Increased Tolerance to Artemisinin in Plasmodium falciparum Is Mediated by a Quiescence Mechanism

Abstract

Artemisinin (ART)-based combination therapies (ACTs) are the first-line drugs-and often the last treatments-that can effectively cure Plasmodium falciparum infections. Unfortunately, the decreased clinical efficacy of artesunate, one of the major ART derivatives, was recently reported along the Thailand-Cambodia border. Through long-term artemisinin pressure in vitro, we have obtained an ART-tolerant strain that can survive extremely high doses of ART. We showed that drug pressure could induce a subpopulation of ring stages into developmental arrest, which can explain the ART tolerance in P. falciparum. We also observed interesting transcriptomic modifications possibly associated with the acquisition of ART tolerance. These modifications include the overexpression of heat shock and erythrocyte surface proteins and the downexpression of a cell cycle regulator and a DNA biosynthesis protein. This study highlights a new phenomenon in the Plasmodium response to ART that may explain the delayed clearance of parasites after artesunate treatment observed on the Thailand-Cambodia border and that provides important information for achieving a better understanding of the mechanisms of antimalarial resistance.Plasmodium falciparum malaria remains the worst parasitic disease in developing countries, with nearly 200 million cases and 800,000 deaths being reported each year (28). Quinolines (mefloquine, amodiaquine, chloroquine, quinine) and antifolate (pyrimethamine, proguanil, sulfadoxine) have been the major antimalarial drugs in areas of endemicity for decades. Nevertheless, the emergence of multidrug-resistant P. falciparum parasites has made these drugs useless in many areas where they are endemic. This situation was particularly threatening, until the introduction of artemisinin (ART) and its derivatives. ART combination therapy (ACT) has become the first-line treatment of uncomplicated falciparum malaria in most countries where it is endemic. More than a million doses of ACT are administered to treat malaria each year, particularly after the recommendation of World Health Organization (WHO) in 2005 (26).Artemisinin is isolated from a plant (Artemisia annua) that has been used for the treatment of fever for more than 2,000 years in Chinese traditional medicine. ART shows rapid antimalarial activity, has few side effects, and is active against parasite strains that are resistant to many traditional antimalarial drugs, such as quinolines and antifolates. To reduce the risk of drug resistance, ART and its derivatives are generally used in combination with other antimalarial agents. Although it is still being debated whether there are parasites that can be considered to have real resistance to ART (23), e.g., greatly increased 50% inhibitory concentrations (IC 50 s), the delayed clearance of parasites has been reported (6,16,17). Delayed clearance suggests that some parasites can survive ART treatment for a longer period of time than expected, but they are eventually killed by the drug, raising the possibility of a surviva...

Artemisinin activity againstPlasmodium falciparumrequires hemoglobin uptake and digestion

“…When studied in culture, artemisinin caused a delay in the development of a subset of ring stage parasites (13,14). Parasite alterations that extend this period of developmental arrest may explain recent observations in Cambodia of delayed clearance of circulating parasites after treatment with artemisinins (12,39).…”

“…Artemisinin tolerant parasites have also been developed in the laboratory (13)(14)(15)(16). In some cases, the parasites remain susceptible to artemisinin in standard growth inhibition assays, but a subpopulation of early ring stage parasites survives by arresting development when exposed to artemisinin (14). To better characterize the antimalarial mechanism of action of artemisinin, we used a flow cytometry-based assay that permitted us to simultaneously analyze the effects of drugs on parasite growth and hemoglobin uptake.…”

Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state

“…However, artemisinin tolerance and quiescence have been reported only for ring forms (23,24), and isoleucine-starved parasites gradually progress past this stage. We conclude that the biological mechanisms for drug-associated dormancy and starvation-induced hibernation may differ, at least in the control of cell-cycle progression.…”

“…Parasites recovered posttherapy remain sensitive to drug. A similar phenomenon has been observed in vitro with parasites cultured in the presence of artemisinin and has given rise to the concept of parasite dormancy, a state that presumably confers drug tolerance (22)(23)(24). To determine whether isoleucine-starved parasites are in a similar dormant state, we cultured parasites in isoleucine-free medium, exposed them to artemisinin, and then assessed viability.…”

Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole

“…Perhaps central to this is the fact that sublethal exposures to artemisinin (and derivatives) in vitro lead to arrest in P. falciparum ring-stages and subsequent transition to a morphologically-distinct 'dormant' state that is able to persist under drug pressure and recover after removal of the treatment [97,98]. Artemisinin-induced 'dormant rings' display reduced susceptibility to artemisinin itself [99,100], but remain susceptible to other drugs targeting the ETC, suggesting that they are still metabolically active [97]. For example, mitochondrion-encoded enzymes of the ETC remain transcribed after treatment with the artemisinin-derivative dihydroartemisinin (DHA), while transcription of the nucleusencoded complex III is completely ablated [101].…”