Geographic structuring of the Plasmodium falciparum sarco(endo)plasmic reticulum Ca2+ ATPase (PfSERCA) gene diversity

Abstract

Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC(50) for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

Figure 1. Predicted domains and localization of the mutations in Pf-SERCA protein sequence.

A) Amino-Acid sequence of Pf-SERCA The functional domains of the protein are located according to the alignment with the rabbit ATPase: A (boxed), P (box-grey) and N (underlined), Plasmodium-specific domains are shown in italic. Hinge between P and N domains are bolded (TNQMT and DPPRK). Introns are located in the gene after L1019 and K1119 (noted by the sign/). Transmembrane domains are bold-underlined. Mutations are boxed in grey (non-synonymous mutation in bold). Three catalytic domains are predicted by PROSITE: cation atpase_N 3 – 81 and ATPase_C 1031–1215 (red), E1_E2 atpase 99 – 348/358-364 (blue), hydrolase 790–935 (braun). The position of the two introns is indicated with a/. B) Prediction of transmembrane domain by HMM (www.PROSITE.org) Ten transmembrane domains were predicted using TMpred with “in to out” (i-o) or “out to in”(o-i) orientation: M1 AA67-85 (o-i), M2 AA94-116 (i-o), M3 AA216-233 (o-i), M4 AA271-290 (i-o), M5 AA298-325 (o-i), M6 AA979-1005 (i-o), M7 AA1053-1073 (o-i), M8 AA1124-1140 (i-o), M9 AA1158-1175 (o-I), M10 AA1183-1206 (i-o). C) Schematic representation of the catalytic domains of Pf-SERCA and localization of the mutations. Mutations are given symbols by continent of origin of the isolates (non synonymous mutations depicted with filled arrows, synonymous mutations depicted with open arrows).

Figure 2. TCS network for the pfserca gene of Plasmodium falciparum.

Each distinct haplotype is represented by an oval with size proportional to its frequency (in parenthesis). Mutational steps are represented by lines. Dots connecting lines represent putative haplotypes that were not sampled. Dashed lines refer to reticulations corrected according to Crandall et al. . Colours correspond to each continent sampled: blue - America (AM: Amazonas; FG: French Guiana; PA: Para), green - Africa (EQ: Equatorial Guinea; SE: Senegal), red - Asia (CA: Cambodia; TH: Thailand).