Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

Nature volume 505, pages 50–55 (2014)Cite this article

Subjects

Abstract

Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Temporal acquisition of mutations in F32-ART5.
Figure 2: Survival rates of Cambodian parasite isolates in the RSA0–3 h, stratified by K13-propeller allele.
Figure 3: Frequency of K13-propeller alleles in 886 parasite isolates in six Cambodian provinces in 2001–2012.
Figure 4: Parasite clearance half-lives.

Similar content being viewed by others

References

  1. Dondorp, A. M. et al. Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 361, 455–467 (2009)

    Article  CAS  Google Scholar 

  2. World Health Organization. Global Report on Antimalarial Drug Efficacy and Drug Resistance: 2000–2010 (World Health Organization, 2010)

  3. Mita, T. et al. Limited geographical origin and global spread of sulfadoxine-resistant dhps alleles in Plasmodium falciparum populations. J. Infect. Dis. 204, 1980–1988 (2011)

    Article  CAS  Google Scholar 

  4. Roper, C. et al. Intercontinental spread of pyrimethamine-resistant malaria. Science 305, 1124 (2004)

    Article  CAS  Google Scholar 

  5. Wootton, J. C. et al. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum . Nature 418, 320–323 (2002)

    Article  CAS  ADS  Google Scholar 

  6. Amaratunga, C. et al. Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. Lancet Infect. Dis. 12, 851–858 (2012)

    Article  Google Scholar 

  7. Kyaw, M. P. et al. Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar. PLoS ONE 8, e57689 (2013)

    Article  CAS  ADS  Google Scholar 

  8. Noedl, H. et al. Evidence of artemisinin-resistant malaria in western Cambodia. N. Engl. J. Med. 359, 2619–2620 (2008)

    Article  CAS  Google Scholar 

  9. Phyo, A. P. et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet 379, 1960–1966 (2012)

    Article  Google Scholar 

  10. Hien, T. T. et al. In vivo susceptibility of Plasmodium falciparum to artesunate in Binh Phuoc Province, Vietnam. Malar. J. 11, 355 (2012)

    Article  CAS  Google Scholar 

  11. Flegg, J. A. et al. Standardizing the measurement of parasite clearance in falciparum malaria: the parasite clearance estimator. Malar. J. 10, 339 (2011)

    Article  Google Scholar 

  12. White, N. J. The parasite clearance curve. Malar. J. 10, 278 (2011)

    Article  CAS  Google Scholar 

  13. Witkowski, B. et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect. Dis. 13, 1043–1049 (2013)

    Article  CAS  Google Scholar 

  14. Cheeseman, I. H. et al. A major genome region underlying artemisinin resistance in malaria. Science 336, 79–82 (2012)

    Article  CAS  ADS  Google Scholar 

  15. Miotto, O. et al. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nature Genet. 45, 648–655 (2013)

    Article  CAS  Google Scholar 

  16. Takala-Harrison, S. et al. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia. Proc. Natl Acad. Sci. USA 110, 240–245 (2013)

    Article  CAS  ADS  Google Scholar 

  17. Lopera-Mesa, T. M. et al. Plasmodium falciparum clearance rates in response to artesunate in Malian children with malaria: effect of acquired immunity. J. Infect. Dis. 207, 1655–1663 (2013)

    Article  CAS  Google Scholar 

  18. Witkowski, B. et al. Increased tolerance to artemisinin in Plasmodium falciparum is mediated by a quiescence mechanism. Antimicrob. Agents Chemother. 54, 1872–1877 (2010)

    Article  CAS  Google Scholar 

  19. Klonis, N. et al. Artemisinin activity against Plasmodium falciparum requires hemoglobin uptake and digestion. Proc. Natl Acad. Sci. USA 108, 11405–11410 (2011)

    Article  CAS  ADS  Google Scholar 

  20. Vigan-Womas, I. et al. An in vivo and in vitro model of Plasmodium falciparum rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype. Infect. Immun. 76, 5565–5580 (2008)

    Article  CAS  Google Scholar 

  21. Cui, L. et al. Mechanisms of in vitro resistance to dihydroartemisinin in Plasmodium falciparum . Mol. Microbiol. 86, 111–128 (2012)

    Article  CAS  Google Scholar 

  22. Leang, R. et al. Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010. Antimicrob. Agents Chemother. 57, 818–826 (2013)

