Altered expression of K13 disrupts DNA replication and repair in Plasmodium falciparum

Background: Plasmodium falciparum has evolved resistance to the artemisinin component of the frontline antimalarial treatment Artemisinin-based Combination Therapy in South East Asia. Millions of lives will be at risk if resistance spreads to Africa. Single non-synonymous mutations in the propeller region of PF3D7_1343700, “K13”are implicated in resistance. In this work, we use transcriptional profiling to characterize a laboratory-generated k13 insertional mutant previously demonstrated to have increased sensitivity to artemisinins to explore the functional role of k13. Results: A set of RNA-seq and microarray experiments confirmed that the expression profile of k13 is specifically altered during the early ring and early trophozoite stages of the mutant intraerythrocytic development cycle. The down-regulation of k13 in this mutant during the early ring stage is associated with a transcriptome shift towards a more trophozoite-like state. To discover the specific downstream effect of k13 dysregulation, we developed a new computational method to search for differential gene expression while accounting for the temporal sequence of transcription. We found that the strongest biological signature of the transcriptome shift is an up-regulation of DNA replication and repair genes during the early ring developmental stage and a down-regulation of DNA replication and repair genes during the early trophozoite stage; by contrast, the expressions of housekeeping genes are unchanged. This effect, due to k13 dysregulation, is antagonistic, such that k13 levels are negatively correlated with DNA replication and repair gene expression. Conclusion: Because a hypothesized mode of action of artemisinins is oxidative stress our results support a role for k13 as a stress response regulator, consistent with the role of its human homolog Keap1, that regulates DNA replication and repair genes in response to oxidative stress. The parasite strains NF54 and PB58 (the K13 mutant) were maintained in identical standard culture conditions and synchronized by 3 rounds of sorbitol synchronization. The time points collected were 6 (n=3), 12 (n=2), 24 (n=5), 38 (n=3), and 48 (n=3 for wild-type and n=2 for K13 mutant) hours after time zero. Time zero was defined as the time when the synchronized culture was half late schizonts and half early rings.