Summary

RMgm-4548
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0925500; Gene model (P.falciparum): PF3D7_1122800; Gene product: calcium-dependent protein kinase 6 (CDPK6)
PhenotypeNo phenotype has been described
Last modified: 22 October 2018, 13:59
  *RMgm-4548
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) : 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
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4548
Principal nameCDPK6-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CDPK6.
The mutant does not contain the hdhfr/yfcu drug-selectable marker cassette. This cassette has been removed by negative selection.

Protein (function)
CDPK6 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.
 

Phenotype
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.

The phenotype of the mutant lacking CDPK6 has not been analysed in detail

From the paper:
'we were not able to generate a triple PKGT619Q-3xHA/CDPK4-KO/CDPK1-KO mutant, while CDPK3 and CDPK6 could be readily knocked out in the PKGT619Q-3xHA/CDPK4-KO background'.

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
To search for genetic interactions between P. berghei protein kinase genes, parasites from a panel of mutant clones lacking a specific kinase were negatively selected for loss of the selection marker and then transfected with a pool of barcoded gene knockout (KO) vectors to inactivate another kinase in the same background. The competitive growth rate of each mutant within the pool was measured during days 4–8 post infection by barcode sequencing. For the background lines, we focussed on the CDPK family and on the two atypical MAP kinases.
In preliminary experiments, we found that a double mutant of map1 and map2 showed normal asexual growth, and the double KO mutant was included in the screen as a single recipient background to identify interactors of either gene. Due to the essential role for PKG in calcium mobilisation upstream of CDPKs, we also included the inhibitor-resistant PKGT(619Q)-3xHA line and its inhibitor-sensitive control, PKG-3xHA. The library of KO vectors was comprised of 37 targeting vectors for protein kinases and 6 characterised vectors targeting unrelated genes for use as references.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0925500
Gene Model P. falciparum ortholog PF3D7_1122800
Gene productcalcium-dependent protein kinase 6
Gene product: Alternative nameCDPK6
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector111850
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 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. Substitution of the GAP40(S148/149A) residue was introduced using primer gap40S148 HA-F instead of gap40 HA-F. Mutations were confirmed by sequencing with primers gap40-QCR1 and GW1. The modified library inserts were released from the plasmid backbone using NotI.
Additional remarks selection procedureThe mutant does not contain the hdhfr/yfcu drug-selectable marker cassette. This cassette 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