Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum

Abstract

Plasmodium falciparum causes the most severe form of malaria in humans. An important class of drugs in malaria treatment is the sulfone/sulfonamide group, of which sulfadoxine is the most commonly used. The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resistance to this drug. In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference. We have expressed eight alleles of P. falciparum PPPK-DHPS in Escherichia coli and purified the functional enzymes to homogeneity. Strikingly, the Ki for sulfadoxine varies by almost three orders of magnitude from 0.14 microM for the DHPS allele from sensitive isolates to 112 microM for an enzyme expressed in a highly resistant isolate. Comparison of the Ki of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds. These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of P. falciparum are central to the mechanism of resistance to sulfones and sulfonamides.

Figure 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 SDS/PAGE 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, Tak9/96-C, K1-C, W2 mef-C, PR145, D10-C/G581, and 3D7-C/E540.

Figure 2

Purification of functional recombinant PPPK-DHPS enzyme from P. falciparum in E. coli. Four liters of either D10-C or K1-C were grown and the PPPK-DHPS activity purified by sequential steps of cation-exchange, gel-filtration, and anion-exchange chromatography. Purification was monitored by enzyme activity and detection with anti-DHPS antibodies. Aliquots of D10-C (A) or K1-C (B) chromatography aliquots were separated by SDS/PAGE, and protein was detected by staining with Coomassie blue. Tracks are as indicated and are in both panels E. coli sonicate, pooled-positive cation-exchange, gel-filtration, and anion-exchange chromatography fractions.

Figure 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 SDS/PAGE 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, Tak9/96-C, K1-C, W2 mef-C, PR145-C, D10-C/G581, and 3D7-C/E540.

Figure 4

Comparison of the K_i values of the purified PPPK-DHPS enzymes for sulfadoxine with sulfathiazole, sulfamethoxazole, and dapsone. The correlation coefficients are sulfadoxine:sulfathiazole, 0.99; sulfadoxine:sulfamethoxazole, 1.0; and sulfadoxine:dapsone, 0.96; and the slopes for each plot are 0.99, 0.87, and 0.89, respectively.