Altered temporal response of malaria parasites determines differential sensitivity to artemisinin

Abstract

Reports of emerging resistance to first-line artemisinin antimalarials make it critical to define resistance mechanisms and identify in vitro correlates of resistance. Here we combine unique in vitro experimental and analytical approaches to mimic in vivo drug exposure in an effort to provide insight into mechanisms of drug resistance. Tightly synchronized parasites exposed to short drug pulses exhibit large stage-dependent differences in their drug response that correlate with hemoglobin digestion throughout most of the asexual cycle. As a result, ring-stage parasites can exhibit >100-fold lower sensitivity to short drug pulses than trophozoites, although we identify a subpopulation of rings (2-4 h postinvasion) that exhibits hypersensitivity. We find that laboratory strains that show little differences in drug sensitivity in standard in vitro assays exhibit substantial (>95-fold) difference in sensitivity when exposed to short drug pulses. These stage-and strain-dependent differences in drug sensitivity reflect differential response lag times with rings exhibiting lag times of up to 4 h. A simple model that assumes that the parasite experiences a saturable effective drug dose describes the complex dependence of parasite viability on both drug concentration and exposure time and is used to demonstrate that small changes in the parasite's drug response profile can dramatically alter the sensitivity to artemisinins. This work demonstrates that effective resistance can arise from the interplay between the short in vivo half-life of the drug and the stage-specific lag time and provides the framework for understanding the mechanisms of drug action and parasite resistance.endoperoxide | Plasmodium | qinghaosu | effective dose model

Artesunate versus Chloroquine Infection–Treatment–Vaccination Defines Stage-Specific Immune Responses Associated with Prolonged Sterile Protection against Both Pre-erythrocytic and Erythrocytic Plasmodium yoelii Infection

“…Artemisinin derivatives such as artesunate (AS) are potent plant-derived antimalaria drugs that act rapidly against the intraerythrocytic BS of P. falciparum and P. vivax infection, and are effective for both CQ-sensitive and CQ-resistant parasites (13). Neither CQ nor AS affects the pre-erythrocytic stages of the malaria parasite; but whereas CQ kills Plasmodium parasites at the trophozoite stage, AS acts on all intraerythrocytic stages of BS development (14,15).…”

Genetic markers of artemisinin resistance in Plasmodium spp. parasites

“…There is very good evidence from laboratory studies of P. falciparum isolates grown in culture that artemisinin susceptibility varies greatly across the cell cycle [26,27]. The patterns as currently understood for wild-type parasites are represented in cartoon form in Figure 1A.…”

“…The mode of action of artesunate in killing malaria parasites is almost certainly multifactorial, as the activated endoperoxide moiety causes widespread non-specific damage to intracellular proteins [26,29]. Thus, artemisinin selection is expected to identify other mechanisms that reduce susceptibility, and indeed, this has been the case.…”

“…It is widely considered that the endoperoxide bridge in artemisinin is strongly activated by haem metabolism in the food vacuole, leading to parasite killing [29], a process that barely occurs during the first few hours of the life cycle, so it is unclear how activation of artemisinin, so lethal to parasites in these first few hours, occurs [18,26]. One plausible hypothesis is that a specific parasite cellular process in the first few hours post-invasion provides a finite burst of activated artemisinin; variant K13 renders ERT resistant to artemisinin simply by preventing this specific event.…”

“…(A) Standard DHA-sensitive cultured laboratory parasite line, as shown for 3D7 by Klonis et al [26]. All stages are susceptible to artemisinin killing; ERT briefly exhibits exquisite susceptibility, while LRT is the least susceptible stage.…”