Research Article

A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites

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Science  03 Jan 2020:
Vol. 367, Issue 6473, pp. 51-59
DOI: 10.1126/science.aax4735

An artemisinin resistance mechanism

Species of the malaria parasite Plasmodium live in red blood cells and possess a highly conserved gene called kelch13. Single point mutations in this gene are associated with resistance to the frontline artemisinin drugs. Birnbaum et al. found that Kelch13 and associated proteins comprise an endocytic compartment associated with feeding on host erythrocytes (see the Perspective by Marapana and Cowman). Hot targets for artemisinin research also occur in this compartment, including the proteins UBP1, AP-2µ, and the parasite homolog of the endocytosis protein Eps15. Inactivation of Kelch13 compartment proteins revealed that these are required for endocytosis of host hemoglobin. Artemisinins are activated by hemoglobin degradation products, so these mutations render the parasite resistant to these drugs to different extents.

Science, this issue p. 51; see also p. 22

Abstract

Artemisinin and its derivatives (ARTs) are the frontline drugs against malaria, but resistance is jeopardizing their effectiveness. ART resistance is mediated by mutations in the parasite’s Kelch13 protein, but Kelch13 function and its role in resistance remain unclear. In this study, we identified proteins located at a Kelch13-defined compartment. Inactivation of eight of these proteins, including Kelch13, rendered parasites resistant to ART, revealing a pathway critical for resistance. Functional analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell. Parasites with inactivated Kelch13 or a resistance-conferring Kelch13 mutation displayed reduced hemoglobin endocytosis. ARTs are activated by degradation products of hemoglobin. Hence, reduced activity of Kelch13 and its interactors diminishes hemoglobin endocytosis and thereby ART activation, resulting in parasite resistance.

Life-threatening malaria is caused by Plasmodium falciparum parasites continuously multiplying in human red blood cells. Artemisinin and its derivatives (ARTs) are the recommended first-line drugs against malaria (1), but their usefulness is threatened by “resistance” (2), defined as reduced clearance of parasites in ART-treated malaria patients (3). ART resistance manifests as a decreased susceptibility of young ring-stage parasites to a short pulse of ART, a property linked to delayed clearance of parasites and recrudescence of disease in patients (46). This can be measured in vitro using a ring-stage survival assay (RSA) (4).

ART resistance is primarily associated with point mutations in the parasite’s Kelch propeller protein Kelch13 (7, 8). At present, there is no coherent picture of the mechanism of ART resistance in the parasite, which, among others, has been connected to increased cellular stress, an activated unfolded protein response, reduced protein translation, altered DNA replication, and increased levels of phosphatidylinositol 3-phosphate (916). Conditional inactivation of Kelch13 causes an arrest of parasites in ring stages (17). However, the cellular function of Kelch13 and its involvement in ART resistance remain enigmatic. Here, we study Kelch13 and identify its interactors and their function. This reveals an entire pathway in ART resistance and a Kelch13-dependent mechanism explaining the reduced susceptibility to ART in resistant parasites.

The Kelch13 compartment and associated proteins defined by a novel BioID approach

We previously found endogenous Kelch13 tagged with green fluorescent protein (GFP) (i.e., expressed from its genomic locus) at foci in close proximity to the parasite’s food vacuole (FV) (17), a lysosome-like compartment where parasites digest endocytosed host cell cytosol (predominantly hemoglobin). To better define the cellular location of Kelch13, we colocalized it with a series of markers (Fig. 1A and fig. S1). This confirmed the proximity of Kelch13 foci to the FV. Although Kelch13 foci rarely overlapped with secretory pathway markers [endoplasmic reticulum (ER) and Golgi], they were frequently in the vicinity of parts of the ER that were near the FV. Kelch13 foci were also found close to the apicoplast, which itself is located in proximity of the FV. Notably, the resistance-conferring form of Kelch13 (Kelch13C580Y; Cys580→Tyr) fully colocalized with episomally expressed wild-type Kelch13 (wtKelch13) (Fig. 1A, fig. S1, and table S1), indicating that resistance does not involve an altered cellular distribution. Kelch13C580Y also colocalized with episomally expressed hemagglutinin (HA)–tagged Kelch13 (fig. S1). Overall, this analysis suggests that Kelch13 is located at an unknown compartment or cellular structure (henceforth, Kelch13 compartment) not identified by general markers of the secretory system.

Fig. 1 Kelch13 location and interactome.

