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Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

ABSTRACT: The widely used antimalarial combination therapy dihydroartemisinin?+?piperaquine (DHA?+?PPQ) has failed in Cambodia. Here, we perform a genomic analysis that reveals a rapid increase in the prevalence of novel mutations in the Plasmodium falciparum chloroquine resistance transporter PfCRT following DHA?+?PPQ implementation. These mutations occur in parasites harboring the K13 C580Y artemisinin resistance marker. By introducing PfCRT mutations into sensitive Dd2 parasites or removing them from resistant Cambodian isolates, we show that the H97Y, F145I, M343L, or G353V mutations each confer resistance to PPQ, albeit with fitness costs for all but M343L. These mutations sensitize Dd2 parasites to chloroquine, amodiaquine, and quinine. In Dd2 parasites, multicopy plasmepsin 2, a candidate molecular marker, is not necessary for PPQ resistance. Distended digestive vacuoles were observed in pfcrt-edited Dd2 parasites but not in Cambodian isolates. Our findings provide compelling evidence that emerging mutations in PfCRT can serve as a molecular marker and mediator of PPQ resistance.

SUBMITTER: Ross LS

PROVIDER: S-EPMC6095916 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

Ross Leila S LS Dhingra Satish K SK Mok Sachel S Yeo Tomas T Wicht Kathryn J KJ Kümpornsin Krittikorn K Takala-Harrison Shannon S Witkowski Benoit B Fairhurst Rick M RM Ariey Frederic F Menard Didier D Fidock David A DA

Nature communications 20180817 1

The widely used antimalarial combination therapy dihydroartemisinin + piperaquine (DHA + PPQ) has failed in Cambodia. Here, we perform a genomic analysis that reveals a rapid increase in the prevalence of novel mutations in the Plasmodium falciparum chloroquine resistance transporter PfCRT following DHA + PPQ implementation. These mutations occur in parasites harboring the K13 C580Y artemisinin resistance marker. By introducing PfCRT mutations into sensitive Dd2 parasites or removing them from r ...[more]

PMID: 30115924

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Project description:BackgroundDue to the widespread resistance of Plasmodium falciparum to chloroquine drug, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment. This study aims to evaluate the extent of chloroquine resistance in P. falciparum infection after the introduction of ACT. This study was carried out based on the mutation analysis in P. falciparum chloroquine resistant transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes. Identification of these molecular markers plays a significant role in monitoring and assessment of drug resistance as well as in designing an effective antimalarial drug policy in India.MethodsSixty blood samples were collected from patients infected with P. falciparum from JIPMER, Puducherry and MKCG Medical College, Odisha. Polymerase chain reaction-restriction fragment length polymorphism was performed, targeting the point mutation of K76T in pfcrt and N86Y in pfmdr1 gene. The PCR products were sequenced, genotyped and further analysed for amino acid changes in these codons.ResultsThe frequency of pfcrt mutation at 76th position was dominant for mutant T allele with 56.7% and wild type K, 43.3%. Majority of pfmdr1 86 allele were wild type, with N (90%) and mutant, Y (10%). Additionally, we found three haplotypes for CQ resistance, SVMNT, CVIET and CVIKT in association with the pfcrt gene. However, a poorly studied SNP in pfmdr1 gene (Y184F) associated with CQ resistance showed high frequency (70%) in P. falciparum isolates.ConclusionsThe point mutation K76T of pfcrt is high in P. falciparum suggesting a sustained high CQ resistance even after five years of the introduction of ACTs for antimalarial therapy. The present study suggests a strong association of CQ resistance with pfcrt T76, but not with the pfmdr1 Y86 mutation. However, sequence analysis showed that Y184F mutation on pfmdr1 gene was found to be associated with high resistance. Also, a new pfcrt haplotype 'CVIKT' associated with CQ resistance was found to be present in Indian strains of P. falciparum. The data obtained from this study helps in continuous monitoring of drug resistance in malaria and also suggests the need for careful usage of CQ in Plasmodium vivax malarial treatment.

Project description:The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.

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Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

Publications

Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

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