    Article  CAS  Google Scholar 

  23. Sidhu, A. B. et al. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 298, 210–213 (2002)

    Article  CAS  ADS  Google Scholar 

  24. Valderramos, S. G. et al. Identification of a mutant PfCRT-mediated chloroquine tolerance phenotype in Plasmodium falciparum . PLoS Pathog. 6, e1000887 (2010)

    Article  Google Scholar 

  25. Bhisutthibhan, J. et al. The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin. J. Biol. Chem. 273, 16192–16198 (1998)

    Article  CAS  Google Scholar 

  26. Eichhorn, T. et al. Molecular interaction of artemisinin with translationally controlled tumor protein (TCTP) of Plasmodium falciparum . Biochem. Pharmacol. 85, 38–45 (2013)

    Article  CAS  Google Scholar 

  27. Sanchez, C. P. et al. Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum . Mol. Microbiol. 70, 786–798 (2008)

    CAS  Google Scholar 

  28. Veiga, M. I. et al. Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance. PLoS ONE 6, e20212 (2011)

    Article  CAS  ADS  Google Scholar 

  29. Raj, D. K. et al. Disruption of a Plasmodium falciparum multidrug resistance-associated protein (PfMRP) alters its fitness and transport of antimalarial drugs and glutathione. J. Biol. Chem. 284, 7687–7696 (2009)

    Article  CAS  Google Scholar 

  30. Anderson, T. J. et al. Are transporter genes other than the chloroquine resistance locus (pfcrt) and multidrug resistance gene (pfmdr) associated with antimalarial drug resistance? Antimicrob. Agents Chemother. 49, 2180–2188 (2005)

    Article  CAS  Google Scholar 

  31. Jambou, R. et al. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 366, 1960–1963 (2005)

    Article  CAS  Google Scholar 

  32. Krishna, S. et al. Artemisinins and the biological basis for the PfATP6/SERCA hypothesis. Trends Parasitol. 26, 517–523 (2010)

    Article  CAS  Google Scholar 

  33. Hunt, P. et al. Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites. Mol. Microbiol. 65, 27–40 (2007)

    Article  CAS  Google Scholar 

  34. Hunt, P. et al. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites. BMC Genomics 11, 499 (2010)

    Article  Google Scholar 

  35. Borges, S. et al. Genome-wide scan reveals amplification of mdr1 as a common denominator of resistance to mefloquine, lumefantrine, and artemisinin in Plasmodium chabaudi malaria parasites. Antimicrob. Agents Chemother. 55, 4858–4865 (2011)

    Article  CAS  Google Scholar 

  36. Chavchich, M. et al. Role of pfmdr1 amplification and expression in induction of resistance to artemisinin derivatives in Plasmodium falciparum . Antimicrob. Agents Chemother. 54, 2455–2464 (2010)

    Article  CAS  Google Scholar 

  37. Chen, N. et al. Deamplification of pfmdr1-containing amplicon on chromosome 5 in Plasmodium falciparum is associated with reduced resistance to artelinic acid in vitro . Antimicrob. Agents Chemother. 54, 3395–3401 (2010)

    Article  CAS  Google Scholar 

  38. Picot, S. et al. A systematic review and meta-analysis of evidence for correlation between molecular markers of parasite resistance and treatment outcome in falciparum malaria. Malar. J. 8, 89 (2009)

    Article  Google Scholar 

  39. Price, R. N. et al. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364, 438–447 (2004)

    Article  CAS  Google Scholar 

  40. Sidhu, A. B. et al. Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J. Infect. Dis. 194, 528–535 (2006)

    Article  Google Scholar 

  41. Yuan, J. et al. Chemical genomic profiling for antimalarial therapies, response signatures, and molecular targets. Science 333, 724–729 (2011)

    Article  CAS  ADS  Google Scholar 

  42. Amambua-Ngwa, A. et al. Population genomic scan for candidate signatures of balancing selection to guide antigen characterization in malaria parasites. PLoS Genet. 8, e1002992 (2012)

    Article  CAS  Google Scholar 

  43. Adams, J. et al. The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol. 10, 17–24 (2000)

    Article  CAS  Google Scholar 

  44. Prag, S. & Adams, J. C. Molecular phylogeny of the kelch-repeat superfamily reveals an expansion of BTB/kelch proteins in animals. BMC Bioinformatics 4, 42 (2003)

    Article  Google Scholar 

  45. Witkowski, B. et al. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia. Antimicrob. Agents Chemother. 57, 914–923 (2013)

