The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background

Zhu, Lei; Tripathi, Jaishree; Rocamora, Frances; Miotto, Olivo; Pluijm, R. van der; Voss, Till S.; Mok, Sachel; Kwiatkowski, Dominic; Nosten, François; Day, Nicholas P. J.; White, Nicholas J.; Dondorp, Arjen M.; Bozdech, Zbynek

doi:10.1038/s41467-018-07588-x

Cited by 49 publications

(86 citation statements)

References 58 publications

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“…Another gene that was upregulated in four of our mutant lines was PHISTa. PHISTfamily proteins are known to have variable expression in resistant parasites (61) and are central to host cell remodeling, along with PfEMP1 (62). We also tested for eIF2␣ phosphorylation in our isogeneic parasite lines, as enhanced phosphorylated eIF2␣ was shown to correlate with high rates of recrudescence following ART (38).…”

Section: Discussionmentioning

confidence: 99%

Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

Siddiqui

Boonhok

Cabrera

et al. 2020

mBio

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Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance. PfK13 is essential for asexual erythrocytic development, but its function is not known. We tagged the PfK13 protein with green fluorescent protein in P. falciparum to study its expression and localization in asexual and sexual stages. We used a new antibody against PfK13 to show that the PfK13 protein is expressed ubiquitously in both asexual erythrocytic stages and gametocytes and is localized in punctate structures, partially overlapping an endoplasmic reticulum marker. We introduced into the 3D7 strain four PfK13 mutations (F446I, N458Y, C469Y, and F495L) identified in parasites from the China-Myanmar border area and characterized the in vitro artemisinin response phenotypes of the mutants. We found that all the parasites with the introduced PfK13 mutations showed higher survival rates in the ring-stage survival assay (RSA) than the wild-type (WT) control, but only parasites with N458Y displayed a significantly higher RSA value (26.3%) than the WT control. After these PfK13 mutations were reverted back to the WT in field parasite isolates, all revertant parasites except those with the C469Y mutation showed significantly lower RSA values than their respective parental isolates. Although the 3D7 parasites with introduced F446I, the predominant PfK13 mutation in northern Myanmar, did not show significantly higher RSA values than the WT, they had prolonged ring-stage development and showed very little fitness cost in in vitro culture competition assays. In comparison, parasites with the N458Y mutations also had a prolonged ring stage and showed upregulated resistance pathways in response to artemisinin, but this mutation produced a significant fitness cost, potentially leading to their lower prevalence in the Greater Mekong subregion. IMPORTANCE Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment.

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Section: Discussionmentioning

confidence: 99%

Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

Siddiqui

Boonhok

Cabrera

et al. 2020

mBio

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“…These data have become a key resource for the epidemiology and population genetics of antimalarial drug resistance [9][10][11][12][13][14][15][16][17][18][19][20][21][22] and an important platform for the discovery of new genetic markers and mechanisms of resistance through genome-wide association studies [23][24][25][26][27] and combined genome-transcriptome analysis. 28 The data have also been used to study gene deletions that cause failure of rapid diagnostic tests 29 ; to characterise genetic variation in malaria vaccine antigens 30,31 ; to screen for new vaccine candidates 32 ; to investigate specific host-parasite interactions 33,34 ; and to describe the evolutionary adaptation and diversification of local parasite populations. 7,9,12,[35][36][37][38][39][40] The Pf Community Project data also provide an important resource for developing and testing new analytical and computational methods.…”

Section: Introductionmentioning

confidence: 99%

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples

Pearson

Amato

Kwiatkowski

2019

Preprint

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MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination. MethodsAll samples in this study were derived from blood samples obtained from patients with P. falciparum malaria, collected with informed consent from the patient or a parent or guardian. At each location, sample collection was approved by the appropriate local and institutional ethics committees. The following local and institutional committees gave ethical approval for the partner studies:Standard laboratory protocols were used to determine DNA quantity and proportion of human DNA in each sample as previously described 7,56 .

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“…Numerous studies conducted by our group as well as others in the last decade showed the potential of utilizing peripheral blood samples from malaria-infected patients to enable studies of in vivo gene expression pro les of malaria parasites including the deadliest species, P. falciparum [10,11,13,15]. For that, it was essential to develop methods for optimal extraction, ampli cation, and detection of RNA in these samples.…”

Section: Resultsmentioning

confidence: 99%

“…Studies on in vivo transcriptomes of P. falciparum isolates from South East Asia have demonstrated that artemisinin drug resistance is associated with distinct transcriptional signature characterized by increased expression of unfolded protein response involving PROSC and TRiC chaperone complexes. In a separate study analyzing the same dataset, transcriptomic and genetic backgrounds that predispose P. falciparum to acquire artemisinin resistance were reported [10,11]. Moreover, two other studies have conducted whole-transcriptome pro ling of severe and acute malaria isolates, aiming to reveal mechanisms driving the systemic pathophysiology of severe malaria and parasite factors that induce apoptosis of human endothelial cells resulting in the disruption of the bloodbrain barrier, respectively [12,13].…”

Section: Introductionmentioning

confidence: 99%

A Comprehensive RNA Handling and Transcriptomics Guide for High-Throughput Processing of Plasmodium Blood-Stage Samples.

Kucharski

Tripathi

Nayak

et al. 2020

Preprint

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BackgroundSequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, we present a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples that can be fully streamlined for high-throughput studies.Results We evaluated the utility of various commercially available RNA-preserving reagents in a range of storage conditions. Similarly, we compared several RNA extraction protocols and established the one most suitable for the extraction of high-quality total RNA from low-parasitemia and low-volume blood samples. Furthermore, we updated the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA. Optimization of SMART-seq2 amplification method to better suit AT-rich P. falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10ng of total RNA and a lower parasitemia limit of 0.05%. Finally, we designed a modified method for depletion of unwanted human hemoglobin transcripts using in vitro CRISPR-Cas9 treatment, thus improving parasite transcriptome coverage in low parasitemia samples. To prove the functionality of the pipeline for both laboratory and field strains, we generated the highest 2-hour resolution RNA-seq transcriptome for Plasmodium falciparum 3D7 intraerythrocytic lifecycle available up-to-date and also applied the entire protocol to create the largest transcriptome data from Southeast Asian field isolates.ConclusionsOverall, our methodology presents an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large scale future transcriptomic studies in the field of malaria.

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The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background

Cited by 49 publications

References 58 publications

Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples

A Comprehensive RNA Handling and Transcriptomics Guide for High-Throughput Processing of Plasmodium Blood-Stage Samples.

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