Variations in the sequence and expression of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and their relationship to chloroquine resistance in vitro

Abstract

Chloroquine has been widely used for malaria treatment and prophylaxis for several decades, but its usefulness has now declined with the emergence of chloroquine resistance. Recent studies showed that the K76T mutation in the PfCRT protein, initially associated to chloroquine-resistant parasites, is sometimes also present in susceptible parasites, suggesting that other factors control the expression of the resistance phenotype. Here, we sought new mutations in the Pfcrt gene and used real-time PCR to investigate variations in the expression level of this gene with respect to the in vitro response to chloroquine. About 40 Cambodian isolates of Plasmodium falciparum were selected on the basis of their response to chloroquine in vitro. The Pfcrt gene was characterised by amplifying and sequencing the full-length cDNA. Twelve point mutations—M74I, N75D/E, K76T, A144F, L148I, I194T, A220S, Q271E, N326S, T333S, I356T and R371I—were detected. Mutations identified at positions 144, 148, 194 and 333 had never been described before. These mutations define six distinct haplotypes, distributed heterogeneously throughout Cambodia. Only the mutations at positions 74–76, 220 and 271 were significantly associated with the in vitro response to chloroquine. Three major haplotypes—MNK/A/Q, IDT/S/E and IET/S/E—accounted for all the isolates examined. The MNK/A/Q haplotype corresponded to susceptible isolates whereas parasites with the IDT/S/E haplotype displayed an intermediate response to chloroquine and those with the IET/S/E haplotype displayed the highest IC₅₀ values. Phylogenic analysis suggested that the IDT and IET haplotypes (positions 74–76) arose independently from the wild-type MNK sequence. We found that the expression level of Pfcrt, evaluated by real-time PCR, had no effect on the response of the parasite to the drug in vitro. Similarly, in a CQ-resistant strain short-term cultured in the presence of CQ, no change was observed in the level of transcripts. These results are discussed in light of recent finding suggesting the possible involvement of other transporters in CQ-resistance.

Introduction

Malaria is responsible for a major parasitic endemic extending over much of the subtropics. Plasmodium falciparum alone accounts for the deaths of more than two million people each year [1]. Moreover, this species has now developed resistance to many anti-malaria drugs, including chloroquine (CQ). This drug has been widely used in the treatment and prophylaxis of malaria for several decades but its usefulness has now declined with the emergence of chloroquine-resistant (CQR) parasites. CQR, which appeared simultaneously in South-East Asia and South America at the beginning of the 1960s, has now reached most of the zones of endemic malaria [2].

Despite important progress in recent years, the molecular basis for CQR remains only partially elucidated. The cg2 gene, located on chromosome 7, was initially thought to be implicated but it was subsequently established that the cg10 gene, located close to cg2, was actually involved [3]. Several mutations in cg10 associated with CQR have been since described in this gene which was therefore renamed P. falciparum chloroquine resistance transporter (Pfcrt) due to its implication in CQR [4], [5]. The CQR phenotype probably results from modifications in the function of the mutated PfCRT protein. The major molecular changes described to date include the replacement of a lysine (K) by a threonine (T) at position 76. This mutation is the most predictive of CQR, both in vitro and in vivo, suggesting that the characterisation of this codon could be used as an epidemiological tool for large-scale studies of CQR in the field [6], [7]. The several studies performed on isolates from Africa [8], [9], [10], [11], [12], Asia [13], [14] and South America [15] have shown that the K76T mutation is present in almost all CQR samples. However, as this mutation is also found in certain strains displaying chloroquine susceptibility (CQS), other mechanisms may be involved in modulating the expression of the CQR phenotype [7], [9], [12], [14].

Two major mechanisms have been evoked to account for the decrease in susceptibility of certain parasites to CQ: (i) reduced access of CQ to its target, heme, rendered insoluble by a decrease in pH in the digestive vacuole consecutive to changes in ion transport via the mutated PfCRT; (ii) direct expulsion of CQ from the digestive vacuole via the mutated PfCRT, resulting from an increase in the affinity of this transporter for CQ [4], [16]. The pumping role attributed to PfCRT could also account for the discrepancies occasionally observed between mutations and susceptibility to drugs. In this way, the parasite modulates its response to CQ by modifying its level of PfCRT production, at least in the allelic replacement systems recently developed for fine functional analysis of the Pfcrt gene [7].

Based on these models, and because the mutations so far reported in the Pfcrt gene are insufficient in themselves to explain the development of CQR, we sequenced the entire Pfcrt gene for wild strains collected in Cambodia. We also used real-time PCR (RT-PCR) to measure the level of expression of the gene. We then compared expression levels with genotype and the susceptibility of isolates to CQ in vitro, to determine the relative importance of the mutations observed and of Pfcrt expression in the development of the CQR phenotype. We found that the structure of the Pfcrt gene had a greater effect on CQR phenotype than did its level of expression in Cambodian isolate, but that other factors should also be considered.