Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness

Abstract

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance in vitro and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping using 34 recombinant haplotypes, and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

Fig. 1.. Antimalarial susceptibilities of the genetic cross parents RF7 × NF54 and the experimental workflow for bulk selection versus individual clone-based linkage mapping.

(A) Bar plots of % survival derived from the DHA ring-stage survival assay (RSA) or the PPQ survival assay (PSA) and the area under the curve (AUC) values, as determined for the genetic cross parents RF7 and NF54. In the RSAs and PSAs, early ring-stage parasites were exposed to the pharmacologically relevant concentrations of 700 nM DHA for 4 hours or 200 nM PPQ for 72 hours, respectively, and survival was measured as a percentage of mock-treated cultures. (B) IC₅₀ values of RF7 and NF54 parasites exposed for 72 hours to first-line antimalarials. Values represent the means ± SE (N = 3 to 17, n = 2). Statistical significance was determined by unpaired Student’s t tests, with a Holm-Sidak post hoc test to correct for multiple comparisons. The IC₅₀ fold shifts are indicated above the bars. *P < 0.05, **P < 0.01, ***P < 0.001. Numbers listed above the statistics indicate the fold differences between parental IC₅₀ values. The table summarizes parental phenotypic and genotypic characteristics. WT, wild-type. Mutant genotypes include the following: Dd2 pfcrt mutations: M74I, N75E, K76T, A220S, Q271E, N326S, I356T, and R371I; quadruple dhfr mutations: N51I, C59R, S108N, and I164L. (C) Overview of the genetic cross pipeline performed in humanized FRG-NOD mice. huRBCs, human red blood cells. (D) Bulk selection approach used for bulk segregant analysis. (E) Individual clone-based linkage approach used to identify genetic determinants of drug resistance. Images were created with BioRender.com.

Fig. 2.. Bulk segregant analyses of progeny pools pressured with PPQ, mdCQ, or PYM.

The significant genetic loci enriched by each drug in individual drug-drug pairwise comparisons are shown with black lines representing G′ values (left), along with their corresponding RF7 parental allele frequencies for the set of 18k SNPs that differ between the two parents (right). The G′ is the smoothed statistical value calculated for individual SNPs and used for detecting QTLs. Regions with a false discovery rate q value < 0.01 (mdCQ versus PYM), < 0.4 (PPQ versus PYM), or < 0.2 (PPQ versus mdCQ) were considered statistically significant QTLs (table S5). Genes enriched by each drug and the RF7 parental allele frequency for the drug-treated samples are colored as follows: mdCQ (orange), PYM (purple), and PPQ (green). Individual allele frequencies for each drug-treated pool are indicated by light dots in the background. The solid colored lines represent the averaged RF7 allele frequency across windows of 100 consecutive SNPs.

Fig. 3.. Genetic analysis of recombinant progeny and enrichment of diverse progeny using drug pulses.

(A) Number and percentage of selfed progeny versus genetic recombinants obtained from cloning in the absence of drug or following drug exposure. Distribution of the 34 unique recombinants obtained with no drug and/or selected by PPQ, mdCQ, or PYM. (B) Allelic map for the 34 recombinant progeny and two parents, grouped by drug treatment condition. Shown are the parental alleles inherited by each progeny for 14,476 SNPs (after excluding SNPs missing in any of the 34 recombinant haplotypes), the genotypes of known resistance markers, and the number of derived progeny clones per haplotype sorted by selection condition. (C) Mean RF7 allele frequency inherited by the 34 recombinant progeny clones for each drug treatment group. (D) Count of KEL1/PLA1/PfPailin haplotypes in progeny clones that were selected by each drug condition. Statistical significance was tested for the drug-selected progeny clones against the “no drug” group, using Fisher’s exact test. **P < 0.01; ***P < 0.001; ns, not significant. (E) Frequency of RF7 parental allele in progeny clones derived posttreatment with PPQ (N = 6), mdCQ (N = 7), or PYM (N = 12) or in the absence of drug (N = 12). Individual allele frequencies (light dots in the background) are reported as a weighted allele frequency across the number of clones for each group. The solid colored lines represent the averaged RF7 allele frequency across windows of 100 consecutive SNPs. The skews in allele frequency at specific loci in the genome are indicated by dashed lines.

Fig. 4.. Phenotypic response of parents and progeny to DHA and PPQ.

