Expression of Plasmodium major facilitator superfamily protein in transporters - Δ Candida identifies a drug transporter

Abstract

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'.

Keywords: Candida glabrata; Plasmodium falciparum; antimalarial; drug discovery; membrane transporter; orthologous system.

Figure 1.

Unveiling PfMFSDT architecture and phylogeny. (A) Overall PfMFSDT architecture as deduced using IBS 1.0.3 [18]. Probable amino acid residues involved in Na⁺- and lysolipid-binding, along with transmembrane helices (H1 to H12) are shown. Blue-colored amino acids represent residues with low consensus value (>50%, <90%), red-colored amino acid represent residues with high consensus value (>90%) and residues marked in green represent residues involved in Na⁺- and lysolipid-binding across kingdom animalia (B) PfMFSDT topology prediction using Protter 1.0 [16]. (C) Maximum likelihood phylogenetic analysis generated using MEGA11 [19] demonstrates a convergent evolutionary pathway of PfMFSDT with MFS proteins from Toxoplasma gondii. Red, blue and orange-filled circles represent MFS proteins from Plasmodium, Toxoplasma and Theileria sp., respectively. MFS: Major Facilitator Superfamily.

Figure 2.

Expression and co-localization of PfMFSDT with PfMSP1. (A) IFA to check the localization of PfMFSDT in the parasite using anti-PfMFSDT_Hydrophillic mouse serum; localization of MSP1 served as a positive control. PfMFSDT localizes toward the parasite cell membrane and co-localizes with PfMSP1. (B) Western blotting confirming PfMFSDT expression during the schizont stage using anti-PfMFSDT_Hydrophilic mouse serum. IFA: Immunofluorescence Assay.

Figure 3.

Expression, localization of PfMFSDT in C. glabrata and drug susceptibility assay. (A) Visualization of PfMFSDT-GFP in transformed C. glabrata strain MSY8 strain using fluorescence microscopy, revealing localization toward the cell membrane and nucleus (i). Western blot using anti-PfMFSDT_Hydrophillic mouse serum confirming expression in transformed C. glabrata strain MSY8. A desired protein band of ∼79 kDa was observed (ii). (B) Drug susceptibility assay was performed by serial dilution spot assay on transformed and untransformed MSY8, in the presence of itraconazole (0.031 μg/ml) and ketoconazole (0.002 μg/ml). PfMFSDT-expressing MSY8 cells showed rescued growth in the presence of itraconazole and ketoconazole.

Figure 4.

Homology modeling, docking studies and antimalarial activity of PfMFSDT inhibitors. (A) Homology modeling of PfMFSDT was done using Prime application in Schrodinger Maestro Suite 2022-4, using chain_A of bacterial oxalate transporter (OxlT 8HPK_A) [24] as a template. The generated model was further refined for its loop refinement and energy minimization using the OPLS4 force field in Schrodinger Prime [25] (i). PfMFSDT model was mapped for its localization in the plasma membrane (ii). (B) Molecular docking of itraconazole and ketoconazole. Molecular docking of itraconazole (i) and ketoconazole (ii) against active site of PfMFSDT using Extra Precision (XP) docking [30–32]. Hydrogen-bonding, hydrophobic and electrostatic interactions were assessed avoiding steric clashes. The Glide Score scoring function was applied to re-score the obtained poses. The MM-GBSA method, utilizing the OPLS4 force field, VSGB solvent model and search algorithms, was used [25] and the binding free energies of ligand-receptor complex were computed. The binding free energy for itraconazole is -64.91 kCal/mol whereas for ketoconazole is -73.8 kCal/mol. MM-GBSA: Molecular Mechanics-Generalized Born Surface Area; OPLS4: Optimized potentials for liquid simulation.

Figure 5.

MD simulation analysis: evaluation of RMSD and RMSF. (A) RMSD of the apoprotein, PfMFSDT (i), PfMFSDT-itraconazole complex (ii) and PfMFSDT-ketoconazole complex (iii). (B) RMSF of the apoprotein, PfMFSDT (i), PfMFSDT-itraconazole complex (ii) and PfMFSDT-ketoconazole complex (iii). MD: Molecular Dynamics, RMSD: Root mean square deviation. RMSF: Root mean square fluctuation.

Figure 6.

MD simulation-based detailed interaction analysis. (A) Interaction of PfMFSDT with itraconazole (i). A schematic of the detailed interactions of itraconazole with PfMFSDT (ii). Interaction properties such as RMSD, rGyr, intermolecular H-bonds, MolSA, SASA and PSA (iii). (B) Interaction of PfMFSDT with ketoconazole (i). A schematic of the detailed interactions of ketoconazole with PfMFSDT (ii). Interaction properties such as RMSD, rGyr, intermolecular H-bonds, MolSA, SASA and PSA (iii). MD: Molecular Dynamics; MolSA: Molecular surface area; PSA: Polar surface area; rGyr: Radius of gyration; RMSD: Root mean square deviation; SASA: Solvent accessible surface area.

Figure 7.

Antimalarial activity of itraconazole and ketoconazole. Pf3D7 (ring stage, at 0.5% parasitemia) was treated with itraconazole and ketoconazole (0.37 to 50 μM) for 72 h. Dose-response curve; IC₅₀ values of itraconazole and ketoconazole were found to be 2.1 μM and 0.6 μM, respectively (i). Giemsa-stained smears showing growth defects in the parasite treated with itraconazole (3.125 μM) and ketoconazole (0.78 μM), as compared with the untreated control (ii).