Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum.
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ABSTRACT:
SUBMITTER: Rohrbach P
PROVIDER: S-EPMC1500988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.
| S-EPMC3571303 | biostudies-literature
Project description:Mefloquine is widely used in combination with artemisinin derivatives for the treatment of falciparum malaria. Mefloquine resistance in Plasmodium falciparum has been related to increased copy numbers of multidrug-resistant gene 1 (pfmdr1). We studied the ex vivo dynamics of pfmdr1 gene amplification in culture-adapted P. falciparum in relation to mefloquine resistance and parasite fitness. A Thai P. falciparum isolate (isolate TM036) was assessed by the use of multiple genetic markers as a single genotype. Resistance was selected by exposure to stepwise increasing concentrations of mefloquine up to 30 ng/ml in continuous culture. The pfmdr1 gene copy numbers increased as susceptibility to mefloquine declined (P = 0.03). No codon mutations at positions 86, 184, 1034, 1042, and 1246 in the pfmdr1 gene were detected. Two subclones of selected parasites (average copy numbers, 2.3 and 3.1, respectively) showed a fitness disadvantage when they were grown together with the original parasites containing a single pfmdr1 gene copy in the absence of mefloquine; the multiplication rates were 6.3% and 8.7% lower, respectively (P < 0.01). Modeling of the dynamics of the pfmdr1 copy numbers over time in relation to the relative fitness of the parasites suggested that net pfmdr1 gene amplification from one to two copies occurs once in every 10(8) parasites and that amplification from two to three copies occurs once in every 10(3) parasites. pfmdr1 gene amplification in P. falciparum is a frequent event and confers mefloquine resistance. Parasites with multiple copies of the pfmdr1 gene have decreased survival fitness in the absence of drug pressure.
| S-EPMC2663078 | biostudies-literature
Project description:BACKGROUND:There 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. METHODS:This 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. RESULTS:The 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. CONCLUSION:Selection 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.
| S-EPMC6208105 | biostudies-literature
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.
| S-EPMC7677662 | biostudies-literature
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.
| S-EPMC4957835 | biostudies-literature
Project description:Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes.
| S-EPMC4873939 | biostudies-literature
Project description:The emergence of artesunate-mefloquine (AS+MQ)-resistant Plasmodium falciparum in the Thailand-Cambodia region is a major concern for malaria control. Studies indicate that copy number increase and key alleles in the pfmdr1 gene are associated with AS+MQ resistance. In the present study, we investigated evidence for a selective sweep around pfmdr1 because of the spread of adaptive mutation and/or multiple copies of this gene in the P. falciparum population in Cambodia.We characterized 13 microsatellite loci flanking (+/-99 kb) pfmdr1 in 93 single-clone P. falciparum infections, of which 31 had multiple copies and 62 had a single copy of the pfmdr1 gene.Genetic analysis revealed no difference in the mean (+/- standard deviation) expected heterozygosity (H(e)) at loci around single (0.75+/-0.03) and multiple (0.76+/-0.04) copies of pfmdr1. Evidence of genetic hitchhiking with the selective sweep of certain haplotypes was seen around mutant (184F) pfmdr1 allele, irrespective of the copy number. There was an overall reduction of 28% in mean H(e) (+/-SD) around mutant allele (0.56+/-0.05), compared with wild-type allele (0.84+/-0.02). Significant linkage disequilibrium was also observed between the loci flanking mutant pfmdr1 allele.The 184F mutant allele is under selection, whereas amplification of pfmdr1 gene in this population occurs on multiple genetic backgrounds.
| S-EPMC3096139 | biostudies-literature
Project description:Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: S(tct)VMNT (91%) and S(agt)VMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites.
| S-EPMC2849383 | biostudies-literature
Project description:The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.
| S-EPMC361573 | biostudies-other
Project description:The 4-aminoquinoline bisquinoline piperaquine is an important partner drug in one of the presently recommended artemisinin combination therapies. Recent clinical trials have confirmed its high efficacy in combination with dihydroartemisinin. Resistance to piperaquine alone has, however, been documented. Amplification in copy number of the Plasmodium falciparum multidrug resistance locus on chromosome 5, containing the pfmdr1 gene, has been shown to confer resistance to structurally unrelated antimalarials. Through the determination of the 50% inhibitory concentrations (IC(50)s) and IC(90)s for piperaquine and chloroquine in a set of 46 adapted P. falciparum cultures originating from the Thai-Burmese border, we have characterized the regions around the pfmdr1 gene and identified a significant association between the presence of pfmdr1 duplications and enhanced sensitivity to piperaquine (P = 0.005 for IC(50) and P = 0.002 for IC(90)) and chloroquine, reaching statistical significance at IC(90)s (P = 0.026). These results substantiate the potential importance of pfmdr1 copy number amplifications in the efficacy of the combination therapy piperaquine-dihydroartemisinin. It supports the rational use of 4-aminoquinolines and artemisinin-based compounds, as they independently select for mutually incompatible combinations of mutations.
| S-EPMC3393437 | biostudies-literature