Paolo Mesén-Ramírez

Bärbel Bergmann

Mourad Elhabiri

Lei Zhu

Heidrun Von Thien

Carolina Castro-Peña

Tim-Wolf Gilberger

Elisabeth Davioud-Charvet

Zbynec Bozdech

Anna Bachmann

Tobias Spielmann
email: spielmann@bni-hamburg.de

The parasitophorous vacuole nutrient pore is critical for drug access in

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Malaria caused by P. falciparum parasites is responsible for more than 400,000 deaths every year [START_REF] Who | World Malaria Report[END_REF]. The symptoms of malaria are caused by continuous cycles of parasite multiplication in red blood cells (RBCs). During their intracellular development malaria parasites propagate in a vacuolar compartment surrounded by the PVM [START_REF] Lingelbach | The parasitophorous vacuole membrane surrounding Plasmodium and Toxoplasma: an unusual compartment in infected cells[END_REF][START_REF] Spielmann | Molecular make-up of the Plasmodium parasitophorous vacuolar membrane[END_REF]. The parasites establish nutrient permeable channels (NPCs) at the PVM that are conductive to small molecules [START_REF] Desai | Pore size of the malaria parasite's nutrient channel[END_REF].

Electrophysiological measurements with parasites extruded from the host cell and isolated membranes demonstrated that the NPCs are mostly in an open state and show low selectivity for molecules of 1.4 kDa or less [START_REF] Desai | A nutrient-permeable channel on the intraerythrocytic malaria parasite[END_REF][START_REF] Desai | Pore size of the malaria parasite's nutrient channel[END_REF][START_REF] Garten | EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX[END_REF]. However, it is unclear for which metabolites this activity is growth relevant. Recent work implicated the PVM proteins EXP2 [START_REF] Garten | EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX[END_REF], EXP1 [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF] and RON3 [START_REF] Low | Deletion of Plasmodium falciparum Protein RON3 Affects the Functional Translocation of Exported Proteins and Glucose Uptake[END_REF] in NPC activity (NPCA). While EXP2 and RON3 are also needed for protein export, inactivation of EXP1 specifically affects the NPCA without interfering with protein export [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF][START_REF] Nessel | EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole[END_REF].

Reduced susceptibility of parasites to artemisinin derivatives (ART) (here simplified as 'ARTresistance') and its partner drugs, such as piperaquine (PPQ), is now common in many regions of South East Asia, causing treatment failures and hampering the efforts to control malaria [START_REF] Amaratunga | Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study[END_REF][START_REF] Dondorp | Artemisinin resistance in Plasmodium falciparum malaria[END_REF][START_REF] Duru | Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations[END_REF][START_REF] Leang | Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment[END_REF][START_REF] Saunders | Dihydroartemisinin-piperaquine failure in Cambodia[END_REF][START_REF] Witkowski | Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia[END_REF][START_REF] Woodrow | The clinical impact of artemisinin resistance in Southeast Asia and the potential for future spread[END_REF]. Upregulation of exp1 transcript levels has been observed in some resistant parasite lines [START_REF] Lisewski | Supergenomic network compression and the discovery of EXP1 as a glutathione transferase inhibited by artesunate[END_REF][START_REF] Rocamora | Oxidative stress and protein damage responses mediate artemisinin resistance in malaria parasites[END_REF]. It was postulated that EXP1 is a membrane glutathione transferase (MGST) that detoxifies heme and has a role in ART resistance by protecting the parasites from ART-mediated oxidative damage [START_REF] Lisewski | Supergenomic network compression and the discovery of EXP1 as a glutathione transferase inhibited by artesunate[END_REF]. However, recent work showed that the proposed MGST catalytic site of EXP1 is not important for its function and no evidence was found for EXP1 as a causal factor in ART resistance [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF][START_REF] Nessel | EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole[END_REF]. Diverse other transcriptional and metabolic alterations have been observed in ART resistant (ART R ) parasites [START_REF] Mok | Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance[END_REF][START_REF] Mok | Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription[END_REF][START_REF] Mok | Artemisinin-resistant K13 mutations rewire Plasmodium falciparum's intra-erythrocytic metabolic program to enhance survival[END_REF][START_REF] Rocamora | Oxidative stress and protein damage responses mediate artemisinin resistance in malaria parasites[END_REF][START_REF] Siddiqui | Multi-omics Based Identification of Specific Biochemical Changes Associated With PfKelch13-Mutant Artemisinin-Resistant Plasmodium falciparum[END_REF][START_REF] Sutherland | Artemisinin susceptibility in the malaria parasite Plasmodium falciparum: propellers, adaptor proteins and the need for cellular healing[END_REF][START_REF] Yang | Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance[END_REF][START_REF] Zhang | Inhibiting the Plasmodium eIF2alpha Kinase PK4 Prevents Artemisinin-Induced Latency[END_REF].

However, the roles of these changes and of EXP1 in ART-resistance remain unclear.

Single point mutations in the propeller domain of the parasite's Kelch13 protein result in ARTresistance [START_REF] Ariey | A molecular marker of artemisinin-resistant Plasmodium falciparum malaria[END_REF][START_REF] Straimer | Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates[END_REF] and such mutations have now also been detected in Africa [START_REF] Uwimana | Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda[END_REF]. Malaria parasites endocytose hemoglobin from the host RBC and digest it in the food vacuole (FV), the parasite's lysosome-like organelle [START_REF] Francis | Hemoglobin metabolism in the malaria parasite Plasmodium falciparum[END_REF][START_REF] Spielmann | Endocytosis in Plasmodium and Toxoplasma Parasites[END_REF], resulting in degradation products that activate ART [START_REF] Klonis | Iron and heme metabolism in Plasmodium falciparum and the mechanism of action of artemisinins[END_REF]. ART R parasites show reduced hemoglobin endocytosis, leading to resistance, likely due to reduced drug activation [START_REF] Birnbaum | A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites[END_REF][START_REF] Yang | Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance[END_REF]. Resistance involves only the first intra-erythrocytic phase, the ring stage [START_REF] Witkowski | Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia[END_REF], and is associated with a fitness cost for the parasite [START_REF] Nair | Fitness Costs and the Rapid Spread of kelch13-C580Y Substitutions Conferring Artemisinin Resistance[END_REF][START_REF] Straimer | Plasmodium falciparum K13 Mutations Differentially Impact Ozonide Susceptibility and Parasite Fitness In Vitro[END_REF]. The reason for the fitness cost of ART resistance is unknown but we previously hypothesised that it might be due to a reduced hemoglobin-derived amino acid supply in ring stages [START_REF] Birnbaum | A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites[END_REF]. Intra-erythrocytic malaria parasites obtain amino acids from two sources: the proteolysis of the globin chains of the hemoglobin endocytosed from the RBC cytosol and free amino acids from the human serum or culture medium [START_REF] Babbitt | Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state[END_REF][START_REF] Liu | Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems[END_REF].

The latter requires transport across the RBC membrane [START_REF] Desai | A voltage-dependent channel involved in nutrient uptake by red blood cells infected with the malaria parasite[END_REF][START_REF] Ginsburg | New permeability pathways induced in membranes of Plasmodium falciparum infected erythrocytes[END_REF]) and the PVM [START_REF] Desai | A nutrient-permeable channel on the intraerythrocytic malaria parasite[END_REF][START_REF] Garten | EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX[END_REF][START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF] but this transport route has not so far been analysed in ART R parasites.

Here we exploited EXP1 mutants to analyse the function of the NPCA in the passage of small solutes across the PVM to reach the parasite and the role of this process in ART R parasites.

The results of this work provide evidence that amino acid deprivation is a reason for the fitness cost in ART R parasites and that Plasmodium parasites can compensate this fitness cost by increasing EXP1 expression to elevate NPC-dependent acquisition of serum-derived amino acids. These findings suggest that the reduced hemoglobin endocytosis in ART R parasites has profound downstream effects that include compensatory adaptations and may explain many changes observed in these parasites.

Results

Nutrients passing through the NPCs

To study the NPCA we took advantage of previously established conditional exp1 knock out (KO) parasites that contain an additional copy of exp1 expressed at low levels [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF]. Induction of the KO (+ rapalog) results in parasites relying on low levels of EXP1 derived from the additional copy (Figure 1A). These EXP1 low parasites show reduced NPCA and are hypersensitive to low amino acid concentrations [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF]. This indicated that the passage of amino acids through the NPC is vital for P. falciparum blood stages and that this genetic approach can be used to assess which nutrients reach the parasite via the NPC and if passage of a particular nutrient through the NPC is relevant for parasite growth.

To further characterise the relationship between EXP1 function and the NPCA we tested whether parasites relying on different modified versions of EXP1 (containing mutations or deletions) showed alterations in the NPCA . Besides previously established complementation constructs [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF] we included a construct with a single point mutation in the EXP1 transmembrane (TM) domain from encoding glycine to the typical TM helix residue leucine (EXP1 TMmut G83L ) which was sufficient to fully inactivate EXP1 . Parasites depending on versions of EXP1 with low complementation activity displayed an aberrant localization of EXP2 (as observed in the EXP1 KO [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF]) and became more sensitive to amino acid deprivation . Linear regression analysis showed that the mis-distribution of EXP2

and the susceptibility to amino acid restriction correlated with the complementation-activity of a given EXP1 version (Figures 1C and 1D). This provides evidence that EXP1 is a contributor to the NPCA and that EXP1 activity directly influences the access of nutrients through the NPC.

Previous work indicated that the NPCs are permeable for monosaccharides [START_REF] Desai | Pore size of the malaria parasite's nutrient channel[END_REF][START_REF] Garten | EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX[END_REF][START_REF] Tanabe | Glucose transport in malaria infected erythrocytes[END_REF]. Malaria parasites require a continuous supply of glucose during propagation within RBCs [START_REF] Kirk | Glucose uptake in Plasmodium falciparum-infected erythrocytes is an equilibrative not an active process[END_REF][START_REF] Woodrow | Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes[END_REF].

