Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii

Abstract

250)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 T. gondii, a related apicomplexan parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing drug concentrations and whole genome sequencing identified several non-conservative 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 acids 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 EC 50 values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondrial genome, including genes encoding cytochrome b and cytochrome oxidase I. Consistent with prior studies in yeast and mammalian tumor cells that implicate the mitochondrion as a target of artemisinins, treatment of wild type parasites with artemisinin decreased mitochondrial membrane potential, and resistant parasites showed altered morphology and decreased membrane potential. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition in T. gondii. Significance (120)Artemisinins provide important therapeutic agents for treatment of malaria and have potential for use in other infections and in cancer. Their use is threatened by the potential for resistance development, so understanding their mechanism of action and identifying genetic changes that alter sensitivity are important for improving clinical outcomes. Our findings suggest that mutations in novel targets can contribute to the emergence of parasites with increased tolerance to artemisinin treatment and that such mutations can confer a fitness advantage even in the absence of a notable shift in EC 50 . Our findings also support the idea that inhibition of mitochondrial function may be an important target in T.gondii, as previously suggested by studies in yeast and human cancer cells.P. falciparum. The ART mechanism of action against tumor cells is also uncertain, but has been linked to oxidative stress, DNA damage, decreased cell replication, and activation of cell death pathways including apoptosis (41, 42). Studies in Baker's yeast (S. cerevisiae) implicate the mitochondria as a target of ART, and consistent with this model, growth on fermentable carbon sources results in genome sequence, as its assembly has been complicated by th...

Restructured mitochondrial-nuclear interaction in Plasmodium falciparum dormancy and persister survival after artemisinin exposure

“…Compared to the results from GB4 parasites, the less dramatic increase of median mitochondrial volume in DHA-exposed 803 parasites at t = 50 hours may be explained by its mixed population of persisters, with large mitochondrial volumes, plus actively replicating ring forms, with smaller mitochondrial volumes. These findings with GB4 and 803 parasites are consistent with the increased mitochondrial volumes that have been reported from other studies of ART-treated Plasmodium and Toxoplasma gondii [30,31].…”

“…In the present study, we have used FACS sorting, ASM, and autofluorescence FLIM-phasor analysis to characterize signature changes in the mitochondria of persisters that develop after exposure of P. falciparum-infected erythrocytes to DHA. [31]. Another study identified the same DegP2 ortholog in a genetic screen, genetic disruption facilitated survival of T. gondii exposed to lethal concentrations of DHA, and deletion of this region in P. falciparum resulted in higher survival in the RSA [44].…”

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

“…The complex II inhibitor TTFA 71,72 has an altered EC 50 when DegP2 is disrupted, which is consistent with DegP2 interacting with the complex II component SDHB, through TGGT1_212930. While this manuscript was in preparation, a study performed using in vitro evolution found that T. gondii parasites with point mutations in DegP2 had decreased ART susceptibility 105 . Interestingly, these researchers also found that such parasites had altered mitochondrial DNA copy number, which in yeast, has been linked to changes in aconitase 106 .…”