Role of Plasmodium falciparum transporters in drug resistance

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

Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5–8 fold elevation in the mutation rate, with an increase of 13–28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an “irresistible” compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this “mutator” parasite can be leveraged to drive P. falciparum resistome discovery

Phosphomimetic substitution at Ser-33 of the chloroquine resistance transporter PfCRT reconstitutes drug responses in Plasmodium falciparum

International audienceThe chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance–conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ∼50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT

The knock-down of the chloroquine resistance transporter PfCRT is linked to oligopeptide handling in Plasmodium falciparum

The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance

Functional screening of selective mitochondrial inhibitors of Plasmodium

Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors. Keywords: Plasmodium falciparum, Plasmodium yoelii, Oxygen consumption, Mitochondrial inhibitors and cytochrom

Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum.

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an irresistible compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this mutator parasite can be leveraged to drive P. falciparum resistome discovery