A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria

Abstract

Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.

Extended Data Figure 1. Ring Parasite PI3P and PI3K: effect of inhibitors, and characterization of antibodies

a, Rings at 6 h post-invasion, were either mock treated or exposed to 4 nM DHA for 4 h. An aliquot was washed in serum-free RPMI and cultured for 24 h. Parasitemia (ring and trophozoite stages) were determined by Giemsa staining and light microscopy. Mean (error bars SD < 2) from three experimental replicates are shown. b, Effect of artemisinins, deoxyartemisinin, PI3K inhibitors wortmannin and LY294002 (and its inactive orthologue LY303511) on PI3P level, as observed by redistribution of the PI3P reporter in transgenic 3D7 parasites expressing SS-EEA1^WT-mCherry (red). Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescence and phase-merged images are as indicated. c, Quantitative analysis of the data in b. Mean (error bars SD < 21) from three experimental replicates are shown. d, Effect of anti-folates and chloroquine on PI3P production, as observed by redistribution of the PI3P reporter in transgenic 3D7 parasites expressing SS-EEA1^WT-mCherry (red). Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescence and phase-merged images are as indicated. e, Quantitative analysis of the data in d. Mean (error bars SD < 27) from three experimental replicates are shown. As indicated in Fig. 1, the PI3P reporter SS-EEA1^WT-mCherry (red) detects PI3P in punctate, perinuclear ER domains in 6 h rings (top panel; ) or reduced PI3P when the reporter diffuses to the PV (periphery). f, Schematic showing structural features of PfPI3K. Custom antibodies were generated in rabbits and guinea pigs against the indicated peptides. Western blots indicate that both antibodies specifically recognize a ~255-kDa protein in infected (IE) but not uninfected (U) erythrocytes and are representative of 10 experimental replicates; raw data in Supplementary Data 2. Indirect immunofluorescence assays using rabbit (top panel) and guinea pig (bottom panel) antibodies confirm the PfPI3K (green) is localized in the parasite. Exp-2 (red) marks the parasite boundary. Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescence and phase-merged images are shown. Data are representative of 10 experimental replicates.

Extended Data Figure 2. Structural analyses of PfPI3K

Sequence alignment of the putative catalytic domain of PfPI3K and its closest human orthologs human VPS34 and Drosophila VPS34. * indicates identity; dots indicate homology. D1889 and Y1915 residues are highlighted in yellow.

Extended Data Figure 3. Structural analyses of PfPI3K and human PI3K, with and without artemisinin derived compounds

a Snapshots from 20 ns MD simulations and chemical structure of artelinic acid (top right), artemisone (bottom left) and deoxyartemisinin (bottom right) bound to PfPI3K. Snapshot from MD simulation of DHA bound to PfPI3K is shown (at the top left) for reference. Due to the lack of the hydroxyl group in artelinic acid and artemisone, no interactions involving D1889 and Y1915 were observed. Instead, the carboxylic group of artelinic acid forms a salt bridge with R1368 and the sulfone moiety of artemisone interacts with R1368. Deoxyartemisinin lacks hydrogen bond donors, so D1889 is not involved in protein-ligand interaction. Instead, the carbonyl oxygen of deoxyartemisinin forms a hydrogen bond with Y1915. Boxed inset shows the change in the overall shape due to the replacement of the endoperoxide with an ether bridge in deoxyartemisinin (green) and compared to DHA (red), thus leading to loss of the shape complementarity and reorientation in the binding site. As shown in Fig. 1 panels a, c, deoxyartemisinin failed to block PI3P production, in transgenic 3D7 parasites expressing SS-EEA1^WT-mCherry (red) or purified PfPI3K. Thus, modeling studies explain a lack of effect of deoxyartemisinin. They would also suggest lack of effect of artelinic acid and artemisone compounds on PfPI3K. It should be noted the target of these compounds is unknown and it remains unclear whether they would share a ring stage target with DHA important for clinical resistance to artemisinins. b, Snapshots of MD simulation of DHA-human Vps34 complex. The initial coordinates of DHA…human Vps34 complex was obtained by the overlay of DHA…PfPI3K model (EDFig. 2b) with the human Vps34 crystal structure (PDB: 3IHY). The obtained complex was then subjected to 20 ns MD simulations. As can be seen, DHA does not interact with D644 and Y670, which correspond to D1889 and Y1915 in PfPI3K, respectively. Instead, the hydroxyl group of DHA now interacts with D761 (left). Snapshot at the right shows an overlay of DHA…human VPS34 complex with the PfPI3K model. In human VPS34 (gray), the loop that contains the D761 residue bends down to interact with DHA. In contrast, D2008 of PfPI3K (cyan), which is in the same position as D761 in human VPS34, has a different orientation which makes it unable to interact with DHA. c, Both D1889 and Y1915 in PfPI3K are conserved in human PI3K p110γ. 20 ns MD simulations of the DHA …p110γ complex (pdb code 4ANV), reveal that although the hydrogen bond between DHA and D841 still remains, the interaction between DHA and Y867 is lost. This suggests a weaker binding of DHA to p110γ compared to PfPI3K. The same argument can be made of p110δ based on sequence homology, but no crystal structure of human p110δ is available. d, Partial sequence alignment of PfPI3K, with human PI3K-C2α, PI3K-C2β, p110δ, p110γ and VPS34. The key residues of PfPI3K (D1889 and Y1915) that interact with DHA and similar positions in the human kinases are colored red. While D1889 in PfPI3K is conserved in both PI3K-C2α and PI3K-C2β, Y1915 in PfPI3K is replaced by F1172 and F1117 in PI3K-C2α and PI3K-C2β, respectively. Therefore, the hydrogen bond of the hydroxyl group in Y1915 suggested to be crucial in the computational analysis of PfPI3K cannot be formed with F. To the best of our knowledge, no crystal structures of PI3K-C2α and PI3K-C2β have been reported. In p110δ, p110γ and VPS34, a proline N-terminal to Y1915 is conserved. It is likely the reason for the different position and low flexibility of this loop observed in the MD simulations of p110δ, and VPS34.

