Double-kill mechanism of artemisinin against Plasmodium
The mechanism by which artemisinin and its derivatives (ARTs) kill malaria parasites remains unclear. Haem or iron activates ARTs to produce free radicals that kill malaria parasites. However, adding iron or haem supply did not enhance, but instead attenuated, the antimalarial effect of ARTs, suggesting that the free-radical effect (FRE) is not the only antimalarial mechanism of ARTs. Here, through the single-cell RNA sequencing analysis of Plasmodium yoelii 17XNL and P. falciparum 3D7 in vivo and in vitro, we found that the sensitive stages to ARTs were associated with the expression of genes related to haem and iron (HI), DNA synthesis, antioxidation and the pentose-phosphate-pathway (PPP). Furthermore, the stages included an ART-sensitive cycle crucial for DNA synthesis that encompassed the release of iron through haem degradation to the activation of the PPP by iron. Additionally, the mechanism of haemozoin formation created a unique condition for interaction between ARTs and HI. In particular, HI can attenuate the antimalarial action of ARTs, strongly suggesting the existence of a HI-use-disturbance effect that combined with FRE to confer ARTs with a double-kill  antimalarial mechanism different from the effect of iron chelators. Overall design: Single-cell RNA sequencing was used to analyze the expression of differentiated genes in P. yoelii yoelii 17XNL and P. falciparum 3D7 treated with artemether or dihydroartemisinin at different time points (0, 3, 9, and 24 hours).