145-C) alleles of PPPK- DHPS in E. coli (Table 1). In addition, two constructs were made to investigate the role of Gly-581 (D10-C͞G581) and Glu-540 (3D7-C͞E540) in resistance to sulfadoxine (Table 1). The eight constructs were transformed into E. coli and expression was analyzed by SDS͞PAGE and an anti-DHPS antibody. A band of 83-kDa was recognized in all constructs, suggesting that the PPPK-DHPS constructs were expressing full-length PPPK-DHPS protein in E. coli (Fig. 1). Purification of P. falciparum PPPK-DHPS from E. coli. Functional P. falciparum PPPK-DHPS was isolated from E. coli by using sequential steps of SP-Sepharose cation exchange, Sephacryl S300HR gel filtration, and Mono Q high-resolution anion-exchange chromatography. The molecular mass esti- FIG. 2. Purification of functional recombinant PPPK-DHPS en- mated by gel filtration ranged from 207 to 246 kDa (mean 222 zyme from P. falciparum in E. coli. Four liters of either D10-C or K1-C kDa), suggesting that the PPPK-DHPS enzyme is a multimer were grown and the PPPK-DHPS activity purified by sequential steps of two or three subunits. Purification was monitored through- of cation-exchange, gel-filtration, and anion-exchange chromatography. Purification was monitored by enzyme activity and detection with out the procedure by the PPPK-DHPS enzyme assay, and anti-DHPS antibodies. Aliquots of D10-C (A) or K1-C (B) chroma- immunoblots of fractions were monitored using the anti- tography aliquots were separated by SDS͞PAGE, and protein was DHPS antibody (Fig. 2). Enzyme activity copurified with a detected by staining with Coomassie blue. Tracks are as indicated and protein of 83 kDa when separated by SDS͞PAGE. Visualiza- are in both panels E. coli sonicate, pooled-positive cation-exchange, tion by Coomassie blue staining of chromatography fractions gel-filtration, and anion-exchange chromatography fractions. Downloaded by guest on September 29, 2021 Medical Sciences: Triglia et al. Proc. Natl. Acad. Sci. USA 94 (1997) 13947

FIG. 3. Purified recombinant PPPK-DHPS from eight different alleles. The eight alleles of PPPK-DHPS were purified using the protocol described in Material and Methods, and aliquots were separated by SDS͞PAGE and either stained with Coomassie blue (A) or electroblotted to nitrocellulose and probed with the anti-DHPS antibodies (B). In both panels the tracks contain the sample as indicated and include, in order, D10-C, 3D7-C, Tak9͞96-C, K1-C, W2 mef-C, PR145-C, D10-C͞G581, and 3D7-C͞E540.

activity of the eight PPPK-DHPS enzymes were obtained to isolates and resistance to sulfadoxine. Therefore, mutations in determine the effect of the different amino acid changes on the the DHPS enzyme are important in the mechanism of resis- catalytic properties. The Km values for pABA were found to tance to this drug. vary more than 10-fold, with D10-C having the lowest and W2 Comparison of the kinetic properties of the different en- mef-C, the highest (Table 2), whereas Vmax for all enzymes zymes not only shows that the amino acid differences are were similar. The different amino acid changes in PPPK-DHPS important in sulfadoxine resistance but also indicates the effect had little effect on catalytic turnover, kcat, varying from 0.024 they have on the magnitude of the Km and Ki. The PPPK-DHPS Ϫ1 Ϫ1 ͞ ͞ s for D10-C to 0.058 s for 3D7-C E540. The kcat Km,or enzyme 3D7-C has Gly-437 rather than Ala-437 in D10-C, and specificity constant of the different enzymes, also varied for the Ki was 10-fold higher for 3D7-C compared with the D10-C the different enzymes, which is essentially a measure of enzyme. However, this amino acid change has a minor effect catalytic efficiency for pABA, by