RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4552
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0615200; Gene model (P.falciparum): PF3D7_0717500; Gene product: calcium-dependent protein kinase 4 (CDPK4)
MutatedGene model (rodent): PBANKA_1008200; Gene model (P.falciparum): PF3D7_1436600; Gene product: cGMP-dependent protein kinase (PKG)
Details mutation: a mutated pkg in which the threonine gatekeeper residue ((T619Q) is mutated to glutamine
TaggedGene model (rodent): PBANKA_1008200; Gene model (P.falciparum): PF3D7_1436600; Gene product: cGMP-dependent protein kinase (PKG)
Name tag: triple-HA
Phenotype Asexual bloodstage;
Last modified: 22 October 2018, 14:57
  *RMgm-4552
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene mutation, 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
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4552
Principal nameCDPK4-KO/pkg(T619Q)-3xHA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageSeverely reduced growth rate of asexual blood stages.

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.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CDPK4 and In this mutant the wild type pkg gene is replaced with a modified allele pkg (T619Q-HA), in which the threonine gatekeeper residue was mutated to a larger glutamine residue together with a C-terminal triple HA epitope tag.
The mutant does not contain the hdhfr/yfcu drug-selectable marker cassette. This cassette has been removed by negative selection.

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 has a role in male gamete formation: in male gametocytes axoneme formation, formation of mitotic spindles and DNA synthesis is inhibited/blocked (see RMgm-12).

Cyclic GMP-dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects. PKG is is a serinethreonine kinase that transfers the γ-phosphate of ATP, in a cGMP-dependent manner, to a variety of substrate proteins. Evidence has been presented that in Plasmodium activation of PKG is critically required to regulate cytosolic Ca2+ levels in multiple life cycle stages. PKG emerges as a universal regulator that controls ookinete gliding, gametocyte activation, and schizont rupture.

Phenotype
Severely reduced growth rate of asexual blood stages.

To verify the negative interaction between pkg and cdpk4, a cdpk4-KO/pkgT619Q-3xHA clonal line was generated and found to have severely reduced growth if compared to either of the single mutants.
.
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.

The follwing evidence is presented for the cdpk4/pkg(T619Q) interaction:
- pkg/cdpk4 interaction is required for merozoite invasion. An ultrastructural analysis of cdpk4-KO/pkg(T619Q)-3xHA schizonts by transmission electron microscopy (TEM) revealed that a significantly higher number of mutant merozoites show a discontinuous IMC with gaps, while single mutants did not.
- pkg/cdpk4 interaction is required for merozoite invasion
- CDPK4 function is revealed by attenuated calcium signals
- CDPK4 is at the interface between the IMC and the glideosome.
- CDPK4 phosphorylates a protein (SOC6) involved in IMC stability
- CDPK4 and 1 functionally interact to control invasion and motility.

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_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) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector087803
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

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1008200
Gene Model P. falciparum ortholog PF3D7_1436600
Gene productcGMP-dependent protein kinase
Gene product: Alternative namePKG
Details of the genetic modification
Short description of the mutationa mutated pkg in which the threonine gatekeeper residue ((T619Q) is mutated to glutamine
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationPoint mutations were introduced with a two-step strategy using l Red-ET recombineering in E. coli. The first step involved the insertion by homologous recombination of a Zeocin-resistance/Phe-sensitivity cassette surrounded by sequences 5' and 3' of the codon of interest amplified using primer pairs specific for the gene of interest (GOI): goi-delF/goi-delR. Recombinant bacteria were then selected on Zeocin. After verification of the recombination event by PCR, a second round of recombination exchanged the Zeocin-resistance/Phe-sensitivity cassette with a PCR product containing the desired mutation surrounding the codon of interest amplified using goi-mutF/goi-mutR primer pairs. Bacteria were selected on YEG-Cl kanamycin plates. Mutations were confirmed by sequencing vectors isolated from colonies sensitive to Zeocin. Generation of knockout and tagging constructs was performed using sequential recombineering and gateway steps as previously described. Lambda red-ET recombineering was first used to introduce a bacterial selection marker amplified into the gDNA insert, such that the target gene is either deleted or prepared for 39-end tagging. The bacterial marker was then replaced with a selection cassette for P. berghei in a Gateway LR Clonase reaction in vitro. The modified library inserts were then released from the plasmid backbone using NotI and used to transfect P. berghei.
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1008200
Gene Model P. falciparum ortholog PF3D7_1436600
Gene productcGMP-dependent protein kinase
Gene product: Alternative namePKG
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/vector-
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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationNo details are provided for the PlasmoGEM vector used
Additional remarks selection procedureThe mutant does not contain a selectable marker. The positive/negative selectable marker cassette has been removed by negative selection with 5-FC.
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