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. 2014 Mar;91(5):918-34.
doi: 10.1111/mmi.12505. Epub 2014 Jan 23.

Structural polymorphism in the promoter of pfmrp2 confers Plasmodium falciparum tolerance to quinoline drugs

Sachel Mok  1 , Kek-Yee Liong, Eng-How Lim, Ximei Huang, Lei Zhu, Peter Rainer Preiser, Zbynek Bozdech
Affiliations

Structural polymorphism in the promoter of pfmrp2 confers Plasmodium falciparum tolerance to quinoline drugs

Sachel Mok et al. Mol Microbiol. 2014 Mar.

Abstract

Drug resistance in Plasmodium falciparum remains a challenge for the malaria eradication programmes around the world. With the emergence of artemisinin resistance, the efficacy of the partner drugs in the artemisinin combination therapies (ACT) that include quinoline-based drugs is becoming critical. So far only few resistance markers have been identified from which only two transmembrane transporters namely PfMDR1 (an ATP-binding cassette transporter) and PfCRT (a drug-metabolite transporter) have been experimentally verified. Another P. falciparum transporter, the ATP-binding cassette containing multidrug resistance-associated protein (PfMRP2) represents an additional possible factor of drug resistance in P. falciparum. In this study, we identified a parasite clone that is derived from the 3D7 P. falciparum strain and shows increased resistance to chloroquine, mefloquine and quinine through the trophozoite and schizont stages. We demonstrate that the resistance phenotype is caused by a 4.1 kb deletion in the 5' upstream region of the pfmrp2 gene that leads to an alteration in the pfmrp2 transcription and thus increased level of PfMRP2 protein. These results also suggest the importance of putative promoter elements in regulation of gene expression during the P. falciparum intra-erythrocytic developmental cycle and the potential of genetic polymorphisms within these regions to underlie drug resistance.

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Figures

Figure 1
Figure 1
Drug inhibitory concentrations of clones 11C and 6A. IC50 and IC90 values (nM) of clones 11C (red) and 6A (blue) when exposed to mefloquine (A) or chloroquine (B) drugs at ring [10 h post invasion (hpi)], trophozoite (24 hpi) and schizont (34 hpi) stages. Values are determined from drug assays done in triplicate. Error bars represent the standard deviations of the IC50/IC90 measurements. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.
Figure 2
Figure 2
Genomic and transcriptomic differences between clone 6A and 11C.A. Distribution of the CGH microarray signal plotted as log2 ratios between clones 6A and 11C over the 14 P. falciparum chromosomes with red peaks representing the statistically significant CNPs. Red lines with log2 ratios > 0 indicates higher copy (above x-axis) number and log2 ratios < 0 (below x-axis) indicate lower copy number in clone 6A relative to 11C. Genes associated with the CNPs are shown in the grey boxes. The double asterisk (**) indicates that the CNP is detected at the non-coding 5′ region of the gene instead of within the coding sequence of the gene.B. Heat maps showing the mean-centred microarray log2 expression ratios of the 4478 P. falciparum genes ordered by fast fourier transformation over the 48 h IDC for clones 6A and 11C.C. Heat map showing the log2 expression ratios of the 23 differentially expressed (DE) genes detected by Statistical Analysis of Microarrays (SAM) with FDR < 5% over the IDC [depicted by hours post invasion (hpi)]. The DE genes with corresponding detected CNPs are indicated with blue or yellow symbols. R: ring; T: trophozoite; S: schizont.
Figure 3
Figure 3
Deletion of the pfmrp2 gene 5′ upstream sequence identified by PCR and sequencing.A. A map of the 5′ upstream region of the 3D7 pfmrp2 gene showing the panel of used primer pairs (purple) and their positions relative to the gene start codon. Listed are the sizes of the PCR products obtained for clones 11C/wt and 6A/mut for the various primer pairs. Bottom panel: Agarose gel images of PCR products obtained for the corresponding primer pairs. The images show full-length PCR products obtained for clone 11C/wt for all primer pairs and the truncated products for the primer pairs 1.9 and 1.12 from 6A/mut.B. Sequence alignment of clone 11C/wt and clone 6A/mut compared to 3D7 sequence (PlasmoDB version 8.1). The sequence alignment depicts the 200 bp coding sequence of the pfmrp2 gene at the 5′ end and its upstream, non-coding region. The grey dotted lines indicate the break points of the 6A/mut deletion. The numbers indicate the base position relative to the start codon. Two DNA constructs of 6A/mut of 0.3 and 0.5 kb in size were sequenced to identify the deletion break point as −4345th bp from the start codon.
Figure 4
Figure 4
Differential promoter activity of pfmrp2 gene in clone 6A/mut and 11C/wt by luciferase reporter assay and expression profiling of pfmrp2.A. Transcriptional activity of PFL1410c (pfmrp2) promoter constructs of clones 6A/mut (blue) and 11C/wt (red) in the ring stage measured by the firefly luciferase reporter assay. Bars represent the firefly luciferase (FL) activity normalized to the level of renilla luciferase (RL) activity for each cell line transfected with each promoter construct of ∼0.3–2.1 kb from the start codon of the pfmrp2 gene. Error bars indicate the standard deviation of the FL activity over three independent transfection experiments.B. Transcriptional profiles of PFL1410c (pfmrp2) and PFL1415w genes in clones 6A/mut and 11C/wt over the 48 h asexual life cycle measured by microarray log2 ratios. Time points in terms of hours post invasion (hpi) for each clone reflect the age of the parasite as calculated from the best fit spearman rank correlations to the reference transcriptome which was previously generated (Foth et al., 2011).C. Schematic diagram depicts the location of the breakpoint sites and the deletion segment (grey dotted box) within the 5′ upstream sequence of the PFL1410c (pfmrp2) gene in clone 6A/mut compared to clone 11C/wt and relative to the neighbouring genesPFL1415w. The numbers represent the distances between the deletion break points and the start codons of both PFL1410c (pfmrp2) and PFL1415w genes. A non-coding RNA, PF12TR011 (light green box), is potentially transcribed within the 1432 bp upstream of the PFL1415w (PlasmoDB version 9).
Figure 5
Figure 5
Characterization of protein expression levels of PfMRP2 in clone 6A/mut and 11C/wt and localization in the parasite by immunofluorescence assay.A. Immunoblot image of the PfMRP2 protein bands detected for each clone sampled at 6 h intervals over the 48 h asexual cycle. Total protein amounts corresponding to equal number of parasites per time point sample were resolved on the SDS-PAGE, blotted and subsequently reacted with the anti-PfMRP2 polyclonal peptide antibody. The same samples were probed with actin as a loading control.B. Relative protein expression levels of PfMRP2 measured by densitometer (Bio-Rad, USA). The protein profiles represent the summed abundance of all bands determined by their optical density (OD) over the IDC for clone 6A/mut (blue) and 11C/wt (red). For this plot the PfMRP2 OD values were normalized by the normalized by OD values for actin obtained identically.C. Immunofluorescence images of PfMRP2 localization labelled with Alexa488 during the development from ring to trophozoite and schizont stages in the wild-type clone 11C parasite. Nucleus was stained with DAPI (4′,6′-diamidino-2-phenylindole). The bottom panel shows dual labelling of the parasite at early schizont stage with anti-PfMRP2 (Alexa488) antibody and digestive vacuole-specific PfCRT antibody (Alexa594).
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