up to 9-fold (Table 2). on the Km. Because this alteration (Gly-437) was present in To test whether the amino acid changes in the DHPS enzyme almost all sulfadoxine-resistant parasites, it suggests that this are important in the mechanism of resistance to sulfadoxine, may be one of the first mutations commonly selected under we measured the inhibitory constant for this drug. Inhibition sulfadoxine pressure. The D10-C͞G581 has Gly-581 rather was by a competitive mechanism for all enzymes, and the Ki for than Ala-581 as in D10-C, and this amino acid change has only sulfadoxine of the eight PPPK-DHPS enzymes was found to a 4-fold effect on the Ki of sulfadoxine, suggesting that this vary significantly by up to 811-fold (Table 2). The enzyme with amino acid change by itself plays a small role in resistance. the lowest Ki was D10-C, which is an enzyme present in However, when Gly-581 is combined with Gly-437, as seen with sulfadoxine-sensitive isolates (8), and the highest was with W2 K1-C, the Ki increased approximately 10-fold compared with mef-C, which is present in highly sulfadoxine-resistant isolates the single amino acid change either at Gly-581 or Gly-437, (8). 3D7-C and Tak9͞96-C are PPPK-DHPS enzymes from showing that the two amino acid changes are synergistic in isolates that have been previously defined as sulfadoxine- their effect on Ki. Similarly, comparison of the Ki for the ͞ sensitive (8). However, the Ki of these enzymes would suggest enzyme D10-C with 3D7-C, Tak9 96-C, and PR145-C shows that these isolates may show some resistance to this drug. that it increases from 0.14 to 1.39, 19.9, and 98.3 ␮M, respec- Indeed, recent analysis of the sulfadoxine sensitivity of 3D7 has tively. The differences between each consecutive enzyme is a shown that this isolate is resistant to medium levels of the drug single amino acid change (Table 1), and each alteration results (22). The PR145-C enzyme, which represents the genotype of in an increase in the Ki of the enzyme for sulfadoxine in DHPS found in 6 of the 10 Thai isolates examined, has a Ki of approximately 10-fold increments. Concomitant with the ␮ 98.3 M, suggesting these isolates are highly resistant to change in Ki is a corresponding increase in the Km of the sulfadoxine. Comparison of the Ki of the different enzymes enzymes for pABA. The effect of the amino acid changes on shows that there is a relationship between the amino acid Ki of the enzymes suggests that an accumulation of mutations differences found in the DHPS enzyme of P. falciparum in DHPS, as a result of selective pressure with sulfadoxine in

Table 2. Kinetic parameters of the PPPK-DHPS enzymes

Vmax, mU͞mg kcat, kcat͞Km, Ϫ1 Ϫ1 Ϫ1 6 Enzyme Km,nM Ki, ␮M protein s s M ϫ 10 D10-C 16.4 Ϯ 1.3 0.14 Ϯ 0.01 17.2 Ϯ 0.3 0.024 1.46 3D7-C 24.2 Ϯ 3.3 1.39 Ϯ 0.23 29.8 Ϯ 1.0 0.042 1.71 Tak9͞96-C 88.9 Ϯ 9.1 19.9 Ϯ 4.0 33.0 Ϯ 0.9 0.046 0.52 K1-C 77.4 Ϯ 7.2 16.2 Ϯ 2.7 27.6 Ϯ 0.7 0.038 0.50 W2mef-C 193.3 Ϯ 13.9 112 Ϯ 10 33.7 Ϯ 0.8 0.047 0.24 PR145-C 112.8 Ϯ 5.8 98.3 Ϯ 7.5 34.4 Ϯ 0.5 0.048 0.42 D10-C͞G581 20.0 Ϯ 2.3 0.55 Ϯ 0.06 32.4 Ϯ 0.9 0.045 2.25 3D7-C͞E540 105 Ϯ 10 27.7 Ϯ 6.1 41.4 Ϯ 1.1 0.058 0.55 mU, milliunits. Downloaded by guest on September 29, 2021 13948 Medical Sciences: Triglia et al. Proc. Natl. Acad. Sci. USA 94 (1997)

