Mitochondrial Membrane Potential in a Small Subset of Artemisinin-Induced DormantPlasmodium falciparumParasites In Vitro

Abstract

Artemisinin-induced dormancy is a proposed mechanism for failures of monotherapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that after dihydroartemisinin treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential marker, and persisted to recovery. RH-positive parasites sorted with fluorescence-activated cell sorting resumed growth at 10,000/well whereas RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was detected only in RH-positive dormant parasites. Importantly, after treatment of dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage.

Novel Plasmodium falciparum metabolic network reconstruction identifies shifts associated with clinical antimalarial resistance

“…Overall, these results indicate that sensitive and resistant parasites are differentially utilizing pathways within these organelles, and have unique requirements for transport of essential substrates. This observation is consistent with previous studies and our enrichment results highlighting the influence of mitochondrial metabolism on survival in the presence of artemisinin [26, 29]. Moreover, oxygen transport into the cell and then into the mitochondria is only essential in sensitive parasites, further predicting differential use of the mitochondria in these parasites as oxygen serves as the terminal step in the electron transport chain.…”

“…For example, resistant models are uniquely enriched with genes involved in pyrimidine biosynthesis and mitochondrial redox reactions. This finding is consistent with the importance of mitochondrial function in surviving artemisinin stress [26, 29] and the physical interactions between artemisinin and proteins involved in glycolysis, nucleotide synthesis, and the mitochondria in mammalian cells and P. falciparum [103–105]. Additionally, the metabolic disruption of the redox reactions in the electron transport chain upon artemisinin treatment (via decreased production of orotate and fumarate, presumably via dihydroorotate dehydrogenase and succinate dehydrogenase enzymes [22, 28, 106]) suggests that changes in these pathways may be important for survival in the presence of the drug.…”

“…This phenotype is correlated with mutations in the P. falciparum Kelch13 gene [13, 14, 16, 17] and changes in both signalling pathways [18–21] and organellar function [22–29]. Overall, due to the complexity of artemisinin’s mechanism of killing (see citations above and [30–36]), it has been challenging to separate the causes and effects of resistance.…”

The Spiroindolone KAE609 Does Not Induce Dormant Ring Stages in Plasmodium falciparum Parasites

“…Both DHA (700 nM) and ART (500 nM) produced dormant ring stages at 24 to 48 h after the start of treatment, with parasites recovering 3 to 5 days after drug exposure. This finding is in accord with the class effect of the artemisinin derivatives resulting in the induction of dormant rings reported elsewhere (38,39), even though the time to recovery of the parasites was shorter than that reported previously (17,39,40). A plausible explanation for the difference is that in the present study, magnetic columns were not used on days 1, 2, and 3 after the start of the experiment to remove growing parasites (late stages) not killed by DHA treatment as well as those emerging from dormant parasites with the short duration of dormancy (24 to 48 h).…”

Inhibiting the Plasmodium eIF2α Kinase PK4 Prevents Artemisinin-Induced Latency

“…We sought to determine the status of eIF2α phosphorylation in malaria parasites treated with ARTs as well as recrudescent parasites post-ART treatment. Two derivatives of artemisinin have been employed to study malaria recrudescence: dihydroartemisinin (DHA) is used in cultured human malaria and artesunate (AS) in rodent malaria (Chen et al, 2014; Codd et al, 2011; LaCrue et al, 2011; Peatey et al, 2015; Shaw et al, 2015; Teuscher et al, 2012; Teuscher et al, 2010). We established recrudescence post-AS treatment in P. berghei ANKA infected Swiss Webster mice (Figure S1 and Table S1).…”