Determining the roles of two key drug resistance proteins in conferring amodiaquine resistance in the malaria parasite

Abstract

Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) and the P. falciparum multidrug resistance protein 1 (PfMDR1) are associated with changes in the parasite's sensitivity to a broad range of drugs, including amodiaquine (AQ). PfCRT and PfMDR1 are located at the membrane of the parasite's digestive vacuole (DV) and their functional expression in Xenopus laevis oocytes has been established as an excellent method for studying their capacity to transport various drugs. There are two distinct types of mutations in PfCRT and PfMDR1 that have been associated with reductions in the parasite's sensitivity to AQ - the GB4-type mutations (common in African and SE Asian parasites) and the 7G8-type mutations (common in parasites from South America and the Philippines). Nevertheless, AQ has remained reasonably effective against GB4-type parasites in Africa but lacks efficacy against 7G8-type parasites which display high-level AQ resistance, both in vitro and clinically. The goal of the research herein was to investigate the role that polymorphisms in PfCRT and PfMDR1 play in conferring AQ resistance in the malaria parasite.

Isoforms of PfCRT were expressed in the oocyte system and their abilities to transport AQ characterised. These measurements revealed that all mutant isoforms of PfCRT mediate AQ transport, whereas wild-type PfCRT3D7 lacks this activity. Moreover, the 7G8-type PfCRT isoforms displayed significantly less capacity for AQ transport compared with GB4-type isoforms. This lower capacity did not appear consistent with the high-level AQ resistance displayed by 7G8-type parasites, particularly since AQ inhibits haemozoin formation in the DV. A kinetic analysis of PfCRT-mediated AQ transport in the oocyte system then revealed that 7G8-type isoforms of PfCRT have a significantly higher affinity for AQ than most of the GB4-type isoforms. It is possible that the high-affinity of 7G8-type PfCRT could allow preferential binding of AQ to the transporter rather than its target, resulting in low concentrations of AQ within the DV.

PfMDR1 isoforms expressed in oocytes have had their capacity to transport AQ measured by Shafik et al. (2022). GB4-type PfMDR1 isoforms had a reduced capacity for AQ transport compared with PfMDR13D7, while 7G8-type isoforms displayed the least capacity. I investigated the impact that D1246Y (found in PfMDR17G8) has on AQ transport when introduced into different field isoforms of PfMDR1. Its presence in GB4-type PfMDR1 isoforms significantly reduced their capacity to transport AQ, potentially resulting in advantageous lower concentrations of AQ in the DV of parasites expressing these mutant PfMDR1 isoforms. This could help explain why the D1246Y mutation in African isolates is often linked with more frequent re-infections following artesunate-AQ treatments.

The PfCRT isoform harboured by Cam734 from Cambodia (PfCRTCam734) confers reduced susceptibility to some antimalarials and an increased fitness or growth rate for the parasite (relative to the wild-type strain). When expressed in oocytes, PfCRTCam734 displayed a high affinity for AQ paired with a high transport capacity. To investigate the evolutionary biochemistry of PfCRTCam734 and its novel mutations, the transport of several drugs were measured via combinatorial mutant isoforms of PfCRT3D7-PfCRTCam734 expressed in oocytes. These results highlighted the importance of 75D and 144F to drug transport, while 148I, 194T, and 333S appeared to modulate drug transport capacity.

This project not only confirms and corroborates previous research into AQ resistance in P. falciparum but has yielded novel insights into the role of PfCRT and PfMDR1 in the parasite's acquisition of AQ resistance. It also highlights mutations important for conferring AQ resistance, the prevalence of which should be monitored. This research can help inform the inclusion of AQ in combination therapies and their future deployment. Show more