The Kennedy phospholipid biosynthesis pathways are refractory to genetic disruption in Plasmodium berghei and therefore appear essential in blood stages

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main membrane phospholipids (PLs) of Plasmodium parasites and can be generated by the de novo (Kennedy) COP-choline and CDP-ethanolamine pathways and by the CDP-diacylglycerol dependent pathway. The Kennedy pathways initiate from exogenous choline and ethanolamine involving choline kinase (CK) and ethanolamine kinase (EK), followed by the choline-phosphate cytidylyltransferase (CCT) and ethanolamine-phosphate cytidylyltransferase (ECT) that catalyse the formation of COP-choline and CDP-ethanolamine. Finally, in Plasmodium, PC and PE are apparently synthesized by a common choline/ethanolamine-phosphotransferase (CEPT). Here, we have studied the essential nature of the Kennedy pathways in Plasmodium berghei, a rodent malaria parasite. Sequence analysis of the P. berghei CEPT, CCT, ECT and CK enzymes revealed the presence of all catalytic domains and essential residues and motifs necessary for enzymatic activities. Constructs were designed for the generation of gene knockout and GFP-fusions of the cept, cct, ect and ck genes in P. berghei. We found that all four genes were consistently refractory to knockout attempts. At the same time, successful tagging of these proteins with GFP demonstrated that the lad were targetable and indicated that these genes are essential in P. berghei blood stage parasites. GFP-fusions of CCT, Ea and CK were found in the cytosol whereas the GFP-CEPT mainly localised in the endoplasmic reticulum. These results indicate that both COP-choline and CDP-ethanolamine de novo pathways are essential for asexual P. berghei development and are non-redundant with other possible sources of PC and PE. (C) 2010 Elsevier B.V. All rights reserved.