(A) Fluorescence microscopy images showing parasites with endogenously GFP-tagged Kelch13 (K13) (17) with coexpressed STVR-SDEL (ER), Sec13p (ER exit sites), P40 (phosphatidylinositol 3-phosphate sensor: food vacuole), ACP (apicoplast), or endogenously GFP-tagged mutated Kelch13 (K13C580Y) with episomal mCherry-Kelch13wt. See fig. S1 for all panels. (Bottom) Most common arrangement of markers in single-nucleus trophozoites. (B) Scheme of DiQ-BioID. (C) Rapalog-dependent recruitment of the biotinylizer (BirA*-FRB) to endogenously GFP- and FKBP-tagged Kelch13 (FKBP-K13). (D) Top-right quadrant of scatter plot of Kelch13 DiQ-BioID, showing proteins enriched (log2 ratio) on rapalog (biotinylizer on target) compared with control (biotinylizer cytoplasmic). Significantly enriched proteins are indicated (short, unique names or PlasmoDB identifiers). False discovery rate (FDR) indicated for the least significant experiment. Asterisks indicate proteins previously suspected in ART resistance. Figure S2 shows full plot and replicas with a different biotinylizer. (E) Fluorescence microscopy of parasites with endogenously 2xFKBP-GFP-2xFKBP–tagged Eps15 episomally expressing mCherry-Kelch13. (F) Immunoprecipitation (IP) of episomally expressed Kelch13 in parasites endogenously expressing GFP-Eps15. I, input extract; P, post IP extract; W, last wash; E, eluate. Full blots and replicas in fig. S4. (G) Top-right quadrant of scatter plot of Eps15 DiQ-BioID, as in (D). Figure S5 shows full plot and independent replicas. (H) Fluorescence microscopy in parasites with endogenously GFP-tagged DiQ-BioID hits (KIC1-10 or short name) colocalized with episomal mCherry-Kelch13. Arrowheads indicate Kelch13 foci overlapping with the KIC signal (all panels in fig. S6). Scale bars, 5 μm. Merge, merged green and red channel; DAPI, 4′,6-diamidino-2-phenylindole (nuclei).

To gain insight into the Kelch13 compartment, we used a novel version of BioID (18), which we call dimerization-induced quantitative BioID (DiQ-BioID), to identify Kelch13 interactors and compartment neighbors in the living cell. In DiQ-BioID, the biotin ligase BirA* is not directly fused to the target protein but is expressed separately (the biotinylizer) and conditionally recruited to the target (using rapalog), where it biotinylates interaction partners (Fig. 1B). The same parasite culture grown without rapalog (i.e., BirA* not on the target) serves as a control (Fig. 1B), permitting highly specific identification of interactors by quantitative mass spectrometry. We used this approach in parasites expressing FK506 binding protein (FKBP)–tagged Kelch13 from the genomic locus (17). Induction of dimerization with rapalog efficiently recruited the biotinylizer to the Kelch13 foci (Fig. 1C), and quantitative mass spectrometry of asynchronous parasites resulted in a list of proteins enriched with very high confidence over control (Fig. 1D, fig. S2, and data S1). Kelch13 was the top hit, confirming that the biotinylizer was successfully recruited to Kelch13, and a band consistent in size with the tagged Kelch13 was detected in anti-biotin blots (fig. S2B). The other hits [designated as Kelch13 interaction candidates (KICs), unless already otherwise named] were predominantly proteins of unknown function. These included proteins that had previously been suspected in ART resistance or were in that context highlighted in genome-wide association studies—for example, an Eps15-like protein (PF3D7_1025000, originally annotated as Formin2) (19); ubiquitin carboxyl-terminal hydrolase 1 (UBP1) (1923); KIC6 (PF3D7_0609700) (20); and MyosinC (MyoC) (19) (Fig. 1D, fig. S2, and data S1)—suggesting that the interactome identified a pathway that may be relevant for ART resistance. Modification of the genomic locus to express the endogenous protein fused to GFP (fig. S3) showed that the top hit of these previously highlighted proteins, the Eps15-like protein (henceforth Eps15), colocalized with Kelch13 (Fig. 1E), and coimmunoprecipitation confirmed that Eps15 interacts with Kelch13 (Fig. 1F and fig. S4).

To further validate the Kelch13 DiQ-BioID results and the method per se, we carried out a reverse DiQ-BioID with the endogenously tagged Eps15 (Fig. 1G and fig. S5). This resulted in a list of high-confidence hits that extensively overlapped with the Kelch13 DiQ-BioID (all 16 top hits were also found in the high-confidence hits of the K13 DiQ-BioID), including Kelch13 and UBP1 (Figs. 1G and 2C and data S1). This suggested that the hits are indeed interactors or compartment neighbors of the Kelch13-Eps15 complex and that the DiQ-BioID experiments reached a high depth of coverage. This was confirmed by tagging the corresponding endogenous genes with the sequence encoding GFP (fig. S3), which showed that, besides Eps15, 10 of 12 tested Kelch13 hits colocalize with Kelch13 compartment foci (Fig. 1H, fig. S6, and table S1), demonstrating the power of the method. Only PFK9 and KIC10 did not show an overlap with Kelch13. Of the 10 colocalizing proteins, eight also colocalized in the resistance-relevant ring stage, whereas the other two (KIC8 and KIC9) were not detected in that stage (fig. S6 and table S1). Thus DiQ-BioID identifies interactors and compartment neighbors in living cells with high specificity and in this study defined a series of proteins proximal to the Kelch13-Eps15 complex.

