A molecular marker of artemisinin-resistant Plasmodium falciparum malaria
Summary (3 min read)
Introduction
- Submitted on 20 Jun 2014 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not.
- The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
- L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.
- Frédéric Ariey, Benoit Witkowski, Chanaki Amaratunga, Johann Beghain, Anne-Claire Langlois, Nimol Khim, Saorin Kim, Valentine Duru, Christiane Bouchier, Laurence Ma, et al.
- Frédéric Ariey, Benoit Witkowski, Chanaki Amaratunga, Johann Beghain, Anne-Claire Langlois, et al., also known as To cite this version.
- Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, the authors associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo.
- The risk of ART-resistant parasites spreading from western Cambodia to the Greater Mekong Subregion and to Africa, as happened previously with chloroquine- and sulphadoxine/ pyrimethamine-resistant parasites3–5, is extremely worrisome.
- One possible explanation is that the parasite clearance half-life is not only determined by the intrinsic susceptibility of a parasite isolate to ART, but also by its developmental stage at the time of ART treatment and host-related parameters such as pharmacokinetics and immunity17.
A candidate molecular marker of ART resistance
- The ART-resistant F32-ART5 parasite line was selected by culturing theART-sensitive F32-Tanzania cloneunder a dose-escalating, 125-cycle *These authors contributed equally to this work.
- 2Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, 75724 Paris Cedex 15, France.
- 6National Center for Parasitology, Entomology andMalaria Control, PhnomPenh, Cambodia.
- Each gene harbours one mutant codon in F32-ART5 compared to F32-TEM, F32-Tanzania or 3D7 (ExtendedData Table 2).
- In contrast, PF3D7_1343700 polymorphism shows a significant associationwith RSA0–3h survival rates (Fig. 2).
EmergenceandspreadofK13-propellermutants inCambodia
- F32-Tanzania parasites exposed to increasing artemisinin concentrations for 120 consecutive cycles18 were analysed by whole-genome sequencing at five time-points (red arrows).
- Data from six provinces were compared (n5 886): Pailin, Battambang and Pursat in the west where ART resistance is established1,6,8,22, Kratie in the southeast where ART resistance has increased in recent years2, and Preah Vihear in the north and Ratanakiri in the northeast where there was virtually no evidence of ART resistance during this time period2.
- TheC580Yallele accounts for85% (189/222) of allmutant alleles observed in 2011–2012 (Extended Data Fig. 2).
- These data indicate that C580Y, R539T and Y493H identify slow-clearing parasites in malaria patients treated with ART.
- Importantly, K13-propeller mutations more accurately identify slowclearing parasites than KH group (Fig. 4b), demonstrating that the association of K13-propeller polymorphism with clinical ART resistance in Cambodia is partially independent of the genetic background of KH subpopulations.
Discussion
- The F32-ART5 lineage acquired aK13-propellermutation as it developed ART resistance, as indicated by its ability to survive a pharmacologically relevantexposure toDHAintheRSA0–3h.
- The K13-propeller shows homology with humanKLHL12 andKLHL2, involved in ubiquitin-based protein degradation, andKEAP1, involved in cell adaptation to oxidative stress (Extended Data Fig. 4).
- Further studies are therefore required to identify additional genetic determinants of ART resistance, which may reside in the strongly selected regions recently identified14,16.
- A, Correlation of parasite clearance half-lives andK13-propeller alleles for parasite isolates in Pursat andRatanakiri in 2009–2010.
METHODS SUMMARY
- The ART-resistant F32-ART5 parasite line was selected by culturing the ARTsensitive F32-Tanzania clone under a dose-escalating regimen of artemisinin for 5 years.
- Reference DNA was extracted from P. falciparum lines 3D7, 89F5PaloAltoUganda andK1992.
- Whole-genome sequencing was performed on F32-Tanzania, F32-TEM, F32-ART5 (4 time points), three reference strains (3D7, 89F5 and K1992) and 21 Cambodian parasite isolates, using an Illumina paired-reads sequencing technology.
- Double-strand sequencing of PCR products was performed byMacrogen.
- Data were analysed with Microsoft Excel and MedCalc version 12.
Published online 18 December 2013.
- Global Report on Antimalarial Drug Efficacy and Drug Resistance: 2000–2010 (World Health Organization, 2010).
- Novel phenotypic assays for the detection of artemisinin- resistant Plasmodium falciparummalaria in Cambodia: in-vitro and ex-vivo drugresponse studies.
- Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations.
- The authors are grateful to the provincial health department directors and other staff of the Cambodian Ministry of Health.
METHODS
- Artemisinin- and mock-pressured P. falciparum F32 lineages.
- Double-strand sequencing of PCR products was performed by Macrogen.
- DNA extraction, Illumina sequencing and SNP genotyping of clinical parasite isolates obtained from malaria patients in Pursat and Ratanakiri provinces, Cambodia, have beenpreviously described15.
- 2FLU), KLHL12 (PDB 2VPJ) and KLHL2 (PDB 2XN4) proteins, respectively, that were used as templates tomodel the 3D-structure of the K13-propeller.
- Data were analysed with Microsoft Excel and MedCalc version 12.
P. falciparum K13 and human KEAP1 proteins and structural 3Dmodel of
- A, Schematic representation of the predicted PF3D7_1343700 protein and homology to human KEAP1.