EXP1 low parasites were hypersensitive to glucose restriction (Figure 1E), indicating that glucose reaches the parasite via NPCs and that its NPC passage is required for efficient parasite growth (Figure 1E). In contrast, EXP1 low parasites were not hypersensitive to low concentrations of the essential primary purine precursor hypoxanthine [START_REF] Asahi | Hypoxanthine: a low molecular weight factor essential for growth of erythrocytic Plasmodium falciparum in a serum-free medium[END_REF] or the essential fatty acids oleic and palmitic acid (that must be scavenged from the host cell [START_REF] Mi-Ichi | Oleic acid is indispensable for intraerythrocytic proliferation of Plasmodium falciparum[END_REF][START_REF] Mitamura | Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum[END_REF]), indicating that these nutrients do not rely on the NPCs to cross the PVM (Figures 1F and1G).

To confirm that our growth assay reflects NPC-dependent passage of the assayed nutrients into the parasite, we used the fluorescently labelled glucose analogue 2-(N-(-7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) [START_REF] Low | Deletion of Plasmodium falciparum Protein RON3 Affects the Functional Translocation of Exported Proteins and Glucose Uptake[END_REF][START_REF] Yamada | A real-time method of imaging glucose uptake in single, living mammalian cells[END_REF] (Figure 1H) to directly monitor glucose uptake in EXP1 low parasites. EXP1 low rings or trophozoites both took up less glucose than controls and EXP1 overexpression (EXP1 high ) restored glucose uptake (Figures 1I,. This is consistent with the results from the growth assays under glucose restriction (Figure 1E) and indicated that the NPCs are already active in ring stages.

Drugs pass through the PVM NPC

Given the limited specificity of the NPCs [START_REF] Desai | Pore size of the malaria parasite's nutrient channel[END_REF] we tested their relevance for access of antimalarial drugs to the parasite. For this we exposed EXP1 low parasites to IC50 96h of different drugs and assessed whether this affected the relative sensitivity of these parasites compared to controls. IC50 96h of the drugs were used to maximise the possible growth difference in either direction. Compared to controls, EXP1 low parasites showed a reduced susceptibility to the 4-aminoquinolines (4-AQ) chloroquine, amodiaquine, and mefloquine (MFQ) as well as to dihydroartemisinin (DHA) (Figures 2A-2D

and S2A-S2D), suggesting that the NPC facilitate access of these drugs to the parasite. In contrast, MMV007839, an inhibitor of the lactate transporter at the parasite plasma membrane [START_REF] Golldack | Substrate-analogous inhibitors exert antimalarial action by targeting the Plasmodium lactate transporter PfFNT at nanomolar scale[END_REF], and epoxomicin, a proteasome inhibitor peptide, did not require the NPC to reach the parasite (Figures 2E and2F).

If the reduced susceptibility of EXP1 low parasites was due to a reduced access of molecules across the PVM, a higher NPCA would be expected to render parasites more sensitive to NPCpermeable drugs. In accordance, parasites overexpressing EXP1 (via an episomal Ty1-tagged copy) became more sensitive to DHA and MFQ (Figure 2G). We also assessed the effect of exposure to IC50 96h DHA in our different complementation lines . In accordance with Figure 2D, parasites relying on EXP1 versions with low activity were less susceptible to DHA (Figures S2E-S2L) and the susceptibility to DHA correlated with the activity of the EXP1 construct the parasites were relying on (Figure 2H). These findings indicated that the amount of DHA reaching the parasite correlates with the level of activity of EXP1.

4-AQ derivatives accumulate in the FV, where they inhibit hemozoin formation [START_REF] Foley | Quinoline antimalarials: mechanisms of action and resistance and prospects for new agents[END_REF] and DHA is activated in the FV by hemoglobin by-products [START_REF] Klonis | Iron and heme metabolism in Plasmodium falciparum and the mechanism of action of artemisinins[END_REF]. It is therefore possible that the passage of other molecules through the NPC indirectly influences the action of these drugs (e.g. by changing drug activation or accumulation in the FV). For instance, glucose depletion influences FV pH [START_REF] Saliba | Acidification of the malaria parasite's digestive vacuole by a H+-ATPase and a H+-pyrophosphatase[END_REF], which affects drug accumulation of some 4-AQ [START_REF] Jida | A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs[END_REF][START_REF] Yayon | Identification of the acidic compartment of Plasmodium falciparum-infected human erythrocytes as the target of the antimalarial drug chloroquine[END_REF]. However, MFQ, which may have a different mode and site of action than other 4-AQ [START_REF] Cowman | Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine[END_REF][START_REF] Wong | Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis[END_REF], was still dependent on the NPCs in our assay , arguing against an indirect effect based on mode of action. To further assess indirect influences, we tested lumefantrine, an aryl amino alcohol unrelated to 4-AQ but with a similar mode of action [START_REF] Combrinck | Insights into the role of heme in the mechanism of action of antimalarials[END_REF][START_REF] Cowell | The genomic architecture of antimalarial drug resistance[END_REF]. Lumefantrine susceptibility was not influenced by NPC levels (Figure 2I), suggesting that the reduced susceptibility for 4-AQ in EXP1 low parasites is independent of indirect effects arising from the mode of action of these drugs. To further explore this issue, we directly monitored uptake of a fluorescently labelled CQ analogue (Fluo-CQ) that has similar properties to CQ [START_REF] Jida | A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs[END_REF] (Figure 2J).

Unexpectedly, the uptake of Fluo-CQ and accumulation in the FV was not affected in EXP1 low parasites (Figures 2J,2K and S3A) and in contrast to CQ there was also no reduced susceptibility to Fluo-CQ in these parasites (Figure 2L), suggesting that Fluo-CQ crosses the PVM independently of the NPC. A related fluorescent CQ analogue (CQ1-NBD) (Figure S3B) that locates to the parasite cytosol behaved similarly to Fluo-CQ (Figures S3C-S3F). Addition of the NBD group tethered to a hydrophobic spacer of 5 methylene groups (Figures 2J and S3B-S3C) likely increased the lipophilicity and membrane permeability of these CQ analogues.

Since Fluo-CQ has the same mode and site of action as CQ but does not depend on the NPC to reach the parasite, this further supports that our growth assay measures the permeability of the analysed 4-AQ through the NPC rather than indirect effects related to drug action or activation.

Overall, these results suggest that the non-specific nature of the NPC renders the PVM permeable to both metabolites important for parasite growth and several antimalarial drugs.

A comparison of the physicochemical properties of all molecules tested here suggests that small water-soluble molecules reach the parasite via the NPCs. Hydrophobic or bulky molecules cross the PVM independent of the NPCs (Table 1).

NPCA influences susceptibility to PPQ

PPQ, a 4-AQ used as partner drug in ART combination therapies, inhibits hemozoin formation in the FV [START_REF] Dhingra | A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine[END_REF]. In contrast to the other tested 4-AQ (Figures 2A-2C), EXP1 low parasites became hypersensitive to PPQ (Figures 3A-3C). This unexpected finding suggested that the mode of action of PPQ synergises with the phenotype of a reduced NPCA. It was previously reported that besides inhibition of hemozoin formation PPQ also inhibits hemoglobin proteolysis [START_REF] Bopp | Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum[END_REF][START_REF] Dhingra | A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine[END_REF][START_REF] Sachanonta | Ultrastructural and real-time microscopic changes in P. falciparum-infected red blood cells following treatment with antimalarial drugs[END_REF] which may starve the parasites from hemoglobin derived-amino acids. Loss of EXP1 and simultaneous inhibition of hemoglobin digestion may together aggravate the reduction of the amino acid supply and render EXP1 low parasites more sensitive to PPQ. Supporting this hypothesis, parasites treated with IC50 96 of PPQ became hypersensitive to amino acid restriction(Figure 3D). Conversely, overexpression of EXP1 improved growth of PPQ-treated parasites under amino acid restriction, suggesting that increased NPC levels restore amino acid supply when hemoglobin digestion is inhibited (Figure 3D). In standard medium parasites overexpressing EXP1 showed a trend for a decreased PPQ susceptibility compared to wt parasites (Figures 3D and 3E). This may indicate that PPQ reaches the parasite independent of the NPCs and that the elevated NPCA increased amino acid availability rather than PPQ passage. These findings support the previously indicated dual mode of action of PPQ [START_REF] Bopp | Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum[END_REF][START_REF] Dhingra | A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine[END_REF] and an important role of the NPCs in the acquisition of amino acids from the host's serum when hemoglobin-derived amino acids are reduced.

Reduced hemoglobin-derived amino acids cause fitness cost in ART R parasites ART R parasites display a fitness cost [START_REF] Nair | Fitness Costs and the Rapid Spread of kelch13-C580Y Substitutions Conferring Artemisinin Resistance[END_REF][START_REF] Straimer | Plasmodium falciparum K13 Mutations Differentially Impact Ozonide Susceptibility and Parasite Fitness In Vitro[END_REF] and ART R field isolates have a lower transition rate from ring to trophozoite stage when amino acid restricted [START_REF] Bunditvorapoom | Fitness Loss under Amino Acid Starvation in Artemisinin-Resistant Plasmodium falciparum Isolates from Cambodia[END_REF]. As ART R parasites show a reduced rate of endocytosis in ring stages a shortened supply of amino acids from hemoglobin endocytosis may be a reason for this fitness cost [START_REF] Birnbaum | A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites[END_REF]. To test this idea, we first analysed the dependency of ART R parasites on amino acids from the culture medium which corresponds to the supply parasites obtain from the host serum in natural infections. 3D7 parasites with a resistanceconferring Kelch13 C580Y mutation (Kelch13 C580Y ) [START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF] were more sensitive to low amino acid levels than parasites with Kelch13wt (Figures 4A and4B). This indicated that ART R parasites have an increased demand for amino acids when isogenic laboratory lines are compared. This effect was specific for amino acids as these parasites behaved similarly to controls under low glucose conditions or if growth was inhibited using NaN3 (Figure 4A).