    Article  CAS  Google Scholar 

  46. Padmanabhan, B. et al. Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. Mol. Cell 21, 689–700 (2006)

    Article  CAS  Google Scholar 

  47. Boyden, L. M. et al. Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities. Nature 482, 98–102 (2012)

    Article  CAS  ADS  Google Scholar 

  48. Li, X., Zhang, D., Hannink, M. & Beamer, L. J. Crystal structure of the Kelch domain of human Keap1. J. Biol. Chem. 279, 54750–54758 (2004)

    Article  CAS  Google Scholar 

  49. Itoh, K. et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13, 76–86 (1999)

    Article  CAS  Google Scholar 

  50. Zhang, D. D. & Hannink, M. Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Mol. Cell. Biol. 23, 8137–8152 (2003)

    Article  CAS  Google Scholar 

  51. Bozdech, Z. & Ginsburg, H. Antioxidant defense in Plasmodium falciparum--data mining of the transcriptome. Malar. J. 3, 23 (2004)

    Article  Google Scholar 

  52. Nesser, N. K., Peterson, D. O. & Hawley, D. K. RNA polymerase II subunit Rpb9 is important for transcriptional fidelity in vivo . Proc. Natl Acad. Sci. USA 103, 3268–3273 (2006)

    Article  CAS  ADS  Google Scholar 

  53. Kettenberger, H., Armache, K. J. & Cramer, P. Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage. Cell 114, 347–357 (2003)

    Article  CAS  Google Scholar 

  54. Dorin-Semblat, D., Sicard, A., Doerig, C., Ranford-Cartwright, L. & Doerig, C. Disruption of the PfPK7 gene impairs schizogony and sporogony in the human malaria parasite Plasmodium falciparum . Eukaryot. Cell 7, 279–285 (2008)

    Article  CAS  Google Scholar 

  55. Tewari, R. et al. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe 8, 377–387 (2010)

    Article  CAS  Google Scholar 

  56. Rosenthal, P. J., McKerrow, J. H., Aikawa, M., Nagasawa, H. & Leech, J. H. A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum . J. Clin. Invest. 82, 1560–1566 (1988)

    Article  CAS  Google Scholar 

  57. Sijwali, P. S. et al. Plasmodium falciparum cysteine protease falcipain-1 is not essential in erythrocytic stage malaria parasites. Proc. Natl Acad. Sci. USA 101, 8721–8726 (2004)

    Article  CAS  ADS  Google Scholar 

  58. Sijwali, P. S., Koo, J., Singh, N. & Rosenthal, P. J. Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum . Mol. Biochem. Parasitol. 150, 96–106 (2006)

    Article  CAS  Google Scholar 

  59. Klonis, N. et al. Altered temporal response of malaria parasites determines differential sensitivity to artemisinin. Proc. Natl Acad. Sci. USA 110, 5157–5162 (2013)

    Article  CAS  ADS  Google Scholar 

  60. Lobo, C. A., Fujioka, H., Aikawa, M. & Kumar, N. Disruption of the Pfg27 locus by homologous recombination leads to loss of the sexual phenotype in P. falciparum. Mol. Cell 3, 793–798 (1999)

    Article  CAS  Google Scholar 

  61. Olivieri, A. et al. The Plasmodium falciparum protein Pfg27 is dispensable for gametocyte and gamete production, but contributes to cell integrity during gametocytogenesis. Mol. Microbiol. 73, 180–193 (2009)

    Article  CAS  Google Scholar 

  62. Sharma, A., Sharma, I., Kogkasuriyachai, D. & Kumar, N. Structure of a gametocyte protein essential for sexual development in Plasmodium falciparum . Nature Struct. Biol. 10, 197–203 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the patients and field staff involved in clinical trials and sample collections. We are grateful to the provincial health department directors and other staff of the Cambodian Ministry of Health. Clinical trials and sample collections were supported in part by the Global Fund Grant Malaria Program Rounds 6 (CAM-607-G10M-CNM3) and 9 (CAM-S10-G14-M), the Bill and Melinda Gates Foundation and USAID (through the World Health Organization), the US DOD Global Epidemic Information System, and the Intramural Research Program, NIAID, NIH. Laboratory work was supported by grants from Banque Natixis (to O.M.-P. and D.M.) and Laboratoire d’Excellence IBEID (Agence Nationale de la Recherche, France) and Institut Pasteur, Division International (ACIP A-10-2010). B.W. was supported by a postdoctoral fellowship from Institut Pasteur, Division International; J.B. by an Institut Pasteur Paris Master-Pro fellowship; and D.M. by the French Ministry of Foreign Affairs. We are grateful to the Wellcome Trust Sanger Institute and the MalariaGEN resource centre for sequencing, genotyping and population structure analysis of some Cambodian clinical samples, funded by the Wellcome Trust (098051; 090770/Z/09/Z) and the MRC (G0600718). We thank the Rotary Club-Versailles for funding computer equipment. P.R., D.M.B. and W.O.R. are staff members of the World Health Organization and the US Navy, respectively. They alone are responsible for the views expressed in this publication, and they do not necessarily represent the decisions, policy or views of the World Health Organization or the US Navy.

