Mutant/mutation
The mutant expresses a GFP-tagged (C-terminal) version of CCT. The single cross-over event result in replacement of the endogenous gene with a cct gene fused to a gfp-tag at the C-terminus.

Protein (function)
See additional information.

Phenotype
Western analysis using GFP-antobodies and fluorescence microscopy showed (low) CCT-GFP expression in (the cytoplasm of) blood stages.

Additional information
The unsuccessful attempts to disrupt the cct gene (see RMgm-335) suggest that this gene is essential for blood stage development (see also below). Tagging of the gene with gfp was successful.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the major phospholipids of cellular membranes. These two phospholipids represent 40–50% and 35–40% of the total phospholipids in Plasmodium. Biosynthesis of PC and PE has been well studied revealing the presence of multiple pathways: de novo CDPcholine (CDP-Cho) and CDP-ethanolamine (CDP-Etn), also called the Kennedy pathways, and the CDP-diacylglycerol (CDP-DAG) dependent pathway.

The Kennedy pathways initiate from exogenous choline and ethanolamine involving choline kinase (CK; EC 2.7.1.32; PF14_0020; PBANKA_104010) and ethanolamine kinase (EK; EC 2.7.1.82), followed by the choline-phosphate cytidylyltransferase (CCT; EC 2.7.7.15; MAL13P1.86; PBANKA_141510) and ethanolamine-phosphate cytidylyltransferase (ECT; EC 2.7.7.14; PF13_0253; PBANKA_136050) 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; 2.7.8.2 (CPT and 2.7.8.1 (EPT); PFF1375c; PBANKA_112700). The de novo Kennedy pathways initiate with the phosphorylation of the polar heads by CK and EK. The phosphorylated polar heads are subsequently coupled to CTP, by CCT and ECT thus generating CDP-Cho and CDP-Etn, respectively.

P. berghei CCT and ECT contain the signature sequence of the cytidylyltransferase family, RxxG(V/I)STT, the CTP binding site HxGH, and the lysine residues involved in P-Cho and in Etn binding. This indicates that PbCCT and PbECT can be expected to carry phosphocholine and phosphoethanolamine transferase activities, respectively.

An additional route termed serine decarboxylation-phosphoethanolamine methylation (SDPM) pathway has been identified in P. falciparum. In this plant-like pathway that connects different routes, the polar head groups of PE and PC are synthesized from serine that is either directly imported from the host or obtained through degradation of host cell haemoglobin. However, phosphoethanolamine N80 methyltransferase (PMT) activity is absent from P. berghei and an ortholog to the P. falciparum pmt gene (MAL13P1.214) could not be identified in the genome of P. berghei and other rodent malaria parasites.

Two different approaches were used to attempt to knockout the cept (RMgm-336), cct (RMgm-335), ect (RMgm-337) and ck (RMgm-334) genes in P. berghei. The first approach was based on double homologous recombination using PCR amplicons to replace the endogenous genes by a selectable marker cassette. The second consisted of gene disruption by single cross-over event leading to duplication of the integrated sequence and the generation of two non-functional copies of the target gene. P. berghei transfections were performed in two independent trials for the double recombination approach and in two independent trials for the gene disruption strategy.

In order to demonstrate that the failure to generate disrupted cept, cct, ect and ck loci was not due to the inaccessibility of these loci to homologous recombination, simultaneously to the gene disruption experiments, transfection experiments were performed with constructs carrying GFP-tagged genes: cept-gfp (RMgm-340), cct-gfp (RMgm-339), ect-gfp (RMgm-341) and ck-gfp (RMgm-338). For all genes, integration in their respective locus was successful generating a full-length version of the gene fused to GFP. These results showed that all these loci were accessible for homologous recombination, but deletion or disruption of the coding sequences could not be obtained.

These results indicate that in P. berghei the CDP-DAG pathway cannot compensate for the absence of the Kennedy routes. When analysing the CDP-Cho and CDP-Etn pathways in P. berghei separately, disruption of neither cct nor ect, could be obtained indicating that each branch of the Kennedy pathways is essential for parasite survival. This indicates that the PC provided by the CDP-Cho branch of the Kennedy pathway cannot be compensated by PE coming from the Kennedy or the CDP-DAG pathway using the PE-methylation enzymes. Most interestingly, de novo synthesized PE cannot be compensated by PE obtained from the CDP-DAG pathway. Again, this is different from yeast where choline kinase or ethanolamine kinase mutants exhibit growth properties similar to the wild type, indicating that none of the Kennedy pathways is essential.

These results suggest an absence of redundancy in the synthesis of PC and PE in Plasmodium what is in contrast to yeast, where each de novo pathway can be compensated by the CDP-DAG pathway.

FIG 1. Biosynthesis pathways for PC, PE and PS in Plasmodium.
Lipids are represented as grey ovals and enzymes in italic and grey. Specific pathways to P. falciparum are marked by blue dotted arrows. The enzymes analysed by gene disruption and gene tagging are boxed. Plasmodium species in which enzyme pathways take place are indicated above the enzyme names.
DAG, diacylglycerol; CDP-DAG, cytidine-diphospho-diacylglycerol; CK, choline kinase; CCT, CTP: phosphocholine cytidylyltransferase; EK, ethanolamine kinase; ECT, CTP: phosphoethanolamine cytidylyltransferase; CEPT, choline/ethanolamine-phosphotransferase; PSS, phosphatidylserine synthase; PSD, phosphatidylserine decarboxylase; PEMT, phosphatidylethanolamine N-methyltransferase; SD, serine decarboxylase; PMT, phosphoethanolamine N-methyltransferase.

Other mutants
RMgm-335: Negative attempts to disrupt the cct gene