PfMFR3: A Multidrug-Resistant Modulator in Plasmodium falciparum.

Frances Rocamora,Emma F Carpenter,Nimisha Mittal,Daniel E Goldberg,Jaeson Calla,Marcus C S Lee,Purva Gupta,Sabine Ottilie,Elizabeth A Winzeler,Madeline R Luth,Edward Owen,Krittikorn Kümpornsin,Manuel Llinás,Krypton Carolino,Eva S Istvan,Erika Sasaki

doi:10.1021/acsinfecdis.0c00676

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.

Highlights

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes
On the basis of the shared structural features across atovaquone, MMV085203, and GNF-Pf-3600 as well as the alteration in sensitivity for all three compounds resulting from the disruption of pf mf r3, we explored the possibility of MMV085203 and GNF-Pf-3600 having the same mechanism of action as atovaquone, a clinically relevant antiparasitic drug that targets the cytochrome bc[1] complex in Plasmodium parasites through competitive inhibition of ubiquinol.[23,24]
We found that overexpressing PfMFR3 rendered the parasite significantly more sensitive to atovaquone (p = 0.0003), demonstrating the opposite phenotype as the line expressing the truncated form of the protein

Summary

Introduction

Chemical genetics is a powerful method for assigning function to uncharacterized genes. As key components of the malaria resistome, transport proteins are often involved in drug response phenotypes[2−4] either as the targets of the drug themselves[5,6] or by helping the parasite evade drug action.[7] Of the ∼120 members of the P. falciparum transportome, many remain unexplored, pending experimental characterization of their specific function and subcellular localization.[4,8] To address this, the forward chemical genetic approach of inducing drug resistance in vitro has revealed a plethora of novel phenotypes associated with drug resistance, which can in turn provide insight into the physiological role of these transport proteins.[2,3,9] In this technique, drug resistance is first selected through prolonged exposure to sublethal concentrations of a compound.

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Results

Conclusion