Role of Plasmodium falciparum transporters in drug resistance

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Although the fight against malaria has achieved a remarkable progress during the last 15 years, there were still 214 million new cases and 438000 estimated deaths caused by malaria worldwide in 2015. Transporters play a crucial role in Plasmodium biology but they can also be considered as double edge swords: on the one hand, they are potential new antimalarial drug targets but on the other hand, they are the main players in the development of drug resistance. PFE0825w is a putative organic cation transporter that has been proposed as the target of the candidate antimalarial drug albitiazolium and the locus where it is localized has been linked to chloroquine transport. In this study, different PFE0825w isoforms were identified and studied using the X. laevis oocyte system. Two of the isoforms were expressed at the oocyte oolemma but no significant transport of putative organic cation substrates was detected, restricting further characterization of this transporter. A better characterized transporter that plays a significant role in resistance against chloroquine (CQ) and quinine (QN) is the chloroquine resistance transporter PfCRT. It is known that this transporter has at least three different phosphorylation sites and that the phosphorylation of one of these sites -T416- is essential for the correct trafficking of PfCRT to the food vacuolar membrane. In this study the role of phosphorylation in the drug-resistance-mediating function of PfCRT was investigated. CQ-resistant parasites treated with the kinase inhibitor ML-7 accumulated more CQ than untreated parasites and showed CQ and QN IC50 values comparable to those of sensitive strains. Along the same line, the mutagenesis of the phosphorylation site S33 to alanine in PfCRT led to reduced CQ and QN IC50 values although no increase in drug accumulation was observed. Furthermore, PfCRTS33A conferred a fitness advantage to the parasites in the absence of CQ and a fitness cost in the presence of the drug. Two protein kinases were analyzed regarding their roles in PfCRT phosphorylation, PfCK2 and PF11_0488, the latter being identified in a Y2H assay. The downregulation of PfCK2 did not have an effect on CQ accumulation, but the overexpression of the C-terminal part of PF11_0488 resulted in reduced levels of CQ accumulation. However, the same fragment did not show any catalytic activity when recombinantly expressed and used in in vitro phosphorylation assays. Downregulation of this kinase was not achievable, most likely due to its essential function. Altogether, these results point to the fact that the parasite susceptibility towards CQ and QN is regulated by phosphorylation, although the exact molecular mechanism needs to be further examined.

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