Isoleucine is the only amino acid absent from hemoglobin that the parasite needs to acquire from the serum [START_REF] Babbitt | Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state[END_REF][START_REF] Liu | Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems[END_REF]. To test whether the growth defect observed in ART R parasites is specific for a diminished supply of hemoglobin-derived amino acids, we cultured sensitive and the 3D7 Kelch13 C580Y ART R parasites in amino acid-restricted medium with or without isoleucine. ART R parasites showed a similar growth defect compared to wild type parasites, irrespective of whether isoleucine was present or not (Figures 4C and4D).

Phosphorylation of the eukaryotic initiation factor 2 α (eIF2α) is a hallmark of the response to amino acid starvation [START_REF] Fennell | PfeIK1, a eukaryotic initiation factor 2alpha kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation[END_REF]. Previous studies showed increased eIF2α phosphorylation in ART R parasites [START_REF] Yang | Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance[END_REF][START_REF] Zhang | Inhibiting the Plasmodium eIF2alpha Kinase PK4 Prevents Artemisinin-Induced Latency[END_REF] or when amino acids were limiting [START_REF] Babbitt | Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state[END_REF][START_REF] Fennell | PfeIK1, a eukaryotic initiation factor 2alpha kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation[END_REF]. To mimic amino acid deprivation in the ring phase of ART R parasites we cultured 3D7 rings under low amino acid conditions. These parasites showed more phosphorylated eIF2α than parasites grown in standard medium . Elevated levels of phosphorylated eIF2α were also detected in ART R parasites grown in amino acid restricted medium. Supplementation with isoleucine alone did not reduce the amount of phosphorylated eIF2α, indicating that the reduced supply of hemoglobin-derived amino acids in ring stages is sufficient to trigger an amino acid starvation response. Altogether, these experiments support the idea that a shortage of hemoglobinderived amino acids is a cause for the fitness cost in ART R parasites.

EXP1 upregulation can compensate the fitness cost in ART R parasites

Previous work had indicated a role of EXP1 in ART resistance and exp1 transcript levels were observed to be upregulated in some resistant parasite lines [START_REF] Lisewski | Supergenomic network compression and the discovery of EXP1 as a glutathione transferase inhibited by artesunate[END_REF][START_REF] Rocamora | Oxidative stress and protein damage responses mediate artemisinin resistance in malaria parasites[END_REF]. Paradoxically, we here found that parasites with low NPCA had a decreased susceptibility to DHA, indicative of a reduced passage of DHA molecules through the PVM (Figures 2D,. Thus, increased EXP1 levels would be expected to sensitise parasites to ART rather than causing resistance. Indeed, overexpression of EXP1 increased the susceptibility to ART (Figure 2G), although this likely is not clinically relevant at these ART concentrations [START_REF] Khoury | Artemisinin Resistance and the Unique Selection Pressure of a Short-acting Antimalarial[END_REF] (see Supplementary Note 1). In agreement, we previously found no evidence for a direct role of EXP1 in ART resistance [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF].

We therefore looked for alternative explanations for the elevated exp1 mRNA levels reported in ART R parasites.

Since the NPCA is critical for amino acid acquisition, EXP1 levels might compensate the reduced amino acid supply in ART R parasites by increasing amino acid access via the NPCs. To test this we overexpressed EXP1 (by an additional episomal copy of exp1) in the 3D7 Kelch13 C580Y parasites (Figures 4F andS4A) which reduced the growth defect under limiting amino acid levels whereas no change was observed in low glucose medium or in the presence of NaN3 (Figures 4A and4B). No difference was observed if EXP1 was overexpressed in Kelch13wt parasites (Figures 4B and S4B). This indicated that specifically the amino acid access through the NPC compensated for the endocytosis reduction in ART R parasites.

Importantly, overexpression of EXP1 restored growth of ART R parasites also in presence of isoleucine (Figures 4C and 4D), connecting the effect to hemoglobin-derived amino acids.

These findings indicate that increased NPC levels compensate the fitness cost of ART resistance by increasing the access to amino acids the parasite would otherwise obtain from hemoglobin digestion. Overexpression of EXP2 (a protein also needed for NPC [START_REF] Garten | EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX[END_REF]) in Kelch13 C580Y parasites did not significantly improve growth under low amino acid conditions (Figures 4B and S4C), suggesting that its upregulation did not sufficiently raise the NPCA to compensate the fitness cost in ART R parasites.

As the resistant parasites used in these experiments had time to adapt to the fitness cost of ART-resistance, we assessed the role of EXP1 in transgenic 3D7 parasites where endocytosis can be conditionally reduced by partially inactivating Kelch13, leading to ART resistance [START_REF] Birnbaum | A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites[END_REF]. The experiments with these parasites (Kelch13-3xNLS) [START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF] confirmed the results with the Kelch13 C580Y parasites: after partial inactivation of Kelch13, the parasites became hypersensitive to low amino acid but not to low glucose levels or to growth inhibition by NaN3. Under amino acid restriction growth of parasites with an inactivated Kelch13 was improved two-fold when EXP1 was overexpressed (+rap, 39.67%) compared to parasites with physiological levels of EXP1 (+rap, 19.37%) (Figures 4G andS4D).

Consistent with the findings in 3D7 parasites (Figures 2D,2G and 2H), upregulation of EXP1 rendered Kelch13 C580Y parasites more sensitive to IC50 96h of DHA (Figure 4H; Supplementary Note 1). Interestingly, Kelch13 C580Y parasites overexpressing EXP1 did not become more sensitive to MFQ (Figure 4H). The reasons for this are unclear, but as also the response to DHA was less than in 3D7, it is possible that resistance already resulted in compensatory changes in these parasites.

NPC upregulation is a natural mechanism to compensate amino acid restriction

Our results indicated that elevated EXP1 levels can compensate the fitness cost in ART R parasites. To test if such a mechanism could be relevant in natural malaria infections, we assessed exp1 mRNA levels in blood samples of malaria patients with known ART resistance characteristics [START_REF] Zhu | The mechanism of artemisinin resistance of <em>Plasmodium falciparum</em> malaria parasites originates in their initial transcriptional response[END_REF] from the TRACII study [START_REF] Van Der Pluijm | Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial[END_REF]. Of these, 577 samples were analysed by microarray and 188 by RNA-Seq. Parasites in patients with prolonged clearance times after ART treatment (indicative of ART resistance) had more exp1 transcripts than ART sensitive controls . Estimation of parasite age in the RNA-Seq samples [START_REF] Lemieux | Statistical estimation of cell-cycle progression and lineage commitment in Plasmodium falciparum reveals a homogeneous pattern of transcription in ex vivo culture[END_REF][START_REF] Zhu | The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background[END_REF] indicated that this effect was not due to a stage difference between the samples (Figure 5B). In addition, exp1 mRNA levels correlated positively with the level of resistance of the parasites in these samples (Figure 5C).

Ring stages of our 3D7 Kelch13 C580Y ART R laboratory parasites also showed elevated exp1

mRNA levels compared to their isogenic control (Figure 5D and S6) whereas ART R trophozoites did not (Figure 5E).

To confirm these findings at the protein level we generated cell lines expressing endogenously HA-tagged EXP1 in resistant (Kelch13 C580Y ) and sensitive (Kelch13 wt ) 3D7 parasites . ART R rings (0-8 hpi) had higher EXP1 protein levels than Kelch13 wt parasites (Figures 5G,. ART R trophozoites (24-32 hpi) also showed an increased amount of EXP1 protein compared to sensitive parasites, presumably due to an accumulation of protein from the entire ring phase. Overall these results indicated that in both, natural infections and in laboratory lines, ART R parasites compensated the reduced hemoglobinderived amino acid supply by increasing exp1 transcription.

Next, we tested if parasites were capable to respond in a similar way when amino acid levels were reduced in the medium. Both sensitive and ART R parasites showed elevated exp1 levels in rings when selected under low amino acid conditions compared to parasites grown in standard medium (Figure 5D), indicating that rings can adapt to low amino acid conditions by increasing NPC levels. ART R trophozoites did not further increase exp1 transcripts in response to amino acid deprivation, likely due to the baseline elevation of exp1 transcripts in the preceding ring, leading to sufficient accumulation of EXP1 protein in the PVM (Figures 5E and5H). Transcript levels of exp2 and several amino acid transporters (assumed to mediate amino acid transport foremost at the parasite plasma membrane as known for ApiAT8 [START_REF] Blomqvist | Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome[END_REF]) were also increased in ART R parasites and after selection in low amino acid medium (Figure S6). While the transcript levels of amino acid transporters showed a mixed profile in patient sample parasites, two were upregulated in parasites with a delayed clearance (Figure S5). The ability of parasites to respond to low amino acid conditions by upregulating EXP1 was also confirmed at the protein level. In parasites with an endogenously HA-tagged EXP1

(Figures S4E-S4G), EXP1 was elevated after growth under amino acid restriction for 4 weeks compared to parasites grown in standard medium (Figures 5I, 5J and S7A-B).

Overall, these results show that ART R parasites have elevated EXP1 levels and that parasites respond in a similar way to growth in low amino acid medium. Since increasing EXP1 levels mitigates the hypersensitivity of ART R parasites to low amino acid conditions we propose that EXP1-dependent upregulation of the NPCA is a natural mechanism in ART R parasites to increase the acquisition of amino acids from the host serum and to compensate the fitness cost caused by their diminished hemoglobin endocytosis (Figure 6).

