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.
Resistance to artemisinin antimalarial drugs is jeopardizing malaria control. This study shows that proteasome-mediated stress responses can be targeted to overcome artemisinin resistance and suggests alternate therapeutic regimens and monitoring strategies.
Resistance to artemisinin antimalarials, some of the most effective antimalarial drugs, has emerged in Southeast Asia, jeopardizing malaria control. We have undertaken a detailed study of artemisinin-sensitive and-resistant strains of Plasmodium falciparum, the parasite responsible for malaria, taken directly from the field in a region where resistance is developing. We compared these strains to lab strains engineered with either mutant or wild-type resistance alleles. We demonstrate that in sensitive P. falciparum, artemisinin induces growth retardation and accumulation of ubiquitinated proteins, indicating that the drugs activate the cellular stress response. Resistant parasites, on the other hand, exhibit reduced protein ubiquitination and delayed onset of cell death following drug exposure. We show that proteasome inhibitors strongly synergize artemisinin activity, offering a means of overcoming artemisinin resistance. We have developed a detailed model of parasite responses and have modelled in vivo clearance profiles. Our data indicate that extending artemisinin treatment from the standard three-day treatment to a four-day treatment will clear resistant parasites, thus preserving the use of this critical therapy in areas experiencing artemisinin resistance.