The NTP Generating Activity of Pyruvate Kinase II is Critical for Apicoplast Maintenance in Plasmodium falciparum

The apicoplast of Plasmodium falciparum parasites is believed to rely on the import of three-carbon phosphate compounds for use in organelle anabolic pathways, in addition to the generation of energy and reducing power within the organelle. We generated a series of genetic deletions in an apicoplast metabolic bypass line to determine which genes involved in apicoplast carbon metabolism are required for blood-stage parasite survival and organelle maintenance. We were able to demonstrate that pyruvate kinase II (PyrKII), the sole predicted source of pyruvate and NTPs within the apicoplast, is essential for organelle maintenance. However, deletions within apicoplast pathways that metabolize pyruvate did not result in organelle disruption, indicating that NTPs generated by PyrKII are the products required for this process. Enzymatic characterization of PyrKII revealed that it was active against all NDPs and dNDPs tested with broad substrate specificity, suggesting that it may be capable of generating any and all NTPs and dNTPs within the organelle. Conditional mislocalization of PyrKII resulted in a decrease in transcript levels within the apicoplast that preceded organelle disruption, suggesting that PyrKII is required for organelle maintenance due to its role in nucleotide triphosphate generation within the apicoplast. PyrKII CLD parasites were cultured for 48 hours with samples taken every eight hours for transcriptomics and processed in triplicate. Nf54 attB parasites without treatment were cultures for 48 hours with samples taken every eight hours and processed in quadruplicate, except for Time 48 Control which is in triplicate. Time 8 for the PyrKII CLD dataset corresponds to the same parasite developmental stage as Time 0 of the Nf54 attB dataset. PyrKII CLD timepoints 8-56 were compared to Nf54 attB timepoints 0-48 for analysis.