plots for Ki of sulfadoxine vs. the Ki for the other compounds gave no significant deviation from linearity with a slope of approximately one, suggesting that each amino acid change had an equal effect on the affinity of binding of each of the inhibitory compounds and that each inhibitor is competing for the same binding site in the active site of the enzyme. Therefore, P. falciparum parasites that are resistant to sulfadoxine are likely to show cross-resistance with these different sulfone and sulfonamide compounds. DISCUSSION The enzyme dihydropteroate synthase is the target of sulfone and sulfonamide drugs (1), which are used extensively in the control of many infections including P. falciparum. Sulfadoxine is the major sulfonamide used for the prophylaxis of P. falciparum infections, and this drug inhibits the enzyme DHPS (23) by direct competition for the substrate binding site. Identification of amino acid differences in the DHPS enzyme of sulfadoxine-resistant compared with sulfadoxine-sensitive P. falciparum isolates suggested that the mechanism of resistance may involve mutations in DHPS that alter the affinity of binding of the drug (7, 8). We have shown in this work that the amino acid differences in DHPS decrease the efficacy of inhibition by sulfadoxine and that such mutations are important for resistance of P. falciparum toward this drug. Sequencing of the DHPS gene from Thai isolates has identified a new allele that was likely to be linked to sulfadoxine resistance. These isolates originate from a geographic area in which there previously has been a heavy use of the combination of sulfadoxine and pyrimethamine, which would favor selection for drug resistance. The DHFR gene of the same Thai isolates have mutations that confer pyrimethamine resistance (18, 19, 24), and therefore, it is highly likely that the differences identified in the DHPS gene of the Thai isolates have evolved in parallel to the DHFR mutations and are important in the mechanism of resistance to sulfadoxine. This was strongly supported by our data showing that the recombinant PPPK-DHPS enzyme from these Thai isolates has an approximately 800-fold higher Ki for sulfadoxine compared with the same enzyme from a sulfadoxine-sensitive isolate. Expression and purification of functional PPPK-DHPS enzyme from P. falciparum in E. coli allowed us to directly address the role of the different amino acids found in alleles of DHPS in the mechanism of resistance to sulfadoxine by analyzing the kinetic properties of the enzyme. The recombinant enzyme was found to have a molecular mass of approximately 222 kDa, suggesting that it consists of a dimer or trimer. This is in agreement with the previously reported size of 200–250 kDa for the P. berghei PPPK-DHPS enzyme (25) FIG. 4. Comparison of the Ki values of the purified PPPK-DHPS enzymes for sulfadoxine with sulfathiazole, sulfamethoxazole, and and also the P. chabaudi enzyme (190 kDa) (16). The D10-C dapsone. The correlation coefficients are sulfadoxine:sulfathiazole, recombinant PPPK-DHPS enzyme had an apparent Km of 16.4 0.99; sulfadoxine:sulfamethoxazole, 1.0; and sulfadoxine:dapsone, nM for pABA compared with a previously measured value for 0.96; and the slopes for each plot are 0.99, 0.87, and 0.89, respectively. the unpurified enzyme in extracts of P. falciparum of 8.1 ␮M (23). Measurement of Km for T. gondii (10) and P. berghei (25) the field, has occurred, resulting in an enzyme with not only PPPK-DHPS enzymes in cell extracts and in purified form much greater Ki, which would confer resistance to this drug, showed that the apparent Km decreased by a factor of 10 upon but also with increased Km. purification. It was suggested that the conformation of the Interaction of DHPS with Sulfa Analogues. Various sulfon- enzyme was altered during purification, which allowed a amides and the sulfone dapsone were tested for their ability to greater access to the active site and thereby enhanced the inhibit the eight different recombinant PPPK-DHPS enzymes. apparent affinity (decreased Km) of the substrate for the The Ki of the