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) CDP-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 CDP-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 loci were targetable and indicated that these genes are essential in P. berghei blood stage parasites. GFP-fusions of CCT, ECT and CK were found in the cytosol whereas the GFP-CEPT mainly localised in the endoplasmic reticulum. These results indicate that both CDP-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.

[1]  M. Nishijima,et al.  Regulation of phosphatidylcholine metabolism in mammalian cells. Isolation and characterization of a Chinese hamster ovary cell pleiotropic mutant defective in both choline kinase and choline-exchange reaction activities. , 1984, The Journal of biological chemistry.

[2]  Eileen Kraemer,et al.  PlasmoDB: a functional genomic database for malaria parasites , 2008, Nucleic Acids Res..

[3]  W. Martin,et al.  Isolation of Plasmodium berghei (malaria) parasites by ammonium chloride lysis of infected erythrocytes. , 1971, Nature: New biology.

[4]  Michael J. Thomas,et al.  Molecular Distinction of Phosphatidylcholine Synthesis between the CDP-Choline Pathway and Phosphatidylethanolamine Methylation Pathway* , 1999, The Journal of Biological Chemistry.

[5]  V. Choubey,et al.  Molecular characterization and localization of Plasmodium falciparum choline kinase. , 2006, Biochimica et biophysica acta.

[6]  A. Heck,et al.  The two biosynthetic routes leading to phosphatidylcholine in yeast produce different sets of molecular species. Evidence for lipid remodeling. , 2003, Biochemistry.

[7]  C. McMaster,et al.  Phosphatidylcholine biosynthesis via the CDP-choline pathway in Saccharomyces cerevisiae. Multiple mechanisms of regulation. , 1994, The Journal of biological chemistry.

[8]  J. Vance.  Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids. , 2008, Journal of lipid research.

[9]  H. Vial,et al.  Prodrugs of bisthiazolium salts are orally potent antimalarials. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[10]  N. Hall,et al.  Comparative genomics of malaria parasites. , 2005, Current opinion in genetics & development.

[11]  Kiaran Kirk,et al.  Membrane Transport in the Malaria-Infected Erythrocyte , 2001 .

[12]  S. Henry,et al.  Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes. , 1999, Progress in lipid research.

[13]  K. Marsh,et al.  Clinical features and pathogenesis of severe malaria. , 2004, Trends in parasitology.

[14]  G. Pessi,et al.  A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Brouwers,et al.  The CDP-ethanolamine Pathway and Phosphatidylserine Decarboxylation Generate Different Phosphatidylethanolamine Molecular Species* , 2007, Journal of Biological Chemistry.

[16]  C. McMaster,et al.  CDP-choline:1,2-diacylglycerol cholinephosphotransferase. , 1997, Biochimica et biophysica acta.

[17]  Gregory D. Schuler,et al.  Database resources of the National Center for Biotechnology Information: update , 2004, Nucleic acids research.

[18]  H. Vial,et al.  Comparison of the cellular and biochemical properties of Plasmodium falciparum choline and ethanolamine kinases. , 2009, The Biochemical journal.

[19]  Shelby L. Bidwell,et al.  Genome Sequence of Babesia bovis and Comparative Analysis of Apicomplexan Hemoprotozoa , 2007, PLoS pathogens.

[20]  Eric Maréchal,et al.  Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways[S] , 2010, Journal of Lipid Research.

[21]  G. Pessi,et al.  In Vivo Evidence for the Specificity of Plasmodium falciparum Phosphoethanolamine Methyltransferase and Its Coupling to the Kennedy Pathway* , 2005, Journal of Biological Chemistry.

[22]  Haiming Wang,et al.  ToxoDB: an integrated Toxoplasma gondii database resource , 2007, Nucleic Acids Res..

[23]  C. Kent.  Regulatory enzymes of phosphatidylcholine biosynthesis: a personal perspective. , 2005, Biochimica et biophysica acta.

[24]  R. Sinden,et al.  Generation of gene targeting constructs for Plasmodium berghei by a PCR-based method amenable to high throughput applications. , 2006, Molecular and biochemical parasitology.

