Evolution of resistance in vitro reveals mechanisms of artemisinin activity in Toxoplasma gondii.

ABSTRACT

Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance in Plasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability of Toxoplasma gondii, a related parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing concentrations and whole-genome sequencing identified several nonconservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acid changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild-type parasites in the presence of drug, despite not changing EC50 values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondria genome, including genes encoding cytochrome b and cytochrome c oxidase I. Prior studies in yeast and mammalian tumor cells implicate the mitochondrion as a target of artemisinins, and treatment of wild-type parasites with high concentrations of drug decreased mitochondrial membrane potential, a phenotype that was stably altered in the resistant parasites. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition of resistance in T. gondii.