- The level of amino-acid identity between the K13propeller and kelch domains of proteins with solved 3D structures, including human KEAP146,47, enabled us to model the 3D structure of the K13-propeller and to map the mutations selected under ART pressure (Extended data Table 5).
- Proteomics data indicate that it is produced by asexual (trophozoites, schizonts, merozoites and rings) and sexual blood stages of P. falciparum, and that it possesses phosphorylated residues in the N-terminal Plasmodium-specific domain (www. plasmodb.org).
- Moreover, ring stages that do not massively digest hemoglobin display a reduced susceptibility to artemisinins 59 .
- The S299T mutation is located within this "unknown" region.
Did you find this useful? Give us your feedback
Citations
1,777 citations
576 citations
570 citations
559 citations
540 citations
Cites background from "A molecular marker of artemisinin-r..."
...Genome-wide association studies and gene candidate-based approaches in P. falciparum are revealing potential mechanisms of resistance to artemisinin (Ariey et al., 2014; Mbengue et al., 2015; Mok et al., 2015; Straimer et al., 2015)....
[...]
...Genome sequencing of parasites from patients that were treated with ACT and demonstrated reduced parasite clearance rates identifiedmutations in the gene encoding a K13 propeller protein, suggesting that this protein was an important determinant of artemisinin resistance (Ariey et al., 2014)....
[...]
...falciparum are revealing potential mechanisms of resistance to artemisinin (Ariey et al., 2014; Mbengue et al., 2015; Mok et al., 2015; Straimer et al., 2015)....
[...]
References
3,166 citations
3,010 citations
1,593 citations
"A molecular marker of artemisinin-r..." refers background in this paper
...2), suggesting strong structural and functional constraints on the protein; (7) the three most-prevalent K13-propeller mutations correlate strongly with RSA0–3h survival rates in vitro and parasite clearance half-lives in vivo at the level of individual parasite isolates andmalaria patients, respectively; and (8) the frequency of mutant alleles correlates strongly with the prevalence of day 3 positivity after ACT treatment at the level of human populations in Cambodia....
[...]
1,281 citations
889 citations
Related Papers (5)
Frequently Asked Questions (9)
Q2. What future works have the authors mentioned in the paper "A molecular marker of artemisinin-resistant plasmodium falciparum malaria" ?
Further studies are therefore required to identify additional genetic determinants of ART resistance, which may reside in the strongly selected regions recently identified14,16.
Q3. How many mutations are sufficient to confer resistance to ART in a typical African parasite?
it is particularlyworrying that as few as two mutations, that is, the K13-propeller M476I and PF3D7_0110400D56V,were sufficient to conferART resistance toF32Tanzania, which has a typical African genetic background.
Q4. What is the significance of the mutations in the K13propeller?
14 as being under recent positive selection, and within the region of top-ranked signatures of selection outlined in ref. 16; (4) multiple mutations, all non-synonymous, are present in the K13propeller, reflecting positive selection rather than a hitchhiking effect or genetic drift; (5)mutations occur in a domain that is highly conserved inP. falciparum, with only onenon-synonymous SNPbeingdocumented in a single parasite isolate from Africa42; (6) all polymorphisms the authors observe in Cambodia are novel and all but one (V568G) occur at positions strictly conserved between Plasmodium species (Supplementary Fig. 1 and Supplementary Fig. 2), suggesting strong structural and functional constraints on the protein; (7) the three most-prevalent K13-propeller mutations correlate strongly with RSA0–3h survival rates in vitro and parasite clearance half-lives in vivo at the level of individual parasite isolates andmalaria patients, respectively; and (8) the frequency of mutant alleles correlates strongly with the prevalence of day 3 positivity after ACT treatment at the level of human populations in Cambodia.
Q5. What is the definition of aK13-propeller polymorphism?
K13-propeller polymorphism fulfils the definition of a molecularmarker of ART resistance for several reasons: (1) there has been a progressive loss of wild-type parasites in western Cambodia during the decade of emerging ART resistance in this region; (2) mutant parasites cluster in Cambodian provinces where ART resistance is well established and are less prevalent where ART resistance is uncommon; (3) PF3D7_1343700 is located 5.9 kilobases upstreamof the 35-kb locus identified in ref.
Q6. How many SNPs are found in PF3D7?
PF3D7_1464500 has 12 SNPs previously reported in older isolates from southeast Asia, including theART-susceptibleDd2 line21, probably reflecting a geographic signature.
Q7. What is the significance of the ART resistance in a wild type parasite?
Allele exchange studies in mutant and wild-type parasites may help to define the contribution of K13-propeller polymorphisms on different genetic backgrounds to theRSA0–3 h survival rate.
Q8. Who obtained the ethical clearances for P. falciparum isolates?
Ethical clearances for parasite isolate collections were obtained from the CambodianNational Ethics Committee for Health Research, the Institutional Review Board of the Naval Medical Research Center, the Technical Review Group of the WHO Regional Office for the Western Pacific, and the Institutional Review Board of the National Institute of Allergy and Infectious Diseases.
Q9. What is the possible explanation for the ART resistance in Cambodia?
One possible explanation is that the parasite clearance half-life is not only determined by the intrinsic susceptibility of a parasite isolate to ART, but also by its developmental stage at the time of ART treatment and host-related parameters such as pharmacokinetics and immunity17.