DOI: 10.6084/m9.figshare.26946693
title: Expression of <i>Plasmodium</i> major facilitator superfamily protein in transporters – Δ <i>Candida</i> identifies a drug transporter

description: <b>Aim:</b> To assess the functional relevance of a putative Major Facilitator Superfamily protein (PF3D7_0210300; ‘<i>Pf</i>MFSDT’) as a drug transporter, using <i>Candida glabrata</i> for orthologous protein expression. <b>Methods:</b> Complementary Determining Sequence encoding <i>Pf</i>MFSDT was integrated into the genome of genetically engineered <i>C. glabrata</i> strain MSY8 via homologous recombination, followed by assessing its functional relevance as a drug transporter. <b>Results & conclusion:</b> The modified <i>C. glabrata</i> strain exhibited plasma membrane localization of <i>Pf</i>MFSDT and characteristics of an Major Facilitator Superfamily transporter, conferring resistance to antifungals, ketoconazole and itraconazole. The nanomolar inhibitory effects of the drugs on the intra-erythrocytic growth of <i>Plasmodium falciparum</i> highlight their antimalarial properties. This study proposes <i>Pf</i>MFSDT as a drug transporter, expanding the repertoire of the currently known antimalarial ‘resistome’. Lethal malaria causing <i>Plasmodium falciparum</i> being an obligate intracellular parasite, relies on various transport mechanisms for its survival. The transporters facilitating the essential nutrient transport for parasite survival represent a repertoire of drug targets. Here, we characterized a member of Major Facilitator Superfamily protein, <i>Pf</i>MFSDT and highlighted its role as a drug transporter in <i>P. falciparum</i>. The complete characterization of <i>Pf</i>MFSDT as a drug transporter has been done using genetically engineered <i>Candida glabrata</i> lacking clinically relevant ABC drug transporter genes. A combination of bioinformatics, cellular-molecular biology and pharmacological approaches has been used to study the potential function of <i>Pf</i>MFSDT. <i>C. glabrata</i> strain MSY8 has been genetically deleted for seven clinically relevant membrane-associated ABC drug transporter genes, along with a gain of function mutation in the <i>PDR1</i> transcription factor resulting in a hyperactivation of <i>CDR1</i> locus has been used as an orthologous expression system for <i>Pf</i>MFSDT characterization. A homologous recombination-based strategy was employed to integrate <i>Pf</i>MFSDT at the CDR1 locus of MSY8 for its orthologous expression. <i>Pf</i>MFSDT-expressing MSY8 strain (MSY8-<i>Pf</i>MFSDT1) was used to assess the efflux of antifungals, itraconazole and ketoconazole, through growth sensitivity assay. The antimalarial properties of ketoconazole and itraconazole was assessed in <i>P. falciparum</i> strain 3D7. <i>C. glabrata</i> strain MSY8 transformed with <i>Pf</i>MFSDT (MSY8-<i>Pf</i>MFSDT1) conferred resistance to antifungal drugs. <i>Pf</i>MFSDT transports antifungal drugs, ketoconazole and itraconazole, as evidenced by the rescued growth of <i>C. glabrata</i> in the presence of both antifungals. Both ketoconazole and itraconazole interact with <i>Pf</i>MFSDT as evaluated by favorable binding energies, implying probable binding interactions with <i>Pf</i>MFSDT in <i>in silico</i> interaction analysis. MD simulation studies revealed that the binding site residues get stabilized and maintain the overall stability of the <i>Pf</i>MFSDT in the presence of ketoconazole and itraconazole. Ketoconazole and itraconazole blocks the growth of the malaria parasite <i>P. falciparum</i> 3D7 strain. <i>Pf</i>MFSDT has been characterized as a drug transporter that is targeted by ketoconazole and itraconazole. <i>Pf</i>MFSDT can act as a target for antimalarial drug development. The strain MSY8-<i>Pf</i>MFSDT1 developed in this study can be further used to identify novel <i>Pf</i>MFSDT-targeting antimalarials.