[25]  G. Carman,et al.  Regulation of Phospholipid Synthesis in the Yeastcki1Δeki1Δ Mutant Defective in the Kennedy Pathway: THECHO1-ENCODED PHOSPHATIDYLSERINE SYNTHASE IS REGULATED BY mRNA STABILITY , 2004 .

[26]  G. Pessi,et al.  Disruption of the Plasmodium falciparum PfPMT Gene Results in a Complete Loss of Phosphatidylcholine Biosynthesis via the Serine-Decarboxylase-Phosphoethanolamine-Methyltransferase Pathway and Severe Growth and Survival Defects* , 2008, Journal of Biological Chemistry.

[27]  G. Carman,et al.  Phosphorylation of Saccharomyces cerevisiae Choline Kinase on Ser30 and Ser85 by Protein Kinase A Regulates Phosphatidylcholine Synthesis by the CDP-choline Pathway* , 2002, The Journal of Biological Chemistry.

[28]  A. Heck,et al.  The Yeast Phospholipid N-Methyltransferases Catalyzing the Synthesis of Phosphatidylcholine Preferentially Convert Di-C16:1 Substrates Both in Vivo and in Vitro* , 2004, Journal of Biological Chemistry.

[29]  C. McMaster,et al.  CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase. , 1997, Biochimica et biophysica acta.

[30]  H. Vial,et al.  Phospholipids in parasitic protozoa. , 2003, Molecular and biochemical parasitology.

[31]  C. Janse,et al.  Stable expression of green fluorescent protein in blood and mosquito stages of Plasmodium berghei. , 1998, Molecular and biochemical parasitology.

[32]  H. Vial,et al.  Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers. , 1984, Biochimica et biophysica acta.

[33]  Ruth H. Hughes,et al.  Characterization of the choline carrier of Plasmodium falciparum: a route for the selective delivery of novel antimalarial drugs. , 2004, Blood.

[34]  S. Henry,et al.  A Role for Phospholipase D (Pld1p) in Growth, Secretion, and Regulation of Membrane Lipid Synthesis in Yeast* , 1998, The Journal of Biological Chemistry.

[35]  Matthew Berriman,et al.  GeneDB: a resource for prokaryotic and eukaryotic organisms , 2004, Nucleic Acids Res..

[36]  J. Jensen,et al.  Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth. , 1985, The Journal of protozoology.

[37]  C. McMaster,et al.  Studies employing Saccharomyces cerevisiae cpt1 and ept1 null mutants implicate the CPT1 gene in coordinate regulation of phospholipid biosynthesis. , 1994, The Journal of biological chemistry.

[38]  Philippa Rhodes,et al.  ApiDB: integrated resources for the apicomplexan bioinformatics resource center , 2006, Nucleic Acids Res..

[39]  G. Carman,et al.  Isolation and Characterization of the Saccharomyces cerevisiae EKI1 Gene Encoding Ethanolamine Kinase* , 1999, The Journal of Biological Chemistry.

[40]  D. Sullivan,et al.  Hemoglobin metabolism in the malaria parasite Plasmodium falciparum. , 1997, Annual review of microbiology.

[41]  R. Tewari,et al.  Calcium and a Calcium-Dependent Protein Kinase Regulate Gamete Formation and Mosquito Transmission in a Malaria Parasite , 2004, Cell.

[42]  K. Matuschewski,et al.  High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. , 2006, Molecular and biochemical parasitology.

[43]  H. Vial,et al.  Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation. , 1997, The Biochemical journal.

[44]  D. Vance,et al.  Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion? , 1988, The Journal of biological chemistry.

[45]  J. Vance.  Molecular and cell biology of phosphatidylserine and phosphatidylethanolamine metabolism. , 2003, Progress in nucleic acid research and molecular biology.

[46]  M. Ferguson,et al.  Phosphatidylethanolamine in Trypanosoma brucei Is Organized in Two Separate Pools and Is Synthesized Exclusively by the Kennedy Pathway* , 2008, Journal of Biological Chemistry.

[47]  C. Kent,et al.  Enzymatic and Cellular Characterization of a Catalytic Fragment of CTP:Phosphocholine Cytidylyltransferase α* , 1999, The Journal of Biological Chemistry.

[48]  R. Wilson,et al.  Separation of viable schizont-infected red cells of Plasmodium falciparum from human blood. , 1978, Annals of tropical medicine and parasitology.