Genome wide adaptations of Plasmodium falciparum in response to lumefantrine selective drug pressure

Abstract

The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC(50) (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1S(LM), obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC(50) of the parental strain. However, after two weeks of culturing V1S(LM) in drug-free medium, the IC(50) returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1S(LM). Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance--they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.

Figure 1. Lumefantrine drug selection regimen.

Number of cycles during which V1S was cultured with varying Lumefantrine (LM) concentrations. In total, parasites were exposed to LM for 166 P. falciparum cycles, finally resulting in LM resistant V1S_LM.

Figure 2. Changes in gene expression profiles between LM resistant V1S_LM and LM sensitive V1S P. falciparum.

A. Heatmap of 589 expressed genes showing Differential Expression (DE) in at least one time point (F adjusted p<0.05). The 2 clusters highlighted with blue and red bars on the right hand side of the heatmap correspond to subtelomeric genes gradually switched off in the presence of LM, and transporters and cell cycle regulators gradually turned on in the presence of LM, respectively. Log2 ratio of V1S_LM vs. V1S expression is indicated by the color key ranging from −6 (blue, under-expression) to 6 (red, over-expression) B. Venn diagram showing the asexual life cycle distribution of DE genes. Analysis was based on linear modeling using Limma package of R/Bioconductor.

Figure 3. Gene Ontology (GO) analysis at main time points of the P. falciparum asexual life cycle.

The time points color legend is indicated at the top left corner of the barplot. GO enrichment is indicated as stacked percentages.

Figure 4. Correlation coefficients of log2 fold changes (FC) by RT-qPCR and microarray analysis.

The linear regression is indicated on the plot, with an r² = 0.7389.

Figure 5. Chromosomes 2 (A) and 10 (B) show over-expression of contiguous probes covering 21 and 22 CDS, respectively.

Amplification of the signals for the left arms of chromosomes 2 (A) and 10 (B) are enlarged for each time point as indicated. Every single coloured dot corresponds to a 25-mer probe: red is for 0 h, blue for 12 h, green for 24 h and yellow for 36 h. Underneath every enlarged chromosomal arm are pink bars indicating 100% robustness of signal amplification at p<0.01 (using SnoopCGH program with Smith–Waterman algorithm implementation). A normal distribution of the log ratios (y-axis) around the zero horizontal line is expected if the expression levels are the same along the chromosome (indicated as kilo base pair [kbp]). The CDS (represented under each chromosome by blue rectangles) contained within each amplified region are indicated on the right with their appropriate annotation (www.genedb.org). The genes marked with an * have been found significant at B>0 in the pairwise comparisons of the microarray data in at least one time point, while the underlined genes have been double checked by qRT-PCR.