Dataset Information

PfMFR3: A Multidrug-Resistant Modulator in Plasmodium falciparum.

ABSTRACT: In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 (pfmfr3) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.

SUBMITTER: Rocamora F

PROVIDER: S-EPMC8042660 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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PfMFR3: A Multidrug-Resistant Modulator in <i>Plasmodium falciparum</i>.

Rocamora Frances F Gupta Purva P Istvan Eva S ES Luth Madeline R MR Carpenter Emma F EF Kümpornsin Krittikorn K Sasaki Erika E Calla Jaeson J Mittal Nimisha N Carolino Krypton K Owen Edward E Llinás Manuel M Ottilie Sabine S Goldberg Daniel E DE Lee Marcus C S MCS Winzeler Elizabeth A EA

ACS infectious diseases 20210314 4

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage <i>P. falciparum</i> parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance eme ...[more]

PMID: 33715347

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Project description:ObjectivesIn the Gambia, chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) is the first-line antimalarial treatment. Plasmodium falciparum parasites carrying mutations associated with resistance to each of these drugs were present in 2001 but did not cause a significant loss of therapeutic efficacy among children receiving the combination CQ/SP. We measured their effect on parasite transmission to Anopheles gambiae mosquitoes.DesignWe conducted a single-blind, randomised, controlled trial with follow-up over 28 d. Mosquito feeding experiments were carried out 7, 10, or 14 d after treatment.SettingThe study took place in the town of Farafenni and surrounding villages in the Gambia.ParticipantsParticipants were 500 children aged 6 mo to 10 y with uncomplicated P. falciparum malaria.InterventionsChildren were randomised to receive CQ, SP, or CQ/SP.Outcome measuresOutcomes related to transmission were determined, including posttreatment gametocyte prevalence and density. Infectiousness was assessed by membrane-feeding A. gambiae mosquitoes with blood from 70 gametocyte-positive patients. Mutations at seven loci in four genes associated with drug resistance were measured pre- and posttreatment and in the midguts of infected mosquitoes.ResultsAfter SP treatment, the infectiousness of gametocytes was delayed, compared to the other two treatment groups, despite comparable gametocyte densities. Among bloodmeal gametocytes and the midguts of infected mosquitoes, the presence of the four-locus multidrug-resistant haplotype TYRG (consisting of mutations pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G) was associated with significantly higher oocyst burdens after treatment with the combination CQ/SP.ConclusionsParasites with a multidrug-resistant genotype had a substantial transmission advantage after CQ/SP treatment but did not have a significant impact on in vivo efficacy of this drug combination. Protocols that include measuring transmission endpoints as well as therapeutic outcomes may be a useful strategy when monitoring the evolution of drug resistance in malaria parasites in vivo.

Project description:The emergence and spread of the Plasmodium falciparum multidrug-resistant 1 (Pfmdr1) allele pose a significant setback to global efforts to control and eradicate malaria infection by diminishing the efficacy of commonly prescribed antimalarial drugs, particularly in Sub-Saharan Africa, where malaria remains endemic. The Pfmdr1 D1246Y mutation is of specific importance due to its potential role in modulating parasite susceptibility to antimalarial medicines and treatment outcomes. This study aimed to determine the presence and prevalence of the wild-type and mutant D1246Y alleles of Pfmdr1 among children in Ibadan, Southwest Nigeria. A total of 133 archived DNA samples collected between March 2016 and June 2021 from children aged 6 to 132 months with varying malaria phenotypes (asymptomatic infection, uncomplicated, and severe malaria) were analyzed. The Pfmdr1 D1246Y allele was amplified via nested PCR, and the mutation was detected using the restriction enzyme EcoRV. The digested nested PCR products were resolved on a 2% agarose gel and visualized under ultraviolet light. All statistical analyses were performed using SPSS version 25, and statistical significance was set at p ≤ 0.05. Among the 133 samples, 97 (72.9%) were successfully genotyped. Of these, 50 (51.55%) carried the wild-type allele, while 47 (48.45%) had the mutant allele. Notably, the Pfmdr1-1246Y mutation was detected in all severe malaria cases (41/41, 100%), whereas its prevalence was significantly lower in asymptomatic (3/36, 8.3%) and uncomplicated malaria cases (3/20, 15%). The difference in mutation prevalence across the malaria phenotypes was statistically significant (p < 0.05). The study provided valuable insight into the coexistence of wild-type and mutant Pfmdr1 D1246Y alleles within the population. It revealed a significantly higher mutation rate in all severe malaria cases, while the wild-type allele remained more prevalent overall. These findings contribute to a deeper understanding of the possible role of the wild-type and mutant D1246Y alleles in the various clinical manifestations of malaria.

Project description:The analysis of in vitro anti-malarial drug susceptibility testing is vulnerable to the effects of different statistical approaches and selection biases. These confounding factors were assessed with respect to pfmdr1 gene mutation and amplification in 490 clinical isolates. Two statistical approaches for estimating the drug concentration associated with 50% effect (EC50 ) were compared: the commonly used standard two-stage (STS) method, and nonlinear mixed-effects modelling. The in vitro concentration-effect relationships for, chloroquine, mefloquine, lumefantrine and artesunate, were derived from clinical isolates obtained from patients on the western border of Thailand. All isolates were genotyped for polymorphisms in the pfmdr1 gene. The EC50 estimates were similar for the two statistical approaches but 15-28% of isolates in the STS method had a high coefficient of variation (>15%) for individual estimates of EC50 and these isolates had EC50 values that were 32 to 66% higher than isolates derived with more precision. In total 41% (202/490) of isolates had amplification of pfmdr1 and single nucleotide polymorphisms were found in 50 (10%). Pfmdr1 amplification was associated with an increase in EC50 for mefloquine (139% relative increase in EC50 for 2 copies, 188% for 3+ copies), lumefantrine (82% and 75% for 2 and 3+ copies respectively) and artesunate (63% and 127% for 2 and 3+ copies respectively). In contrast pfmdr1 mutation at codons 86 or 1042 were associated with an increase in chloroquine EC50 (44-48%). Sample size calculations showed that to demonstrate an EC50 shift of 50% or more with 80% power if the prevalence was 10% would require 430 isolates and 245 isolates if the prevalence was 20%. In conclusion, although nonlinear mixed-effects modelling did not demonstrate any major advantage for determining estimates of anti-malarial drug susceptibility, the method includes all isolates, thereby, potentially improving confirmation of candidate molecular markers of anti-malarial drug susceptibility.

Dataset Information

PfMFR3: A Multidrug-Resistant Modulator in Plasmodium falciparum.

Publications

PfMFR3: A Multidrug-Resistant Modulator in <i>Plasmodium falciparum</i>.

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