Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations

Abstract

Plasmodium falciparum chloroquine resistance is a major cause of worldwide increases in malaria mortality and morbidity. Recent laboratory and clinical studies have associated chloroquine resistance with point mutations in the gene pfcrt. However, direct proof of a causal relationship has remained elusive and most models have posited a multigenic basis of resistance. Here, we provide conclusive evidence that mutant haplotypes of the pfcrt gene product of Asian, African, or South American origin confer chloroquine resistance with characteristic verapamil reversibility and reduced chloroquine accumulation. pfcrt mutations increased susceptibility to artemisinin and quinine and minimally affected amodiaquine activity; hence, these antimalarials warrant further investigation as agents to control chloroquine-resistant falciparum malaria.

Fig. 1

pfcrt mutations confer chloroquine resistance and altered susceptibility to other heme (ferriprotoporphyrin IX)–binding antimalarials in P. falciparum. (A to F) Results of in vitro drug assays with pfcrt-modified and control lines. Each line was tested in duplicate against each antimalarial drug at least four times, in the presence or absence of 0.8 μM verapamil (VP). IC₅₀ values (shown as the mean + SE for each line) correspond to the concentration at which incorporation of [³H]hypoxanthine was half-maximal and were derived from the inhibition curves generated as a function of drug concentration. Mann-Whitney tests were used to assess statistically significant differences between the reference line C2^GC03 and the others (*P < 0.05, **P <0.01). IC₅₀ and IC₉₀ values and compound structures are detailed in table S2 and fig. S3. The finding that pfcrt mutations conferred increased susceptibility to both quinine and mefloquine provides an intriguing contrast with the recent report that the introduction of mutations into pfmdr1 led to decreased susceptibility to quinine and increased susceptibility to mefloquine (17). (G) Chloroquine accumulation assays (using 30 nM [³H]chloroquine) confirm that pfcrt mutations confer a CQR phenotype. The saturable component of [³H]chloroquine accumulation, measured as femtomoles of drug per 10⁶ infected red blood cells, was calculated by subtracting the nonsaturable accumulation (measured in the presence of 10 μM unlabeled chloroquine) from the total accumulation (measured in the absence of unlabeled drug). The mean + SE, calculated from five experiments performed in duplicate, is shown for each line. Accumulation values in the pfcrt-modified clones C3^Dd2 and C6^7G8 showed a statistically significant difference from values observed in C2^GC03 (**P < 0.01). The chloroquine data in (A) and (G) prove a central role for pfcrt point mutations in conferring CQR to the GC03 line, although they do not rule out the possibility that other loci can contribute to this phenotype. It remains to be established whether mutant pfcrt alleles can confer CQR to multiple, genetically distinct chloroquine-sensitive lines. One hypothesis under consideration is that pfcrt confers CQR and altered susceptibility to other heme-binding antimalarials through a combination of direct effects (which may involve drug-protein interactions) and indirect effects on parasite physiological processes (which may influence drug accumulation and formation of drug-heme complexes).