Abstract
BackgroundDrug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.ResultsLoci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.ConclusionsQuantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.
Highlights
Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria
We denoted the CQ responses of these clones as CQ sensitive (CQ-S), chloroquine resistance (CQ-R) or high level chloroquine resistance (CQ-hiR), respectively. These data are consistent with a previous proposal that multiple mutations confer CQ-hiR [18] in this lineage, and suggest a suitable range of CQ doses for dissecting the critical genetic loci in linkage group selection (LGS) experiments, below
We expected that parasites surviving 3 mg CQ kg-1 day-1 would be enriched with parasites having CQ-R phenotypes, while those surviving 10 mg CQ kg-1 day-1 would be preferentially enriched with CQ-hiR parasites only
Summary
Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. For chloroquine resistance (CQ-R), genetic linkage studies [1,2], other experimental approaches [3] and phenotype/genotype associations in parasites from natural infections [3,4,5] have mapped and identified the K76T mutation in the chloroquine resistance transporter, pfCRT, as the dominant genetic determinant in the most important human parasite Plasmodium falciparum. Specific point mutations in the multidrug resistance gene (pfmdr1) encoding an ABC transporter (P-glycoprotein homologue, Pgh-1) have been shown to modulate the level of resistance in CQ-R parasites in transfection experiments [10,11] and in association studies using parasites from natural infections [12,13] These two genes neither account for the full variation of in vitro CQ responses, including high-level CQ-R (CQ-hiR) [4,14] nor the appearance of CQ-R in another major human pathogen, P. vivax [15]
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