Global RNA expression profiling of k13-edited mutant and isogenic wild-type Plasmodium falciparum malaria parasites during the parasite's intra-erythrocytic developmental cycle and the parasite's response to dihydroartemisinin (DHA).

K13 mutations are causal for artemisinin resistance in Plasmodium falciparum human malaria. The objective of our study is to characterize gene expression signatures associated with K13 mutations by comparing transcriptional profiles and response to DHA of K13 mutant (C580Y, R539T) and isogenic wild-type lines that were generated by zinc finger nuclease (ZFN) based editing in a long-term adapted (Dd2) and a contemporary laboratory-adapted clinical isolate (Cam3.II). We employed microarray technology to establish the global gene expression profiles of these five parasite lines (Dd2 K13 WT, Dd2 K13 R539T, Dd2 K13 C580Y, Cam3.II K13 R539T, Cam3.II K13 WT) from early ring to late schizont stage over the 48h intra-erythrocytic developmental cycle. Samples were collected at 0, 3, 6, 16, 24, 32, 48h time points for Dd2 lines and 0, 8, 16, 24, 32, 40, 48h for Cam3.II lines. The RNA expression profiles of the K13 mutant and isogenic wild type parasites were compared to identify changes at basal levels. Parasites were also treated with either 700nM dihydroartemisinin (DHA) or 0.1% DMSO vehicle for 6h and sampled at 3, 6, 24, 32 and 48h post-treatment to identify differences in transcriptional response to DHA associated with K13 mutations. Three independent experiments were performed for each parasite strain and condition, except for Cam3.II DHA treatment which was only done once. Extraction of total RNA, synthesis and amplification of target DNA was carried out as described (in Z. Bozdech, S. Mok & A. P. Gupta (2013) DNA microarray-based genome-wide analyses of Plasmodium parasites. Methods in molecular biology 923: 189-211). Cy5-labelled cDNA samples were hybridized against a common Cy3-labelled pool consisting of RNA from mixed 3D7 asexual stages on a custom microarray platform.