Nature. 2014 Jan 2;505(7481):50-5. doi: 10.1038/nature12876. Epub 2013 Dec 18.

A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.

Ariey F¹, Witkowski B², Amaratunga C³, Beghain J¹, Langlois AC⁴, Khim N², Kim S², Duru V², Bouchier C⁵, Ma L⁵, Lim P⁶, Leang R⁷, Duong S⁷, Sreng S⁷, Suon S⁷, Chuor CM⁷, Bout DM⁸, Ménard S⁹, Rogers WO¹⁰, Genton B¹¹, Fandeur T¹², Miotto O¹³, Ringwald P¹⁴, Le Bras J¹⁵, Berry A⁹, Barale JC¹, Fairhurst RM¹⁶, Benoit-Vical F¹⁷, Mercereau-Puijalon O¹⁸, Ménard D¹⁹.

Author information

1: 1] Institut Pasteur, Parasite Molecular Immunology Unit, 75724 Paris Cedex 15, France [2] Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, 75724 Paris Cedex 15, France [3] 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 Institut Pasteur, Unité de Biologie et Génétique du Paludisme, Team Malaria Targets and Drug Development, 75724 Paris Cedex 15, France (J.-C.B.).
2: Institut Pasteur du Cambodge, Malaria Molecular Epidemiology Unit, Phnom Penh, Cambodia.
3: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
4: 1] Institut Pasteur, Parasite Molecular Immunology Unit, 75724 Paris Cedex 15, France [2] Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, 75724 Paris Cedex 15, France.
5: Institut Pasteur, Plate-forme Génomique, Département Génomes et Génétique, 75724 Paris Cedex 15, France.
6: 1] Institut Pasteur du Cambodge, Malaria Molecular Epidemiology Unit, Phnom Penh, Cambodia [2] Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA [3] National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia.
7: National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia.
8: SSA WHO, Drug Monitoring in Cambodia, National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia.
9: 1] Service de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Toulouse, 31059 Toulouse Cedex 9, France [2] 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 Institut Pasteur, Unité de Biologie et Génétique du Paludisme, Team Malaria Targets and Drug Development, 75724 Paris Cedex 15, France (J.-C.B.).
10: Naval Medical Research Unit #2 Detachment, Phnom Penh, Cambodia.
11: Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland.
12: 1] Institut Pasteur, Parasite Molecular Immunology Unit, 75724 Paris Cedex 15, France [2] Institut Pasteur du Cambodge, Malaria Molecular Epidemiology Unit, Phnom Penh, Cambodia.
13: 1] MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK [2] Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok 10400, Thailand [3] Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
14: Global Malaria Program, World Health Organization, 1211 Geneva, Switzerland.
15: Centre National de Référence du Paludisme, CHU Bichat-Claude Bernard, APHP, PRES Sorbonne Paris Cité, 75018 Paris, France.
16: 1] Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA [2].
17: 1] Centre National de la Recherche Scientifique, Laboratoire de Chimie de Coordination UPR8241, 31077 Toulouse Cedex 4, France [2] Université de Toulouse, UPS, Institut National Polytechnique de Toulouse, 31077 Toulouse Cedex 4, France [3].
18: 1] Institut Pasteur, Parasite Molecular Immunology Unit, 75724 Paris Cedex 15, France [2] Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, 75724 Paris Cedex 15, France [3].
19: 1] Institut Pasteur du Cambodge, Malaria Molecular Epidemiology Unit, Phnom Penh, Cambodia [2].

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.

Comment in

Malaria: Resistance nailed. [Nature. 2014]

PMID:: 24352242
PMCID:: PMC5007947
DOI:: 10.1038/nature12876

[Indexed for MEDLINE]

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Extended Data Figure 4

Schematic representation of homology between P. falciparumK13 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 . The level of amino-acid identity between the K13-propeller and kelch domains of proteins with solved 3D structures, including human KEAP1^,, enabled us to model the 3D structure of the K13-propeller and to map the mutations selected under ART pressure (). 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 structure closed by the interaction between a C-terminal beta-sheet and the N-terminal blade^,. The first domain has three β-sheets, the fourth one being contributed by an extra C-terminal β-sheet called β’1 in . The human KEAP1 kelch propeller scaffold is destabilized by a variety of mutations affecting intra- or inter-blade interactions in human lung cancer and hypertension. The positions of the various mutations are indicated by a sphere, colour-coded as in –. The M476 residue mutated in F32-ART5 is indicated in dark grey. Like the mutations observed in human KEAP1^,, 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 modules. 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 genes^,. 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 highest. Its regulation is still poorly understood and no Nrf2 orthologue could be identified in the Plasmodium genome.

A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

Nature. 2014 Jan 2;505(7481):50-55.