Identification and functional validation of the novel antimalarial resistance locus PF10_0355 in Plasmodium falciparum

Abstract

The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (∼ 1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.

Figure 1. Parasite global population structure and genetic diversity versus divergence.

(A) Population structure is visualized using the first two principal components of genetic variation for 57 parasites. Solid circles represent individual parasites, with colors assigned by reported origin: Africa in red, America in blue, and Asia in green. The nine strains used for ascertainment sequencing are indicated with (*). (B) Genetic diversity (SNP π) in Senegal versus divergence (F_ST) between Senegal and Thailand is reported for 688 genes containing >3 successfully genotyped SNPs. Blue diamonds: enzymes, acyl-CoA synthetases (ACS) or transporters; red diamonds: antigens, vars, rifins, stevors or surfins; gray diamonds: all other genes. Gene IDs (PlasmoDB.org) for highlighted genes are listed in Table S7. A gene with unknown function is flagged with (*) to indicate that SNP π is off-scale (0.014).

Figure 2. Genome-wide association study (GWAS) results.

(A) Genome-wide significant associations were found for five antimalarials (out of thirteen tested) using EMMA and HLR tests. They include pfcrt (chromosome 7) associated with chloroquine resistance and eleven novel associations with resistance to several drugs, listed in Table 1. (B) Quantile-quantile plots for the P-value distributions in (A) show no significant confounding from population structure. Bonferroni-corrected genome-wide significance is marked with a dashed line; Benjamini-Hochberg significance is marked with a dotted line. (C-D) Close-ups are shown of the GWAS signal (top) and haplotypes (bottom) for resistance to (C) chloroquine (CQ) around the gene pfcrt and (D) halofantrine (HFN) around the gene PF10_0355. Yellow: sensitive allele; red: resistant allele; Blue: no data. Isolates are ordered by IC₅₀, with the highest IC₅₀ on the bottom.

Figure 3. Overexpression of PF10_0355 decreases parasite susceptibility to halofantrine (HFN) and related antimalarials.

Parasite susceptibility to six antimalarials was measured by ³H-hypoxanthine incorporation. Comparisons were made between Dd2 (HFN-sensitive strain) and SenP08.04 (HFN-resistant strain), as well as 4 transfected lines. “Dd2+Dd2”: Dd2 parasites overexpressing PF10_0355 from Dd2; “Dd2+P08”: Dd2 parasites overexpressing PF10_0355 from SenP08.04. Overexpression of PF10_0355 decreases parasite susceptibility to (A) HFN and structurally related (B) mefloquine (MFQ) and (C) lumefantrine (LUM). Overexpression of PF10_0355 does not alter parasite susceptibility to (D) chloroquine (CQ), (E) artemisinin (ARTS) or (F) atovaquone (ATV). Mean IC₅₀ ± standard error is shown. Significance levels: *: p<0.05, **: p<0.01, ***: p<0.001.

Figure 4. Correlations between antimalarial drugs tested.

(A) Pearson correlation values (r) between log₁₀(IC₅₀) values are rendered as a color in a symmetric correlation matrix (red: correlated; white-uncorrelated, blue: inversely correlated). Thirteen antimalarials are measured: artemether (ARTM), artesunate (ARTN), artemisinin (ARTS), dihydroartemisinin (DHA), halofantrine (HFN), lumefantrine (LUM), mefloquine (MFQ), quinine (QN), chloroquine (CQ), amodiaquine (ADQ), atovaquone (ATV), piperaquine (PIP), and halofuginone (HFG). Drugs are grouped by structural relatedness. (B–F) Correlation plots are given with a linear regression line for HFN compared to the 5 other drugs tested for antimalarial resistance with PF10_0355 overexpression: (B) LUM, (C) MFQ, (D) ATV, (E) CQ, and (F) ARTS.

Figure 5. Copy number variation at PF10_0355 is associated with HFN resistance.

(A) Mean PF10_0355 copy number (± standard deviation for three replicates) in the parent Dd2 and transfected lines from qPCR analysis. Dd2+Dd2: Dd2 parasites overexpressing PF10_0355 from HFN-sensitive Dd2; Dd2+P08: Dd2 parasites overexpressing PF10_0355 from HFN-resistant SenP08.04. Copy number was compared to the reference locus PF07_0076. (B) Increased hybridization intensity at PF10_0355 on the high-density SNP array, measured by Z-scores for normalized and background-corrected data, for the HFN-resistant isolate SenP19.04. (C) Strains with increased copy number of PF10_0355 (as measured by qPCR >1.2x 3D7) show a significantly higher resistance to HFN (p = 0.02, Student t-test).