Identification of a Mutant PfCRT-Mediated Chloroquine Tolerance Phenotype in Plasmodium falciparum

James W Kazura,Stephanie G Valderramos,Lise Musset,Lisa A Purcell,Juan-Carlos Valderramos,Odile Mercereau-Puijalon,Eric Legrand,David A Fidock

doi:10.1371/journal.ppat.1000887

Abstract

Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.

Highlights

The massive use of chloroquine (CQ) in the 20th century heralded substantial gains in the global fight against malaria
Generation of recombinant lines expressing mutant pfcrt To define the impact of mutant pfcrt on CQ response in diverse genetic backgrounds, we developed an allelic exchange strategy based on a single round of homologous recombination and singlesite crossover integration (Figure 1A), and applied this to the CQsensitive P. falciparum strains 3D7, D10 (Papua New Guinea), and GC03
We provide evidence that the genetic background of P. falciparum determines whether expression of mutant pfcrt allele confers a full CQ resistance (CQR) phenotype, as defined by CQ IC50 values that exceed the in vitro CQR threshold [30], or instead mediates increased tolerance to CQ, as evidenced by dose-response shifts manifesting primarily at the IC90 level

Summary

Introduction

The massive use of chloroquine (CQ) in the 20th century heralded substantial gains in the global fight against malaria. These advances were later lost as CQ resistance (CQR) arose and spread throughout malaria-endemic areas [1,2]. Recent findings suggest the possibility of reintroducing CQ-based combination therapies into African regions where an extended hiatus from CQ use has led to the dominance of CQ-sensitive Plasmodium falciparum parasites that have outcompeted the less-fit CQ-resistant strains [6]. CQ is thought to act by accumulating to low millimolar concentrations in the acidic digestive vacuole of asexual intra-erythrocytic Plasmodium parasites, wherein it interferes with the detoxification of iron-bound heme moieties produced as a result of hemoglobin degradation [7]

Methods

Results

Conclusion