Mechanisms of Artemisinin Resistance in the Rodent Malaria Pathogen Plasmodium yoelii

Abstract

Artemisinin and its derivatives are important new antimalarials which are now used widely in Southeast Asia. Clinically relevant artemisinin resistance has not yet been reported but is likely to occur. In order to understand how the malaria parasite might become resistant to this drug, we studied artemisinin resistance in the murine malaria parasite Plasmodium yoelii. The artemisinin-resistant strain (ART), which is approximately fourfold less sensitive to artemisinin than the sensitive NS strain, accumulated 43% less radiolabeled drug in vitro (P < 0.01). Within the parasite, the drug appeared to react with the same parasite proteins in both strains. The translationally controlled tumor protein, one of the artemisinin target proteins, did not differ between the strains. No DNA sequence difference was found, but the resistant strain was found to express 2.5-fold-more protein than the sensitive strain (P < 0.01). Thus, the phenotype of artemisinin resistance in P. yoelii appears to be multifactorial.Drug-resistant malaria is a major worldwide public health problem. In Southeast Asia, for example, Plasmodium falciparum strains have become resistant to all of the classical antimalarials (12). Fortunately, these strains are still susceptible to the artemisinin derivatives; derivatives such as artemether and artesunate are now widely used in this region (11).Artemisinin was originally isolated from Artemisia annua, an herb used as an ancient Chinese herbal remedy. All of the artemisinin compounds contain stable endoperoxide bridges. Evidence from a variety of labs suggests that the antimalarial activity of artemisinin is dependent on the cleavage of the endoperoxide by intraparasitic heme. The cleaved endoperoxide ultimately becomes a carbon-centered free radical which then functions as an alkylating agent, reacting with both heme and parasite proteins (but not DNA) (7,11,14). In P. falciparum, one of the principal alkylation targets is the translationally controlled tumor protein (TCTP) homolog (4). Some intraparasitic TCTP is situated in the membrane surrounding the heme-rich food vacuole (5), where heme could catalyze the formation of drug-protein adducts.Clinically relevant resistance to artemisinin derivatives has not yet been reported, although P. falciparum isolates may vary two-to fourfold in their in vitro sensitivities to these drugs (2, 3, 18). However, since the drugs are being widely used, artemisinin resistance is likely to develop in the near future. Recently, a strain of the murine malaria parasite Plasmodium yoelii with unstable resistance to artemisinin was obtained (13). Here we attempted to characterize the mechanisms of resistance in this strain. MATERIALS AND METHODSParasites. Plasmodium yoelii strains ART and NS (13) were passaged and grown in male ICR mice (Harlan, Indianapolis, Ind.) by intraperitoneal injection of 10 6 parasitized erythrocytes. Each mouse infected with the resistant strain was given a subcutaneous injection of artemisinin (150 mg/kg of body weight in sesame oil). When parasi...

Role ofpfmdr1Amplification and Expression in Induction of Resistance to Artemisinin Derivatives inPlasmodium falciparum

“…Several proteins have been shown to interact with artemisinin. The translationally controlled tumor protein (TCTP) binds to radioactively labeled dihydroartemisinin (5) and is overexpressed in rodent Plasmodium yoelii parasite lines with decreased susceptibility to artemisinin (44). Another protein that may interact with artemisinins is the sarcoplasmic reticulum Ca 2ϩ ATPase 6 (PfATP6); this enzyme, when expressed in Xenopus oocytes, was specifically inhibited by artemisinin derivatives containing an endoperoxide bridge (11).…”

Immunoprecipitation of [ 3 H]Dihydroartemisinin Translationally Controlled Tumor Protein (TCTP) Adducts from Plasmodium falciparum -Infected Erythrocytes by Using Anti-TCTP Antibodies

“…Since the food vacuole is rich in heme (29), it is possible that artemisinin might react with the TCTP molecules associated with this structure. Artemisinin-resistant strains of Plasmodium yoelii express higher levels of TCTP than do artemisinin-sensitive strains (33). However, there is no direct evidence that the alkylation of TCTP by artemisinin is responsible for the antimalarial effects of the drug.…”

Proteomic analysis of tumor tissue in CT-26 implanted BALB/C mouse after treatment with ascorbic acid

“…Overexpression of TCTP in mammalian cells resulted in slow growth and a delay in cell cycle progression [31]. An increase in TCTP levels was reported to be associated with increased chemoresistance [34,35]. Overexpression of mammalian TCTP results in microtubule stabilization and alteration of cell morphology [31].…”