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

<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 &amp; 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.**研究目的**：以光滑念珠菌（*Candida glabrata*）作为同源蛋白表达宿主，评估推定的主要促进因子超家族（Major Facilitator Superfamily, MFS）蛋白PF3D7_0210300（简称*Pf*MFSDT）作为药物转运蛋白的功能相关性。**研究方法**：通过同源重组将编码*Pf*MFSDT的互补确定序列（Complementary Determining Sequence）整合至基因工程改造的光滑念珠菌MSY8菌株的基因组中，随后评估其作为药物转运蛋白的功能相关性。**结果与结论**：经改造的光滑念珠菌菌株可使*Pf*MFSDT定位于质膜，并表现出主要促进因子超家族转运蛋白的特性，同时赋予菌株对抗真菌药物酮康唑及伊曲康唑的耐药性。这两种药物对恶性疟原虫（*Plasmodium falciparum*）红细胞内增殖的纳摩尔级抑制活性，凸显了其抗疟活性。本研究将*Pf*MFSDT鉴定为药物转运蛋白，扩充了当前已知的抗疟“耐药组（resistome）”范围。致死性疟疾病原体恶性疟原虫为专性胞内寄生虫，依赖多种转运机制维持生存；参与寄生虫生存必需营养物质转运的转运蛋白，构成了一类药物靶点库。本研究对主要促进因子超家族蛋白成员*Pf*MFSDT进行了功能鉴定，并阐明其在恶性疟原虫中作为药物转运蛋白的作用。本研究利用敲除了临床相关的ATP结合盒式（ATP-binding cassette, ABC）药物转运蛋白基因的基因工程光滑念珠菌，完成了*Pf*MFSDT作为药物转运蛋白的完整功能鉴定。研究结合生物信息学、细胞分子生物学及药理学方法，探究了*Pf*MFSDT的潜在功能。光滑念珠菌MSY8菌株经基因改造，敲除了7个临床相关的膜结合型ABC药物转运蛋白基因，同时携带*PDR1*转录因子功能获得性突变，导致*CDR1*位点过度激活，该菌株被用作*Pf*MFSDT功能鉴定的同源表达系统。本研究采用基于同源重组的策略，将*Pf*MFSDT整合至MSY8菌株的*CDR1*位点以实现同源表达。通过生长敏感性实验，对表达*Pf*MFSDT的MSY8菌株（MSY8-*Pf*MFSDT1）进行抗真菌药物伊曲康唑与酮康唑的外排功能评估。在恶性疟原虫3D7菌株中评估了酮康唑与伊曲康唑的抗疟活性。转染*Pf*MFSDT的光滑念珠菌MSY8菌株（MSY8-*Pf*MFSDT1）可获得抗真菌药物耐药性。光滑念珠菌在两种抗真菌药物存在下的生长恢复现象证实，*Pf*MFSDT可转运酮康唑与伊曲康唑这两种抗真菌药物。通过结合能分析可知，酮康唑与伊曲康唑均可与*Pf*MFSDT发生相互作用，这一结果在计算机模拟（in silico）相互作用分析中提示二者与*Pf*MFSDT存在潜在结合互作。分子动力学（Molecular Dynamics, MD）模拟研究显示，在酮康唑与伊曲康唑存在时，*Pf*MFSDT的结合位点残基可保持稳定，并维持该蛋白的整体结构稳定性。酮康唑与伊曲康唑可抑制疟原虫恶性疟原虫3D7菌株的增殖。*Pf*MFSDT已被鉴定为可被酮康唑与伊曲康唑靶向结合的药物转运蛋白。*Pf*MFSDT可作为抗疟药物开发的潜在靶点。本研究构建的MSY8-*Pf*MFSDT1菌株，可进一步用于筛选靶向*Pf*MFSDT的新型抗疟药物。