Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility

Clare R Harding,Nina F Gnädig,Sebastian Lourido,Saima M Sidik,Elizabeth A Boydston,Kurt E Ward,Boryana Petrova,David A Fidock,John Okombo,Alice L Herneisen,Benedikt M Markus

doi:10.1038/s41467-020-18624-0

Abstract

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.

Highlights

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; resistance threatens to undermine global control efforts
Despite species-specific differences that could impact ART susceptibility—such as the lack of substantial hemoglobin uptake by T. gondii—here, we demonstrate that a point mutation in K13, homologous to the canonical P. falciparum K13C580Y, reduced the susceptibility of T. gondii to dihydroartemisinin (DHA)
Genome-wide screens in T. gondii further identified mutants in a putative porphyrin transporter (Tmem14c) that are more susceptible to DHA, as well as mutations in several genes involved in mitochondrial metabolism that decreased drug susceptibility

Summary

Introduction

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; resistance threatens to undermine global control efforts. Deleting the homologous gene in P. falciparum, PfDegP, lowers heme levels and DHA susceptibility These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA. Directed evolution coupled with whole-genome sequencing has identified targets and resistance pathways for many antiparasitic compounds[16,27,28,29] Such approaches can be time-consuming, may fail to detect minor mutations or those that negatively impact parasite fitness, and can only be used for positive selection schemes. Genome-wide screens in T. gondii further identified mutants in a putative porphyrin transporter (Tmem14c) that are more susceptible to DHA, as well as mutations in several genes involved in mitochondrial metabolism that decreased drug susceptibility. In keeping with this observation, ΔDegP2 parasites have alterations in the electron transport chain (ETC) and the tricarboxylic acid cycle (TCA)—two iron–sulfur cluster-dependent processes

Methods

Results

Conclusion