Fig. 2 Kelch13 complex locates with AP-2μ but is distinct to clathrin complex.

(A) Fluorescence microscopy images of parasites endogenously expressing 2xFKBP-GFP–tagged clathrin heavy chain (CHC) or AP-2μ with episomal mCherry-Kelch13. Arrows mark overlap, and arrowheads mark similar region of Kelch13 foci. Scale bar, 5 μm. Merge, merged green and red channel; DAPI (nuclei). (B) Top-right quadrant of scatter plot of CHC DiQ-BioID. Figure S7 shows full plot and replicas with a different biotinylizer. (C) Heatmap of four Kelch13, Eps15, and CHC DiQ-BioID experiments showing proteins enriched in at least three of four experiments with FDR < 1%. K-means clustering into two clusters highlights overlap of Kelch13 and Eps15 hits (dark green cluster) compared with CHC hits (light green clusters). Gray blocks indicate not identified or no ratio due to missing label. Red font indicates DiQ-BioID baits, and orange font indicates proteins further analyzed.

Kelch13-Eps15 marks an AP-2 compartment devoid of clathrin

Because Eps15 in other organisms is typically involved in endocytosis (24, 25), we hypothesized that the Kelch13 compartment might also be involved in this process. In malaria blood stages, endocytosis mediates the large-scale uptake of host cell cytosol (consisting predominantly of hemoglobin), but the molecular details of this process are poorly understood (26). In model organisms, the most common and best studied form of endocytosis is mediated by clathrin, which depends on the AP-2 adaptor complex (27, 28). Although P. falciparum Eps15 contains regions specific to malaria parasites, it shows typical hallmarks of Eps15 proteins, including AP-2 binding sites (fig. S5B). To test whether the Kelch13-Eps15 complex marks an endocytic structure, we colocalized Kelch13 with endogenously GFP-tagged clathrin heavy chain (CHC) or AP-2μ (fig. S3). AP-2μ–GFP foci colocalized with (Fig. 2A, arrowhead) or were found in the same region as Kelch13 foci (Fig. 2A, arrow), indicating an endocytosis role for the Kelch13-Eps15 compartment. Unexpectedly, CHC was present in foci that did not overlap with Kelch13 (Fig. 2A). This showed that Kelch13 colocalizes with a compartment that contains AP-2 but is devoid of clathrin. Whereas clathrin typically has additional functions besides endocytosis where it associates with other adaptors, AP-2 is generally associated with clathrin-dependent endocytosis (27, 28). AP-2 independent of clathrin is a highly unusual configuration that, to our knowledge, was so far only observed in Aspergillus nidulans (29). Of note, AP-2μ has previously been suspected to be involved in ART resistance (30, 31).

To probe this distinction between clathrin and the Kelch13-Eps15 complex and further assess the specificity of the DiQ-BioID procedure, we performed DiQ-BioID with the CHC (fig. S7). This resulted in a list of high-confidence hits differing from the Kelch13 and Eps15 DiQ-BioIDs and included known CHC interactors such as the putative clathrin light chain (even more enriched than the CHC itself) and subunits of the AP-1 adaptor complex (Fig. 2B, fig. S7, and data S1). Unexpectedly, subunits of AP-4—in other organisms considered a clathrin-independent adaptor of the trans-Golgi network (32, 33)—were also enriched, whereas AP-3 was only very mildly enriched, not reaching statistical significance (data S1). AP-4 is not well studied to date, but it is known to interact with tepsin (34, 35). P. falciparum tepsin (34) was a prominent hit in the CHC DiQ-BioID, validating the AP-4 hits. This suggests that CHC associates with AP-4 in malaria parasites, either indicating differences compared to other organisms or that DiQ-BioID uncovers interactions not retained using previously used approaches in other organisms. Hence, DiQ-BioID reveals a credible CHC interactome that is clearly distinct from the Kelch13-Eps15 interactomes. The CHC DiQ-BioID also detected a different Kelch protein (PF3D7_1205400), potentially indicating that different trafficking complexes harbor distinct Kelch proteins. Only PFK9, HSP90 (heat shock protein 90), and FKBP35 were hits overlapping with the Kelch13 and Eps15 DiQ-BioID experiments, suggesting that these hits might result from the method rather than being biologically meaningful (Fig. 2, B and C). This was supported by DiQ-BioID using the biotinylizer alone (fig. S8 and data S2), and a lack of colocalization with Kelch13 was experimentally confirmed in the case of PFK9 (Fig. 1G and fig. S6). Therefore, we did not analyze these proteins further.

Overall, these findings show that Kelch13 defines proteins at a compartment that colocalizes with the classical endocytosis adaptor AP-2μ but that is entirely distinct from clathrin, which typically associates with the AP-2 complex in other organisms. This may indicate the existence of an unusual clathrin-independent endocytosis pathway in malaria parasites.

