Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Abstract

Background: The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results: Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions: We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.

Figure 1

Amplification event surrounding pfmdr1 on chromosome 5 in Dd2. The log₂ ratio of the intensity of each unique probe in Dd2 was divided by the intensity of each unique probe in 3D7 to generate the plot. The probe log ratios were colored by the moving average over a 500 base pair window as indicated in the color bar. A normal distribution around zero was expected if the copy number was the same. The ends of chromosome 5 are highly polymorphic subtelomeric regions and thus showed much lower probe intensity in Dd2 when compared with the reference 3D7. The amplification containing 14 genes including pfmdr1 (marked with an asterisk) is enlarged in the inset. Mb, megabases.

Figure 2

SNPs in Dd2. (a) Plot of -log₁₀ P values for z-test (blue line) performed with Dd2 versus the 3D7 reference; detected are all of the reported single nucleotide polymorphisms (SNPs) in the pfcrt gene that were represented in the microarray (red triangles). Two SNPs were not detected (black triangles) because of the lack of unique probes to these positions. The thin and thick black arrows correspond to SNPs indicated in panel b. The P-value cutoff was 1 × 10^-8 (black line). (b) Visualization of probe intensity log ratios in Dd2 versus 3D7. Each probe is shown as a single pixel and is colored based on its log ratio. Continuous strips of dimmer pixels correspond to SNPs that map to multiple unique probes, as indicated by the thin and thick white arrows. (c) Probes were tiled through the genome with an offset of two to three base pairs of alternating strands. The position marked in red is the position of the SNP marked by the thick arrows in panels a and b. The base pair positions on the left indicate the start of the probe with respect to the plus strand of chromosome 5 and its orientation.

Figure 3

Mutation in 3D7^attB histone 2B. (a) Plot of -log P-value for z-test (blue line) performed with 3D7^attB versus the 3D7 reference detected a polymorphism in the histone 2B gene with a P-value cutoff of 1 × 10^-8 (black line). (b) Sequencing of the histone 2B gene in 3D7^attB revealed two consecutive point mutations resulting in a coding nonsynonymous mutation.

Figure 4

Fosmidomycin resistance. (a) Fosmidomycin 50% inhibitory concentration (IC₅₀) values (means and standard deviations) were calculated from independent [³H]hypoxanthine assays in duplicate; the number of assays (n) is indicated in parenthesis. Drug susceptibility profiles of Dd2 (wild-type) (n = 7), FOS-R^Dd2-CL1 (n = 5), FOS-R^Dd2-CL2 (n = 3), and control FOS-R^Dd2-CL1-no drug (n = 3). Tests for significant differences between the parental and drug-resistant isolates were performed using analysis of variance with Bonferroni posttests with α = 0.001; significant differences (P value < 0.001) are marked with three asterisks (***). (b) Effect of fosmidomycin on 1-deoxy-D-xylulose 5-phosphate (DOXP) and 2-C-methyl-D-erythritol-4-phosphate (MEP) biosynthesis in intact erythrocyte-free parasites exposed to fosmidoymicin for 24 hours and then labeled with [2-¹⁴C]pyruvic acid as a metabolic precursor. Means and standard deviations were calculated from four independent experiments performed in duplicate. Differences between control (no drug) Dd2 and fosmidomycin-treated Dd2 were significant by Student's t-test for DOXP (1 μmol/l drug: P value 1 × 10^-8; 2 μmol/l drug: P value 1 × 10^-6) and MEP (1 μmol/l drug: P value 4 × 10^-6; 2 μmol/l drug: P value 5 × 10^-8) and are marked with three asterisks (***). (c) To generate the plot, the log₂ ratio of the intensity of each unique probe in FOS-R^Dd2-CL1 was divided by the intensity of each unique probe in Dd2. The probe log ratios were colored by the moving average over a 500 base pair window as indicated in the color bar. The amplification was approximately 100 kb and contained 23 genes including pfdxr (marked with an asterisk). (d) Quantitative RT-PCR analysis of pfdxr transcript level and quantitative PCR analysis gene copy number in FOS-R^Dd2-CL1 compared with Dd2. The means and standard deviations of two independent quantitative RT-PCR and quantitative PCR assays are represented. Significance differences (P value < 0.001) in ΔCt values were determined by Wilcoxon rank sum test and are marked with three asterisks (***).