Genetic alteration of ferredoxin NADP⁺-reductase or cysteine desulfurase in piperaquine-resistant Plasmodium berghei restores susceptibility to lumefantrine and abolishes the impact of probenecid, verapamil, and cyproheptadine

Abstract

The ability of the human malaria parasite, Plasmodium falciparum to develop resistance against mainstay drugs remains a public health problem. Currently, the antimalarial drugs, lumefantrine (LM), and piperaquine (PQ) are essential components of the mainstay artemisinin-based therapies used for the treatment of malaria globally. Here, we used a model parasite Plasmodium berghei, to investigate the mechanisms of LM and PQ resistance. We employed known resistance reversing agents (RA): probenecid, verapamil, or cyproheptadine to study the mechanisms of LM and PQ resistance in the standard 4-day suppressive test. We then employed reverse genetics to assess the impact of deleting or over-expressing plausible genes associated with the metabolism and transport of drugs. We show that only, cyproheptadine at 5mgkg-1 restored LM activity by above 65% against LM-resistant parasites (LMr) but failed to reinstate PQ activity against PQ-resistant parasites (PQr). Whereas the PQr had lost significant susceptibility to LM, the three RA, cyproheptadine verapamil, and probenecid restored LM potency by above 70%, 60%, and 55% respectively against the PQr. We thus focused on the mechanisms of LM resistance in PQr. Here we show the partial deletion of the cysteine desulfurase (SUFS) and overexpression of the Ferredoxin NADP+ reductase (FNR) genes in the PQr parasite achieved two results; i) abolished the impact of RA on LM activity; ii) restored the susceptibility of PQr to LM alone. Our findings associated SUFS and FNR protein with the action of LM and RA action in P. berghei. We demonstrate that the incorporation of any of the RA into an antimalarial combination that comprises LM would augment LM activity and concomitantly antagonize the emergence of LM resistance derived from PQ pressure. The impact of RA, deletion of SUFS, and overexpression of FNR on LM activity need to be tested in Plasmodium falciparum.Author summaryLumefantrine (LM) and piperaquine (PQ) are essential drugs for the treatment of malaria globally. Here, we used Plasmodium berghei, a model parasite that infects rodents to study how parasites escape killing by PQ and LM. We first used a second drug: probenecid, verapamil, or cyproheptadine to enhance the activity of LM or PQ. We show that cyproheptadine restores LM activity against LM-resistant parasites (LMr) but failed to reestablish PQ activity against PQ-resistant parasites (PQr). Since PQr is resistant to LM, combining LM with either cyproheptadine, verapamil, or probenecid reinstates LM activity against PQr. We then focused mainly on LM resistance in PQr. After genetically manipulating the PQr, we reveal that cysteine desulfurase (SUFS) and ferredoxin NADP+reductase (FNR) regulate LM capacity to kill parasites. Decreasing the level of SUFS or increasing FNR levels in the PQr makes the parasites susceptible to LM but abolishes the impact of probenecid, verapamil, and cyproheptadine on LM activity. Overall, we provide clues on the link between SUFS and FNR in the action of LM and RA in P. berghei. This study provides a basis for an in-depth analysis of how LM mediates parasites kill and how the parasite escapes LM action in Plasmodium falciparum.