Fitness Costs and the Rapid Spread of
            <i>kelch13</i>
            -C580Y Substitutions Conferring Artemisinin Resistance

Nair, Shalini; Li, Xue; Arya, Grace A; McDew-White, Marina; Ferrari, Marco; Nosten, François; Anderson, Tim

doi:10.1128/aac.00605-18

Cited by 68 publications

(101 citation statements)

References 39 publications

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“…4). We used amplicon sequencing (Nair et al 2018) to determine the frequencies of mtDNA from the two parents in those samples for which we used sWGA (Fig. 2C; Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, we do not see evidence for selection against the kelch13 -C580Y allele (chr 13) that underlies resistance to ART treatment. We previously used CRISPR/Cas9 editing to add the C580Y substitution to a wild type parasite (Nair et al 2018). Head-to-head competition experiments revealed strong fitness costs ( s = 0.15/asexual cycle) associated with this substitution.…”

Section: Discussionmentioning

confidence: 99%

“…Most studies of Plasmodium falciparum to date focus only on the asexual erythrocytic stages (Rosario et al 1978; Walliker et al 2005; Petersen et al 2015; Straimer et al 2017; Nair et al 2018), because they can be easily cultured in vitro in red blood cells, circumventing the need for humans or great apes, the natural hosts for this parasite. Two new research tools now allow us to examine selection across the complete life cycle of P. falciparum .…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we measured skews in allele frequencies across the genome in the progeny of a genetic cross between artemisinin resistant (ART-R, kelch13- C580Y) and ART sensitive (ART-S, kelch13 -WT) parasites throughout the life cycle to identify genes that influence parasite fitness in parasite stages infecting both the mosquito and vertebrate host. We selected ART-R and ART-S parental parasites in order to examine loci contributing to fitness and compensation for deleterious effects of ART-R alleles (Nair et al 2018). We used the humanized mouse model to allow parasite liver stage development of the genetic cross progeny, sWGA to enrich parasite DNA from host contaminations and pooled sequencing to determine temporal change in allele frequency and characterize genomic regions under selection.…”

Section: Introductionmentioning

confidence: 99%

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Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Kumar

McDew-White

et al. 2019

Preprint

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28Malaria is transmitted through female Anopheline mosquitoes where gamete fusion and meiosis 29 occurs, and humans where parasites proliferate asexually. We describe a powerful approach to 30 identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-31 cycle stages in human malaria parasite Plasmodium falciparum using bulk segregant analysis 32 (BSA). We combined experimental genetic crosses using humanized mice, with selective whole 33 genome amplification and BSA at multiple developmental stages in both mosquito and vertebrate 34 host to examine parasite competition and identify genomic regions under selection. We generated 35 crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, 36 kelch13-WT) parasite clones recently isolated from Southeast Asian patients. We then quantified 37 genome-wide changes in allele frequency in the parasite progeny population from infected midgut 38 and salivary glands of Anopheles stephensi mosquitoes, infected livers, emerging merozoites and 39 aliquots of in vitro cultured progeny parasites at intervals over 30 days. Three striking results 40 emerge: we observed (i) a strong skew (>80%) towards alleles from the ART-R parent in the 41 mosquito stage, that dropped to ~50% in the blood stage as selfed ART-R parasites were selected 42 against; (ii) highly repeatable skews in allele frequencies across the genome in blood stage 43 parasites; (iii) particularly strong selection (selection coefficient (s) ≤ 0.18/asexual cycle) against 44 alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 45 containing ARPS10. This approach robustly identifies selected loci and has strong potential for 46 identifying parasite genes that interact with the mosquito vector or compensatory loci involved in 47 drug resistance. 48 49 3 50 Parasitic organisms frequently use multiple hosts and have several morphologically and 51 transcriptionally distinctive life cycle stages. Within each host, parasites must circumvent immune 52 defenses and navigate to new tissues. There are frequently extreme bottlenecks in parasite numbers 53 during transmission (Hopp et al. 2015), with rapid proliferative growth within hosts, and intense 54 competition between co-infecting parasite genotypes. For example, the life cycle of malaria 55 parasites involves successive infection of two hosts: female Anopheles mosquitoes, where gamete 56 fusion, meiosis and recombination occurs, and humans in which parasites travel from the skin, 57 develop in the liver and then proliferate asexually in the blood stream. Ideally, we would like to 58 understand how natural selection operates across the complete life cycle and document the genes 59 subject to selection pressures at each life cycle stage: during erythrocytic growth, gametocyte 60 production, oocyst development in the mosquito midgut, migration of sporozoites to the salivary 61 glands, transmission from the salivary glands, sporozoite survival in the skin, and est...

