Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Schumann, Robin; Bischoff, Eileen; Klaus, Severina; Möhring, Sophie; Flock, Julia; Keller, Sandro; Remans, Kim; Ganter, Markus; Deponte, Marcel

doi:10.1016/j.redox.2021.102177

Cited by 10 publications

(15 citation statements)

References 57 publications

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“…As artificially titrating K13 or KIC7 levels had a similar effect it can be inferred that this is due to a proportional reduction in endocytosis. The same effect on resistance was observed in a K13 titration recently 15 and a role of K13 protein abundance in resistance is also supported by the finding that increasing levels of K13 C580Y reverts resistant parasite back to sensitive 7 . Interestingly, for E252Q, which is located outside the propeller domain, protein levels were not reduced.…”

Section: Discussionsupporting

confidence: 67%

“…Resistant clinical isolates and laboratory parasites carrying the resistance-associated mutations k13 R539T and k13 C580Y contain reduced amounts of K13 (7,(12)(13)(14). Artificially modulating K13 levels indicated that the reduced K13 levels lead to resistance (7,12,15). However, the impact of mutations on K13 levels and the relationship to resistance has not been systematically analysed and it is unclear whether other k13 mutations confer resistance through the same mechanism or affect the functionality of K13.…”

Section: Introductionmentioning

confidence: 99%

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Impact of different mutations on Kelch13 protein levels, ART resistance and fitness cost inPlasmodium falciparumparasites

Behrens

Schmidt

Peigney

et al. 2022

Preprint

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Combination therapies containing Artemisinin derivatives (ART) are the first line treatment of malaria but their effectiveness is reduced by resistance due to a fraction of ring stage Plasmodium parasites surviving high ART levels. Resistance-causing kelch13 (k13) mutations are common in South East Asia but were only recently and sparsely detected in Africa, which experiences the highest malaria burden. Kelch13 shares a cellular compartment with other proteins (KICs) several of which cause resistance when inactivated or, in a few cases, when mutated. To see if the sparse detection of resistance mutations in Africa is due to unknown mutations, we tested 135 k13 and kic mutations detected mostly in African field isolates. No kic mutation caused resistance but two in k13, V520A and V589I, did. These mutations were geographically much more widespread in Africa but conferred lower levels of resistance than known ART resistance mutations. A dissection of the mechanism using isogenic parasites with different k13 mutations and parasites that we selected for even higher ART resistance, showed that resistance is a function of K13 protein levels in ring-stage parasites and correlates with the fitness cost. This indicated that hyper-resistance is unlikely to arise in the field. Double mutations in k13 had not even additive effects and combinations including a non-k13 mutation led to high fitness costs, suggesting that such combinations also pose no risk for higher resistance in the field. Overall, our results indicate that resistance is restricted by a proportional fitness cost but that incidence-lowering measures may favor high-resistance mutations.SignificanceOur findings indicate that hyper-resistant parasites are unlikely to occur in endemic settings due to the proportional fitness cost. Mutations within k13 were not additive and mutations outside k13 had a disproportionally high fitness cost. The strong influence of the fitness cost may have favored moderate frequencies of the resistance mutations with low fitness cost detected in many African settings that we characterized in this study. These mutations, so far gone unnoticed, may be optimal in high endemicity regions, where relative drug use is presumably low but frequent multiple infections increase competition. A given endemic setting may thus favor variants with K13 levels for an optimal resistance-fitness balance which is relevant for incidence-lowering interventions.

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Section: Discussionsupporting

confidence: 67%

Section: Introductionmentioning

confidence: 99%

Impact of different mutations on Kelch13 protein levels, ART resistance and fitness cost inPlasmodium falciparumparasites

Behrens

Schmidt

Peigney

et al. 2022

Preprint

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“…We here refer to this in vitro measurable phenomenon as "in vitro ART resistance" when the survival of parasites is above 1% in an RSA as previously defined [23]. Point mutations in the gene encoding the parasite protein Kelch13 (K13) are the main cause for the reduced ART susceptibility of laboratory and field parasite isolates [24,25] and was shown to lead to decreased K13 protein levels [26][27][28][29][30]. This results in reduced hemoglobin uptake via endocytosis, which is assumed to cause less ART activation through hemoglobin degradation products, resulting in the reduced parasite ART susceptibility [26,31].…”

Section: Introductionmentioning

confidence: 99%

“…Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of endocytosis in malaria parasites.of laboratory and field parasite isolates [24,25] and was shown to lead to decreased K13 protein levels [26][27][28][29][30]. This results in reduced hemoglobin uptake via endocytosis, which is assumed to cause less ART activation through hemoglobin degradation products, resulting in the reduced parasite ART susceptibility [26,31].…”

mentioning

confidence: 99%

The Kelch13 compartment is a hub of highly divergent vesicle trafficking proteins in malaria parasites

Schmidt

Wichers

Behrens

et al. 2022

Preprint

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Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility ofP. falciparumparasites to Artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in ART resistance. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins are among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by identifying four novel proteins at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these, and previously identified proteins, showed that canonical vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance and endocytosis, strengthening the link to endocytosis. Despite this, most showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation. A second group of proteins did not influence endocytosis or ART resistance and was characterised by a lack of vesicle trafficking domains. We here identified the first essential protein of the second group and showed that it is needed in late-stage parasites. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of the endocytosis in malaria parasites.

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“…The survival of K MT F MT A MT was no different from that of MRA1240 following 700 nM DHA treatment which was unexpected because MRA1240 contains the k13-R539T allele expected to confer a higher ART resistance than k13-C580Y. Interestingly, following 20 nM DHA treatment the K MT F MT A MT parasite showed greater sensitivity than MRA1240; k13-R539T may lead to greater loss of K13 function in parasites and consequently greater ART resistance, although both R539T and C580Y have a similar effect on K13 folding in vitro [19]. Perhaps it also reflects the complexity of the mode of action of ART and the resistance mechanisms evolved by the parasite.…”

Section: Discussionmentioning

confidence: 91%

Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum

Kampoun

Srichairatanakool²,

Prommana³

et al. 2022

Malar J

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Background The resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, is of great concern. Mutations in the kelch13 (k13) gene (for example, those resulting in the Cys580Tyr [C580Y] variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a k13 mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2), and crt (chloroquine resistance transporter), in addition to k13 gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13. Methods The reported mutations were introduced, together with a mutation to produce the k13-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0−3 h ring survival assay (RSA0−3 h) was investigated. Results and conclusion Introducing both fd-D193Y and arps10-V127M into a k13-C580Y-containing parasite, but not a wild-type k13 parasite, increased survival of the parasite in the RSA0−3 h. The results suggest epistasis of arps10 and k13, with arps10-V127M a modifier of ART susceptibility in different k13 allele backgrounds.

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Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Cited by 10 publications

References 57 publications

Impact of different mutations on Kelch13 protein levels, ART resistance and fitness cost inPlasmodium falciparumparasites

Impact of different mutations on Kelch13 protein levels, ART resistance and fitness cost inPlasmodium falciparumparasites

The Kelch13 compartment is a hub of highly divergent vesicle trafficking proteins in malaria parasites

Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum

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