RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1040100; Gene model (P.falciparum): PF3D7_1401800; Gene product: choline kinase (CK)
Name tag: GFP-mutant3
Phenotype Asexual bloodstage;
Last modified: 25 May 2010, 21:53
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20478340
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherS. Déchamps; K. Wengelnik; H.J. Vial; L. Gannoun-Zaki
Name Group/DepartmentDynamique des Interactions Membranaires Normales et Pathologiques
Name InstituteUniversité Montpellier
Name of the mutant parasite
RMgm numberRMgm-338
Principal nameCK-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Asexual blood stageWestern analysis using GFP-antibodies and fluorescence microscopy showed CK-GFP expression in (the cytoplasm of) blood stages.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

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

Protein (function)
See additional information.

Western analysis using GFP-antibodies and fluorescence microscopy showed CK-GFP expression in (the cytoplasm of) blood stages.

Additional information
The unsuccessful attempts to disrupt the ck gene (see RMgm-334) 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; PF14_0020; PBANKA_104010) and ethanolamine kinase (EK; EC, followed by the choline-phosphate cytidylyltransferase (CCT; EC; MAL13P1.86; PBANKA_141510) and ethanolamine-phosphate cytidylyltransferase (ECT; EC; 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; (CPT and (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.

In P. berghei CK, the Brenner's phosphotransferase motif FCHNDLQENN is found. The choline kinase motif hhDhExxxxNxxxxDhxNhhxE and the conserved hydrophobic residues (Trp394, Trp397, and Tyr416) are present and make it likely that the ck gene encodes a choline kinase enzyme.

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-334: Negative attempts to disrupt the ck gene

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1040100
Gene Model P. falciparum ortholog PF3D7_1401800
Gene productcholine kinase
Gene product: Alternative nameCK
Details of the genetic modification
Name of the tagGFP-mutant3
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid EcoRV
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe single cross-over event result in replacement of the endogenous gene with a ck gene fused to a GFP-tag at the C-terminus.

To generate the pDR-CK-GFP construct, the nucleotide sequence was modified without changing the amino acid sequence to add an EcoRV site and amplified using genomic DNA as a template with the following primer pairs: p5/p6, and p7/p8. Amplification using primers p5 and p8 gave the final fragment of 1110 bp (nucleotides 214-1323 of the Pbck ORF) that was inserted in pDR009.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Additional information primer 1p5; endogenous ck F / GFP target (KpnI)
Additional information primer 2p6; endogenous ck R / GFP target (EcoRV)
Additional information primer 3p7; endogenous ck F / GFP target (EcoRV)
Additional information primer 4p8; endogenous ck R / GFP target (ApaI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6