Discussion

Here we show that growth-relevant amounts of energy and carbon sources pass through the PVM NPCs and that this activity is directly linked to the level of EXP1 activity. While the pore size of the NPCs was already assessed electrophysiologically with dextrans and solutes in freed parasites or isolated membranes [START_REF] Desai | A nutrient-permeable channel on the intraerythrocytic malaria parasite[END_REF][START_REF] Desai | Pore size of the malaria parasite's nutrient channel[END_REF], our growth assays show which molecules pass through the NPCs in living parasites and whether this activity is relevant for parasite growth or not. Hypoxanthine and fatty acids were NPCindependent, agreeing with the high passive membrane permeability of hypoxanthine [START_REF] Whittam | The high permeability of human red cells to adenine and hypoxanthine and their ribosides[END_REF] and the fact that fatty acids are not typically transported by non-selective channels [START_REF] Schwenk | Fatty acid transport across the cell membrane: regulation by fatty acid transporters[END_REF]. The NPCs are already active in ring stages, as previously postulated [START_REF] Low | Deletion of Plasmodium falciparum Protein RON3 Affects the Functional Translocation of Exported Proteins and Glucose Uptake[END_REF][START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF] and this may be the reason for the delayed ring stage in EXP1 parasites [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF].

It should be noted that the EXP1 low parasites still have partial NPCA and hence the observed effect is smaller than the actual contribution of the NPCs to generate access for a particular nutrient. However, as complete removal of exp1 is lethal [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF], full KOs are unsuitable for these experiments. While the standard RPMI medium contains an excess of nutrients, the concentrations used in our limiting media are below those typical for human serum [START_REF] Brown | From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro[END_REF][START_REF] Kumar | Linking nutrient sensing and gene expression in Plasmodium falciparum blood-stage parasites[END_REF] (Supplementary Table 1). Such conditions were previously used to show the importance for nutrient passage through the RBC membrane which only becomes apparent if nutrients are limiting [START_REF] Pillai | Solute restriction reveals an essential role for clag3-associated channels in malaria parasite nutrient acquisition[END_REF].

Critically, we provide evidence that several antimalarial drugs pass through the NPCs. The properties for NPC passage (Table 1) might inform medicinal chemistry approaches for lead compounds with poor membrane permeability. While we found that modulation of NPC levels alters drug access, this is constrained by the essential nutrient passage function. A diminished NPCA is therefore unlikely to be an option for parasites to become resistant to drugs but the NPCs may themselves represent a drug target that due to the broad functionality may not be prone to resistance.

It was shown that non-ionised CQ passively passes into uninfected RBCs, indicating that this may be the main mode of how CQ crosses membranes (Ferrari andCutler, 1990, 1991).

However, only a very small fraction of all CQ molecules is in the non-ionised form at physiological pH and evidence of ion-trapping in uninfected RBCs suggests that once in the RBC, the proportion of ionised CQ may be even higher [START_REF] Fitch | Erythrocyte surface: novel determinant of drug susceptibility in rodent malaria[END_REF][START_REF] Macomber | Chloroquine: physiological basis of drug resistance in Plasmodium berghei[END_REF]. The predominance of this poorly membrane permeable ionised form of CQ could explain our finding that CQ depends on the NPC to reach the parasite. This scenario likely applies also for the other 4-AQ with the possible exception of PPQ for which the additional mode of action precluded assessing NPC dependence with certainty in our assay. However, as parasites do not become more sensitive to PPQ if EXP1 is overexpressed (in contrast to MFQ), PPQ may be NPC-independent and this hypothesis fits with the larger molecular weight and higher lipophilicity of PPQ compared to other 4-AQ (Table 1). Interestingly, Fluo-CQ, a more hydrophobic analogue of CQ, did not to depend on the NPCs in both, our growth assay and when microscopically monitoring uptake into the parasite. Hence, Fluo-CQ, as suggested previously [START_REF] Jida | A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs[END_REF], has better passive membrane permeability but may be too bulky for the NPC. As Fluo-CQ retains the mode of action of CQ [START_REF] Jida | A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs[END_REF], the findings with this molecule show that our assay was not influenced by the mode of action of 4-AQ, excluding such effects as a reason for the reduced susceptibility of EXP1 low parasites to 4-AQ.

Overall, our data therefore support the idea that 4-AQ, once in the RBC cytosol, do not efficiently cross the PVM in a passive way but require the NPCs.

Our findings reveal a critical role for the parasite´s amino acid supply in drug action and resistance. ART R parasites show diminished hemoglobin endocytosis in ring stages which is expected to reduce amino acid levels [START_REF] Birnbaum | A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites[END_REF]. The hypersensitivity of ART R parasites to low amino acid levels observed here as well as in previous work [START_REF] Bunditvorapoom | Fitness Loss under Amino Acid Starvation in Artemisinin-Resistant Plasmodium falciparum Isolates from Cambodia[END_REF] and the reversal of this effect by increasing the parasite's NPCA, lend experimental evidence that amino acid deprivation contributes to the fitness cost in ART R parasites. Accordingly, we found evidence for elevated exp1 mRNA levels in ART R parasites from human infections in South East Asia. Together with our in vitro work this indicates that increased EXP1 levels, and possibly other means to increase amino acid acquisition, are a compensatory mechanism of the parasite to reduce the fitness cost of ART resistance (Figure 6). Reducing either amino acid concentration in the medium, or Kelch13 levels (resulting in ART resistance) or EXP1 levels, each prolong the ring stage [START_REF] Birnbaum | A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites[END_REF][START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF] and ART R parasites also have a prolonged ring stage [START_REF] Hott | Artemisinin-resistant Plasmodium falciparum parasites exhibit altered patterns of development in infected erythrocytes[END_REF][START_REF] Mok | Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription[END_REF], congruent with our conclusion that amino acid deprivation is an important factor responsible for the fitness cost in ART R parasites and that NPC levels influences this factor. This fitness cost and the compensatory mechanisms in ART R parasites are likely most relevant in untreated hosts where Kelch13 wild-type parasites have a growth advantage over ART R parasites. In contrast, resistance itself is the deciding selection advantage over sensitive parasites during the short times when the parasites are exposed to ART.

Our findings also support work indicating inhibition of hemoglobin proteolysis (thereby reducing amino acid levels) as an additional mode of action for PPQ compared to that of other 4-AQ [START_REF] Bopp | Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum[END_REF][START_REF] Dhingra | A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine[END_REF]. Hence, ART resistance and the frequently used ART partner drug PPQ might pose a synergistic selection pressure to increase the amino acid supply from sources other than hemoglobin and may have favoured compensatory changes facilitating the development of resistance with this drug combination.

Distinguishing cause, effect and compensatory mechanisms in pathogen resistance is often not straight forward. By modulating NPC levels and using restricted media this work indicates an increased dependency on serum-derived amino acids as a downstream effect of ART resistance and upregulation of EXP1 as a compensation mechanism, rather than being itself a cause of resistance. Amino acids are critical for protein synthesis and have central functions in cellular metabolism [START_REF] Krishnan | Amino Acid Metabolism in Apicomplexan Parasites[END_REF]. Isoleucine is the only amino acid absent from hemoglobin. Reduced hemoglobin endocytosis in ART R parasites is therefore not expected to resemble isoleucine starvation [START_REF] Babbitt | Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state[END_REF]. Nevertheless, the reduction of hemoglobin-derived amino acids also resulted in a general amino acid deprivation response as indicated by the eIF2α phosphorylation. These results may explain the basal eIF2α phosphorylation in ART R parasites [START_REF] Yang | Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance[END_REF][START_REF] Zhang | Inhibiting the Plasmodium eIF2alpha Kinase PK4 Prevents Artemisinin-Induced Latency[END_REF]. However, the deprivation of hemoglobin-derived amino acids will also result in effects specific to the metabolic function of each kind of amino acid. For instance glutamine [START_REF] Divo | Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth[END_REF] is the main carbon input of the mitochondrial TCA cycle in asexual blood stage parasites [START_REF] Macrae | Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum[END_REF] and is known from other organisms to be required for the synthesis of many metabolites, including glutathione and nucleotides [START_REF] Krishnan | Amino Acid Metabolism in Apicomplexan Parasites[END_REF][START_REF] Yoo | Glutamine reliance in cell metabolism[END_REF]. The reduction of hemoglobin-derived amino acids indicated by our work could therefore be one reason for some of the wide-ranging differences seen in ART R parasites compared to sensitive parasites [START_REF] Mok | Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance[END_REF][START_REF] Mok | Artemisinin-resistant K13 mutations rewire Plasmodium falciparum's intra-erythrocytic metabolic program to enhance survival[END_REF][START_REF] Sutherland | Artemisinin susceptibility in the malaria parasite Plasmodium falciparum: propellers, adaptor proteins and the need for cellular healing[END_REF][START_REF] Yang | Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance[END_REF][START_REF] Zhang | Inhibiting the Plasmodium eIF2alpha Kinase PK4 Prevents Artemisinin-Induced Latency[END_REF].

It is possible that the trade-off between ART resistance and acquisition of amino acids limits the extent of resistance achievable with the Kelch13-based resistance mechanism and provides one possible explanation why full ART resistance has so far not arisen. In this scenario the maximum capacity to obtain amino acids via the NPCs (and possibly other components influencing amino acid levels in the parasite) limits the level of ART resistance, because endocytosis cannot be further reduced without critically reducing amino acid levels in the parasite (Figure 6). This also raises the prospect that host nutritional status, a factor affecting the amino acid concentrations in the serum [START_REF] Semba | Child Stunting is Associated with Low Circulating Essential Amino Acids[END_REF], may influence the fitness cost of ART resistance and the efficacy of PPQ in P. falciparum infected individuals.

Knowing the factors incurring the fitness cost in ART R parasites and the mechanisms of the parasite to mitigate these effects could help identify further strategies that could contribute to the current efforts to extend the usefulness of ART [START_REF] Schallig | Randomised controlled trial of two sequential artemisinin-based combination therapy regimens to treat uncomplicated falciparum malaria in African children: a protocol to investigate safety, efficacy and adherence[END_REF]van der Pluijm et al., 2020).

STAR Methods

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Tobias Spielmann spielmann@bni-hamburg.de

Materials availability

All unique reagents generated in this study can be obtained upon reasonable request to the Lead Contact.

