Artemisinin-resistant K13 mutations rewire Plasmodium falciparum’s intra-erythrocytic metabolic program to enhance survival

Abstract

The emergence and spread of artemisinin resistance, driven by mutations in Plasmodium falciparum K13, has compromised antimalarial efficacy and threatens the global malaria elimination campaign. By applying systems-based quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we provide evidence that K13 mutations alter multiple aspects of the parasite’s intra-erythrocytic developmental program. These changes impact cell-cycle periodicity, the unfolded protein response, protein degradation, vesicular trafficking, and mitochondrial metabolism. K13-mediated artemisinin resistance in the Cambodian Cam3.II line was reversed by atovaquone, a mitochondrial electron transport chain inhibitor. These results suggest that mitochondrial processes including damage sensing and anti-oxidant properties might augment the ability of mutant K13 to protect P. falciparum against artemisinin action by helping these parasites undergo temporary quiescence and accelerated growth recovery post drug elimination.

Plasmodium falciparum resistance to ACTs: Emergence, mechanisms, and outlook

“…Transcriptomic analysis of clinical ART-resistant parasite isolates revealed upregulation of genes involved in the UPR pathway including the ER-located reactive oxidative stress complex (e.g., cyclophilin 19B, BiP, ERp72, GRP94) and the cytoplasmic T-complex protein 1 ring complex (TRiC), allowing the resistant parasites to better tolerate the proteotoxic effects of ARTs ( Mok et al, 2015 ). Proteomic analysis of laboratory engineered isogenic parasites further confirmed that K13 mutations are associated with upregulated chaperones, including the cyclophilin 19B protein ( Mok et al, 2021 ). Increased expression of genes involved in adaptive responses against cellular damage has also been documented in in vitro selected ART-resistant parasites ( Rocamora et al, 2018 ).…”

“…Alteration in cellular processes involving hemoglobin endocytosis and digestion, FV integrity, and heme detoxification all can lead to changes in ART activation. A peptidomics study revealed that the K13 mutants had lower levels of hemoglobin-derived peptides than the sensitive isolate at the trophozoite stage ( Siddiqui et al, 2017 ), but a more recent study did not find such a difference at the ring stage ( Mok et al, 2021 ). Several proteins that are associated with ART resistance including K13, AP-2μ, UBP-1, PfPI3K (phosphatidylinositol-3-kinase), Coronin, and Falcipain 2 may play roles in hemoglobin uptake and digestion ( Sutherland et al, 2020 ; Xie et al, 2020 ).…”

“…Repeated drug pressure and dormancy over time may select for fast-recovering rings that can withstand the drug exposure without undergoing dormancy ( Cheng et al, 2012 ). ART appears to temporally slow down the parasite growth and alter the periodicity of the cell cycle, and the resistant parasites are able to withstand and overcome this growth retardation with accelerated post-drug recovery ( Dogovski et al, 2015 ; Klonis et al, 2011 ; Mok et al, 2021 ).

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“…However, a large-scale transcriptomic analysis of clinical parasites with ART resistance from the GMS did not find altered k13 expression at the transcript level ( Mok et al, 2015 ), while a time-course analysis of wild-type k13 expression after AL treatment identified a negative correlation between k13 expression and PC 1/2 ( Silva et al, 2019 ). A comparative transcriptomic and proteomic analysis of isogenic parasites demonstrated that the impact of the K13 mutations on the K13 protein levels depended on the mutations and developmental stages, although they all showed elevated transcripts at the ring stage ( Mok et al, 2021 ). Specifically, compared to the wild-type K13 protein level in the Cam3.II genetic background, the R539T-K13 protein levels were lower at both ring and trophozoite stages, whereas the C580Y–K13 level was reduced only at the trophozoite stage.…”

“…In addition, another sign of global translation arrest is the reduced tRNA expression. K13 mutant parasites may also possess a higher ability to suppress tRNA expression after ART treatment as found during amino acid starvation, thus enhancing recovery from cell-cycle arrest ( McLean and Jacobs-Lorena, 2017 ; Mok et al, 2021 ). K13 mutant parasites display lesser ubiquitination of proteins, which may prevent the overwhelming of the proteasome response pathway ( Bridgford et al, 2018 ; Dogovski et al, 2015 ; Siddiqui et al, 2020 ).…”