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Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum.

ABSTRACT: The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome. We identified two distinct Fluo-4 staining phenotypes: preferential staining of the food vacuole versus a more diffuse staining of the entire parasite. Genetic, positional cloning and pharmacological data causatively link the food vacuolar Fluo-4 phenotype to those Pgh-1 variants that are associated with altered drug responses. On the basis of our data, we propose that Pgh-1 imports solutes, including certain antimalarial drugs, into the parasite's food vacuole. The implications of our findings for drug resistance mechanisms and testing are discussed.

SUBMITTER: Rohrbach P

PROVIDER: S-EPMC1500988 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum.

Rohrbach Petra P Sanchez Cecilia P CP Hayton Karen K Friedrich Oliver O Patel Jigar J Sidhu Amar Bir Singh AB Ferdig Michael T MT Fidock David A DA Lanzer Michael M

The EMBO journal 20060622 13

The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome. We identified two distinct Fluo-4 staining phenotypes: preferential sta ...[more]

PMID: 16794577

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Project description:BackgroundThere are concerns that resistance to artemisinin-based combination therapy might emerge in Kenya and sub-Saharan Africa (SSA) in the same pattern as was with chloroquine and sulfadoxine-pyrimethamine. Single nucleotide polymorphisms (SNPs) in critical alleles of pfmdr1 gene have been associated with resistance to artemisinin and its partner drugs. Microsatellite analysis of loci flanking genes associated with anti-malarial drug resistance has been used in defining the geographic origins, dissemination of resistant parasites and identifying regions in the genome that have been under selection.MethodsThis study set out to investigate evidence of selective sweep and genetic lineages in pfmdr1 genotypes associated with the use of artemether-lumefantrine (AL), as the first-line treatment in Kenya. Parasites (n = 252) from different regions in Kenya were assayed for SNPs at codons 86, 184 and 1246 and typed for 7 neutral microsatellites and 13 microsatellites loci flanking (± 99 kb) pfmdr1 in Plasmodium falciparum infections.ResultsThe data showed differential site and region specific prevalence of SNPs associated with drug resistance in the pfmdr1 gene. The prevalence of pfmdr1 N86, 184F, and D1246 in western Kenya (Kisumu, Kericho and Kisii) compared to the coast of Kenya (Malindi) was 92.9% vs. 66.7%, 53.5% vs. to 24.2% and 96% vs. to 87.9%, respectively. The NFD haplotype which is consistent with AL selection was at 51% in western Kenya compared to 25% in coastal Kenya.ConclusionSelection pressures were observed to be different in different regions of Kenya, especially the western region compared to the coastal region. The data showed independent genetic lineages for all the pfmdr1 alleles. The evidence of soft sweeps in pfmdr1 observed varied in direction from one region to another. This is challenging for malaria control programs in SSA which clearly indicate effective malaria control policies should be based on the region and not at a country wide level.

Project description:BackgroundLumefantrine and mefloquine are used worldwide in artemisinin-based combination therapy (ACT) of malaria. Better understanding of drug susceptibility and resistance is needed and can be obtained from studies of genetic crosses.MethodsDrug response phenotypes of a cross between Plasmodium falciparum lines 803 (Cambodia) and GB4 (Ghana) were obtained as half-maximal effective concentrations (EC50s) and days to recovery (DTR) after 24 h exposure to 500 nM lumefantrine. EC50s of mefloquine, halofantrine, chloroquine, and dihydroartemisinin were also determined. Quantitative trait loci (QTL) analysis and statistical tests with candidate genes were used to identify polymorphisms associated with response phenotypes.ResultsLumefantrine EC50s averaged 5.8-fold higher for the 803 than GB4 parent, and DTR results were 3-5 and 16-18 days, respectively. In 803 × GB4 progeny, outcomes of these two lumefantrine assays showed strong inverse correlation; these phenotypes also correlated strongly with mefloquine and halofantrine EC50s. By QTL analysis, lumefantrine and mefloquine phenotypes mapped to a chromosome 5 region containing codon polymorphisms N86Y and Y184F in the P. falciparum multidrug resistance 1 protein (PfMDR1). Statistical tests of candidate genes identified correlations between inheritance of PfK13 Kelch protein polymorphism C580Y (and possibly K189T) and lumefantrine and mefloquine susceptibilities. Correlations were detected between lumefantrine and chloroquine EC50s and polymorphisms N326S and I356T in the CVIET-type P. falciparum chloroquine resistance transporter (PfCRT) common to 803 and GB4.ConclusionsCorrelations in this study suggest common mechanisms of action in lumefantrine, mefloquine, and halofantrine responses. PfK13 as well as PfMDR1 and PfCRT polymorphisms may affect access and/or action of these arylaminoalcohol drugs at locations of hemoglobin digestion and heme metabolism. In endemic regions, pressure from use of lumefantrine or mefloquine in ACTs may drive selection of PfK13 polymorphisms along with versions of PfMDR1 and PfCRT associated with lower susceptibility to these drugs.

Project description:BackgroundAnti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance.MethodsHere, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast.ResultsExposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene.DiscussionThis data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria.ConclusionArtemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.

Dataset Information

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum.

Publications

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum.

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