Structure-function studies of multidrug resistance transporters of the malaria parasite.

Many genetic determinants of drug resistance in the malaria parasite Plasmodium falciparum encode transporters. Ascertaining the role of these proteins in drug action and drug resistance has historically been impeded due to the difficulties in achieving their expression in heterologous systems, such as Xenopus laevis oocytes. Indeed, several technical challenges must be overcome when expressing functional P. falciparum proteins in X. laevis oocytes, including the disparity in codon usage between the two species. This disparity can be overcome by codon harmonisation, the recoding of AT-rich P. falciparum coding sequences to emulate the codon usage of X. laevis. Moreover, if the P. falciparum transporter typically resides in intracellular membranes, the identification and removal of trafficking motifs may be necessary to facilitate its expression at the oocyte's surface, as required for directly measuring the transport of substrates by the protein. The functional expression of the P. falciparum chloroquine resistance transporter (PfCRT) in X. laevis oocytes shed light on its role in the parasite's acquisition of chloroquine resistance. The success of this endeavour relied on the codon harmonisation of the pfcrt coding sequence and the removal of putative trafficking motifs. Here, the contribution of these two sequence modifications to the functional expression of PfCRT in X. laevis oocytes was investigated by determining which pfcrt coding sequences resulted in optimal expression in X. laevis oocytes, using western blot analyses and chloroquine influx assays. This work revealed that codon harmonisation of the coding sequence and the removal of two motifs were essential to achieving the functional expression of PfCRT in oocytes. These findings were used to establish a robust expression system for the P. falciparum multidrug resistance protein 1 (PfMDR1) in oocytes. Mutations in PfCRT and PfMDR1 are associated with the development of multidrug resistance and collateral sensitivity - the phenomenon whereby acquiring resistance to one drug results in concomitant sensitivity to a second drug - in the malaria parasite. However, the mechanisms by which PfCRT and PfMDR1 modulate the parasite's susceptibility to many drugs are poorly understood. Here, the drug transport properties of novel PfCRT variants were characterised using the X. laevis oocyte system to understand the role(s) of the transporter in altering the parasite's susceptibility to chloroquine, quinine, quinidine, mefloquine, and amantadine. From these analyses, two molecular mechanisms for PfCRT-induced collateral sensitivity were elucidated: (1) the potent inhibition of a specific PfCRT variant by quinine, and (2) the delivery of drugs to their site of action via PfCRT. These analyses also provided insights into the role of residues at positions 72, 75, 76, 163, 329, 352, and 356 in altering the substrate specificity and transport capacity of PfCRT. The roles of conserved residues within Loop 7 and transmembrane domains 5 and 10, including G186, T193, C289, C301, C309, C312, T380, and G387, in PfCRT's structure and function were investigated. This work highlighted that these residues play vital roles in maintaining the structural and functional integrity of PfCRT. The interactions of several PfMDR1 variants with chloroquine, quinine, and lumefantrine were investigated, following confirmation that the proteins were equally expressed at the oocyte's surface. The findings from these transport assays indicate that PfMDR1 modulates the parasite's susceptibility to drugs by redistributing them within the parasite, and thus altering their access to their antiplasmodial targets, analogous to the mechanism of PfCRT-induced collateral sensitivity here described. Exploiting these mechanisms of collateral drug sensitivity may lead to strategies that prevent or retard the spread of drug-resistant malaria