Kelch13 compartment proteins are involved in endocytosis

To investigate a possible role of the Kelch13-Eps15 compartment in endocytosis, suggested by the presence of Eps15 and colocation with Ap-2μ, we first carried out correlative light and electron microscopy (CLEM) with the parasites expressing endogenously GFP-tagged Eps15. This revealed that Eps15 resides in proximity of host cell cytosol-filled membranous structures in the parasite in three of three analyzed cells and in five of five foci in these cells (Fig. 3A and fig. S9, A to C). Hence, it can be inferred that Kelch13, Eps15, AP-2, UBP1, and the nine colocalizing KICs are present proximal to host cell cytosol-filled structures in the parasite, congruent with a role of the Eps15-Kelch13 complex in endocytosis of host cell cytosol. In support of this, we noted that in >90% of cells with vesicle-like structures visible in differential interference contrast (DIC), Kelch13 foci were found at such structures (fig. S9D), suggesting that these may represent the host cell cytosol-filled structures observed by CLEM and likely are cytostomes.

Fig. 3 Kelch13 compartment proteins are involved in endocytosis of host cell cytosol.

(A) CLEM with a cell expressing endogenously tagged Eps15-GFP: confocal image (scale bar, 2 μm), EM section and magnification thereof (scale bars, 0.5 μm). Arrowheads indicate vesicles containing material of host cell density. N, nucleus; FV, food vacuole. Details and two more cells in fig. S9. (B) Summary of Kelch13 colocalization and SLI-TGD screen of DiQ-BioID hits. nd, not done. (C) Growth curves of parasites after induction of KS. One of three independent experiments. Figure S10 shows replicas and details on KS. (D) Example DIC images showing bloated (control) and nonbloated (inhibited endocytosis) FVs (partly highlighted: dashed line and arrows) after KS (rapalog) and E64 treatment. (E) Quantification of number of cells with bloated FVs after E64 treatment for control (no rapalog) and KS (rapa) for 8 hours (n for control/rapa: UPB1, 33/33; KIC7, 33/33; Kelch13, 32/33; AP-2μ, 33/32; Eps15, 33/33; 2102, 33/33; 3D7 NaN3, 34/34). Fisher’s exact test. Replicas in fig. S10. (F) Growth (cell diameter) of cells scored for bloated FVs after KS compared with control, data pooled from all three (five for NaN3) independent experiments, with a total of n = 99/97 cells for no rapalog/rapa with UBP1, 101/97 with KIC7, 134/134 with Eps15, 99/97 with AP-2μ, 100/100 with 2102, and 170/170 for 3D7 ± NaN3. Unpaired, two-tailed t test. ns, not significant.

To more directly assess the function of KICs, we first carried out a selection-linked integration gene disruption (SLI-TGD) screen (17) to establish which of the Kelch13 compartment proteins are essential for parasite survival. Of the 13 tested genes, only the ones encoding Eps15, UBP1, and KIC7 were refractory to disruption and hence are likely essential for parasite survival (Fig. 3B and fig. S3). Knock sideways (KS) (17) to conditionally inactivate these proteins, as well as AP-2μ, confirmed their importance for parasite growth (Fig. 3, B and C, and fig. S10). Note that the smaller effect on growth after inactivating Eps15 likely is the result of inefficient KS (Fig. 3C and fig. S10). To test whether Kelch13, UBP1, KIC7, Eps15, and AP-2μ are involved in endocytosis, we conditionally inactivated them by KS (fig. S10) and assessed hemoglobin uptake into the parasite over 8 hours using a previously established assay, which results in a bloated FV phenotype if endocytosis is operational (36). Inactivation of UBP1, KIC7, Eps15, and AP-2μ significantly reduced transport of hemoglobin to the FV, whereas the controls or inactivation of Kelch13 or an unrelated essential protein [PF3D7_0210200 (17), here called 2102] did not (Fig. 3D). Inactivation of KIC7, AP-2μ, and to a lesser extent Eps15, reduced growth over the 8-hour assay time. However, the growth defect itself was not the reason for the phenotype, as inducing an unrelated growth defect using azide did not impair endocytosis (Fig. 3E). Surprisingly, inactivation of Kelch13 significantly increased parasite size compared with the control, and inactivation of UBP1 showed a clear reduction of endocytosis but no significant reduction in cell size over the 8-hour assay time (Fig. 3, D to F). We conclude that proteins of the Kelch13 compartment play a role in endocytic uptake of hemoglobin, but Kelch13 itself does not appear to be necessary for this process in trophozoites. The lack of effect in trophozoites agrees with the previous finding that inactivation of Kelch13 specifically affected the ring stage only (17).