(A and B) Dose-response curves for RF7 and NF54 parents across a range of DHA and PPQ concentrations (N = 3, n = 2). The percent survival at the RSA and PSA concentrations of 700 nM DHA and 200 nM PPQ for 4 or 72 hours, respectively, were used to determine DHA and PPQ resistance levels, whereas AUC values were measured as total survival across a range of concentrations (22 nM to 2.8 μM for DHA and 1.6 nM to 25.6 μM for PPQ). (C and D) DHA and PPQ response measured by %RSA and DHA AUC values (C) and %PSA and PPQ AUC values (D), respectively, in the 34 independent recombinant haplotypes and two parents. Progeny clones were obtained either in the absence of drug pressure (gray) or after selection with PYM (purple), mdCQ (orange), or PPQ (green). Data are plotted alongside RF7 (red arrow) and NF54 (blue arrow). Each bar represents the mean percent survival ± SE or AUC ± SE for a recombinant haplotype profiled in four independent experiments with technical duplicates. Haplotypes are ordered by the parasites’ resistance levels for each drug metric. For certain haplotype groups in which two or more identical clones were obtained, multiple points depict phenotypic results for more than one sibling progeny.

Fig. 5.. QTL mapping of DHA responses in progeny identifies k13 as the primary DHA resistance locus.

(A and B) LOD plots for %RSA and AUC levels showing the significant QTLs above the 95% probability threshold (red line). (C) List of QTL segments for %RSA and AUC levels. (D) Genes in QTL segment on chr13, with those having nonsynonymous mutations between RF7 and NF54 colored in orange (n = 20) or gray where mutations were absent in RF7 and NF54. Gene names are as listed. uf, unknown function. (E) The %RSA and AUC levels in recombinant progeny segregated by k13 parental allele, C580Y (in RF7) and WT (in NF54). Significance was tested using Mann-Whitney U tests. ***P < 0.001. (F) DHA responses in k13-edited isogenic RF7 clones showed that the k13 WT allele reduces %RSA and AUC levels. Bars represent the means ± SE (N = 4, n = 2). Significance was tested using Mann-Whitney U tests. *P < 0.05. (G and H) Linkage of chr1 (G) and chr14 (H) segments with pfcrt from QTL analysis using the pfcrt genotype as an outcome. Shown are the significant QTLs above the 95% probability threshold for each analysis. (I) LOD plot for AUC levels after adjusting for k13 as a covariate suggests independent inheritance of the chr14 segment with k13 and coinheritance of the chr1 segment with k13. (J) Scatterplot of AUC levels in progeny segregated by k13, mrp1 (chr1), and arps10 (chr14) genotypes, showing that the RF7 chr14 segment is associated with increased AUC levels. We note that none of the progeny harbored MUT mrp1 and WT k13 alleles. Significance between groups was tested using Mann-Whitney U tests. *P < 0.05, ***P < 0.001. MUT, mutant.

Fig. 6.. QTL mapping of PPQ resistance in progeny and response in pfcrt-edited progeny reveal an association of PPQ resistance with mutant Dd2 + M343L pfcrt and multicopy pm2/3.

(A and B) LOD plots for %PSA and AUC levels showing QTLs detected above the 95% probability threshold (red line). (C) List of QTL segments for %PSA and AUC levels. (D and E) Scatterplot of the %PSA or AUC levels for 34 independent recombinant progeny and parents, segregated by pm2/3 copy and pfcrt genotypes. NF54, 3D7 pfcrt; RF7, Dd2 + M343L pfcrt. Significant differences in PPQ response between the recombinant groups harboring different pm2/3 copies were tested by Mann-Whitney U tests. *P < 0.05, ***P < 0.001. (F) PPQ response in isogenic RF7 clones containing one versus three copies of pm2/3 in the mutant Dd2 + M343L pfcrt background, showing that single-copy pm2/3 reduces %PSA and AUC levels. Bars represent the means ± SE (N = 4, n = 2). Significance was tested using Mann-Whitney U tests. *P < 0.05. (G) PPQ %PSA and (H) AUC levels in pfcrt-edited HapD progeny (3D7 versus Dd2 + M343L alleles) with single pm2/3 copy, and in pfcrt-edited HapU progeny (Dd2 versus Dd2 + M343L alleles) with either one, two, or three copies of pm2/3. Bars represent the means ± SE (N = 4, n = 2). Significance between the isogenic edited progeny lines was tested using Mann-Whitney U tests. *P < 0.05. The colored key for parasite lines applies to (G) to (L). (I to L) Dose-response curve in RF7 clones [I; colored the same as (F)], HapD (J), unedited HapU (K), and pfcrt-edited HapU (L) progeny, showing that multicopy pm2/3 and mutant pfcrt are necessary for the PPQ biphasic response. Each line depicts the mean percent parasite survival ± SE (N = 3 to 4, n = 2). In parallel studies, serum was noted to lower %PSA (fig. S8, B and C).

Fig. 7.. Impact of mutant k13 and multicopy pm2/3 on parasite asexual fitness.

