Project description:Concerns about impending failure of artemisinin compounds (ART) have grown with global use of ART-based combination therapy (ACT) against malaria. WHO has defined Plasmodium falciparum resistance to ART as prolonged parasite clearance half-life in vivo (t1/2) plus the presence of certain K13 Kelch-propeller substitutions, e.g. C580Y. Recrudescences and fever clearance times after ART monotherapy, however, have not correlated well with these criteria. We have crossed K13 C580 wild-type and 580Y-mutant parasites for ART studies in Aotus. Artesunate treated C580- but not 580Y-infections recrudesced requiring retreatment, and K13 type had little or no effect on t1/2. These results challenge K13 and t1/2 variations as markers of increased resistance to ART per se and emphasize the need for effective partner drugs in ACTs.
Project description:Plasmodium falciparum intraerythrocytic stage transcription four hour time course for NF54 and PB58 (an isogenic line with a piggyBac transposon in the 5’ UTR of K13 of K13 that alters K13 expression).
Project description:Plasmodium falciparum intraerythrocytic stage transcription four hour time course for NF54 and PB58 (an isogenic line with a piggyBac transposon in the promotor of K13 that alters K13 expression)
Project description: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).
Project description: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.
Project description:ChIP-seq experiments were performed to profile PfH3.3 (PF3D7_0617900) in the malaria parasite Plasmodium falciparum. Sequencing of ChIP samples showed enrichment of PfH3.3 at GC-rich coding sequences and subtelomeric repetitive regions throughout the intraerythrocytic life cycle and additionally in intergenic regions during trophozoite stages. Also the promoter and the coding sequence of the active and poised var2CSA gene were marked (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1)
Project description:K13 mutations are causal for artemisinin resistance in Plasmodium falciparum human malaria. We characterized changes in protein abundance associated with K13 mutations during the parasite's 48h intra-erythrocytic developmental cycle by comparing protein expression profiles of K13 mutant (C580Y, R539T) and isogenic wild-type lines that were generated by zinc finger nuclease (ZFN) based editing in a laboratory-adapted clinical isolate (Cam3.II). For each parasite line, we harvested tightly synchronized ring and trophozoite parasites on two independent occasions, except for the C580Y line which was harvested only once at trophozoite stage.
Project description:Epigenetic mechanisms have been poorly understood in Plasmodium falciparum, the causative agent of malaria. To elucidate stage specific epigenetic regulations in P. falciparum, we performed genome-wide mapping of various histone modifications, nucleosomes and RNA Polymerase II. Our comprehensive analysis suggest that transcription initiation and elongation are distinct in Plasmodium. In this study, by analyzing histone modifications, nucleosome occupancy and RNA Polymerase II (Pol II) at three different IEC developmental stages of Plasmodium; ring, trophozoite and schizont, we tried to unravel the epigenetic mechanism associated with gene regulation. Examination of H3K27me3, H3K4me3, H3K9me3, H3K14ac, H3K4me1, H3K79me3, H3K27ac, H3K4me2, H3K9ac, H4ac, RNA Pol II and Histone H3 at three different stages of Plasmodium falciparum
Project description:The emergence of artemisinin resistance in Southeast Asia, dictated by mutations in the Plasmodium falciparum k13 gene, has compromised antimalarial efficacy and created a core vulnerability in the global malaria elimination campaign. Applying quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we observe that K13 mutations reprogram multiple aspects of intra-erythrocytic parasite biology. These changes impact its cell cycle periodicity, the unfolded protein response and protein degradation, vesicular trafficking and endocytosis, and mitochondrial functions including the TCA cycle, the electron transport chain, and redox regulation. Ring-stage artemisinin resistance mediated by the K13 R539T mutation was neutralized using atovaquone, an electron transport chain inhibitor. Our data suggest that modification of mitochondrial physiology, accompanied by other processes to reduce artemisinin’s proteotoxic effects, help protect parasites against this pro-oxidant drug, allowing resumption of growth once the rapidly-cleared artemisinins have reached sub-therapeutic levels.