A detailed comparison of the stage-specific essentiality of Kelch13, UBP1, and KIC7 revealed that all three proteins caused a similar phenotype when inactivated in rings (severe delay of ring stage with limited progression to the trophozoite stage). In contrast, inactivation in early trophozoites prevented development into schizonts in KIC7 and UBP1, but not in Kelch13 (fig. S11). This suggests a similar function of these proteins but that Kelch13 is not essential for this function in trophozoites. A previously established KS cell line that only partially inactivates Kelch13 (Kelch13 3xNLS) and that does not noticeably affect parasite growth in a 4-day growth assay (17) showed a mild delay of the ring stage (fig. S11). Hence, while not lethal, partial inactivation of Kelch13 (3xNLS line) leads to a much milder, but similar, effect compared with full inactivation of Kelch13 (1xNLS line).

Kelch13 influences endocytosis and resistance in ring stages

Hemoglobin by-products activate ART, and decreased hemoglobin digestion reduces ART susceptibility (3739). Hence, our data might indicate that reduced levels of endocytic uptake of host cell cytosol could, by controlling the supply of hemoglobin available for digestion and ART activation, be the mechanism of ART resistance. However, this would need to take place in the resistance-relevant ring stage (4). As UBP1 and KIC7 affected endocytosis in trophozoites and showed a similar phenotype in rings to Kelch13 (fig. S11), we reasoned that Kelch13 might influence endocytosis specifically in ring stages. However, as it is not clear whether young ring stages endocytose hemoglobin at all (3941), we decided to first clarify this. Because of the overabundance of hemoglobin in the parasite culture, we used host cells loaded with fluorescent dextrans, a well-established tool for studying endocytic uptake in malaria parasites (36, 40). These experiments demonstrated uptake of host cell material into young ring stages (Fig. 4A).

Fig. 4 A Kelch13-defined pathway in endocytosis and resistance.

(A) Fluorescence microscopy images of saponin-released young ring stages (examples of spherical, partial, and full amoeboid parasite) grown for 3 to 6 hours in red blood cells preloaded with fluorescent dextran (Alexa647) show internalized host cell cytosol (arrowheads). Scale bar, 5μm. (B to D) Quantification of dextran uptake into 3-to-6-hour rings after inactivation (rapa) of Kelch13 by KS in the 3xNLS line (17) (B) or KIC7 (C) compared with control (no rapa) or in the K13mut parasites compared with wild type (3D7) (D). Each point shows the amount of fluorescence internalized into one cell (arbitrary fluorescence units). Data pooled from three independent experiments (individual experiments in fig. S12) with a total of n = 138, 140, 132, 130, 122, and 114 cells for Kelch13 KS control, rapa, KIC7 control, KIC7 rapa, 3D7, and K13mut, respectively. Unpaired, two-tailed t test. P values and percent reduction of the mean are indicated. (E) RSAs after inactivating Kelch13 by KS (rapa) compared with control with Kelch13 3xNLS parasites (17). (F) RSAs with the TGD lines indicated. (G) Growth rate of TGD lines. (H) RSAs after KS (rapa) to inactivate the proteins indicated compared with control or in 3D7 after inhibiting growth using NaN3. (I) Heatmap of transcription levels in 1-hour intervals across asexual blood stage development [values from (42)], clustered according to similarity for the proteins analyzed in this study (see also fig. S13). Note that KIC2 was not part of the dataset. Bottom shows data of colocalization, RSA, and endocytosis experiments (table S1; Fig. 4, B, F, and H; and Fig. 3E; respectively) as heatmaps (color shading indicated; gray, not applicable). Asterisk indicates ring-stage endocytosis assay. (J) RSA with parasites harboring a mutated ubp1 gene encoding a change from Arg to His at amino acid position 3138 (19). Resistance in RSA defined as mean survival above 1% [green line in (F), (H), and (J)], a previously set standard (4). [(B) to (H), and (J)] Error bars show SD (not shown if close to mean). P values indicated [only selected P values in (F) and (G)]; ns, not significant. [(E) to (H), and (J)] Unpaired, two-tailed t test with Welch’s correction. Each point indicates an independent experiment.

Next, we tested the role of Kelch13 in endocytosis in 0-to-6-hour young rings [the time the drug pulse is applied in ART susceptibility assays (4)] using the Kelch13 3xNLS parasites (the cell line leading to only partial inactivation of Kelch13, to avoid detrimental effects on parasite growth). Inactivation of Kelch13 significantly reduced endocytic uptake in these rings (Fig. 4B and fig. S12). Inactivation of KIC7 similarly reduced endocytosis in rings (Fig. 4C and fig. S12), congruent with its similar KS phenotype to Kelch13 in rings and its role in endocytosis in trophozoites (Fig. 3E). Hence, KIC7 affects endocytosis in both rings and trophozoites and, together with the similar stage-specific phenotypes, suggests that the Kelch13 compartment proteins are needed for endocytosis in all asexual blood stages, with the exception of Kelch13, which is only needed for this process in rings.

