Overexpression of plasmepsin II and plasmepsin III does not directly cause reduction in Plasmodium falciparum sensitivity to artesunate, chloroquine and piperaquine

Abstract

Artemisinin derivatives and their partner drugs in artemisinin combination therapies (ACTs) have played a pivotal role in global malaria mortality reduction during the last two decades. The loss of artemisinin efficacy due to evolving drug-resistant parasites could become a serious global health threat. Dihydroartemisinin-piperaquine is a well tolerated and generally highly effective ACT. The implementation of a partner drug in ACTs is critical in the control of emerging artemisinin resistance. Even though artemisinin is highly effective in parasite clearance, it is labile in the human body. A partner drug is necessary for killing the remaining parasites when the pulses of artemisinin have ceased. A population of Plasmodium falciparum parasites in Cambodia and adjacent countries has become resistant to piperaquine. Increased copy number of the genes encoding the haemoglobinases Plasmepsin II and Plasmepsin III has been linked with piperaquine resistance by genome-wide association studies and in clinical trials, leading to the use of increased plasmepsin II/plasmepsin III copy number as a molecular marker for piperaquine resistance. Here we demonstrate that overexpression of plasmepsin II and plasmepsin III in the 3D7 genetic background failed to change the susceptibility of P. falciparum to artemisinin, chloroquine and piperaquine by both a standard dose-response analysis and a piperaquine survival assay. Whilst plasmepsin copy number polymorphism is currently implemented as a molecular surveillance resistance marker, further studies to discover the molecular basis of piperaquine resistance and potential epistatic interactions are needed.

Keywords: Artemisinin; Drug resistance; Malaria; Piperaquine; Plasmepsin.

Graphical abstract

Fig. 1

Overexpression of plasmepsin genes. (A) Three transgenic lines were generated for this study with the 3D7 parasites transfected with a blank vector, Plasmepsin II vector or Plasmepsin III vector, designated as Blank-3D7, PM2-3D7 and PM3-3D7, respectively. The increase in the gene expression level was presented as fold change. The level of expression of plasmepsin 1-4 was monitored by RT-qPCR (shown as PM1 to PM4). The fold change values were compared to those of Blank-3D7, giving the fold change value of 1 for the controls. The fold change values of PM2 and PM3 expression were significantly increased in PM2-3D7 and PM3-3D7, respectively (*p-value < 0.05). (B) Western analysis of Plasmepsin proteins. The extract from synchronized trophozoite-stage parasites was used for the analysis and probed with the antibodies against PM2 (α-PM2) and PM3 (α-PM3) with the antibodies against ERD2 (α-ERD2) as a loading control. Signal quantification with Image Studio Lite was normalized with the ERD2 control and presented as fold increase in comparison to Blank-3D7 (shown as numbers under the α-PM3 panel). The fold increase value for PM2 was not available since the PM2 band was not visible in Blank-3D7 even after extended exposure time.

Fig. 2

Effect of plasmepsin overexpression on piperaquine and chloroquine susceptibility. The IC₅₀ values for piperaquine (A) and chloroquine (B) in the transgenic lines were determined. The information is reported in Table 1, and the dose-response curves are shown in Supplementary Fig. 1. (C) Dose-response curves at high piperaquine concentrations. The dose-response curve showing the bimodal pattern was not found even when the drug concentrations and the experiments were done according to the previous report presenting the bimodal pattern (Bopp et al., 2018).

Fig. 3

Effect of plasmepsin overexpression on artemisinin susceptibility. (A) Effect of drug exposure time on LICA. The graphs represent the difference in percent survival at specific drug concentrations. The drug exposure times were set at 1, 3 and 5 h. The difference between sensitive and resistant strains is maximized at the drug exposure time of 3 h. A short exposure time (1 h) is not enough to differentiate between strains. Longer exposure time (5 h) affected the ability to differentiate between sensitive and resistant strains. (B) Strong correlation between the artemisinin susceptibility values obtained from TMI and LICA. Pearson's correlation coefficients between methods are statistically significant in the assays performed with the 3D7 and ANL2 P. falciparum lines. (C) Effect of plasmepsin overexpression on artesunate susceptibility. The artesunate susceptibility IC₅₀ values were determined in the three transgenic lines without any significant difference. Each experiment was done in duplicate. Two independent biological parasite samples were tested. The information is reported in Table 1 and the dose-response curves are shown in Supplementary Fig. 1.