Data and code availability

Examples

The published article includes all data sets generated or analysed during this study.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Parasite cell lines

Asexual intra-erythrocytic P. falciparum parasites (strain 3D7) were cultured in human erythrocytes (O+) (transfusion blood, Universität Klinikum Eppendorf, Hamburg). Cultures were grown in RPMI complete medium (Applichem, Darmstadt, Germany) containing 0.5% Albumax (Invitrogen, Carlsbad, CA), 20 mM glucose and 200 mM hypoxanthine according to standard procedures and maintained at 37˚C in a microaerophilic atmosphere of 1% O2, 5% CO2, and 94% N2. Transgenic cell lines were maintained under drug selection with 2 μg/ml Blasticidin S (Life Technologies) (for expression of the DiCre from pSkipFlox [START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF], 0.9 μM DSM1 (BEI Resources; https://www.beiresources.org) (for expression of EXP1 complementation constructs) and 400 μg/ml G418 (Sigma-Aldrich).

Human blood samples

Samples to drive the expression of EXP1-TMmut G83L and yDHODH as resistance marker.

To To modify the endogenous exp1 locus in GFP-Kelch13 wt and GFP-Kelch13 C580Y parasites [START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF] (which are WR99210 and DSM1 resistant) via SLI, the last 467 bp of the exp1 gene were amplified with primers to append a triple HA tag. The neomycin phosphotransferase gene was amplified with primers to append upstream a T2A skip peptide (See Figure S4E). Both fragments were cloned into a vector containing blasticidine deaminase as resistance marker(Mesen-Ramirez et al., 2016) using NotI and XmaI to generate a promoterless plasmid (pSLI2-BSD).

Parasite transfection

For transfection of episomal constructs, mature schizonts were enriched with 60% Percoll and resuspended in transfection buffer containing 50 μg of plasmid DNA and electroporated using a Nucleofector II (Lonza). For generation of complementation cell lines(Mesen-Ramirez et al., 2019) condΔEXP1 parasites were transfected with pEXP1comp plasmids containing yDHODH as resistance marker and selection was done with 0.9 μM DSM1 and BSD. Transgenic Kelch13 C580Y and Kelch13+3NLS parasites [START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF] were transfected with pEXP1comp-Neo and pEXP2comp-Neo plasmids and selected with 400 μg/ml G418 (Sigma-Aldrich) to obtain Kelch13 C580Y +EXP1, Kelch13+3NLS+EXP1 and Kelch13 C580Y +EXP2.

Preparation of nutrient-limited media

To obtain amino acid-restricted RPMI medium, RPMI-1640 complete medium was added in a 1:20 dilution to amino acid-free RPMI medium 1640 (US Biological) containing 5% Albumax, 5 mM glucose and 200 µM hypoxanthine. This resulted in a final concentration of 6 mM glucose and 1:20 of the concentration of every amino acid found in standard RPMI complete (Supplementary Table 1). For growth experiments in presence of isoleucine we adjusted the amino acid restricted medium to a level that parasites would grow ~50% (compared to standard medium) when isoleucine was added. For this RPMI-1640 complete medium was added in a 1:50 dilution to amino acid-free RPMI and further supplemented with fresh isoleucine to a final concentration of 382 µM.

To prepare glucose-restricted medium, glucose-free RPMI medium (Gibco) containing 5%

Albumax and 200 µM hypoxanthine was supplemented with D-(+) glucose monohydrate (Merck) to a final concentration of 2.5 mM. For hypoxanthine-restricted medium RPMI complete medium containing 5% Albumax was supplemented with hypoxanthine to a final concentration of 500 nM. Lipid-free medium [START_REF] Brancucci | Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum[END_REF] was generated by complementing RPMI-1640 complete (prepared as described above, without Albumax) with 0.39% fatty acid-free BSA (Sigma). To generate fatty acid-restricted medium oleic and palmitic acids were added to this medium at a final concentration of 30 μM each (Sigma-Aldrich). Lyso PC (Avanti Polar Lipids) was added to this medium to a final concentration of 20 μM from 30 mM ethanol-solved stocks as previously done [START_REF] Brancucci | Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum[END_REF]. A second medium containing 50 μM of oleic and palmitic acids each was used as control medium containing non-limiting amounts of fatty acids. Optimal nutrient concentrations to obtain a parasite growth of ~50 % were determined experimentally (Figure S8H andS8I).

Conditional EXP1 knock-out via DiCre-mediated excision in complementation cell lines

Ring stages of P. falciparum transgenic and condEXP1 cell lines expressing Ty-tagged EXP1

with different modifications under different promoters (condEXP1 low , condEXP1 mid and condEXP1 high ) were synchronised twice with 5% sorbitol with a development stage window a time window of 6 hours as described previously [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF]. Parasite cultures were divided into two culture dishes and rapalog (Clontech) was added to a final concentration of 250 nM to one of the two dishes. The dish without rapalog served as control culture. The medium was changed daily and the parasites further cultured in the presence of rapalog for 48 hours. This resulted in EXP1 low , EXP1 mid and EXP1 high parasites (designated 'rapalog') that relied on the episomally expressed construct. These parasites were compared to the matched controls that did not receive rapalog and still contained the genomic exp1 gene (designated 'control') and used for growth, uptake and immunofluorescence assays.

Imaging

Microscopy of live parasites and immunofluorescence assay was performed with a Zeiss Axioscope M1 equipped with a 100X/1.4 numerical aperture oil immersion objective. A Hamamatsu Orca C4742-95 camera and the Zeiss Axiovision software version 4.7 were used for collecting images. Images were processed and overlaid with Corel PHOTO-PAINT X6

(https://www.coreldraw.com). Fluorescence quantifications were performed with Image J version 1.46r [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF].

Visualization of glucose, Fluo-CQ and CQ-NBD1 uptake

Synchronised ring stages of EXP1 low (ΔEXP1 parasites relying on lowly expressed complementing EXP1-Ty) or EXP1 high parasites (containing a highly expressed complementing EXP1-Ty) obtained after one cycle with or without rapalog (see conditional diCre-based excision of complemented knock-outs) were allowed to progress to trophozoites. For glucose uptake, these trophozoites (24-30 hours post invasion) were washed once with RPMI medium containing 1 mM glucose and then incubated in RPMI medium containing 1 mM glucose and 100 µM 2-NBDG (Invitrogen) for 15 minutes at 37°C protected from light. The 2-NBDG stock (10 mM in ethanol) was stored at -80 C and freshly diluted before each assay. For uptake in ring stages, 4-12 hours post invasion rings were prepared similarly to the trophozoites but cells were incubated for 2 hours at 37°C with 100 µM 2-NBDG. For uptake of fluorescent CQ analogues, trophozoites were washed twice with RPMI medium complete, followed by incubation in RPMI medium containing 500 nM Fluo-CQ or 500 nM CQ1-NBD for 20 minutes at 37°C protected from light. The DMSO-solved stock (5 mM) of each compound was stored at -80 C and freshly diluted before each assay. After incubation of the parasites with 2-NBDG or CQ analogues, the parasites were washed twice with RPMI medium containing 1 mM glucose (glucose uptake) or RPMI complete (CQ uptake) and immediately imaged. All micrographs were recorded blinded to the nature of the sample with the same acquisition settings and exposure time. The fluorescence (as integrated density) in the parasite cytosol and area of fluorescence in µm 2 were measured with Image J version 1.46r [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] and the background fluorescence was subtracted. For Fluo-CQ, the fluorescence in the food vacuole (as integrated density) was measured. Images were processed with Corel PHOTO-PAINT X6 (https://www.coreldraw.com) and the integrated density and the area values were plotted using GraphPadPrism version 6.07.

Flow cytometry (FC)

Parasitemia of parasite cultures was determined as previously described using flow cytometry [START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF]. For this, 20 μl resuspended parasite culture was incubated in RPMI medium containing 4.5 μg/ml dihydroethidium (Cayman Chemical) and 5 μg/ml Hoechst (Cheomdex) for 20 minutes at room temperature protected from light. After this, the cells were fixed with RPMI containing 0.003% glutaraldehyde. For every sample, 100,000 events were recorded using a LSRII flow cytometer (Beckton Dickinson), and the parasitemia was determined with the FACS Diva Software. Gating strategy for infected RBCs is shown in Figure S8J.

Determination of 96 hours IC50 for antimalarial drugs and nutrients

To determine the IC50 after two growth cycles for the drugs used in this study condΔEXP1 parasites were sorbitol synchronised and the start parasitemia was adjusted to approximately 0.1% in RPMI complete medium with 5% haematocrit. Equivalent volumes of cells were cultured in 2 ml dishes containing RPMI complete with increasing concentrations of The final parasitemia for every drug concentration was measured at day five by FC and the start parasitemia was subtracted from the final parasitemia to obtain the adjusted final parasitemia. The IC50 values were calculated using GraphPadPrism version 6.07.

Growth assays under nutrient limiting conditions and in presence of drug at IC50 96h

To evaluate growth under low nutrient conditions, EXP1wt low , EXP1wt mid and EXP1wt high ring stage parasites (i.e. after excision of the endogenous genes from the condΔEXP1 parasites in the previous cycle to obtain parasites relying on the complementing constructs) and the matched controls grown without rapalog (see conditional EXP1 knock-out via DiCre-mediated excision in complementation cell lines) were diluted in RPMI complete and the parasitemia was adjusted to 0.1% (control) and 0.5% (rapalog), respectively. The cultures were washed twice with the corresponding RPMI nutrient-restricted medium (glucose, amino acid, hypoxanthine or lipid-free medium). Equivalent volumes of the washed cells were transferred to 2 ml dishes containing either RPMI complete medium or the corresponding nutrientrestricted medium. For drug susceptibility, the cells were transferred to RPMI complete without drugs or RPMI containing the IC50 96h determined for each drug: 15 nM chloroquine diphosphate, 7 nM MFQ hydrochloride (diluted from a 48.3 mM DMSO-solved stock), 2.5 nM amodiaquine, 1nM DHA (diluted from a 250 µM DMSO-solved stock), 33 nM lumefantrine (diluted from a 5 mM DMSO solved stock), 3 nM piperaquine tetraphosphate tetrahydrate (diluted from a water-solved 5.25 mM stock), 160 nM MVV007839, 2.5 nM epoxomicin (diluted from a 180 µM DMSO-solved stock), 10 nM Fluo-CQ and 8 nM CQ1-NBD, diluted from a 5 mM DMSO-solved stock). The curves for determination of IC50 96 are shown in Figure S2A-S2D and S8A-S8I.

