Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

Gnädig, Nina F.; Stokes, Barbara H.; Edwards, Rachel L.; Kalantarov, Gavreel F.; Heimsch, Kim C.; Kuderjavy, Michal; Crane, Audrey; Lee, Marcus; Straimer, Judith; Becker, Katja; Trakht, Ilya; John, Audrey Odom; Mok, Sachel; Fidock, David A.

doi:10.1371/journal.ppat.1008482

Cited by 63 publications

(131 citation statements)

References 88 publications

(132 reference statements)

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“…Increased artemisinin resistance has been found in P. falciparum parasites with Pfkelch13 mutations ( Miotto et al, 2015 ; Ariey et al, 2014 ; Ghorbal et al, 2014 ; Straimer et al, 2015 ; Mbengue et al, 2015 ). A previous study found that PfKelch13 could modulate the level of a signaling molecule phosphatidylinositol 3-phosphate (PI(3)P) through interaction with PfPI3K ( Mbengue et al, 2015 ), while other studies did not detect the interaction between PfKelch13 and PfPI3K ( Siddiqui et al, 2020 ; Gnädig et al, 2020 ; Birnbaum et al, 2020 ). PfKelch13 mutations have been linked to the accumulation of PI(3)P in P. falciparum -infected RBCs and this increased PI(3)P level is highly correlated with parasite resistance to artemisinin ( Mbengue et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Pasaje

Srivastava

et al. 2020

eLife

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Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin and environmental factors. However, PI(3)P function during the Plasmodium stress response was unknown. Here, we used PI3K inhibitors and antimalarial agents to examine the importance of PI(3)P under thermal conditions recapitulating malarial fever. Live cell microscopy using chemical and genetic reporters revealed that PI(3)P stabilizes the digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. A chemoproteomic approach identified PfHsp70-1 as a PI(3)P-binding protein. An Hsp70 inhibitor and knockdown of PfHsp70-1 phenocopy PI(3)P-deficient parasites under heat shock. Furthermore, PfHsp70-1 downregulation hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1 and present a novel PI(3)P function in DV stabilization during heat stress.

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Section: Introductionmentioning

confidence: 99%

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Pasaje

Srivastava

et al. 2020

eLife

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“…Intriguingly, the most prevalent SEA K13 mutation, C580Y, was fitness neutral in vitro when gene edited into recent Cambodian clinical isolates, whereas it displayed a significant growth defect when introduced into ART-susceptible parasites isolated before ARTs were widely deployed (34,36). Recently, it has been demonstrated that PF K13 localizes to the parasite cytostomes and other intracellular vesicles and plays a role in parasite hemoglobin endocytosis and trafficking to the lysosome-like digestive vacuole (37)(38)(39). K13 mutations are thought to lead to a partial loss of protein function, which subsequently impairs hemoglobin endocytic uptake, thereby lessening ART activation and conferring ART resistance (37).…”

Section: Introductionmentioning

confidence: 99%

Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That is Reversed by Proteasome Inhibition

Simwela

Stokes

Aghabi

et al. 2020

Preprint

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The recent emergence of Plasmodium falciparum (PF) parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the PF K13 protein, which enhance survival of early ring stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo. However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous PF K13 mutations into the K13 gene of an artemisinin-sensitive, P. berghei (PB) rodent model of malaria. Introduction of the orthologous PF K13 F446I, M476I, Y493H and R539T mutations into PB K13 produced gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-hour in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant PB K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into PB while the equivalent of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these PB K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome ART resistance. Taken together, our work provides clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro, and most importantly under in vivo conditions.IMPORTANCERecent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon treatment with the drug. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, P. berghei. Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo, which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y, I543T) into the corresponding amino acid residues, while other introduced mutations (M476I, R539T equivalents) carried a pronounced fitness cost. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines.

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“…Intriguingly, recent data confirm the association of key resistance-mediator K13 with Rab-GTPases [25], adding to the repertoire of proteins comprising K13-mediated endocytic vesicles, and by extension supporting the role of prenylation in K13-mediated processes associated with ART MOA.…”

Section: Discussionmentioning

confidence: 61%

The apicoplast link to fever-survival and artemisinin-resistance in the malaria parasite

Zhang¹,

Wang²,

Oberstaller³

et al. 2020

Preprint

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BackgroundThe emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. We developed a new large-scale phenotypic screening pipeline and used it to carry out the first large-scale forward-genetic phenotype screen in P. falciparum to identify genes that allow parasites to survive febrile temperatures.ResultsScreening identified more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants were more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin included highly essential, conserved pathways associated with protein-folding, heat-shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the parasite’s algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general were up-regulated in response to heat shock, as were other Plasmodium genes with orthologs in plant and algal genomes.ConclusionsPlasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiotic cyanobacterium-related ancestral genes in the parasite’s genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors.

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Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

Cited by 63 publications

References 88 publications

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death

Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That is Reversed by Proteasome Inhibition

The apicoplast link to fever-survival and artemisinin-resistance in the malaria parasite

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