Author information

Author notes

  1. Frédéric Ariey, Johann Beghain, Sandie Ménard, Antoine Berry & Jean-Christophe Barale

    Present address: Present addresses: Institut Pasteur, Genetics and Genomics of Insect Vectors Unit, 75724 Paris Cedex 15, France (F.A.); Institut Pasteur, Functional Genetics of Infectious Diseases Unit, 75724 Paris Cedex 15, France (J.B.); Centre de Physiopathologie de Toulouse-Purpan, Institut National de la Santé et de la Recherche Médicale UMR1043, Centre National de la Recherche Scientifique UMR5282, Université Toulouse III, 31024 Toulouse Cedex 3, France(S.M. & A.B.); Institut Pasteur, Unité de Biologie et Génétique du Paludisme, Team Malaria Targets and Drug Development, 75724 Paris Cedex 15, France (J.-C.B.).,

  2. Rick M. Fairhurst, Françoise Benoit-Vical, Odile Mercereau-Puijalon and Didier Ménard: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Pasteur, Parasite Molecular Immunology Unit, 75724 Paris Cedex 15, France ,

    Frédéric Ariey, Johann Beghain, Anne-Claire Langlois, Thierry Fandeur, Jean-Christophe Barale & Odile Mercereau-Puijalon

  2. Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, 75724 Paris Cedex 15, France ,

    Frédéric Ariey, Johann Beghain, Anne-Claire Langlois, Jean-Christophe Barale & Odile Mercereau-Puijalon

  3. Institut Pasteur du Cambodge, Malaria Molecular Epidemiology Unit, Phnom Penh, Cambodia ,

    Benoit Witkowski, Nimol Khim, Saorin Kim, Valentine Duru, Pharath Lim, Thierry Fandeur & Didier Ménard

  4. Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Chanaki Amaratunga, Pharath Lim & Rick M. Fairhurst

  5. Département Génomes et Génétique, Institut Pasteur, Plate-forme Génomique, 75724 Paris Cedex 15, France,

    Christiane Bouchier & Laurence Ma

  6. National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia ,

    Pharath Lim, Rithea Leang, Socheat Duong, Sokunthea Sreng, Seila Suon & Char Meng Chuor

  7. SSA WHO, Drug Monitoring in Cambodia, National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia ,

    Denis Mey Bout

  8. Service de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Toulouse, 31059 Toulouse Cedex 9, France ,

    Sandie Ménard & Antoine Berry

  9. Naval Medical Research Unit #2 Detachment, Phnom Penh, Cambodia ,

    William O. Rogers

  10. Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland ,

    Blaise Genton

  11. MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK ,

    Olivo Miotto

  12. Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok 10400, Thailand ,

    Olivo Miotto

  13. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK ,

    Olivo Miotto

  14. Global Malaria Program, World Health Organization, 1211 Geneva, Switzerland ,

    Pascal Ringwald

  15. Centre National de Référence du Paludisme, CHU Bichat-Claude Bernard, APHP, PRES Sorbonne Paris Cité, 75018 Paris, France ,

    Jacques Le Bras

  16. Centre National de la Recherche Scientifique, Laboratoire de Chimie de Coordination UPR8241, 31077 Toulouse Cedex 4, France ,