For determination of susceptibility to low amino acid conditions, 3D7 wild type, 3D7+EXP1

and the transgenic Kelch13 cell lines (Kelch13 C580Y , Kelch13 C580Y +EXP1, Kelch13 C580Y +EXP2, Kelch13+3xNLS and Kelch13+3xNLS+EXP1) were synchronised twice with 5% sorbitol to obtain a development stage window of 5 hours. Ring stages of the next cycle were diluted to 0.1% parasitemia in complete RPMI medium and washed twice with RPMI low AA or with RPMI low glucose (depending on the nutrient assayed). Equal volumes of these cells were transferred to 2 ml dishes with RPMI complete with and without rapalog (250 nM), RPMI without AA, RPMI without AA ± 382 µM isoleucine, RPMI low AA (1:50) ± 382 µM isoleucine, low AA (1:20) ± rapalog, RPMI low glucose ± rapalog and RPMI complete containing 75 μM NaN3 (Sigma-Aldrich) ± rapalog. Cultures were fed daily with fresh medium containing the drugs to maintain the genetic modifications of the respective cell lines. For induction of knock sideways in Kelch13+3xNLS cell lines, fresh rapalog was added each day and parasites were maintained in the corresponding medium in presence or absence of rapalog for two growth cycles.

Initial (day 0) and final parasitemia (day 5) were measured using FC and initial parasitemia was subtracted from the final parasitemia to obtain the adjusted final parasitemia. Relative growth for each cell line and condition was calculated as the adjusted final parasitemia in restricted medium or medium containing IC50 96 of each drug versus the adjusted final parasitemia in RPMI complete medium without drug.

Immunofluorescence assays

To assess localization of EXP2 in the EXP1 complementation cell lines, the parasites were prepared for IFA as follows: ring stage parasites relying on the complementing constructs (i.e.

after excision of the endogenous genes from the condΔEXP1 parasites in the previous cycle, see conditional EXP1 knock-out via DiCre-mediated excision in complementation cell lines)

and the matched controls grown without rapalog were sorbitol synchronised and allowed to develop to 24-30 h.p.i trophozoites. Thereafter the cells were washed once in 1x PBS and fixed with 4% paraformaldehyde/0.0075% glutaraldehyde as described [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF][START_REF] Tonkin | Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method[END_REF] and used for IFA with the cells in suspension.

To evaluate the localization of the Ty-tagged EXP1-TMmut G83L complementation construct the cells were prepared for IFA as follows to distinguish protein localized at the PPM from that localized at the PVM as previously established(Mesen-Ramirez et al., 2019): Compound 2 [START_REF] Taylor | The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony[END_REF]) (a kind gift of Mike Blackman, the Francis Crick Institute, London) was added to trophozoite stages at 1 μM final concentration and grown overnight to obtain arrested-schizonts that were fixed in 4% paraformaldehyde/0.0075% glutaraldehyde as described [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF][START_REF] Tonkin | Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method[END_REF] and used for IFA with cells in suspension.

The IFA with the fixed cells was carried out essentially as described [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF][START_REF] Tonkin | Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method[END_REF]. The cells were permeabilised with 0.5% Triton X-100 in PBS, blocked with 3% BSA in PBS and incubated in suspension for 1 hour with primary antibodies: Fluor 488 or Alexa Fluor 594 conjugated secondary antibodies specific for mouse and rabbit IgG (Invitrogen) diluted 1:2,000 in 3% BSA in PBS and containing 1 μg/ml DAPI. Before imaging cells were washed five times with PBS.

To quantify the percentage of cells with a correct or aberrant localization of EXP2 at the PVM, parasites were counted (operator blinded to the identity of the sample) and classified as correct when EXP2 exhibited more than 50 % circumferential localization of the signal and as aberrant when less than 50% of the cell was surrounded by fluorescence.

For localization of EXP1-Ty in Kelch13 and 3D7 cell lines asynchronous parasite cultures were harvested, washed twice with PBS, air-dried as thin monolayers on 10-well slides (Thermo Fischer) and fixed in 100% acetone for 30 minutes at room temperature as described [START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF][START_REF] Spielmann | etramps, a new Plasmodium falciparum gene family coding for developmentally regulated and highly charged membrane proteins located at the parasite-host cell interface[END_REF]. After rehydration with PBS, the cells were labelled with mouse α-Ty (Sigma) (1:20,000) and α-GFP rabbit (Thermo Fischer) (1:500) for detection of GFP-tagged endogenous Kelch13wt and Kelch13 C580Y . Washes and incubation with secondary antibodies was performed as described above but carried out on the slide that was kept in a humid chamber. Slides were sealed with a cover slip using mounting medium (Dako).

Total protein extracts

For detection of EXP1wt-Ty1 in total parasite extracts of Kelch13 cell lines, an asynchronous culture was incubated with 0.03% saponin in PBS for 10 minutes on ice to lyse the host cells of the parasites and uninfected RBCs (saponin pellets). After three washes with DPBS proteins were extracted from the parasite pellet with protein lysis buffer (0.5x PBS/4% SDS/0.5% Triton X-100) containing protease cocktail inhibitor (Roche) and 1mM PMSF. After centrifugation at 16,000g for 5 minutes, the supernatant was transferred to a fresh tube and pellet and equivalent amounts of the supernatant were diluted with reducing SDS sample buffer and analysed by SDS-PAGE immunoblotting.

Solubility assays

To distinguish PV soluble proteins from parasite-internal and membrane-associated proteins, trophozoites of EXP1-TMmut G83L-Ty1 (from 5 ml culture with a parasitemia of ~5%) were enriched in a Percoll gradient [START_REF] Heiber | Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export[END_REF], washed twice with PBS and incubated on ice for 10 minutes with 100 μl PBS containing 0.015% saponin (Sigma) and centrifuged at 16,000g for 5 minutes. The supernatant (PV and host cell soluble proteins) was transferred to a new tube and mixed with protease cocktail inhibitor (Roche), 1 mM PMSF and reducing SDS sample buffer. The parasite pellet (membrane proteins and soluble proteins within the parasite) was resuspended in 100 μl of protein lysis buffer containing complete protease inhibitor cocktail and 1 mM PMSF. The pellet lysate was cleared by centrifugation at 16,000g

for 5 minutes, transferred to a new tube and mixed with reducing SDS sample buffer.

Equivalent volumes of the pellet lysate and the supernatant containing the soluble proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting.

Immunoblotting

Protein samples separated by SDS-PAGE were transferred to Amersham Protran membranes (GE Healthcare, Germany) in a tankblot device (Bio-Rad) using transfer buffer (0.192 M glycine, 0.1% SDS, 25 mM Tris-HCl pH=8.0) with 20% methanol. Membranes were blocked for 30 minutes with PBS containing 5% skim milk. Primary antibodies were diluted in PBS containing 5% skim milk and incubated with membranes overnight in the following dilutions: mouse α-Ty (Sigma), 1:20,000; mouse α-GFP (Roche), 1:1000; rabbit α-SBP1(C) [START_REF] Mesen-Ramirez | Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity[END_REF], 1:2,000;rabbit anti-BIP [START_REF] Struck | Re-defining the Golgi complex in Plasmodium falciparum using the novel Golgi marker PfGRASP[END_REF], 1:2,000; rabbit anti-phospho eIF2α (Ser51) (Cell Signaling), 1:1000 and mouse anti-GAPDH [START_REF] Daubenberger | Identification and recombinant expression of glyceraldehyde-3-phosphate dehydrogenase of Plasmodium falciparum[END_REF], 1:2000. After three washes with PBS, horseradish peroxidase-conjugated secondary antibodies goat α-mouse (Dianova), 1:3,000 or donkey α-rabbit (Dianova), 1:2,500 were added to the membrane in PBS containing 5% skim milk and incubated for 2 hours. Detection was done after three washes with PBS using enhanced chemiluminescence (Bio-Rad/Thermo Fischer), and signals were recorded with a ChemiDoc XRS imaging system (Bio-Rad).

Quantitative reverse transcription real-time PCR

Kelch13wt-GFP and Kelch13 C580Y -GFP [START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF] The LightCycler 480 (Roche) was used for quantitative real-time PCR analysis using the provided software version 1.5. cDNA template was mixed with QuantiTect SYBR Green PCR reagent (Qiagen) and 0.3 µM sense and antisense primer in a final volume of 10 µl. Reactions were incubated at 95°C for 15 minutes, then subjected to 40 cycles of 95°C for 15 seconds and 60°C for 1 minute and a subsequent melting step (60-95°C). The specificity of each primer pair was confirmed after each RT-qPCR run by dissociation curve analysis. Ct calculation was done using the fit points analysis method provided by the software. Relative quantification of exp1 and exp2 expression levels normalized to the expression of fructose-bisphosphate aldolase (PF3D7_1444800) was performed using primer sets described in Supplementary Table 2. To control for slight stage differences between the different cell lines, primer pairs targeting the putative mitochondrial import receptor subunit tom22 (PF3D7_0524700), the Tcomplex protein 1 subunit theta (PF3D7_0214000), early transcribed membrane protein 5 (etramp5, PF3D7_0532100), apical membrane antigen 1 (ama1, PF3D7_1133400) as well as the housekeeping gene arginyl-tRNA synthetase (PF3D7_1218600) were included (Supplementary Table 2). Relative expression data (RELATEXP) were corrected for amplification efficiency of each primer pair, which was determined by dilution of a single cDNA from 3D7 over 5-6 logs of concentration (Supplementary Table 2).

