Artesunate tolerance in transgenic Plasmodium falciparum parasites overexpressing a tryptophan-rich protein

Abstract

Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins.

Fig. 1.

Structure of Pf10_0026 gene and tryptophan-rich domain. (A) Two-exon structure and localization of Pf10_0026 on chromosome 10. The transmembrane domain is encoded at the 5′ end of exon 1 and the tryptophan-rich (W-rich) domain by the 3′ region of exon 2. The region corresponding to the W-rich domain encoded by residues 755 to 965 is shown on the mRNA structure. (B) HMM profile based on 79 genomic sequences of Plasmodium species showing a tryptophan-rich motif. The frequency of each amino acid is represented by the size of the amino acid one-letter code.

Fig. 2.

Dose-dependent increase in expression of PArt upon drug pressure. (A) Expression ratio of PArt/histone H3 proteins measured from a Western blot of parasites exposed to increasing concentrations of artesunate for 3 h, using mouse antibodies raised against the W-rich domain of PArt. The analysis of Western blot seen in Fig. S1 in the supplemental material, using Quantity One (Bio-Rad) and histone H3 as a reference, shows a 2- to 6-fold increase of expression of PArt protein after a 3-h exposure to doses ranging from 40 to 400 ng/ml of artesunate, respectively. (B) Expression ratio of PArt/HSP70 proteins measured as described above, with PfHSP70 as the reference protein. (C) PArt/PFI0425w RNA expression ratio calculated from quantitative RT-PCR performed with parasites exposed to increasing concentrations of artesunate for 3 h. The mRNA expression level of PArt is normalized to the expression level of PFI0425w. The means from three measurements (bars indicate standard deviations) shows that a 2.2- and 3.4-fold increase of mRNA expression is observed after a 3-h exposure to 200 and 400 ng/ml of artesunate, respectively. Asterisks indicate statistical significance as determined by a one-sided Wilcoxon rank-sum test (P = 0.05).

Fig. 3.

Kinetics of tagged PArt expression protein during the intraerythrocytic parasite cycle. (A) Western blot analysis of the expression of PArt at the trophozoite stage (30 h postinvasion) in the wild type (FUP/CB) and in the overexpressing line (PArt/myc). Western blots were probed with mouse antibodies raised against the W-rich portion of PArt and with antibody against HSP70 as a loading control (left) or with a mouse monoclonal anti-myc antibody (right). (B) mRNA expression level of PArt during the development cycle of the overexpressing line PArt/myc relative to PArt expression in wild-type FUP/CB. An increase of RNA is observed during the first 24 h postinvasion. The expression ratio was normalized to the mRNA level of PFI0425w. Error bars indicate standard deviations.

Fig. 4.

Localization of PArt in FUP/CB and PArt/myc lines. Immunofluorescence assays were performed with the wild-type (FUP/CB) (A) or the overexpressing parasites (PArt/myc) (B). PArt localization with a polyclonal mouse antibody raised against the W-rich portion of the protein is shown in green. Nuclei are labeled blue with Hoescht 33342. Pictures were taken under identical exposure conditions.

Fig. 5.

Tolerance of the PArt/myc line to a 3-h exposure to artesunate. (A) Identical development of the wild type (FUP/CB) and the overexpressing transgenic line (PArt/myc) during 72 h in the absence of artesunate. Parasitemia and stage distribution were determined by the optical microscopy of Giemsa-stained blood films made every 24 h of parasite development. White bars indicate ring stages, i.e., ring-shaped parasites, uninucleated with thin cytoplasm; gray bars indicate trophozoites, i.e., uninucleated parasites with fleshy cytoplasm; black bars indicate schizonts, i.e., multinucleated parasites. (B) Artesunate induced a dose-dependent delay in the development of the wild type (FUP/CB) and the overexpressing transgenic line (PArt/myc). Parasitemia and stage distribution were determined as described above at time zero and at 24, 48, and 72 h after exposure to artesunate. Parasite reinvasion is totally completed only at 72 h due to a drug-induced delay in development. Bars are defined as described above. (C) Survival of the wild type (white bars), control transfected line (light gray bars), or overexpressed transgenic line (dark gray bars) to a 3-h incubation with increasing doses of artesunate was determined by 72 h after the end of drug exposure (pooled results from four independent experiments). The survival of drug-exposed parasites is expressed as the percentage of the parasitemia reached at 72 h in untreated cultures. A one-sided rank-sum Wilcoxon test was used to determine drug concentrations for which the survival rate of the PArt/myc line was significantly increased compared to that of controls. Doses leading to significant survival differences (20 and 40 ng/ml) are indicated by an asterisk. Error bars indicate standard deviations.