    Françoise Benoit-Vical

  17. Université de Toulouse, UPS, Institut National Polytechnique de Toulouse, 31077 Toulouse Cedex 4, France ,

    Françoise Benoit-Vical

Authors
  1. Frédéric Ariey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benoit Witkowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chanaki Amaratunga
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johann Beghain
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anne-Claire Langlois
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nimol Khim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Saorin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Valentine Duru
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Christiane Bouchier
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Laurence Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Pharath Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rithea Leang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Socheat Duong
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Sokunthea Sreng
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Seila Suon
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Char Meng Chuor
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Denis Mey Bout
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Sandie Ménard
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. William O. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Blaise Genton
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Thierry Fandeur
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Olivo Miotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Pascal Ringwald
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Jacques Le Bras
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Antoine Berry
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Jean-Christophe Barale
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Rick M. Fairhurst
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Françoise Benoit-Vical
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Odile Mercereau-Puijalon
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Didier Ménard
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.W., S.M., A.B. and F.B.-V. produced the F32-ART5 and F32-TEM clonal lines and analysed their survival rates. F.A. and J.B. developed computational components of the whole-genome sequence analysis. C.B. and L.M. performed whole-genome sequencing. F.A., C.A., S.K., V.D., P.L., R.L., S.D., Se.S., So.S., C.M.C., D.M.B., W.O.R., B.G., T.F., P.R., J.L.B., R.M.F. and D.M. conducted clinical studies and collected parasite isolates. A.-C.L., N.K., S.K., V.D., S.M. and A.B. performed PCR and sequencing analyses. B.W., F.B.-V., V.D. and D.M. performed in vitro assays (RSA0–3 h). O.M. provided genotyping and population structure data for Cambodian parasite isolates. J.-C.B. and O.M.-P. performed three-dimensional structure modelling. F.A., R.M.F., F.B.-V., O.M.-P. and D.M. conceived of the study, supervised the project, processed the data and wrote the manuscript with contributions from B.W., C.A., A.B. and J.-C.B.

Corresponding authors

Correspondence to Frédéric Ariey, Rick M. Fairhurst, Françoise Benoit-Vical, Odile Mercereau-Puijalon or Didier Ménard.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

The following reagents have been deposited to the MR4/BEI by D.M.: MRA-1236 (Plasmodium falciparum IPC 3445 Pailin Cambodia 2010), MRA-1237 (Plasmodium falciparum IPC 3663 Pailin Cambodia 2010), MRA-1238 (Plasmodium falciparum IPC 4884 Pursat Cambodia 2011), MRA-1239 (Plasmodium falciparum IPC 5188 Ratanakiri Cambodia 2011), MRA-1240 (Plasmodium falciparum IPC 5202 Battambang Cambodia 2011) and MRA-1241 (Plasmodium falciparum IPC 4912 Mondulkiri Cambodia 2011).

Extended data figures and tables

Extended Data Figure 1 SNP-calling algorithm and sequence and coverage of SNPs.

a, SNP-calling algorithm of the whole-genome sequence comparison of F32-ART5 and F32-TEM. b, Sequence and coverage of SNPs in seven candidate genes differing in F32-TEM and F32 ART5.

Extended Data Figure 2 Geographic distribution of K13-propeller alleles in Cambodia in 2011–2012.

Pie charts show K13-propeller allele frequencies among 300 parasite isolates in ten Cambodian provinces. Pie sizes are proportional to the number of isolates and the different alleles are colour-coded as indicated. The frequencies (95% confidence interval) of mutant K13-propeller alleles are: Pailin (95%, 88–99, n = 84), Battambang (93%, 87–99, n = 71), Pursat (89%, 67–99, n = 19), Kampot (83%, 52–98, n = 12), Kampong Som (71%, 29–96, n = 7), Oddar Meanchey (76%, 58–89, n = 33), Preah Vihear (16%, 3–40, n = 19), Kratie (71%, 44–90, n = 17), Mondulkiri (67%, 9–99, n = 3) and Ratanakiri (6%, 1–19, n = 35).

Extended Data Figure 3 Correlation between the frequency of wild-type K13-propeller alleles and the prevalence of day 3 positivity after ACT treatment in eight Cambodian provinces.

The frequency of day 3 positivity is plotted against the frequency of wild-type K13-propeller alleles. Data are derived from patients treated with an ACT for P. falciparum malaria in 2010–2012 in eight Cambodian provinces (Extended Data Figure 2): Pailin (n = 86, 2011 WHO therapeutic efficacy study, artesunate-mefloquine); Pursat (n = 32, 2012 WHO therapeutic efficacy study, dihydroartemisinin-piperaquine); Oddar Meanchey (n = 32, 2010 NAMRU-2 therapeutic efficacy study, artesunate-mefloquine); Kampong Som/Speu (n = 7, 2012 WHO therapeutic efficacy study, dihydroartemisinin-piperaquine); Battambang (n = 18, 2012 WHO therapeutic efficacy study, dihydroartemisinin-piperaquine); Kratie (n = 15, 2011 WHO therapeutic efficacy study, dihydroartemisinin-piperaquine); Preah Vihear (n = 19, 2011 WHO therapeutic efficacy study, dihydroartemisinin-piperaquine); Ratanakiri (n = 32, 2010 WHO therapeutic efficacy study, dihydroartemisinin-piperaquine). Spearman’s coefficient of rank correlation (8 sites): r = −0.99, 95% confidence interval −0.99 to −0.96, P < 0.0001.

