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.

FIG. 1.

Stepwise drug selection of an artemisinin-tolerant Plasmodium falciparum parasite. Dots represent the number of 24-h cycles of drug pressure applied to F32-Tanzania to select for strain F32-ART. The level of parasitemia was fixed to 5 to 7% before drug exposure, and new pressure was reapplied when the level of parasitemia reached 5% or higher. The duration of the experiment was defined by the time scale at the top.

FIG. 2.

Epifluorescence observations of parasites with or without drug pressure. (A) Before drug pressure. Ring forms are present (R123 positive; not detectable with DAPI; no malaria pigment). (B) After 24 h of drug pressure. Quiescent ring forms are the only viable stage present (R123 positive; not detectable with DAPI; no malaria pigment). (C) After 48 h of drug pressure. Quiescent ring forms are the only viable stage present (R123 positive; not detectable with DAPI; no malaria pigment). (D and D′) Restoration of the parasite cycle with the presence of mature trophozoites and young schizonts, respectively, 24 h after drug removal (R123 positive; nuclei detectable with DAPI; presence of malaria pigment). (E) The presence of mature schizonts 30 h after drug removal (R123 positive; multiple nuclei detectable with DAPI; presence of malaria pigment).

FIG. 3.

Flow cytometry analysis of strain F32-ART submitted to 9 μM artemisinin treatment. Yellow dots (white gate) correspond to DAPI-positive events, and blue dots correspond to DAPI-negative events (early rings and erythrocytes). The progressive increase in the number of DAPI-positive events during pressure (B and C) was due to dead forms, which incorporate DAPI better than ring forms; however, fluorescence was limited to 10³, while schizont forms (D) exhibited high levels of DAPI incorporation, with fluorescence ranging from 2 × 10³ to 2 × 10⁴ (red gate). Dead forms progressively disappeared from the culture after pressure, corresponding to the relative decrease in the numbers of DAPI-positive events after the release of the artemisinin pressure. (A) Before drug pressure. Ring forms (0 to 12 h; 5% parasitemia) are the selected stage. (B) After 24 h of drug pressure. Quiescent ring forms are the only stage present with dead parasites. (C) After 48 h of drug pressure. Quiescent ring forms are the only stage present with dead parasites. (D) Twenty-four hours after drug removal. Restoration of the parasite cycle with the presence of trophozoites and schizonts is shown (parasites in the red square).