sulfonamides (sulfadoxine, sulfamethoxazole, enzyme (10). However, it is more likely that an inhibitor or a and sulfathiazole) varied for each enzyme by approximately modulator is present in the unpurified enzyme preparations, 5-fold, with sulfathiazole being the most potent among them which would explain the lower Km values in more purified (Fig. 4). The sulfone dapsone was found to be the most potent extracts. The low Km for pABA would suggest that the P. compound tested, which is in agreement with previous results falciparum enzyme would be capable of efficient catalysis at obtained by assaying DHPS enzyme activity in extracts of P. very low concentrations of pABA. falciparum isolates (23). The amino acid changes in the PPPK-DHPS enzymes had a The different amino acid changes in the eight DHPS en- large effect on both the apparent Km for pABA and the Ki for zymes had similar effects on the magnitude of the Ki for the sulfadoxine of the PPPK-DHPS enzyme. Because sulfadoxine sulfone and sulfonamide compounds (Fig. 4). The slope of the is a structural analogue of pABA, the increasing Ki and Km for Downloaded by guest on September 29, 2021 Medical Sciences: Triglia et al. Proc. Natl. Acad. Sci. USA 94 (1997) 13949

each amino acid change suggests that most of the alterations This study clearly has shown that the different amino acids are affecting the interaction of the homologous moiety of the seen in the DHPS enzyme of sulfadoxine-resistant P. falcipa- inhibitor, rather than the side group, with the active site of the rum are important in the mechanism of resistance to this enzyme. The DHPS enzyme, when compared with other compound and other sulfone and sulfonamide drugs. How- organisms, is not highly conserved (7, 8); however, the muta- ever, it does not rule out involvement or alterations in other tions linked to sulfadoxine resistance in the DHPS enzyme of genes contributing to the level of resistance. Analysis of the P. falciparum are located in or very near to small regions of genetic cross between a sulfadoxine-resistant and a sulfadox- highly conserved amino acids, and it is likely that many of the ine-sensitive parent has shown that mutations in DHPS are mutations identified are at or very close to the pABA binding important in the cloned line tested, but it has also implicated site in the catalytic site of the DHPS enzyme. The large effect a second gene (22). It is difficult to extrapolate the profound of the amino acid mutations on Km of the DHPS enzyme for differences in Ki of sulfadoxine of the purified PPPK-DHPS pABA would suggest that a parasite expressing some of these enzymes to the effect the amino acid changes would have in mutant enzymes would be unable to efficiently utilize this vivo on the magnitude of resistance to the drug, but it is highly substrate at low cytosolic pABA concentrations. However, the likely that they are of major importance in the level of parasites that express this enzyme do not show any obvious resistance to sulfadoxine that is seen in P. falciparum. deleterious growth effects, suggesting that they are able to cope with a less efficient enzyme or that the concentration of We thank S. Caruana and M. Swift for culture of P. falciparum pABA is sufficiently high even compared with the highest K . parasites and Dr. S. Thaithong for providing the isolates from Thai- m land. We also thank Drs. H. Ginsburg, L. Tilley, and B. Scopes for The single amino acid change, Ala-437 to Gly-437, is found in helpful advice. This research was supported by the Australian National all of the DHPS sequences from sulfadoxine-resistant isolates, Health and Medical Research Council, the United Nations Develop- suggesting that