Artemisinins are the corner stone of anti-malarial drugs1. Emergence and spread of resistance to them2–4 raises risk of wiping out recent gains achieved in reducing world-wide malaria burden and threatens future malaria control and elimination on a global level. Genome wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance5–10. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase as well as its lipid product phosphatidylinositol 3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signaling, where transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.
Artemisinin resistance threatens worldwide malaria control and elimination. Elevation of phosphatidylinositol-3-phosphate (PI3P) can induce resistance in blood stages of The parasite unfolded protein response (UPR) has also been implicated as a proteostatic mechanism that may diminish artemisinin-induced toxic proteopathy. How PI3P acts and its connection to the UPR remain unknown, although both are conferred by mutation in Kelch13 (K13), the marker of artemisinin resistance. Here we used cryoimmunoelectron microscopy to show that K13 concentrates at PI3P tubules/vesicles of the parasite's endoplasmic reticulum (ER) in infected red cells. K13 colocalizes and copurifies with the major virulence adhesin PfEMP1. The PfEMP1-K13 proteome is comprehensively enriched in multiple proteostasis systems of protein export, quality control, and folding in the ER and cytoplasm and UPR. Synthetic elevation of PI3P that induces resistance in absence of K13 mutation also yields signatures of proteostasis and clinical resistance. These findings imply a key role for PI3P-vesicle amplification as a mechanism of resistance of infected red cells. As validation, the major resistance mutation K13C580Y quantitatively increased PI3P tubules/vesicles, exporting them throughout the parasite and the red cell. Chemical inhibitors and fluorescence microscopy showed that alterations in PfEMP1 export to the red cell and cytoadherence of infected cells to a host endothelial receptor are features of multiple K13 mutants. Together these data suggest that amplified PI3P vesicles disseminate widespread proteostatic capacity that may neutralize artemisinins toxic proteopathy and implicate a role for the host red cell in artemisinin resistance. The mechanistic insights generated will have an impact on malaria drug development.
The export of numerous proteins to the plasma membrane of its host erythrocyte is essential for the virulence and survival of the malaria par-
SummaryThe pathogenicity of the most deadly human malaria parasite, Plasmodium falciparum, relies on the export of virulence factors to the surface of infected erythrocytes. A novel membrane compartment, referred to as Maurer's clefts, is transposed to the host erythrocyte, acting as a marshal platform in the red blood cell cytoplasm, for exported parasite proteins addressed to the host cell plasma membrane. We report here the characterization of three new P. falciparum multigene families organized in 9 highly conserved clusters with the Pfmc-2tm genes in the subtelomeric regions of parasite's chromosomes and expressed at early trophozoite stages. Like the PfMC-2TM proteins, the PfEPF1, 3 and 4 proteins encoded by these families are exported to the Maurer's clefts, as peripheral or integral proteins of the Maurer's cleft membrane and largely exposed to the red cell cytosolic face of this membrane. A promoter titration approach was used to question the biological roles of these P. falciparumspecific exported proteins. Using the Pfepf1 family promoter, we observed the specific downregulation of all four families, correlating with the inefficient release of merozoites while the parasite intra-erythrocytic maturation and Maurer's clefts morphology were not impacted.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.