Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

Serena Pulcini,David A Fidock,Daniel A Daley,Henry M Staines,Sanjeev Krishna,Roland A Cooper,Guillaume Bouyer,Catherine M Moore,Lindsey M Altenhofen,Jianbing Mu,Manuel Llinás,Sarah H Shafik,Heather J Painter,Andrew H Lee,David J P Ferguson,Matthew J Hoke,Rowena E Martin

doi:10.1038/srep14552

Abstract

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

Highlights

CQ is a diprotic weak base that accumulates in the parasite’s acidic food vacuole (FV) by diffusion and subsequent trapping by protonation
The native function of PfCRT is not clear, it has been postulated to be involved in hemoglobin catabolism, possibly by mediating the transport of hemoglobin-derived peptides/amino acids from the FV12, a hypothesis consistent with recent heterologous expression and metabolomics studies[7,13,14]
We investigated the effect of these changes on the parasite’s sensitivity to other antimalarial classes, such as the artemisinins, that some have considered to act in the FV of the parasite[20,21]

Summary

Introduction

CQ is a diprotic weak base that accumulates in the parasite’s acidic food vacuole (FV) by diffusion and subsequent trapping by protonation. A mutation at position c814t in the pfcrt coding sequence, resulting in the amino acid mutation L272F, was detected in the selected line, designated 3D7L272F, and was absent in its parent.

Results

Conclusion