Transcriptional profiling of P. falciparum parasites from patient blood

Samples from uncomplicated P. falciparum malaria patients had previously been collected during a published large epidemiological survey monitoring the spread of artemisinin resistance conducted thought the greater Mekong Sub regions (GMS) between 2016and 2018[START_REF] Van Der Pluijm | Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial[END_REF]. This survey was monitored by the Mahidol-Oxford Tropical Medicine Research Unit Clinical Trials Support Group, had written informed consent from all participants before the study was carried out and the study protocol was approved by the medium. Cell pellet was divided into 4 dishes and hematocrit adjusted to 5%. One dish corresponding to the first time point (0-8 h.p.i) was incubated with DPBS containing 0.03% saponin and proteins were extracted from the parasite pellet with protein lysis buffer (0.5x PBS/4% SDS/0.5% Triton X-100) as described above for total protein extracts. The remaining dishes were further cultured under standard conditions with the corresponding medium, harvested sequentially every eight hours to obtain the time points 8-16, 16-24 and 24-32 h.p.i.

and lysed as described above. Samples were stored at-20 C until all samples were collected and analysed in parallel by immunoblotting. For densitometric analysis, detection was done by enhanced chemiluminescence (Bio-Rad/Thermo Fischer) and signals were recorded with a ChemiDoc XRS imaging system (Bio-Rad). Intensity of HA signal of EXP1-HA and BIP for every time point was quantified with Image Lab software 5.2 (Bio-Rad). HA signal was normalized to the BIP signal and the ratio under low AA was compared to that under complete, which was set to 100%.

Detection of eIF2α phosphorylation

Schizont stages of 3D7 and Kelch13 C580Y -GFP parasites were Percoll purified and allowed to invade fresh RBCs with shaking for 30 minutes at 37 C, cultured for three hours in standard medium and in RPMI low AA (1:50) ± 382 µM isoleucine. The 0-3 h.p.i old ring stages were sorbitol synchronised and washed twice with the corresponding media. Ring stages were further cultured for five hours in the corresponding media and saponin pellets were prepared from 5-8 h.p.i old ring stages. Samples were analysed by Western Blot to detect phosphorylated eIF2α and GAPDH.

QUANTIFICATION AND STATISTICAL ANALYSIS

Unless otherwise indicated, statistical significance was calculated using a two-tailed unpaired t-test performed using GraphPad Prism v6.

  The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost Paolo Mesén-Ramírez, Bärbel Bergmann, Mourad Elhabiri, Lei Zhu, Heidrun von Thien, Carolina Castro-Peña, Tim-Wolf Gilberger, Elisabeth Davioud-Charvet, Zbynek Bozdech, Anna Bachmann, et al. To cite this version: Paolo Mesén-Ramírez, Bärbel Bergmann, Mourad Elhabiri, Lei Zhu, Heidrun von Thien, et al.. The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost. Cell Host & Microbe, 2021, 29 (12), pp.1774-1787.e9. 10.1016/j.chom.2021.11.002. hal-03544974

Figure 1 .

 1 Figure 1. NPC-dependence of nutrients (A) Rapalog-dependent generation of parasites relying on low levels of EXP1 (EXP1 low ). (B) Phenotypes of parasites relying on modified EXP1 versions. Left, EXP2 PVM distribution (n=58 and n=80 from 2 independent experiments; P values, Fischer's exact test) and immunofluorescence assay (IFA) images (middle) of trophozoites relying on EXP1 TMmut G83L (rapalog) compared to controls. Right, growth of these parasites (+rap) and controls (-rap) in low amino acid versus complete medium. α-HA: endogenous EXP1-HA. DAPI, nuclei. (C-D) Linear regression analysis of EXP2 distribution (C) or growth under low amino acids (D) of parasites relying on the EXP1 constructs indicated in (B) or Figures S1A-G plotted against

Figure 2 .

 2 Figure 2. NPC levels influence susceptibility to antimalarial drugs (A-G) Growth of control (-rap) and EXP1 low (+rap) parasites or 3D7 and EXP1 overexpressing (3D7+EXP1) parasites exposed to IC50 96h of the drugs indicated versus growth without drug. (H) Linear regression of growth after IC50 96h DHA exposure versus no DHA (see Figure S2E-S2F) plotted as in Figure 1C and 1D. (I) Growth of EXP1 low (+rap) and control parasites (-rap) exposed to lumefantrine IC50 96h versus no drug. (J) Live cell images of Fluo-CQ uptake in condEXP1 low parasites with low (rapalog) or control levels of EXP1. DIC, differential interference contrast; scale bars, 5 μm. (K) Quantification of Fluo-CQ fluorescence in the FV of parasites in (J). Mean (green line), n=73 (control) and n=67 (rapalog) parasites from 3 independent experiments. (L) Growth of EXP1 low (+rap) and control parasites (-rap) exposed to Fluo-CQ IC50 96h versus growth without Fluo-CQ. A-G, I and L, mean (green line) of ≥6 biological replicas from ≥2 occasions. Error bars, SD; P values, two-tailed unpaired t test.

Figure 3 .

 3 Figure 3. NPCA influences susceptibility to PPQ

Figure 4 .

 4 Figure 4. Increased EXP1 levels compensate amino acid deficit in ART R parasites (A-B) Growth of the indicated parasite lines under the indicated conditions (low AA, 1:20 AA) compared to complete medium (high) or with or without sodium azide (NaN3). (C) Growth of the indicated parasites in amino acid-depleted (no AA) or limited medium (low AA, 1:50 AA) versus growth in standard medium (high) ± isoleucine (Ile). (D) Growth of indicated parasites relative to 3D7 (red line) calculated from C. Points relative to 3D7 in each experiment. (E) Fold change of phosphorylated eIF2α (P) in 3D7 and Kelch13 C580Y parasites grown in limited medium (low AA, 1:50) ± Ile versus standard medium (high AA, red line) quantified by densitometry normalized to GAPDH. Blots in Figures S7C-S7D; n=3 independent experiments. (F) IFA images of GFP-Kelch13 C580Y and GFP-Kelch13wt cell lines expressing EXP1-Ty high . α-GFP, endogenous Kelch13; α-Ty1, episomal EXP1-Ty1. Scale bars, 5 μm. (G-H) Growth of the indicated parasites in low AA versus growth in standard (G) or DHA or MFQ (IC50 96 ) containing medium (H) versus growth without drug. (G) +rap, partial Kelch13 inactivation (Birnbaum et al., 2020). A-E and G-H, mean (green line); A-D and G-H n≥6 independent replicates. Error bars, SD; P values, two-tailed unpaired t test (B-D, G) or nonparametric (Mann-Whitney) t test (A, G).

Figure 5 .

 5 Figure 5. EXP1 upregulation is a natural mechanism to compensate amino acid shortage (A) Violin plot of normalized exp1 transcript levels in parasite samples from patients with the indicated parasite clearance half-life (pch) after ART treatment. P values, one sided Mann-

Figure 6 .

 6 Figure 6. Model for amino acid supply in ART R and sensitive parasites (A) P. falciparum-infected RBC showing sources of amino acids for the parasite. (B) Changes in the balance of amino acid supply in ART R and sensitive parasites.

  episomally express EXP1wt in the SLI-generated Kelch13 cell lines the recodonised exp1 gene(Mesen-Ramirez et al., 2019) was PCR amplified using primers to append the Ty1 epitope tag sequence and cloned into the plasmid pEXP1comp(Mesen-Ramirez et al., 2019) using XhoI and XmaI to place it under the control of the hsp86 promoter. Next, the neomycin phosphotransferase gene was PCR amplified from pSLI-2xFKBP(Birnbaum et al., 2017) and used to exchange the yDHODH resistance marker using BamHI and HindIII. This resulted in pEXP1comp-NeoR. To episomally express EXP2-Ty1 in the SLI-generated Kelch13 cell lines a recodonised exp2 gene(Mesen-Ramirez et al., 2019) (GenScript) was PCR amplified and cloned into pEXP1comp-NeoR using XhoI and AvrII.To generate parasites expressing HA-tagged endogenous EXP1 via SLI[START_REF] Birnbaum | A genetic system to study Plasmodium falciparum protein function[END_REF], the last 467 bp of the exp1 gene were amplified from 3D7 genomic DNA and cloned into pSLI-HA[START_REF] Mesen-Ramirez | Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity[END_REF] using NotI and SalI to obtain pSLI-exp1-HA.

  chloroquine diphosphate (Sigma-Aldrich) [0-100 nM], mefloquine (MFQ) hydrochloride (Sigma-Aldrich) [0-250 nM], DHA (Adipogen) [0-10 nM], amodiaquine (Santa Cruz Biotechnology) [0-50 nM], lumefantrine (Sigma-Aldrich) [0-250 nM], piperaquine tetraphosphate tetrahydrate (Sigma-Aldrich) [0-50 nM], MVV007839 (Medicines for Malaria Ventures (www.mmv.org), kindly provided by Eric Beitz, Christian-Albrechts-Universität, Kiel)[0-500 nM], epoxomicin (Sigma-Aldrich) [0-100 nM], Fluo-CQ[START_REF] Jida | A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs[END_REF] and CQ1-NBD [0-50 nM]. For determination of the concentration when nutrient concentration limited growth to 50% after 96h ('IC50 96h ' of nutrients), the parasite cells were cultured with increasing concentrations of hypoxanthine [0-4000 µM] (Sigma-Aldrich) added to hypoxanthine free medium, oleic acid/palmitic acid [0-70 µM] (Sigma-Aldrich) added to lipid free medium and glucose [0-50 mM] added to medium without glucose. Before culturing in these media, the parasites were first washed twice with the corresponding nutrient-depleted medium (for nutrient-free media see preparation of nutrient-limited media). The curves for determination of IC50 96 are shown in FigureS2A-S2D and S8A-S8G.To compare IC50 values of DHA and MFQ between parasites expressing physiological levels of EXP1 and EXP1 low parasites, synchronous ring stages of EXP1wt low parasites (i.e. after rapalog-mediated excision of the genomic exp1, see conditional EXP1 knock-out via DiCremediated excision in complementation cell lines) and the matched controls grown without rapalog were diluted to 1% (rapalog) and 0.1 % (control parasites) parasitemia in RPMI complete medium. Equivalent volumes of cells were cultured with RPMI complete medium containing increasing concentrations of MFQ hydrochloride (Sigma-Aldrich) [0-300 nM] and DHA (Adipogen) [0-10 nM] and the selection drugs to maintain the genetic alterations and the episomal plasmid of the used cell lines. The start parasitemia was determined by FC and the medium of the cultures changed daily for four days (thereby adding fresh drug every day).