Extended Data Figure 4 Schematic representation of homology between P. falciparum K13 and human KEAP1 proteins and structural 3D model of the K13-propeller domain.

a, Schematic representation of the predicted PF3D7_1343700 protein and homology to human KEAP1. Similar to KEAP1, PF3D7_1343700 contains a BTB/POZ domain and a C-terminal 6-blade propeller, which assembles kelch motifs consisting of four anti-parallel beta sheets. b, Structural 3D model of the K13-propeller domain showing the six kelch blades numbered 1 to 6 from N to C terminus and colour-coded as in Supplementary Fig. 1. The level of amino-acid identity between the K13-propeller 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). The accuracy of the K13-propeller 3D model was confirmed by Modeller-specific model/fold criteria of reliability (see Methods). We predict that the K13-propeller folds into a 6-bladed β-propeller structure48 closed by the interaction between a C-terminal beta-sheet and the N-terminal blade46,48. The first domain has three β-sheets, the fourth one being contributed by an extra C-terminal β-sheet called β'1 in Supplementary Fig. 1. The human KEAP1 kelch propeller scaffold is destabilized by a variety of mutations affecting intra- or inter-blade interactions in human lung cancer46 and hypertension47. The positions of the various mutations are indicated by a sphere, colour-coded as in Figs 2–4. The M476 residue mutated in F32-ART5 is indicated in dark grey. Like the mutations observed in human KEAP146,47, many K13-propeller mutations are predicted to alter the structure of the propeller or modify surface charges, and as a consequence alter the biological function of the protein. Importantly, the two major mutations C580Y (red) and R539T (blue) observed in Cambodia are both non-conservative and located in organized secondary structures: a β-sheet of blade 4 where it is predicted to alter the integrity of this scaffold and at the surface of blade 3, respectively. The kelch propeller domain of KEAP1 is involved in protein–protein interactions like most kelch containing modules43. KEAP1 is a negative regulator of the inducible Nrf2-dependent cytoprotective response, sequestering Nrf2 in the cytoplasm under steady state. Upon oxidative stress, the Nrf2/KEAP1 complex is disrupted, and Nrf2 translocates to the nucleus, where it induces transcription of cytoprotective ARE-dependent genes49,50. We speculate that similar functions may be performed by PF3D7_1343700 in P. falciparum, such that mutations of the K13-propeller impair its interactions with an unknown protein partner, resulting in a deregulated anti-oxidant/cytoprotective response. The P. falciparum anti-oxidant response is maximal during the late trophozoite stage, when haemoglobin digestion and metabolism are highest51. Its regulation is still poorly understood and no Nrf2 orthologue could be identified in the Plasmodium genome.

Extended Data Table 1 Sequence of the primers used to amplify the genes containing nonsynonymous single-nucleotide polymorphisms in F32-ART5
Full size table
Extended Data Table 2 Description of the eight nonsynonymous, single-nucleotide polymorphisms acquired in the F32-ART5 compared to the F32-TEM lineage during an effective 5-year discontinuous exposure to increasing concentrations of artemisinin
Full size table
Extended Data Table 3 Reported characteristics of the genes mutated in F32-ART5 parasites
Full size table
Extended Data Table 4 Geographic origin and year of collection of archived blood samples studied for K13-propeller polymorphism
Full size table
Extended Data Table 5 Polymorphisms observed in the K13-propeller in Cambodian P. falciparum isolates collected in 2001–2012 and in The Gambia (ref. 42)
Full size table
Extended Data Table 6 Association between polymorphisms observed in the K13-propeller and KH subpopulations (ref. 15) in 150 P. falciparum isolates collected in 2009–2010 in Pursat (n = 103) and Ratanakiri (n = 47) provinces, Cambodia
Full size table

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-2. (PDF 530 kb)

Supplementary Data

This file contains the details for the wildtype 3D7 sequence. (XLSX 13 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ariey, F., Witkowski, B., Amaratunga, C. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 (2014). https://doi.org/10.1038/nature12876

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12876

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing