RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1114900; Gene model (P.falciparum): PF3D7_0515300; Gene product: phosphatidylinositol 3-kinase (PI3K)
PhenotypeNo phenotype has been described
Last modified: 10 September 2010, 19:18
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 4
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20709789
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).
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherTawk, L; Vial H; Wengelnik, K
Name Group/DepartmentCNRS UMR5235
Name InstituteUniversité Montpellier 2

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1114900
Gene Model P. falciparum ortholog PF3D7_0515300
Gene productphosphatidylinositol 3-kinase
Gene product: Alternative namePI3K
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPCR construct
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption Since no sequence information for the 5' end of the PI3K gene was available (corresponding to the first 387 aa of the P. chabaudi sequence), a PI3K knock-out construct was generated that upon double homologous recombination would lead to deletion of the accessory and catalytic domains generating a truncated protein without a functional kinase domain
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt the gene PIK3 (phosphatidylinositol 3-kinase, putative) indicate an essential role of this protein during asexual blood stage development.

The PI3-kinase gene was disrupted using a PCR-based method. The gene deletion was achieved by transfection with linear DNA obtained by PCR amplification without prior cloning. In the transfection construct, the resistance gene is flanked by two short regions homologous to the target locus. Two sequential PCR reactions were performed. Two 500 bp PI3K homologous sequences were amplified with primers 161/162 and 168/164. The obtained amplicons were then combined with linearised vector pDEFhDHPEA that supplied the human DHFR resistance cassette and PCR amplified with primers 161 and 164 to yield a final KO construct of 3 kbp. In four independent transfections, pyrimethamine resistant parasites were obtained suggesting successful transfection. However, none of the populations showed integration of the recombinant DNA into the PI3K locus, as analysed by PCR.

In order to control that targeting of the PI3K locus was possible, three additional constructs were generated for single homologous recombination. Two constructs aimed at tagging PI3-kinase at the C-terminal end with either green fluorescent protein (GFP) or a triple hemagglutinin (3HA) tag. A third construct was identical to the 3HA-tag plasmid except that the stop codon of the PI3K open reading frame was maintained, thus not changing the coding sequence upon integration while introducing the same genetic control elements as with the authentic tagging constructs. Correct genome integration was only achieved with the control construct that maintained the stop codon, while modifying the PI3-kinase sequence by adding a GFP- or 3HA-tag was unsuccessful in four independent transfections.

In an Abstract presented at Woods Hole in 2007 it has been reported that it was impossible to knock out PI3K in P. berghei, indicating that this kinase is essential for blood stage development (see below for the Abstract). In the abstract no details of the knock-out attempts and DNA constructs used for gene disruption were available. The Woods Hole Abstract is cited in the following paper Vaid et al.( 2010) PfPI3K, a Phosphatidylinositol-3 kinase from Plasmodium falciparum, is exported to the host erythrocyte and is involved in hemoglobin trafficking. Blood 115, 2500-07.

2007 Molecular Parasitology: Abstracts
2007 MPM - Abstract #269A
Phosphatidylinositol 3-Phosphate in Plasmodium blood stage development
Lina Tawk, Bernard Payrastre, Henri Vial, Christian Roy, Kai Wengelnik

The inositol head group of the membrane lipid phosphatidylinositol can be phosphorylated at several positions by specific kinases producing phosphoinositides (PIPs) that play important roles in many cellular processes. PI3P (phosphatidylinositol 3-monophosphate), the product of PI3-kinase, is involved in vesicular trafficking from the endosome to the lysosome in mammalian cells or to the vacuole in yeast. We are interested in studying the function of PI3P in Plasmodium. PFE0765w encodes a PI3-kinase of class III, an extremely long protein of 2133 amino acids containing numerous highly repetitive sequences. It was impossible to knock out this gene in P. berghei indicating that this kinase is essential for blood stage development. Phospholipids of P. falciparum-infected red blood cells were extracted and the individual PIPs analysed by HPLC. The PI3P was found to be in relatively high amounts, indicating that PI3P may play an important role in the life cycle of the parasite. Only PI4P and PI(4,5)P2 were detected in uninfected red blood cells. PI3P is known to be bound specifically by proteins containing FYVE and PX domains. Surprisingly, the P. falciparum genome codes for just two proteins containing a PX domain and one protein containing a FYVE domain. These three domains were expressed in E. coli in fusion with GST. One PX domain specifically bound PI3P in vitro. We are currently trying to determine the subcellular localization of PI3P in Plasmodium by using PI3P specific probes either expressed as GFP fusions in the parasite or used as recombinant proteins on fixed cells. The intraparasitic localization of PI3P and the characterization of PI3P-binding proteins will shed light on the implication of PI3P in Plasmodium blood stage development.

See also the abstract of the paper of Vaid et al.( 2010) PfPI3K, a Phosphatidylinositol-3 kinase from Plasmodium falciparum, is exported to the host erythrocyte and is involved in hemoglobin trafficking. Blood 115, 2500-07.

Polyphosphorylated phosphoinositides (PIPs) are potent second messengers, which trigger a wide variety of signaling and trafficking events in most eukaryotic cells. However, the role and metabolism of PIPs in malaria parasite Plasmodium have remained largely unexplored. Our present studies suggest that PfPI3K, a novel phosphatidylinositol-3-kinase (PI3K) in Plasmodium falciparum, is exported to the host erythrocyte by the parasite in an active form. PfPI3K is a versatile enzyme as it can generate various 3'-phosphorylated PIPs. In the parasite, PfPI3K was localized in vesicular compartments near the membrane and in its food vacuole. PI3K inhibitors wortmannin and LY294002 were effective against PfPI3K and were used to study PfPI3K function. We found that PfPI3K is involved in endocytosis from the host and trafficking of hemoglobin in the parasite. The inhibition of PfPI3K resulted in entrapment of hemoglobin in vesicles in the parasite cytoplasm, which prevented its transport to the food vacuole, the site of hemoglobin catabolism. As a result, hemoglobin digestion, which is a source of amino acids necessary for parasite growth, was attenuated and caused the inhibition of parasite growth.
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
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6