Expression of <i>Plasmodium</i> major facilitator superfamily protein in transporters – Δ <i>Candida</i> identifies a drug transporter

Aim: To assess the functional relevance of a putative Major Facilitator Superfamily protein (PF3D7_0210300; ‘PfMFSDT’) as a drug transporter, using Candida glabrata for orthologous protein expression. Methods: Complementary Determining Sequence encoding PfMFSDT was integrated into the genome of genetically engineered C. glabrata strain MSY8 via homologous recombination, followed by assessing its functional relevance as a drug transporter. Results & conclusion: The modified C. glabrata strain exhibited plasma membrane localization of PfMFSDT 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 Plasmodium falciparum highlight their antimalarial properties. This study proposes PfMFSDT as a drug transporter, expanding the repertoire of the currently known antimalarial ‘resistome’. Lethal malaria causing Plasmodium falciparum 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, PfMFSDT and highlighted its role as a drug transporter in P. falciparum. The complete characterization of PfMFSDT as a drug transporter has been done using genetically engineered Candida glabrata 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 PfMFSDT. C. glabrata 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 PDR1 transcription factor resulting in a hyperactivation of CDR1 locus has been used as an orthologous expression system for PfMFSDT characterization. A homologous recombination-based strategy was employed to integrate PfMFSDT at the CDR1 locus of MSY8 for its orthologous expression. PfMFSDT-expressing MSY8 strain (MSY8-PfMFSDT1) 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 P. falciparum strain 3D7. C. glabrata strain MSY8 transformed with PfMFSDT (MSY8-PfMFSDT1) conferred resistance to antifungal drugs. PfMFSDT transports antifungal drugs, ketoconazole and itraconazole, as evidenced by the rescued growth of C. glabrata in the presence of both antifungals. Both ketoconazole and itraconazole interact with PfMFSDT as evaluated by favorable binding energies, implying probable binding interactions with PfMFSDT in in silico interaction analysis. MD simulation studies revealed that the binding site residues get stabilized and maintain the overall stability of the PfMFSDT in the presence of ketoconazole and itraconazole. Ketoconazole and itraconazole blocks the growth of the malaria parasite P. falciparum 3D7 strain. PfMFSDT has been characterized as a drug transporter that is targeted by ketoconazole and itraconazole. PfMFSDT can act as a target for antimalarial drug development. The strain MSY8-PfMFSDT1 developed in this study can be further used to identify novel PfMFSDT-targeting antimalarials.