Oxidative stress and protein damage responses mediate artemisinin resistance in malaria parasites

Rocamora, Frances; Zhu, Lei; Liong, Kek Yee; Dondorp, Arjen M.; Miotto, Olivo; Mok, Sachel; Bozdech, Zbynek

doi:10.1371/journal.ppat.1006930

Cited by 151 publications

(198 citation statements)

References 100 publications

(187 reference statements)

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“…Resistance to artemisinin has long been thought to be multigenic, consistent with our model presented above 14,53 . So far, laboratory evolution of artemisinin resistance has mostly generated parasites with K13 mutations that are different from those most prevalent in the field, or mutations in other loci altogether, such as the recently-described pfcoronin 6,14,47 . Similarly, the AP-2µ(I592T) mutation described in this study has not been observed in human infections.…”

Section: Discussionmentioning

confidence: 99%

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

Henrici

Edwards

Zoltner

et al. 2019

Preprint

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The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the -subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2 we identified interacting partners including AP-2 AP-1/2 AP-2 and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

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Section: Discussionmentioning

confidence: 99%

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

Henrici

Edwards

Zoltner

et al. 2019

Preprint

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“…About 10% of the parasites are primed for stress adaptation under normal conditions. Stress responsiveness is an important determinant for drug resistance in P. falciparum [14,15]. This is also evident from the fact that even a clonal population of resistant parasites do not show complete resistance (varying Ring Survival Assay (RSA) levels, a measure of resistance against artemisinin [44].…”

Section: Discussionmentioning

confidence: 99%

“…This is attributed to the fact that during heat stress there is an increase in virulence (var) gene expression which helps in the binding of infected RBCs to the endothelial receptors resulting in cytoadherence [12]. Recent reports also suggest stress responses as crucial mediator of artemisinin resistance in P. falciparum [14,15].…”

Section: Introductionmentioning

confidence: 99%

Single Cell RNA-sequencing reveals cellular heterogeneity, stage transition and antigenic variation during stress adaptation in synchronized Plasmodium falciparum

Rawat

Srivastava

Gupta

et al. 2019

Preprint

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The malaria parasite has a complex life cycle exhibiting phenotypic and morphogenic variations in two different hosts. Phenotypic cell-to-cell variability can be an important determinant of cellular adaptation, stress tolerance and immune evasion in the host. To investigate cellular heterogeneity, we performed single cell RNA-sequencing (scRNA-seq) of 4949 and 6873 synchronized Plasmodium cells in control and under temperature stress condition (phenocopying the cyclic bouts of fever experienced during malarial infection). High-resolution clustering of scRNA-seq datasets and a combination of gene signatures allow identification of cellular heterogeneity and stage transition during stress adaptation. We identified a subset of parasites primed for gametogenesis and another subset primed for stress adaptation. Interestingly, temperature stress inducted the process of gametogenesis by upregulation of master regulator (AP2-G) of sexual conversion. Moreover, pseudotime analysis indicated bifurcation for cell-fate decision to gametogenesis at two different stages of intra-erythrocytic cycle. Furthermore, we identified a rare population of cells, which is only emerged during the stress condition, showing the reactive state of the pathogen against the temperature stress condition. Interestingly, genes associated with the gametogenesis, chaperon activity and maintenance of cellular homeostasis showed maximum variation under temperature stress condition. We also developed an online exploratory tool (website: http://bit.ly/plasmo_sync), which will provide new insights into gene function under normal and physiological stress condition.Thus, our study suggests that the variability and versatility of the maintenance of cellular homeostasis should enable cells to survive under different stress conditions, and may act as an important stimulator of development of drug-resistance in Plasmodium falciparum.

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“…A further 65 genes were significantly enriched in individual screens ( Supplementary Table 4). Because K13 resistance alleles retain function and have not been recovered by selection in culture, the absence of K13 from our loss-of-function screens was expected 12,13,17,18,20,21 .…”

Section: Genome-wide Screens Implicate Tca and Heme Biosynthesis Pathmentioning

confidence: 99%

“…The mechanism underlying this effect remains under scrutiny, but it is thought to involve changes in the unfolded protein response, autophagy, and PI3K activity [14][15][16][17] . Decreased ART sensitivity has also been attributed to mutations unrelated to K13, such as coronin 18 , clathrin adaptors [19][20][21] , and cysteine proteases 22 . P. falciparum strains without identified causative mutations have displayed decreased clearance as well 23,24 .…”

Section: Introductionmentioning

confidence: 99%

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

Harding

Sidik

Petrova

et al. 2019

Preprint

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Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria, however, resistance threatens to undermine global control efforts. To explore artemisinin resistance in apicomplexan parasites broadly, we used genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncovered the putative porphyrin transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provided evidence that mitochondrial metabolism can modulate resistance. We show that disruption of a top candidate from the screens, the mitochondrial protease DegP2, lowered levels of free heme and decreased DHA susceptibility, without significantly altering fitness in culture. Deletion of the homologous gene in P. falciparum, PfDegP, similarly lowered heme levels and DHA susceptibility. These results expose the vulnerability of the heme biosynthetic pathway for genetic perturbations that can lead to survival in the presence of DHA. We go on to show that chemically reducing heme biosynthesis can decrease the sensitivity of both T. gondii and P. falciparum to DHA, suggesting guidelines for developing combination therapies.

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Oxidative stress and protein damage responses mediate artemisinin resistance in malaria parasites

Cited by 151 publications

References 100 publications

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

Single Cell RNA-sequencing reveals cellular heterogeneity, stage transition and antigenic variation during stress adaptation in synchronized Plasmodium falciparum

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

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