RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1432300; Gene model (P.falciparum): PF3D7_1216600; Gene product: cell traversal protein for ookinetes and sporozoites (CelTOS; cell traversal protein for ookinetes and sporozoites)
Name tag: triple-HA
Phenotype Fertilization and ookinete;
Last modified: 23 October 2018, 16:11
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) : 30315162
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/ResearcherFang H, Billker O, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine, Faculty of Medicine
Name InstituteUniversity of Geneva
Name of the mutant parasite
RMgm numberRMgm-4558
Principal namePbcelTOS-3xHA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookinetePbcelTOS-3xHA expression in micronemes of ookinetes
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal HA-tagged version of celTOS.

Protein (function)
CelTOS is localized to the micronemes of ookinetes and (salivary gland) sporozoites and is secreted. It plays a role in migration of ookinetes through cells of the mosquito midgutwall and in hepatocyte traversal of sporozoites.

PbcelTOS-3xHA expression in micronemes of ookinetes

The mutant in this study was generated to screen for genetic interactions among protein kinases. In this study a role of CDPK4 during erythrocytic (asexual blood stage) proliferation has been found, which was dependent on a negative interaction with PKG.

From the paper:
In P. falciparum schizonts and P. berghei sporozoites, PKG controls microneme secretion, a process that is also critical to sustain gliding in ookinetes. The PKG-inhibitor C2 blocks the secretion of the ookinete microneme protein CelTOS-3xHA into the culture supernatant, specifically in the inhibitor-sensitive line, indicating that signalling through PKG is required for microneme secretion also in ookinetes. However, depletion of CDPK1 and chemical inhibition of CDPK4 does not affect secretion of CelTOS-3xHA either individually, or in combination. In marked contrast, deletion of CDPK3, an ookinete-specific CDPK needed for optimal gliding, does reduce secretion of CelTOS-3xHA. Complementation of cdpk3 deletion ascertained that this effect was due to the absence of CDPK3 expression. Furthermore, CDPK3 does not appear to interact functionally with CDPK4, since addition of inhibitor 1294 does not decrease motility further in the CDPK3-KO. Altogether, this suggests that the main function of CDPK3 is to control microneme secretion downstream of PKG but independently of CDPK4, while CDPK1 and CDPK4 perform complementary functions in supporting efficient gliding.

From the Abstract:
Most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito.

Additional information

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1432300
Gene Model P. falciparum ortholog PF3D7_1216600
Gene productcell traversal protein for ookinetes and sporozoites
Gene product: Alternative nameCelTOS; cell traversal protein for ookinetes and sporozoites
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector066761
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPreparation of targeting vectors. 3xHA tagging, knockout and allelic replacement constructs in P. berghei were generated using phage recombineering in Escherichia coli tryptic soy agar (TSA) bacterial strain with PlasmoGEM vectors (http://plasmogem.sanger.ac.uk/). For final targeting vectors not available in the PlasmoGEM repository, generation tagging constructs was performed using sequential recombineering and gateway steps. For each gene of interest (goi), the Zeocin-resistance/Phe-sensitivity cassette was introduced using oligonucleotides goi HA-F x goi HA-R for 3xHA tagging. The modified library inserts were released from the plasmid backbone using NotI.
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