(A) Experimental design showing the generation of the panel of isogenic RF7 lines used in the coculture competitive fitness assays. (B) Pairwise competitive growth assays showing the proportion of k13 C580Y or WT alleles in the RF7 line harboring either three copies or one copy of pm2/3 and the proportion of RF7 lines carrying either three copies or one copy of pm2/3. Values shown are the averaged percentages from four independent experiments with two technical replicates per pairwise competition assay. (C) Fitness cost per generation, showing the relative change in percentage of the k13 and pm2/3 genotypes for each pairwise comparison.

Fig. 8.. QTL mapping of LMF and MFQ response identifies multiple peaks including mutant Dd2 + M343L pfcrt.

(A) LMF IC₅₀ and (B) MFQ IC₅₀ values of the 34 unique recombinant haplotypes and two parents. Each bar represents the mean ± SE IC₅₀ for a recombinant haplotype (N = 4, n = 2). Haplotypes were ordered by LMF IC₅₀ levels. For certain haplotype groups in which identical clones were obtained, multiple points depict data for more than one sibling progeny. Insets depict LMF and MFQ dose-response curves for RF7 (red) or NF54 (blue). Each curve represents the mean ± SD for an independent experiment. (C and D) LOD plots showing QTLs for LMF and MFQ above or near the 95% probability threshold (red line). (E and F) LMF and MFQ IC₅₀ values for the 34 recombinant progeny and two parents, grouped by their pfcrt haplotypes (E) or grouped by their atg18 and pfcrt haplotypes (F). MUT, mutant T38I. (G) LMF IC₅₀ and (H) MFQ IC₅₀ values in pfcrt-edited HapD progeny (3D7 versus Dd2 + M343L) having single pm2/3 copy, and in edited HapU progeny (Dd2 versus Dd2 + M343L) having either one, two, or three copies of pm2/3. Bars represent the means ± SE (N = 3 to 4, n = 2). The colored key for parasite lines applies to (G) to (K). (I) LMF and (J) MFQ dose-response curves in edited HapD progeny showing that mutant Dd2 + M343L pfcrt confers increased tolerance to these drugs. Each line depicts the mean ± SE percent parasite survival (N = 3 to 4, n = 2). (K) MFQ dose-response curve in pfcrt-edited HapU progeny with variable pm2/3 copies, showing that single pm2/3 copy associates with reduced MFQ sensitivity. Each line depicts the mean ± SE percent parasite survival (N = 4, n = 2). Statistical significance was determined by Mann-Whitney U tests (A, B, E, and F) or unpaired Student’s t tests (G and H). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 9.. Phenotypic response of genetic cross parents, RF7 × NF54, to preclinical compounds and identification of pfcrt, abci3, pm2/3 as markers of MMV665939 and MMV675939 resistance.

(A) Seventy-two–hour drug susceptibility IC₅₀ values for RF7 × NF54. IC₅₀ fold shifts are indicated above the bars. Values represent the means ± SE (N = 3 to 7, n = 2). (B) Dose-response curves, showing a sigmoidal survival curve for MMV665939 and a biphasic response for MMV675939 in RF7. Each line represents the mean ± SD percent parasite survival per experiment (N = 4, n = 2). (C and D) LOD plots for MMV665939 IC₅₀ and MMV675939 AUC levels showing QTLs above the 95% confidence threshold. (E) MMV665939 IC₅₀ and (F) MMV675939 AUC levels for the 34 recombinant progeny and two parents, grouped by pm2/3 copy, pfcrt, and abci3 genotypes. (G) MMV665939 IC₅₀ and (H) MMV675939 AUC levels in pfcrt-edited HapD progeny (3D7 versus Dd2 + M343L) and in edited HapU progeny (Dd2 versus Dd2 + M343L) having either one, two, or three copies of pm2/3. Values represent the means ± SE (N = 4, n = 2). The colored key for parasite lines applies to (G) to (K). (I) MMV675939 dose-response curves in unedited HapD (3D7) versus edited HapD (Dd2 + M343L) progeny, showing that mutant pfcrt is sufficient to generate a biphasic curve on a single-copy pm2/3 background. For (I) to (K), each line depicts the mean ± SE percent parasite survival (N = 4, n = 2). (J) MMV675939 dose-response curves of HapU (Dd2 + M343L) with one to three copies of pm2/3, showing that multicopy pm2/3 can augment the biphasic response. (K) MMV675939 dose-response curves in pfcrt-edited HapU (Dd2) progeny with one to three copies of pm2/3, showing that the M343L mutation is not required to generate a biphasic response on a Dd2 pfcrt background. Statistical significance was determined by unpaired Student’s t tests adjusted by multiple testing (A) or Mann-Whitney U tests (E to H). *P < 0.05, **P < 0.01, ***P < 0.001.