Summary

RMgm-4155
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0615200; Gene model (P.falciparum): PF3D7_0717500; Gene product: calcium-dependent protein kinase 4 (CDPK4)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 22 May 2017, 18:42
  *RMgm-4155
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28481199
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, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine
Name InstituteUniversity of Geneva
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4155
Principal nameCDPK4-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNo male gametes are formed after activation of male gametocytes.
Fertilization and ookineteNo male gametes are formed after activation of male gametocytes (no exflagellation).
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CDPK4 (and is drug-selectable marker free).

Protein (function)
CDPK4 belongs to an expanded family of Ca2+ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. In plants, CDPKs translate Ca2+ signals generated by external stimuli into cellular responses, thereby regulating cell division and differentiation, the development of tolerance to stress stimuli and the specific defense responses to pathogens.

CDPK4 plays a role in male gamete formation after activation of male gametes. It plays a role in male gametocytes axoneme formation, formation of mitotic spindles and DNA synthesis. Evidence has been presented for an additional role in sporozoites: reduced invasion of hepatocytes (RMgm-1510).  See PF3D7_0717500 for other CDPK4 mutants

Phenotype
No male gametes are formed after activation of male gametocytes (no exflagellation).
Through analysis of a number of mutants expressing mutated CDPK4 proteins, combined with inhibitor studies, evidence is presented for the following: 'CDPK4 controls three processes essential to progress from a single haploid microgametocyte to the release of eight flagellated microgametes in Plasmodium berghei. A myristoylated isoform is activated by Ca2+ to initiate a first genome replication within twenty seconds of activation. This role is mediated by a protein of the SAPS-domain family involved in S-phase entry. At the same time, CDPK4 is required for the assembly of the subsequent mitotic spindle and to phosphorylate a microtubule-associated protein important for mitotic spindle formation. Finally, a non-myristoylated isoform is essential to complete cytokinesis by activating motility of the male flagellum. This role has been linked to phosphorylation of an uncharacterised flagellar protein. 

Additional information

Other mutants
See PF3D7_0717500 for other CDPK4 mutants

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0615200
Gene Model P. falciparum ortholog PF3D7_0717500
Gene productcalcium-dependent protein kinase 4
Gene product: Alternative nameCDPK4
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-087803
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe hdhfr/yfcu drug-selectable marker has been removed by negative selection
Additional remarks selection procedureThe hdhfr/yfcu drug-selectable marker has been removed by negative selection
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