this may be the first mutation in response to drug ment Program͞World Bank͞World Health Organization Special Pro- pressure. The 3D7 and Tak9͞96 isolates are resistant to medium gram for Research, and Training in Tropical Diseases. A.F.C. is levels of sulfadoxine, and subsequent selection would result in supported by an International Research Scholar’s award from the additional mutations that have an additive effect on the Ki and, Howard Hughes Medical Institute. therefore, the level of resistance of the parasite to the drug. This 1. Brown, G. M. (1971) Adv. Enzymol. Relat. Areas Mol. Biol. 35, is similar to the results found in resistance of pyrimethamine for 35–77. the enzyme DHFR in P. falciparum (24). 2. Schellenberg, K. A. & Coatney, G. R. (1961) Biochem. Pharma- The amino acid changes in DHPS have a profound effect on col. 6, 143–152. the Ki of sulfadoxine, proving that these alterations are im- 3. Krungkrai, J., Webster, H. K. & Yuthavong, Y. (1989) Mol. portant in the mechanism of resistance to this drug. The P. Biochem. Parasitol. 32, 25–38. falciparum line 3D7, which has a DHPS sequence identical to 4. Ferone, R. (1970) J. Biol. Chem. 245, 850–854. D10 except for the single alteration Gly-437, has been previ- 5. Chulay, J. D., Watkins, W. M. & Sixsmith, D. G. (1984) Am. J. ously classed as sensitive (8). However, more recent data have Trop. Med. Hyg. 33, 325–330. shown that the 3D7 parasite line was resistant to low levels of 6. Landgraf, B., Kollaritsch, H., Wiedermann, G. & Wernsdorder, W. H. (1994) Trans. R. Soc. Trop. Med. Hyg. 88, 440–442. sulfadoxine (22). This is consistent with a higher Ki of the 7. Triglia, T. & Cowman, A. F. (1994) Proc. Natl. Acad. Sci. USA 91, 3D7-C PPPK-DHPS enzyme compared with D10-C. A major 7149–7153. problem with the classification of P. falciparum with respect to 8. Brooks, D., Wang, P., Read, M., Watkin, W., Sims, P. & Hyde, resistance to sulfadoxine is the difficulty of the in vitro parasite J. (1994) Eur. J. Biochem. 224, 397–405. IC50 assay. This is exemplified by the large variation in IC50 that 9. Volpe, F., Dyer, M., Scaife, J. G., Darby, G., Stammers, D. K. & has been reported for this drug, probably as a result of differing Delves, C. J. (1992) Gene 112, 213–218. concentrations of pABA and folate in the growth medium that 10. Allegra, C. J., Boarman, D., Kovacs, J. A., Morrison, P., Beaver, can confound the assay (8, 26). The line Tak9͞96 has also been J., Chabner, B. A. & Masur, H. (1990) J. Clin. Invest. 85, 371–379. classified as sensitive to sulfadoxine (8), but because of the 11. Dallas, W. S., Gowen, J. E., P. H., R., Cox, M. J. & Dev, I. K. 174, high K of the Tak9͞96-C enzyme it is likely that it is also (1992) J. Bacteriol. 5961–5970. i 12. Lopez, P., Espinosa, M., Greenberg, B. & Lacks, S. A. (1987) J. resistant to sulfadoxine. The PPPK-DHPS enzymes PR145-C Bacteriol. 169, 4320–4326. and W2 mef-C are from highly sulfadoxine-resistant parasites, 13. Fermer, C., Kristiansen, B.-E., Skold, O. & Swedberg, G. 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Agents Che- i mother. 35, 267–271. suggesting that they are all competing for the pABA binding 24. Sirawaraporn, W., Sirawaraporn, R., Cowman, A. F., Yuthavong, site. Therefore, it is likely that the different P. falciparum Y. & Santi, D. V. (1990) Biochemistry. 29, 10779–10785. isolates with DHPS genotypes of sulfadoxine-resistant para- 25. Ferone, R. (1973) J. Protozool. 20, 459–463. sites would be cross-resistant to the other sulfone and sulfon- 26. Watkins, W. M., Sixsmith, D. G., Chulay, J. D. & Spencer, H. C. amide compounds tested here. (1985) Mol. Biochem. Parasitol. 14, 55–61. Downloaded by guest on September 29, 2021