RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4535
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0826700; Gene model (P.falciparum): PF3D7_0925900; Gene product: conserved Plasmodium protein, unknown function (parasitophorous vacuole protein 5, PV5)
Name tag: mCherry-3xMyc
Transgene
Transgene not Plasmodium: GFP fused to the signal peptide of PBANKA_081890
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: Not available; Gene product: Not available
Insertion locus: Gene model: PBANKA_0826700; Gene product: conserved Plasmodium protein, unknown function (parasitophorous vacuole protein 5, PV5)
Phenotype Asexual bloodstage; Sporozoite;
Last modified: 25 June 2020, 11:01
  *RMgm-4535
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30232409
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherMatz JM, Matuschewski K
Name Group/DepartmentDepartment of Molecular Parasitology
Name InstituteInstitute of Biology, Humboldt University
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4535
Principal namePBANKA_0826700-tagged
Alternative namePV5-tagged
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageExpression in blood stages. Evidence for parasitophorous vacuole location.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteExpression in liver stages. Evidence for parasitophorous vacuole location.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mCherry tagged version of PBANKA_0826700 (PV5) and expresses GFP under the constitutive hsp70 promoter. GFP is fused to the signal peptide of PBANKA_081890 and consequently GFP is localized to the parasitophorous vacuole  (see RMgm-1333).

Protein (function)
The protein was selected in a screen for putative PV (parasitophorous vacuole) proteins.
Proteins are targeted to the PV by means of default protein secretion, which is initiated by the recognition and cleavage of an amino-terminal signal peptide (SP). To identify novel PV candidates, we searched the Plasmodium genome database (PlasmoDB) for SP-containing proteins and sequentially removed proteins containing predicted transmembrane domains, export motifs, apicoplast targeting peptides, endoplasmic reticulum (ER) retention signals and GPI anchors, which could potentially redirect the protein to other locations. Twelfe proteins were selected. Five (PV1-5) proteins were shown to have a PV location in blood- and liver-stages.

Phenotype
Expression in blood stages. Evidence for parasitophorous vacuole location.
Expression in liver stages. Evidence for parasitophorous vacuole location.
PV5 is essential for blood stage development (see RMgm-4540).

Additional information
Five (PV1-5; PBANKA_0919100; PBANKA_1441700; PBANKA_1350500; PBANKA_1349200; PBANKA_0826700) proteins were shown to have a PV location in blood- and liver-stages.
All fve proteins were found to be associated with the parasite periphery and with PV-derived tubules and vesicular structures throughout the entire asexual cycle. Strikingly, a substantial fraction of PV4 was found in the erythrocyte cytoplasm during ring and early trophozoite stages. This exported protein fraction progressively disappeared during parasite growth and was not detected in mature trophozoites or schizonts. During the schizont stage, all five proteins localised around the formed merozoites and co-localised with the hemozoin crystals of the digestive vacuoles. We next investigated whether the PV proteins are indeed associated with the vacuolar lumen rather than the parasite surface. To that end, we imaged ruptured schizonts, which have already disintegrated the iRBC membrane and the PV, thus leading to the loss of all soluble PV contents. Upon merozoite egress all fve proteins localised almost exclusively to the digestive vacuoles. Upon inspection of individual emerging merozoites, PV2-5 were hardly detected, whereas PV1 was found to be associated with a distinct intraparasitic structure, possibly refecting localisation to parasite dense granules, as previously demonstrated for other PV proteins23. In no instance were the PV proteins detected on the surface of emerging merozoites further supporting the notion that they are indeed mostly luminal.
Inoculation of in vitro cultivated hepatoma cells with the transgenic sporozoites revealed that all fve proteins are highly expressed in the liver stage PV. PV5 was not expressed during early pre-erythrocytic growth, but was only present in the PV of more mature parasite stages.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0826700
Gene Model P. falciparum ortholog PF3D7_0925900
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameparasitophorous vacuole protein 5, PV5
Details of the genetic modification
Name of the tagmCherry-3xMyc
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 usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor endogenous tagging of the PV candidates, a GFP(PV) co-localisation plasmid was constructed by fusing the bacterial backbone and adjacent mCherry-3xMyc tag sequence of the pBAT vector with the Plasmodium-specific fraction of the pGFP-PV plasmid using the KpnI and PvuII restriction sites. The sequences directly upstream of the stop codons were amplified from genomic DNA (ranging in size from 758 to 1,628 bp) and inserted into the GFP(PV) co-localisation plasmid using HpaI in combination with EcoRI or SacII. For double homologous recombination, 3′ fragments were amplified and cloned into the intermediate vectors as described for the gene deletion constructs.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP fused to the signal peptide of PBANKA_081890
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double 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 parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionInsertion locus
Gene Model of Parasite PBANKA_0826700
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameparasitophorous vacuole protein 5, PV5
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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