Extended Data Figure 4. Transgenic P. falciparum expressing PfKelch13^C580Y mutation in two different strains of P. falciparum using distinct approaches and markers

a, Strategy 1: CRISPR-Cas9 used to introduce a single point mutation in the P. falciparum NF54 genome as described elsewhere. Both parent and mutant strains were sequenced to ensure that the only change detected was PfKelch13^C580Y. Western blots using anti-PfKelch13 antibodies confirm that mutation does not change the level of PfKelch13 protein. b, Strategy 2 expresses a dominant negative PfKelch13^C580Y and wild type PfKelch13^WT tagged with HA, and driven by the constitutive cam promoter in P. falciparum 3D7. Western blots confirm that anti-HA antibodies detected tagged forms of PfKelch13 in transfected but not non-transfected 3D7. Comparable levels of wild type and mutant transgenes are expressed in resulting two strains of parasites (as shown). In a-b, BiP, an ER marker serves as a parasite loading control in western blots. Molecular weight standards (in kDa) are as indicated. c, Western blot indicate that antibodies to PfKelch13 specifically recognize a ~83-kDa protein in infected (IE) but not uninfected erythrocytes (U). Molecular weight standards (in kDa) are as indicated. In a-c: data are representative of three experimental replicates; raw data are in Supplementary Data 2.

Extended Data Figure 5. An in vitro ring-stage survival assay (RSA) level is associated with PI3P elevation in the presence and absence of Kelch mutations

a, Schematic of the RSA assay. b, Summary of RSA, PI3P, PfKelch13 mutation and PfPI3K polymorphisms in clinical and laboratory strains utilized in this study. Means are shown for three experimental replicates. RSA SD < 0.5; PI3P SD < 8 (as shown by error bars in Fig. 3c). c–d, Construction of 3D7 transgenic parasites expressing myc-tagged forms of human VPS34, the mammalian ortholog of PfPI3K. The resulting Pf^VPS34-myc1/2 express the vps34 transgene in ring stage parasites, as detected in indirect immunofluorescence assays (Pf^{VPS34-MUTANT-myc} data not shown in IFA). Exp-2 (red) marks the parasite boundary. Blue, parasite nucleus, P, parasite, E, erythrocyte; scale bar, 5 μm. Fluorescent and merged images are shown. Whole genome Illumina sequencing indicated VPS34-myc1 was inserted into PF3D7_1363900 and VPS34-myc2 is inserted in PF3D7_0718800. Both PF3D7_1363900 and PF3D7_0718800 encode for unknown function and neither has been reported in GWAS on artemisinin resistance. e, Construction of the strain 3D7-SS-EEA1^R1374A-mCherry (used as transfection control) and western blot to confirm expression of SS-EEA1^R1374A-mCherry (arrow). Molecular weight standards (in kDa) are as indicated. f, Construction of Pf^VPS34-myc1: PfKelch13^WT-HA and Pf^VPS34-myc1: PfKelch13^C580Y-HA parasites and western blots confirming expression of the transgenes. Raw data for western blots in e-f are shown in Supplementary Data 2. Molecular weight standards (in kDa) are as indicated. In d-f, data are representative of three experimental replicates.

Extended Data Figure 6. Evidence that the PI3K-AKT pathway is responsive to DHA

a, Western blot shows specificity of anti-PfAKT antibody that recognizes a band of expected size in infected (IE) but not uninfected (U) erythrocytes (1x 10⁷ total cells loaded in each lane). Raw data is in Supplementary Data 2. Data are representative of three experimental replicates. b, Strategy and construct for generating transgenic 3D7 parasites expressing PfAKT-GFP.