To assess whether this could be the reason for resistance, we next tested hemoglobin uptake into rings of resistant parasites containing a mutated genomic Kelch13C580Y (17). The resistant parasites also displayed reduced endocytic uptake (Fig. 4D and fig. S12), suggesting this as a reason for reduced susceptibility to ART. To further confirm this hypothesis, we inactivated Kelch13 (again using the Kelch13 3xNLS line) and measured whether this can render parasites ART-resistant using a standard RSA (4). When Kelch13 was inactivated, parasites showed levels of resistance comparable to the resistant Kelch13C580Y parasite line (Fig. 4E). Thus, Kelch13 inactivation reduces endocytosis of host cell material and increases resistance. We conclude that early ring stages already endocytose hemoglobin, and that this process is reduced in parasites with a resistance-conferring mutation in Kelch13 or in which Kelch13 is inactivated, and that this correlates with ART resistance.

Kelch13 compartment proteins are involved in ART resistance

Having established that inactivation of Kelch13 induces ART resistance, we next assessed whether other Kelch13 compartment proteins also play a role in ART resistance. First, we used the cell lines with the disrupted, nonessential KICs. Whereas parasites with disrupted KIC1, 2, 3, 6, 8, and 9, as well as KIC10 (which is not Kelch13 compartment–associated), did not show an altered response to ART, disruption of KIC4, KIC5, and MCA-2 led to a reduced susceptibility in a standard RSA (Fig. 4F). While the reduced susceptibility correlated with a low growth rate of the KIC5 and MCA-2 disruption lines (suggesting some importance of these proteins for efficient parasite growth), there was little correlation overall between growth and ART resistance (Fig. 4G), excluding growth levels as a factor in resistance. For instance, the line with a disruption of KIC10, the only non–Kelch13 compartment KIC (Fig. 1H), had the third lowest growth rate but did not show resistance (Fig. 4, F and G).

To assess the role of the essential proteins AP-2μ, Eps15, UBP1, and KIC7 in ART resistance, we partially inactivated them (using KS) before and during the ART drug pulse of the RSA in a manner that did not kill the parasites. Inactivation of KIC7, AP-2μ, Eps15, and UBP1 induced ART resistance (Fig. 4H). In contrast, reducing parasite viability with azide or inactivation of an unrelated essential protein [2102 (17)] did not affect the outcome of the RSA, indicating that the effect on ART susceptibility is specific for the function of Kelch13 and its compartment proteins (Fig. 4H). We conclude that inactivation of more than half (7 of 13) of the tested Kelch13 compartment proteins, as well as Kelch13 itself, reduces the responsiveness to ART. Notably, all of the Kelch13 compartment proteins affecting endocytosis (Fig. 3, D and E) also rendered parasites resistant, establishing a strong link between hemoglobin uptake and ART resistance (table S1). We conclude that Kelch13 compartment proteins are part of an ART resistance pathway, and the correlation of reduced endocytosis and resistance indicates that this is the mechanism of this mode of resistance. To test whether the corresponding genes belong to a coregulated pathway, we assessed their stage-specific transcription patterns across asexual blood stage development (42). Except for mca2, all genes involved in resistance clustered into one group when compared with (i) the other validated Kelch13 compartment hits (Fig. 4I), (ii) all significant Kelch13 interactome hits (fig. S13A), and (iii) all hits significant in any of the BioIDs (fig. S13B). In contrast, the other hits showed widely differing transcription patterns, reflecting the asynchronous nature of the parasites used as input for the DiQ-BioID. Overall, the coexpression lends independent support to the idea that the genes experimentally identified here belong to a coregulated pathway involved in hemoglobin endocytosis.

To show that the resistance pathway of the Kelch13 interactors is clinically relevant, we chose a mutation in UBP1 (R3138H) that was identified as a possible contributor to ART resistance by genomic surveillance of parasite field samples (19). We changed this position in UBP1 in 3D7 parasites and found that this rendered these parasites resistant to ART (Fig. 4J) at a level comparable to the resistance demonstrated when UBP1 was inactivated by KS (Fig. 4H). These findings demonstrate that the pathway identified here can be clinically relevant and that mutations in the UBP1 can modulate ART resistance.

