How can we identify parasite genes that underlie antimalarial drug resistance?

Abstract

This article outlines genome-scale approaches that can be used to identify mutations in malaria (Plasmodium) parasites that underlie drug resistance and contribute to treatment failure. These approaches include genetic mapping by linkage or genome-wide association studies, drug selection and characterization of resistant mutants, and the identification of genome regions under strong recent selection. While these genomic approaches can identify candidate resistance loci, genetic manipulation is needed to demonstrate causality. We therefore also describe the growing arsenal of available transfection approaches for direct incrimination of mutations suspected to play a role in resistance. Our intention is both to review past progress and highlight promising approaches for future investigations.

Figure 1. Linkage group selection

The progeny from a cross between sensitive (white) and resistant (blue) parasites infect mice. Drug sensitive (white) progeny are removed by drug selection, enriching for drug-resistant alleles. The enriched region of the genome can be detected by measuring the proportion of alleles from the resistant parent at markers across the genome. The graph shows a hypothetical allele frequency plotted across the genome in the control (red line) and drug-treated (black line) groups. The drug selection step could be performed in culture flasks for Plasmodium falciparum or in rhesus macaques for Plasmodium vivax. QTL: Quantitative trait loci.

Figure 2. Recombination rate and linkage disequilibrium differ between populations

The decay in linkage disequilibrium (LD) is plotted for parasite isolates from three locations. In Senegal, there is no detectable LD between markers separated by more than 1 kb, and LD is weak at 250 bp. At the other extreme in a low-transmission setting in Brazil, LD remains high between markers separated by more than 10 kb, while Thai populations with low to moderate transmission show intermediate levels of LD. Hence, a lower density of polymorphic markers is required to detect associations between traits and marker loci in Brazil in comparision to Senegal. The statistic r² is useful for calculation of sample sizes needed to detect association: twice as many samples are needed to detect associations between a marker and a trait locus with r² of 0.5 compare with r² = 1. Data taken from [29].

Figure 3. Marker density for linkage and association analyses

Both linkage and association mapping aim to identify marker loci that are close to loci underlying resistance. The central difference lies in the numbers of times that genomes are reshuffled by recombination. In linkage mapping, parents and progeny are separated by a single round of recombination in controlled laboratory crosses. Hence, the size of haplotype blocks retained from the parents is quite large, and relatively sparse marker spacing (5–10 cM, 75–150 kb) is needed to detect genome regions containing trait loci. In comparison, genome-wide association studies use parasite samples from natural populations that are separated by an unknown number of generations and recombination events. As a consequence, their genomes show much greater levels of reshuffling, and dense marker spacing is required to detect association between marker loci and trait loci. The bars represent chromosomal sequences inherited from a resistant (dark blue) or sensitive (light blue) parasite parent.

Figure 4. Population structure and false-positive association

(A) Population subdivision. Parasites are sampled from two different populations (circles and squares), that differ in allele frequency at many loci. Resistance (blue shading) is common in the circles but rare in the squares, generating false-positive associations between resistance and loci that differentiate the two populations. (B) Relatedness. Within a single population, some parasites are related or identical (linked by lines), and thus tend to carry the same alleles. Inclusion of related isolates can generate false-positive associations between resistance and alleles that happen to be segregating within parasite ‘families’.

Figure 5. Clone tree of Plasmodium chabaudi

The arrows indicate the selection regimens at each stage. The boxed clones were sequenced using Illumina (CA, USA) short read methods. Mutations differentiating these clones (red text) are marked on the tree to show where they occur. Two mutations occurred in ubp1. These were observed independently within the CQ-selected lineage. The approximate position of the changes that were associated with resistance was determined by linkage group selection (see Figure 1 and text). The 400-kb duplication contains pcmdr1 and is translocated from chromosome 12 to chromosome 4 [142]. CQ: Chloroquine; MEF: Mefloquine; PYR: Pyrimethamine. Adapted from [38].

Figure 6. Strategies that can be used to study the function of candidate drug-resistance genes in Plasmodium falciparum

(A) Allelic exchange. Integration of the allelic exchange plasmid via a single crossover homologous recombination results in the introduction of candidate drug resistance mutations (black knobs) into the targeted gene, which remains under the control of the endogenous promoter (black arrow). Integration of the allelic exchange plasmid simultaneously renders the wild-type allele nonfunctional by separating it from its promoter and/or by truncating the coding sequence. Concatemerized plasmids will integrate as tandem copies (not shown). (B) Gene disruption. Integration of a gene targeting construct by single crossover homologous recombination results in the disruption of a gene of interest. No sequences are removed from the genome and recombination between homologous regions can potentially restore the wild-type locus. Plasmids that have concatemerized prior to integration will be inserted as tandem copies (not shown). (C) Gene deletion. Gene deletion constructs contain a positive selectable marker flanked by regions of homology (H) and a negative selectable marker on the plasmid backbone. Negative selection pressure kills all parasites that possess the negative selectable marker, and thus, selects for the rare double crossover homologous recombination event that has resulted in loss of the plasmid backbone and the irreversible removal of a candidate gene sequence from the genome. Double crossover homologous recombination may also occur via a single crossover integration intermediate followed by looping out of the plasmid backbone (not shown). (D) Bxb1 integrase-mediated transgene integration. Bxb1 mycobacteriophage integrase (provided on a helper plasmid; not shown) mediates site-specific recombination between a chromosomal attB (here shown in the cg6 gene as reported in [115]) and a plasmid-contained attP site, resulting in the rapid integration of plasmid-borne sequences into the P. falciparum genome. hDHFR and bsd are positive selectable markers. Note that approaches (B) and (C) are not possible for genes with an essential role in the (haploid) asexual blood stages.