Figure 1. DHA targets PfPI3K

a, SS-EEA1^WT-mCherry detects ring PI3P in punctate (ER) domains. Mutant SS-EEA1^R1374A-mCherry secretes to the PV (second row; ). 4 nM DHA redistributes SS-EEA1^WT-mCherry to the PV. Washout restores ER-PI3P. 4 nM deoxyartemisinin, no effect. Blue, nucleus; scale, 5 μm; P, parasite; E, Erythrocyte. Mean (SD) with three experimental replicates with image analysis from 400 optical sections. b-d, Effects of DHA on (b) PI3P mass (c) immunopurified PfPI3K (raw data in Supplementary Data 2) and (d) mammalian PI3Kinases. Mean from three experimental replicates (each with triplicate data points). For (b), SD < 3; (c) upper graph, SD < 1.5; lower graph SD < 5; (d) SD < 0.5. e, Overlay of the model of PfPI3K (cyan) and human class III PI3K VPS34 (grey, pdb code 3IHY) with active site marked (asterisk). f, DHA in PfPI3K model (cyan) binding site. g, Surface representation of f. Additional details in Extended Data Figs. 1–3.

Figure 2. Proteostatic regulation of PfPI3K by PfKelch13

a, PfKelch13 ‘Kelch’ propeller domains. b, PfPI3K, PfKelch13 polymorphisms, PfPI3K levels in clinical (ANL) resistant (2, 4, and 9) and sensitive (1, 7) strains and laboratory strain 3D7; loading control PfFKBP. ANL-7, contaminated with resistance mutation R539T. c–d, Western blots show PfPI3K in NF54-PfKelch13^C580Y and NF54-PfKelch13^WT,; 3D7-PfKelch13^C580Y-HA and 3D7-PfKelch13^WT-HA; loading controls, BiP, PfFKBP. e, Panels: (Top left) PfKelch13 (arrow) binding to PfPI3K is reduced by PfKelch13^C580Y. In the PfKelch13^WT background, IP PfPI3K displays K48-ubiquitination (top center, arrow), atypical poly-ubiquitination (lower left, *) and degradation (lower right), all reduced by PfKelch13^C580Y. K63-ubiquitination was not observed (top right). IP Lysate input PfFKBP. f, PfKelch13 but not PfKelch13^C580Y binds PfPI3K (arrow). Loading control, PfFKBP. Molecular weights, in kDa. In b-f, data are representative of three experimental replicates. g, A model for Fig. 2. Additional details in Extended Data Fig. 4. Raw data for b-f, in Supplementary Data 2.

Figure 3. Elevated PI3P and artemisinin-resistance in presence and absence of PfKelch13 mutations

a, RSA vs. PI3P levels in clinical (triangle) and laboratory (circle) strains. 1–4: sensitive strains ANL-1, 3D7, NF54-PfKelch13^WT, 3D7-PfKelch13^WT-HA with RSA ≤1. * ANL-7 contaminated with (R539T) resistant parasites show intermediate PI3P/RSA levels. 3D7-PfKelch13^C580Y-HA contains a chromosomal wild type copy of PfKelch13 and thus shows lower PI3P and RSA (49 and 9) than NF54-PfKelch13^C580Y (59 and ~ 13). b, 3D7 expressing transgenic myc-tagged VPS34 (left) or VPS34-catalytically dead mutant (right); loading control PfFKBP; raw data in Supplementary Data 2. Molecular weights, in kDa. Data are representative of three experimental replicates. c, PI3P levels in indicated strains. d, PI3P, RSA in VPS34 strains respond to PfKelch13. 1–4, sensitive strains ANL-1, Pf^{VPS34-MUTANT-myc}, SS-EEA1^R1374A-mCherry and 3D7. In a, c, d, Mean from three experimental replicates (each in triplicate). (a) RSA, SD < 0.5; (c) PI3P, SD < 8 (shown by error bars). e, a model for PI3P-induced artemisinin resistance. Further details in Extended Data Fig. 5.

Figure 4. PI3P-mediated resistance linked to GWAS

a–b Effect of DHA on a, cellular and b, immunopurified, PfAKT. c, Expression of PfAKT-GFP in Pf 3D7. Loading controls, PfFKBP, erythrocyte Band 3. Molecular weights in kDa. RSA, PI3P, as indicated. In a-c, mean of three experimental replicates (each with triplicate data points). AKT activity and RSA SD < 0.5; PI3P SD < 5. In b-c: western blots are representative of three experimental replicates; raw data in Supplementary Data 2. d, PI3P-mediated resistance pathways. Bold script: Lipid (PI3P) or protein components from this study and GWAS; Plain: GWAS only ^,. Solid lines, established relationships; broken/dotted lines, partial functional validation; broad grey arrows, in silico prediction. Additional details in Extended Data Fig. 6.