Plasmodium falciparum Falcipain-2a Polymorphisms in Southeast Asia and Their Association With Artemisinin Resistance

Abstract

Background: Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and in vitro selection for artemisinin resistance selected for an FP2a nonsense mutation.

Methods: To investigate associations between FP2a polymorphisms and artemisinin resistance and to characterize the diversity of the enzyme in parasites from the China-Myanmar border, we sequenced the full-length FP2a gene in 140 P falciparum isolates collected during 2004-2011.

Results: The isolates were grouped into 8 different haplotype groups. Haplotype group I appeared in samples obtained after 2008, coinciding with implementation of artemisinin-based combination therapy in this region. In functional studies, compared with wild-type parasites, the FP2a haplotypes demonstrated increased ring survival, and all haplotype groups exhibited significantly reduced FP2a activity, with group I showing the slowest protease kinetics and reduced parasite fitness.

Conclusions: These results suggest that altered hemoglobin digestion due to FP2a mutations may contribute to artemisinin resistance.

Figure 1.

Falcipain-2a (FP2a) haplotypes of 140 China-Myanmar Plasmodium falciparum isolates. (A) Domain organization of the FP2a protein showing functional cysteine protease domain ([CP] amino acids [AA], 261–482), inhibitor domain ([ID] AA, 165–222), and the transmembrane domain ([TM] AA, 35–57). (B) Twenty-five distinct haplotypes identified in the 140 isolates were categorized into 8 haplotype groups based on sequence phylogeny. The left panel shows each haplotype groups and their frequency in 140 samples. The bar graph (right) shows the proportions of haplotype groups over time.

Figure 2.

Three-dimensional modeling of Pf3D7-falcipain-2a (FP2a) (amino acids, 245–484) using 3PNR.pdb. (A) Single-nucleotide polymorphisms in the protease domain (T343P, D345G, A353T, V393I, A400P, and Q414E) and the prodomain (M245I, E248D, E249A, K255R, and N257E) are highlighted. (B) A353, A400, and Q414 lie in the binding interface of FP2a and its inhibitor. A353 is partially buried, whereas A400 is fully buried in the interface and located in the middle of a short helix. (C) Q414 forms a hydrogen bond with the inhibitor. Abbreviation: ICP, Plasmodium berghei homolog of falstatin (inhibitor of cysteine proteases).

Figure 3.

Phenotypic characterization of representative isolates. (A) Mean survival rates from ring-stage survival assay of 140 isolates grouped according to falcipain-2a (FP2a) haplotype groups (I–VIII). Wild type (WT) = 3D7 and 3 field isolates with WT K13 and FP2a sequences. ALL = mean for all 137 isolates with FP2a single-nucleotide polymorphisms (SNPs). (B) Mean growth in amino acid-depleted complete medium (CM-AA) of 29 representative isolates. ALL = mean growth in CM-AA for all 27 isolates with FP2a SNPs. (C) Average number of merozoites (N_avg) in the mature schizonts of 29 representative isolates. ALL = mean N_avg for all 27 isolates with FP2a SNPs. *, P < .05 (t test) vs WT in A–C. (D) Michealis-Menten curve-fits of Z-LR-AMC hydrolysis by trophozoite extracts from WT and parasites in haplotype groups I–VIII.