Spontaneous mutations in the Plasmodium falciparum sarcoplasmic/ endoplasmic reticulum Ca2+-ATPase (PfATP6) gene among geographically widespread parasite populations unexposed to artemisinin-based combination therapies

Abstract

Recent reports on the decline of the efficacy of artemisinin-based combination therapies (ACTs) indicate a serious threat to malaria control. The endoplasmic/sarcoplasmic reticulum Ca(2+)-ATPase ortholog of Plasmodium falciparum (PfSERCA) has been suggested to be the target of artemisinin and its derivatives. It is assumed that continuous artemisinin pressure will affect polymorphism of the PfSERCA gene (serca) if the protein is the target. Here, we investigated the polymorphism of serca in parasite populations unexposed to ACTs to obtain baseline information for the study of potential artemisinin-driven selection of resistant parasites. Analysis of 656 full-length sequences from 13 parasite populations in Africa, Asia, Oceania, and South America revealed 64 single nucleotide polymorphisms (SNPs), of which 43 were newly identified and 38 resulted in amino acid substitutions. No isolates showed L263E and S769N substitutions, which were reportedly associated with artemisinin resistance. Among the four continents, the number of SNPs was highest in Africa. In Africa, Asia, and Oceania, common SNPs, or those with a minor allele frequency of ≥0.05, were less prevalent, with most SNPs noted to be continent specific, whereas in South America, common SNPs were highly prevalent and often shared with those in Africa. Of 50 amino acid haplotypes observed, only one haplotype (3D7 sequence) was seen in all four continents (64%). Forty-eight haplotypes had frequencies of less than 5%, and 40 haplotypes were continent specific. The geographical difference in the diversity and distribution of serca SNPs and haplotypes lays the groundwork for assessing whether some artemisinin resistance-associated mutations and haplotypes are selected by ACTs.

FIG. 1.

Polymorphism in the P. falciparum SERCA (PfATP6) genes from worldwide parasite populations unexposed to artemisinin-based combination therapies. Nonsynonymous substitutions and synonymous substitutions observed in 656 samples examined in this study are highlighted in pink and blue, respectively, alongside the 3D7 sequence (PlasmoDB, PFA0310c). Substitutions reported by other investigators are underlined. Sequence regions for 10 transmembrane domains, 6 cytoplasmic domains, 5 luminal domains, putative calcium-binding sites (*), and a phosphorylation site (boxed D at 358) were inferred from the rabbit serca gene (Swiss-Prot Protein Data Bank [PDB] code P04191). The L at position 263, where experimental substitution to E results in abrogation of inhibition of PfSERCA by artemisinin (32), is indicated by $. The S769N substitution, which has been shown to be associated with an increased artemether IC₅₀ in French Guiana (13), is indicated by #. Dashes between positions 465 and 466 and at position 844 denote deletions.

FIG. 2.

Frequency distributions of SNPs and amino acid haplotypes in serca of P. falciparum isolates from Africa, Asia, Oceania, and South America. (a) Alleles in SNPs are divided into those with a minor allele frequency of <5% (open bars) and those with a minor allele frequency of ≥5% (shaded bars). (b) SNPs are divided into those that are continent specific (open bars) or not (shaded bars). (c) Amino acid haplotypes are divided into those with a minor haplotype frequency of <5% (open bars) and those with a minor haplotype frequency of ≥5% (shaded bars). (d) Amino acid haplotypes are divided into those that are continent specific (open bars) or not (shaded bars). The vertical axes represent the number of isolates.

FIG. 3.

Amino acid haplotypes of P. falciparum serca observed in 656 worldwide samples. Amino acid positions are numbered according to the 3D7 serca sequence (PFA0310c). Deletions of N next to position 465 are dashed. “Others,” haplotypes of cultured parasites: 1, 3D7 and Dd2; 9, two Sudanese isolates; 10, one Sudanese isolate; 50, HB3.