RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1230200; Gene model (P.falciparum): PF3D7_0615500; Gene product: cdc2-related protein kinase 5 (CRK5)
Name tag: GFP
Phenotype Gametocyte/Gamete; Oocyst;
Last modified: 21 August 2020, 17:14
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) : 32568069
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/ResearcherBalestra AC, Brochet M
Name Group/DepartmentFaculty of Medicine
Name InstituteUniversity of Geneva
Name of the mutant parasite
RMgm numberRMgm-4847
Principal nameCRK5-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot tested
Gametocyte/GameteThe tagged protein was localised to the nucleus in non-activated gametocytes and at 1 min after activation of male gametocytes showed an additional location at the mitotic spindle as suggested by co-localisation with tubulin
Fertilization and ookineteNot tested
OocystGFP expression observed in oocysts
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal GFP-tagged version of CRK5 

Protein (function)
From the Abstract: 'Cell cycle transitions are generally triggered by variation in the activity of cyclindependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.'

Expression not tested in detail. The tagged protein was localised to the nucleus in non-activated gametocytes and at 1 min after activation of male gametocytes showed an additional location at the mitotic spindle as suggested by co-localisation with tubulinExpression observed in oocysts. The mutant is used for mass-spec analyses of co-precipitated proteins in activated male gametocytes.

Additional information
- Previous attempts to disrupt P. berghei crk5 had suggested the gene is essential for asexual blood-stage proliferation (Tewari et al., 2010). However, using long sequence homology regions to replace crk5 with a T. gondii DHFR/TS resistance marker resistant KO-parasites were obtained.
- We tagged the endogenous crk5 gene with an AID/HA epitope tag to degrade the fusion protein in presence of auxin in a strain expressing the Tir1 protein (Philip and Waters, 2015). Addition of the AID/HA tag to the CRK5 C-terminus imposed a significant fitness cost, with a 2-fold decrease in exflagellation in the absence of auxin, but importantly, depletion of CRK5-AID/HA by one hour of auxin treatment of mature gametocytes prior to activation resulted in a dramatic reduction in exflagellation.

Evidence in this study is presented that:
- CRK5 is a key regulator of gametogony and sporogony in the mosquito
- Phosphoproteome kinetics point to direct phosphorylation of the pre-replicative complex by CRK5
- CRK5 is required for both S- and M- phases during P. berghei gametogony
- We observed an average 2-fold fewer polyploid (>2N) gametocytes 1 min post-activation , demonstrating a dependency on CRK5 for DNA replication during male gametogony.
- Cells depleted of CRK5 are unable to correctly assemble or maintain the spindle, with a greater than two-fold decrease of visible spindles at 1 min pa in absence of CRK5
- CRK5 is part of an atypical nuclear cyclin/CDK complex
- CRK5, SOC2 ((PBANKA_1442200)and CDKrs (PBANKA_0824400; (CDK regulatory subunit, CDKrs) have a similar location and complementary functions during gametogony. CDKrs is a protein related to Cks1 and CksHs2, two CDK-associated proteins in Saccharomyces cerevisiae and and human, respectively.
- CRK5, SOC2 and CDKrs have a similar location and complementary functions during gametogony
- SOC2 expression does not follow a temporal cyclin pattern during gametogony and the CSC complex (CRK5/SOC2/CDKrs complex) is stable during the first round of mitosis
- CSC is dynamically phosphorylated during the first round of replication

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1230200
Gene Model P. falciparum ortholog PF3D7_0615500
Gene productcdc2-related protein kinase 5
Gene product: Alternative nameCRK5
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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