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“…4). We used amplicon sequencing (Nair et al 2018) to determine the frequencies of mtDNA from the two parents in those samples for which we used sWGA (Fig. 2C; Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Kumar

McDew-White

et al. 2019

Preprint

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“…Amplicon analysis in Plasmodium has been adapted to multiple sequencing platforms depending on the desired cost, sample size, and sequence length [3,[9][10][11]. Because of this high resolution and flexibility, amplicon-based methods have been utilized in a range of applications, including studies of allele-specific vaccine efficacy [1], disease severity [10], clearance rate [12], within-host competition [13], relapse rate [9], drug resistance [5][6][7], host selection [8], and population structure [8,14]. Amplicon sequencing has high sensitivity for the detection of minority parasite lineages within an infection, and is of particular interest in longitudinal studies that track intra-host dynamics [3,4].…”

Section: Introductionmentioning

confidence: 99%

Detection of low-density Plasmodium falciparum infections using amplicon deep sequencing

Early

Daniels

Farrell

et al. 2018

Preprint

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Background: Deep sequencing of targeted genomic regions is becoming a common tool for understanding the dynamics and complexity of Plasmodium infections, but its lower limit of detection is currently unknown. Here, a new amplicon analysis tool, the Parallel Amplicon Sequencing Error Correction (PASEC) pipeline, is used to evaluate the performance of amplicon sequencing on low-density Plasmodium DNA samples. Illumina-based sequencing of two P. falciparum genomic regions (CSP and SERA2) was performed on two types of samples: in vitro DNA mixtures mimicking low-density infections (1-200 genomes/μl) and extracted blood spots from a combination of symptomatic and asymptomatic individuals (44-653,080 parasites/μl). Three additional analysis tools-DADA2, HaplotypR, and SeekDeep-were applied to both datasets and the precision and sensitivity of each tool were evaluated.. Results:Amplicon sequencing can contend with low-density samples, showing reasonable detection accuracy down to a concentration of 5 Plasmodium genomes/μl. Due to increased stochasticity and background noise, however, all four tools showed reduced sensitivity and precision on samples with very low parasitemia (<5 copies/μl) or low read count (<100 reads per amplicon). PASEC could distinguish major from minor haplotypes with an accuracy of 90% in samples with at least 30 Plasmodium genomes/μl, but only 61% at low Plasmodium concentrations (<5 genomes/μl) and 46% at very low read counts (<25 reads per amplicon). The four tools were additionally used on a panel of extracted parasitepositive blood spots from natural malaria infections. While all four identified concordant patterns of complexity of infection (COI) across four sub-Saharan African countries, the COI values obtained for individual samples differed in some cases. Conclusions:Amplicon deep sequencing can be used to determine the complexity and diversity of low-density Plasmodium infections. Despite differences in their approach, four state-of-the-art tools resolved known haplotype mixtures with similar sensitivity and precision. Researchers can therefore choose from multiple robust approaches for analyzing amplicon data, however, error filtration approaches should not be uniformly applied across samples of varying parasitemia. Samples with very low parasitemia and very low read count have higher false positive rates and call for read count thresholds that are higher than current recommendations.

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The newly discovered role of endocytosis in artemisinin resistance

Behrens

Schmidt

Spielmann

2021

Medicinal Research Reviews

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Artemisinin and its derivatives (ART) are the cornerstone of malaria treatment as part of artemisinin combination therapy (ACT). However, reduced susceptibility to artemisinin as well as its partner drugs threatens the usefulness of ACTs. Single point mutations in the parasite protein Kelch13 (K13) are necessary and sufficient for the reduced sensitivity of malaria parasites to ART but several alternative mechanisms for this resistance have been proposed.Recent work found that K13 is involved in the endocytosis of host cell cytosol and indicated that this is the process responsible for resistance in parasites with mutated K13.These studies also identified a series of further proteins that act together with K13 in the same pathway, including previously suspected resistance proteins such as UBP1 and AP-2μ. Here, we give a brief overview of artemisinin resistance, present the recent evidence of the role of endocytosis in ART resistance and discuss previous hypotheses in light of this new evidence. We also give an outlook on how the new insights might affect future research.

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Fitness Costs and the Rapid Spread of kelch13 -C580Y Substitutions Conferring Artemisinin Resistance

Cited by 68 publications

References 39 publications

Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Detection of low-density Plasmodium falciparum infections using amplicon deep sequencing

The newly discovered role of endocytosis in artemisinin resistance

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