Antimalarial Quinolines and Artemisinin Inhibit Endocytosis in Plasmodium falciparum

Abstract

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.

Artemisinin activity againstPlasmodium falciparumrequires hemoglobin uptake and digestion

“…This finding is in agreement with previous work showing that artemisinin treatment leads to an accumulation of undigested hemoglobin in the parasite (22). We also showed that hemoglobin uptake and subsequent hydrolysis are required for potent artemisinin activity.…”

“…P. falciparum takes up and degrades host hemoglobin in an acidic digestive vacuole in a process that begins in the mid ring stage and reaches a peak in the mid trophozoite stage (20,21). Some antimalarials, including artemisinin, are reported to inhibit hemoglobin uptake (22), but it is not clear if this is a direct effect, or a consequence of parasite killing. To monitor hemoglobin uptake and parasite viability simultaneously we established a correlative flow cytometric assay employing parasites that had invaded resealed RBCs containing a fluorescent marker (21).…”

“…RBC lysate was prepared (22) and the hemoglobin content determined (43). Packed midtrophozoite-infected RBCs (1-3% parasitemia, 3D7) were lysed in 10 volumes of EB containing 0.02% saponin, washed and resuspended at 10-50;000 parasites∕μL in EB or RBC lysate, containing 50 μM fluorescein-dextran and dilutions of artemisinin or DHA.…”

A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum

“…Because of the similarities between the current model for hemoglobin transport and the endocytic pathway in higher eukaryotes (Goldberg et al, 1990;Hoppe et al, 2004;Langreth et al, 1978;Saliba et al, 2003), we investigated the role of actin dynamics in hemoglobin transport to the FV. Actin has been shown to play various regulatory roles in endocytosis, including recruiting endocytic components to the plasma membrane, initiating plasma membrane invaginations, vesicle scission and transport to endocytic compartments (Kaksonen et al, 2006;Qualmann et al, 2000;Smythe and Ayscough, 2006).…”

Antimalarial Drugs – What is in Use and What is in the Pipeline

“…Arylaminoalcohols (Fig. 4) seem to interfere with the heme digestion [35,36]. Amplification of the pfmdr1 gene appears to be the main factor in arylaminoalcohol resistance [37 -42].…”