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.

Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

“…In contrast to published data 36 , we also found endogenously GFP-tagged Pf ApiAT10 localizing at the PPM. To re-probe into the apparent PPM localization of endogenously GFP-tagged ApiAT10 we also over-expressed this gene as a GFP and mCherry fusion protein using two different promotors ( crt 40 or ama1 41 ).…”

“…Recent work using episomally overexpressed PfApiAT10-GFP implied an association of this transporter with the mitochondrial membrane 36 in PfDd2 parasites. This localization differs from the observed PPM localization of endogenously GFP-tagged PfApiAT10 in both, 3D7 as well as 3D7-iGP parasites (Figure 1B, 4C, S2A) reported in this study.…”

“…The observed PPM localization of the investigated ApiATs is in concordance with published data, since i) the P. berghei ApiAT8 homologue was shown to be a general cationic amino acid transporter of the PPM 29,30 , ii) Pf ApiAT8 has recently been localized to the PPM using an overexpression approach 31 , and iii) in T. gondii several ApiATs ( Tg ApiAT1, Tg ApiAT2, Tg ApiAT3-1, Tg ApiAT3-2, Tg ApiAT3-3, Tg ApiAT5-3, Tg ApiAT6-1 and Tg ApiAT6-3) have been located at the PPM as well 25,29,30,32,33,44 . Recent work using episomally overexpressed Pf ApiAT10-GFP implied an association of this transporter with the mitochondrial membrane 36 in Pf Dd2 parasites. This localization differs from the observed PPM localization of endogenously GFP-tagged Pf ApiAT10 in both, 3D7 as well as 3D7-iGP parasites (Figure 1B, 4C, S2A) reported in this study.…”

“…Additionally, sporozoite development likely requires PbApiAT2, PbApiAT4, PbApiAT9 and PbApiAT10 35 and a lack of PbApiAT2 further impairs oocyst development. Furthermore, an association of episomally overexpressed PfApiAT10-GFP (termed MFR3-GFP) with the mitochondrion has been reported 36 .…”

Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance

“…Therefore, to find compounds with activity against yeast at physiologically relevant concentrations, we used a modified S. cerevisiae strain, termed the “ABC 16 -Green Monster” (GM), in which 16 ABC transporters have been replaced with a green fluorescent protein (GFP) 25 . This has proven an excellent platform for in vitro drug selection and target identification experiments because of lower compound requirements 26 – 30 .…”