Lack of association of the S769N mutation in Plasmodium falciparum SERCA (PfATP6) with resistance to artemisinins - PubMed (original) (raw)

Lack of association of the S769N mutation in Plasmodium falciparum SERCA (PfATP6) with resistance to artemisinins

Long Cui et al. Antimicrob Agents Chemother. 2012 May.

Abstract

The recent emergence of artemisinin (ART) resistance in Plasmodium falciparum in western Cambodia, manifested as delayed parasite clearance, is a big threat to the long-term efficacy of this family of antimalarial drugs. Among the multiple candidate genes associated with ART resistance in P. falciparum, the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase PfATP6 has been postulated as a specific target of ARTs. The PfATP6 gene harbors multiple single-nucleotide polymorphisms in field parasite populations, and S769N has been associated with decreased sensitivity to artemether in parasite populations from French Guiana. In this study, we used an allelic exchange strategy to engineer parasite lines carrying the S769N mutations in P. falciparum strain 3D7 and evaluated whether introduction of this mutation modulated parasite sensitivity to ART derivatives. Using three transgenic lines carrying the 769N mutation and two transgenic lines carrying the wild-type 769S as controls, we found that S769N did not affect PfATP6 gene expression. We compared the sensitivities of these parasite lines to three ART derivatives, artemether, artesunate, and dihydroartemisinin, in 18 biological experiments and detected no significant effect of the S769N mutation on parasite response to these ART derivatives. This study provides further evidence for the lack of association of PfATP6 with ART resistance.

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Figures

Fig 1

Fig 1

Development of transgenic lines in 3D7 with the PfATP6 S769N mutation. (A) Schematic representation of single-crossover event at the Pfatp6 locus. (Top) The Pfatp6 locus on chromosome 1. Solid lines represent introns or intergenic regions, and filled boxes indicate the coding regions. (Middle) Plasmid pHD22y-pfatp6-769N, showing the Pfatp6 genomic fragment and the drug selection cassette hDHFR. (Bottom) Predicted single-crossover events at the Pfatp6 locus. The fragment within the bracket indicates the scenario when integration of concatemerized plasmid occurs. The C-terminal fragment of PfATP6 cloned in the transfection plasmid is shown as filled boxes. The open and filled lozenges indicate the locations of the wild-type and mutant amino acids at position 769, respectively. Restriction enzyme KpnI sites and the expected sizes of DNA fragments after KpnI digestion are illustrated. The positions and orientations of the primers on chromosome 1 and the plasmid are shown. Primer pairs P1 and P2 were used for integration-specific PCR, whereas primers P3 and P4 were used for determining the copy number of the integrated plasmid. The position of the probe used for genomic Southern blotting is also marked. (B) Integration-specific PCR products, based on used of primer P1 on chromosome 1 and primer P2 on the plasmid, showing 32 positive and 2 negative clones (lanes 28 and 32). The PCR products of 32 positive clones were sequenced, and asterisks indicate the three clones with the S769N mutation. (C) Copy numbers of the integrated plasmid or concatemers at the Pfatp6 locus as determined by real-time PCR using primer pairs P3 and P4. Shown here are five transgenic clones, with two containing the wild-type residue (739S-9 and 769S-17) and three with the 769N mutation (769N-2, 769N-7, and 769N-31). (D) Genomic Southern blot of DNA isolated from 3D7 and five clones with plasmid integration at the Pfatp6 locus. Genomic DNA was digested with KpnI and separated in a 1% agarose gel. The blot was hybridized with the probe marked in panel A, which revealed a ca. 10-kb fragment in 3D7 and 5.8-kb fragment in the recombinant Pfatp6 locus.

Fig 2

Fig 2

Pfatp6 expression in wild-type and S769N mutant clones. Pfatp6 expression levels are shown in the ring (12 h), trophozoite (30 h), and schizont (38 h) stages. The relative expression level of Pfatp6 was determined by real-time PCR analysis using primers P3 and P4. A housekeeping gene, seryl-tRNA synthetase (PF07_0073), was used as an internal control. There were no significant differences in mRNA levels among the parasite clones at each development time point (P > 0.05, ANOVA).

Fig 3

Fig 3

Scatter plot of IC50s of 3D7 and five transgenic clones carrying either 769N (2, 17, and 31) or 769S (9 and 17), assayed with ATM (top), ATS (middle), and DHA (bottom). Each value indicates the mean from three technical replicates. The means ± standard deviations were calculated from the means of 18 biological experiments. For statistical comparison, data were normalized using natural logarithm transformation. For each drug, there were no significant differences among the parasite lines after controlling for multiple tests (P > 0.05, unpaired t test).

Fig 4

Fig 4

Dose-response curves of 3D7 and five parasite clones with either 769N (2, 17, and 31) or 769S (9 and 17) assayed against ATM (top), ATS (middle), and DHA (bottom). The results were obtained from 18 independent experiments, each with three technical replicates. Percent inhibition values are shown as means ± standard deviations.

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