Reduced levels of Kelch13 explain ART resistance

To determine how mutated Kelch13 influences resistance, we first assessed whether the interaction profile of mutated Kelch13 was altered compared with wtKelch13. However, DiQ-BioID with the Kelch13C580Y parasite line revealed no marked differences to the DiQ-BioID with wtKelch13 (Fig. 5A, fig. S14, and table S1). The only exception was KIC10, which was less enriched in the Kelch13C580Y DiQ-BioID, but this is the only KIC not colocalizing with Kelch13 and has no role in resistance (Figs. 1H and 4F and table S1). As these findings indicated that there is no change in the interaction profile of Kelch13C580Y compared with wtKelch13, we hypothesized that mutating Kelch13 does not change a specific quality of its function but reduces its overall activity. This idea is also suggested by our finding that reduced activity of Kelch13 causes resistance (Fig. 4E). In addition, previous work indicated that resistant parasites harbor less Kelch13 protein (43). In agreement with this observation, we found ~30 to 50% less Kelch13 in parasites with the resistance-conferring form in Western blots and by measuring Kelch13 levels directly in living cells (Fig. 5, B to D). To test whether increased expression of Kelch13 can lead to the loss of resistance, we episomally expressed different versions of Kelch13 on the resistant genomic kelch13C580Y background. Adding wtKelch13 (leading to expression of episomal wtKelch13 and endogenous Kelch13C580Y) rendered these resistant parasites fully sensitive to dihydroartemisinin (DHA) again (Fig. 5E). To test whether this was due to an additional property held by wtKelch13 that is lacking in Kelch13C580Y, we episomally expressed Kelch13C580Y on the resistant background (resulting in parasites expressing episomal Kelch13C580Y and endogenous Kelch13C580Y, in effect only raising the abundance of Kelch13C580Y). This also reverted the parasites from resistant to sensitive again (Fig. 5E), demonstrating that reduced protein levels (or reduced activity) alone explains resistance, congruent with the finding that partially inactivating Kelch13 has a similar effect (Fig. 4E). In contrast, Kelch13 missing the Plasmodium-specific region (PSR), a modification that led to a loss of the correct location of this construct (Fig. 5E), did not revert the resistance phenotype when added on top of the kelch13C580Y background (Fig. 5E). This demonstrated the specificity of the effect with episomal Kelch13C580Y and indicated that the BTB and Kelch13 domain without the PSR are not functional.

Fig. 5 Reduced Kelch13 activity leads to ART resistance.

(A) Heatmap of DIQ-BioID data of the four Kelch13 and the two Kelch13C580Y experiments [hits with FDR < 1% in at least two of four (Kelch13) or two (Kelch13C580Y) experiments]. Color intensities: 2nd to 98th percentile of log2 rapalog/control normalized ratios of all proteins in this analysis. NaN (data S3) values in gray. Rows were clustered and gene identifiers were color coded as in Fig. 2C. (B) Immunoblot-based quantification of wtKelch13 (K13wt) and Kelch13C580Y (K13C580Y) levels in parasites expressing the otherwise-identical GFP-fused protein from the endogenous locus. (Left) An example band pair with Kelch13 levels relative to the loading control (aldolase) shown as bars below the blot (axis shows intensity ratio). (Right) K13C580Y levels compared with K13wt (n = 3 independent experiments; each experiment is shown by a point that derives from the average of a serial dilution of K13wt and K13C580Y run on the same gel and normalized relative to the respective aldolase control). (C and D) Quantification of Kelch13 foci fluorescence intensity in trophozoites (n = 105, 102 foci) and rings (n = 47, 51 foci) in K13wt and K13C580Y parasites, respectively. Data pooled from three independent experiments; percent reduction of the mean is indicated. Example fluorescence images of ring stages are shown. (E) Fluorescence microscopy images of parasites with endogenous Kelch13C580Y episomally expressing the construct indicated (PSR, plasmodium-specific region; L, linker) and RSA with the respective cell lines (right). Cell lines expressing mChe-K13wt and mChe-K13C580Y were imaged after smearing, mChe-K13ΔPSR live. Size bar, 5 μm. (F) Model of resistance and summary of results of this study. Asterisk indicates only in ring stages; nd, not determined (see also table S1). Bars show SD, mean is indicated. Unpaired, two-tailed t test with Welch's correction. P values are indicated [in (E), only for selected pairs]; ns, not significant.

Overall, these results show that reduced activity of Kelch13 is the most likely mechanism of how Kelch13 mutation causes resistance and that this reduced activity, at least in part, is due to decreased Kelch13 levels in resistant parasites. A generally reduced function also explains why inactivation of several Kelch13 compartment proteins renders parasites resistant, as each will reduce the output of a common pathway that, based on our functional data, is endocytosis.

Discussion

Here, we show that Kelch13 defines an endocytosis pathway required for the uptake of host cell hemoglobin and that this pathway is critical for ART resistance. Although it is well established that ARTs are activated by the parasite’s digestion products of hemoglobin and that this is a prerequisite for ART action (3739), this process was not considered to be involved in resistance in parasites with Kelch13 mutations (15, 16). Instead, potential roles of Kelch13 downstream of ART activation, e.g., in mitigating the ART-induced cellular stress response, are the current target of interest and central to hypotheses on the mechanism of resistance (15, 16). However, our data indicate that Kelch13 and its compartment proteins mediate resistance upstream of both, drug activation and action. We propose a model where Kelch13 and its compartment proteins control endocytosis levels, thereby influencing the amount of hemoglobin available for degradation and hence the concentration of active drug (Fig. 5F). This mechanism explains the slowed development of ring stages observed in ART-resistant parasites (9, 44), as the reduction in hemoglobin endocytosis in rings diminishes the supply of amino acids. This agrees with the finding that ART-resistant parasites depend more on exogenous amino acids to mature from rings to trophozoites than wild-type parasites (45) and that removing functions needed for amino acid access of the parasite prolongs the ring stage (46). Reduced levels of amino acids could also account for some of the changes observed in ART-resistant parasites, such as elevated cellular stress responses (9, 15). It also indicates that the fitness cost incurred by resistance-conferring Kelch13 mutations (47, 48) is a direct result of the resistance mechanism. Hence, there is a trade-off between resistance and growth levels in ring stages (Fig. 5F), creating a pressure for compensatory adaptations that may explain the importance of the genetic background in parasite resistance and fitness (8, 48).

Altered endocytosis as a mechanism of ART resistance reconciles aspects of previous findings relating to ART resistance, e.g., a role of phosphatidylinositol 3-phosphate (and its generating kinase) (12), a membrane signature specific for endosomes (49) that is also present on the endolysosomal system of malaria parasites (36, 50), or of coronin (51) that could influence endocytosis via actin. We also validated the suspected role of AP-2μ in resistance (30, 31) and show that, as in other organisms (24), this protein is involved in endocytosis. This indicates that Kelch13 marks a clathrin-independent endocytosis pathway that unexpectedly still contains the typical clathrin adaptor AP-2μ. Besides Eps15 and AP-2μ, KIC4 shows homology to alpha adaptins [according to HHPred (52)], adding an additional protein typical for endocytosis in a generally highly derived pathway.

In contrast to its interactors, inactivation of Kelch13 impaired endocytosis only in rings, not in trophozoites. This likely is the reason why Kelch13, among all the proteins influencing endocytosis, is the one frequently found mutated in resistant parasite isolates (53). Complete inactivation of Kelch13 arrests growth in ring stages (17), and a similar phenotype was here observed with KIC7 and UBP1. The stage-specific effects of inactivating Kelch13 are observed in a milder form in ART-resistant parasites that display a prolonged ring stage followed by an accelerated development in later stages (9, 44), and this observation was here recapitulated by partial inactivation of Kelch13. This indicates that resistance-conferring mutations partially reduce Kelch13 function, a hypothesis supported by our finding that partial inactivation of Kelch13 in rings induces parasite resistance and that additional expression of Kelch13C580Y on a resistant background with the same mutation is sufficient to render parasites fully sensitive again. As resistant parasite field isolates harbor less Kelch13 (43), which we also found in our resistant laboratory line here, but transcript levels appear to be unchanged (9), it can be assumed that resistance-conferring mutations alter Kelch13 protein stability.

We envisage that the mechanism of ART resistance indicated by this work will aid in finding ways to antagonize it. It may also inform the choice of ART partner drugs, particularly as hemoglobin digestive processes are the target of existing drugs. Finally, the here-identified proteins of the resistance pathway are candidates for novel parasite markers influencing ART resistance that now can be assessed in population studies, as illustrated by the identification of a resistance-conferring mutation in UBP1. It should, however, be noted that resistance-causing mutations are most likely to occur in proteins that have the least impact on trophozoite survival, such as Kelch13.

Supplementary Materials

science.sciencemag.org/content/367/6473/51/suppl/DC1

Materials and Methods

Fig. S1 to S14

Table S1

References (5478)

Data S1 to S4

References and Notes

Acknowledgments: This article is dedicated to Hendrik Herrmann. We thank L. Tilley for suggestions of the timing of Kelch13 KS induction for RSAs. We thank Jacobus Pharmaceuticals for supplying WR99210. DSM1 (MRA-1161) was received from MR4/BEI Resources, NIAID, NIH. Funding: E.J., S.F., A.B.S., and T.S. acknowledge funding by the Research Training Group (GRK 1459) of the German Research Foundation (DFG). S.Schm. acknowledges funding by the German Center for Infection Research (TTU 03.806), M.S. thanks the Jürgen Manchot Stiftung and S.Scha. the Claussen-Simon Stiftung for funding. W.A.M.H. obtained financial support from Netherlands Organization for Scientific Research (NWO-VENI, 722.012.004). Author contributions: J.B. designed research, carried out experiments, analyzed data, prepared figures, and wrote the methods section. S.Scha., S.Schm., E.J., and S.F. designed and carried out experiments, analyzed data, and prepared figures. M.S., R.S., B.B., U.F., P.M.-R., and A.B.S. carried out experiments. W.A.M.H. and C.G.T. conducted the mass spectrometry experiments, analyzed data, and prepared figures. R.B. supervised the mass spectrometry experiments and analyzed data. T.S. conceived of and supervised the project, designed experiments, carried out imaging and image analysis, prepared figures, and wrote the paper with critical input from all authors. Competing interests: The authors declare no competing interests. Data and materials availability: All data are available in the manuscript or the supplementary materials, and the Kelch13, Eps15, and CHC mass spectrometry data are available via ProteomeXchange with identifier PXD008219.
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