Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.

Artemisinin and its derivatives (collectively referred to as ARTs) rapidly reduce the parasite burden in Plasmodium falciparum infections, and antimalarial control is highly dependent on ART combination therapies (ACTs). Decreased sensitivity to ARTs is emerging, making it critically important to understand the mechanism of action of ARTs. Here we demonstrate that dihydroartemisinin (DHA), the clinically relevant ART, kills parasites via a two-pronged mechanism, causing protein damage, and compromising parasite proteasome function. The consequent accumulation of proteasome substrates, i.e., unfolded/damaged and polyubiquitinated proteins, activates the ER stress response and underpins DHA-mediated killing. Specific inhibitors of the proteasome cause a similar build-up of polyubiquitinated proteins, leading to parasite killing. Blocking protein synthesis with a translation inhibitor or inhibiting the ubiquitin-activating enzyme, E1, reduces the level of damaged, polyubiquitinated proteins, alleviates the stress response, and dramatically antagonizes DHA activity.

Highlights d K13 artemisinin resistance-conferring mutations lead to reduced K13 abundance d K13 is localized to cytostome-like structures at the parasite periphery d Reduced K13 abundance impairs hemoglobin catabolism and lessens artemisinin activation d Reduced artemisinin activation limits cell damage, permitting parasite survival

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.