  rabbit α-HA (Cell Signaling Technology) (1:500), mouse α-Ty (Sigma) (1:20,000) and rabbit α-MSP1 (PPM marker [1:1,000]) (MRA-34, BEI Resources; https://www.beiresources.org) //www.malariaresearch.eu) (1:2,000) diluted in 3% BSA in PBS. After incubation with the primary antibody the cells were washed three times with PBS with intermittent centrifugations at 16,000g to change solutions and further incubated for 1 hour with Alexa

  parasites were cultured in parallel in RPMI complete and amino acid restricted medium for 4 months (~60 development cycles). Tightly synchronised 4-12 h.p.i old rings and 24-32 h.p.i old trophozoites were harvested and centrifuged at 2000 r.p.m for 5 minutes. The cell pellets were thoroughly resuspended in five volumes TRIzol (Life Technologies), incubated for 5 minutes at 37 C and stored at -80 C until RNA purification. RNeasy Mini (Qiagen) with on-column DNase I (Qiagen) treatment was used for RNA purification. Absence of gDNA was checked for each sample using 50 ng RNA and the sbp1 primer set. If necessary, DNasing was repeated until the sample was free of any DNA contamination by qPCR. Afterwards, cDNA was synthetized with the SuperScript III Reverse Transcriptase (Invitrogen) primed with random hexamers (Invitrogen) at 50°C for 1 hour.

  Figure S1. Correlation of EXP1 activity with nutrient permeability and EXP2 phenotypes. Related to Figure 1. (A-G) Left to right, distribution of EXP2 at the PVM (phenotype quantification and representative IFA images) of age-matched trophozoites relying on the schematically shown modified EXP1 (rapalog) compared to the controls still expressing endogenous EXP1. Right, relative growth after 96 h under low amino acids compared to standard medium of parasites relying on the indicated modified EXP1. α-HA: floxed endogenous EXP1-HA; α-EXP2 endogenous EXP2. DIC, differential interference contrast; scale bars, 5 µm; DAPI, nuclei. EXP2 distribution: mean of n=52 control and n=67 rapalog-treated parasites

Figure S2 .

 S2 Figure S2. Activity of NPCs influences sensitivity to antimalarial drugs. Related to Figure 2. (A-D) Curves of dose-dependent survival after 96 h of control condEXP1 low and EXP1 low (rapalog) parasites treated with increasing concentrations (log) of MFQ (0-300 nM) (A) and DHA (0-10 nM) (C). Mean of n=3 independent experiments. MFQ and DHA IC50 96 values (B, D) calculated from (A) and (C)

Figure S3 .

 S3 Figure S3. EXP1 levels compensate amino acid deficit in ART R parasites. Related to Figures 4 and 5. (A) Immunoblots of protein extracts from saponin pellets from 3D7 and Kelch13 C580Y ring stages cultured in medium with high AA concentration (RPMI standard), low AA (1:50 amino acid concentration of RPMI) ± isoleucine (Ile) (382 µM) probed with α-eIF2α (P) (phosphorylated eIF2α) and α-GAPDH (loading control). (B) Fold change of eIF2α (P) under low AA ± Ile relative to high AA (red line) quantified by

Table 1 . Properties of substances tested Nutrient MW (kDa) H2O solubility (g/L) Charge # NPC-dependence

 1

	Glucose	180.16	900 (soluble)	0	+
	2-NBDG	342.26	10 (soluble)	0	+
	Hypoxanthine 136.11	0.7 (insoluble)	0	-
	Isoleucine	131.18	22.3 (soluble)	0	+
	Oleic acid	282.47	1.5x10 -5 (insoluble) -	-
	Palmitic acid 256.43	4x10 -5 (insoluble)	-	-
	Amodiaquine 355.87	47 (soluble)	++	+
	Chloroquine	319.88	50 (soluble)	++	+
	CQ1-NBD	540.02	soluble*	+	-
	DHA	284.35	0.1 (insoluble)	0	+
	Epoxomicin	554.37	insoluble*	0	-
	Fluo-CQ	583.09	soluble*	+	-
	Lumefantrine 528.94	>0.00005 (insoluble) 0	-
	Mefloquine	378.32	1.8 (soluble)	+	+
	MMV007839 312.19	n.t.	0	-
	PPQ	535.52	13 (soluble)	+	-

*considered soluble or insoluble but no exact value available # at pH=7 n.t.: not tested

  TMmut G83L , a recodonised exp1 gene[START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF] was PCR amplified with overlapping primers (see Supplementary Table2for all primers used in this study) to introduce a single point mutation (G83L) in the transmembrane domain of EXP1 and cloned into plasmid pEXP1comp containing the nmd3 promoter[START_REF] Mesen-Ramirez | EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites[END_REF]

	To generate EXP1-
	informed consent was obtained from all participants before the study was carried out and the
	study protocol was approved by the Oxford Tropical Research Ethics Committee and for each
	site by the appropriate institutional review board and/or the national ethics committee (van
	der Pluijm et al., 2019; van der Pluijm et al., 2020). The study was registered
	with ClinicalTrials.gov, number NCT02453308.
	METHOD DETAILS
	Cloning

from uncomplicated P. falciparum malaria patients were collected during a published large epidemiological survey monitoring the spread of artemisinin resistance conducted thought the greater Mekong Sub regions (GMS) between 2016 and 2018. Information related to age, sex, and gender identity (if known) of the subjects and sample size was already provided

[START_REF] Van Der Pluijm | Determinants of dihydroartemisininpiperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study[END_REF][START_REF] Van Der Pluijm | Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial[END_REF]

. This survey was monitored by the Mahidol-Oxford Tropical Medicine Research Unit Clinical Trials Support Group. Written

Acknowledgements

We thank Ralf Krumkamp for help with statistical analysis and data presentation. This project was funded by the German research foundation grant SP1209/4-1. M.E. and E.D.C. thank the International Center for Frontier Research in Chemistry in Strasbourg and the Laboratoire d'Excellence ParaFrap (grant LabEx ParaFrap ANR-11-LABX-0024 (E.D.C.)). Z.B. and Z.L. acknowledge funding by the Singapore National Medical Research Council grant #NMRC/OFIRG/0040/2017. α-EXP2 antibodies were obtained from The European Malaria Reagent Repository (http://www.malariaresearch.eu). We thank Claudia Daubenberger for α-GAPDH antibodies, Mike Blackman for Compound-2, Jacobus Pharmaceuticals for WR99210 and MR4/BEI Resources, NIAID, NIH for DSM1 (MRA-1161) and rabbit α-MSP1 (MRA-34).

SUPPLEMENTAL INFORMATION

Supplemental information can be found online

Declaration of interests

The authors declare no competing interests.

Oxford Tropical Research Ethics Committee and for each site by the appropriate institutional review board and/or the national ethics committee [START_REF] Van Der Pluijm | Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial[END_REF]. The mRNA profiling was conducted with total RNA extracted from the parasitized blood samples collected from the patients using DNA microarrays and Next generation Sequencing (RNA-Seq) as described [START_REF] Kucharski | A comprehensive RNA handling and transcriptomics guide for highthroughput processing of Plasmodium blood-stage samples[END_REF]. Data analysis for the Transcriptome-Wide Association Studies of artemisinin resistance encompassing the entire dataset is currently under preparation [START_REF] Zhu | The mechanism of artemisinin resistance of <em>Plasmodium falciparum</em> malaria parasites originates in their initial transcriptional response[END_REF]

Developmental stage estimation of field samples

The method of age estimation for the field parasite samples was described previously [START_REF] Lemieux | Statistical estimation of cell-cycle progression and lineage commitment in Plasmodium falciparum reveals a homogeneous pattern of transcription in ex vivo culture[END_REF][START_REF] Zhu | The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background[END_REF]. In brief, it was determined by a mixture linear regression model presented in the following formula:

Where Eg is a gene's expression value in a parasite field sample, xg(hr) is that gene's reference expression level at the asexual stage of hpi hr, zg denote the average reference expression of sexual stages (from the 5 th to 12 th day during gametocytes developing), α is the proportion of gametocytes to the total parasite count (sum of sexual and asexual parasites) and εg is the associated error term. The reference asexual transcriptomes were generated during asexual intraerythrocytic cycle in P. falciparum 3D7 strain with two hours intervals of 48 hours (GEO accession number GSE149865). The reference gametocyte transcriptomes are accessible via GEO accession number GSE121505. The result was obtained based on the maximum loglikelihood values evaluated over a grid of mixtures for varying the gametocyte proportion α, and hpi hr, for each sample. The details are available in the paper under review [START_REF] Zhu | The mechanism of artemisinin resistance of <em>Plasmodium falciparum</em> malaria parasites originates in their initial transcriptional response[END_REF].

Time resolved expression of EXP1 under low amino acid conditions

To analyse differences in protein levels of EXP1 parasites expressing endogenous tagged EXP1-HA were cultured four weeks in RPMI low AA and RPMI complete. Percoll-purified schizonts of every condition were allowed to invade fresh RBCs with shaking for 30 minutes at 37 C and cultured for eight hours under standard conditions. 0-8 hours post invasion (h.p.i) old ring stages were sorbitol synchronised and washed twice with the corresponding RPMI

Supplemental information

The parasitophorous vacuole nutrient channel is p critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost