Summary

RMgm-4568
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1003600; Gene model (P.falciparum): PF3D7_0404500; Gene product: 6-cysteine protein (P36p; Pb36p; P52)
Transgene
Transgene not Plasmodium: RFP
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PY17X_1003500; Gene product: 6-cysteine protein (P36)
Phenotype Liver stage;
Last modified: 1 January 2019, 13:23
  *RMgm-4568
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30547015
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherArredondo SA, Kappe SHI
Name Group/DepartmentCenter for Global Infectious Disease Research
Name InstituteSeattle Children’s Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4568
Principal namePyΔp52
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageThree knockout parasite lines were created: ΔP36, ΔP52 and ΔP52ΔP36.
None of the 17 mice injected with ΔP52 sporozoites became patent, and only 2 in 17 mice injected with the ΔP36 line became patent, the latter exhibited a patency delay of 1 day. In addition, only 2 in 45 mice injected with ΔP52ΔP36 parasites showed a blood infection, and patency in these animals was further delayed by an extra day.
In all 3 mutants no PVM was observed in infected Hepa 1–6 hepatoma cells.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of P52 and expresses RFP under control of the constitutive eef1α promoter.

Protein (function)
P52 (P36p) and P36 are members of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins (Thompson J. et al., Mol. Biochem. Parasitol. (2001)118, 147-54). The proteins are characterised by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organisation throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.
Both genes have been shown to play a role in invasion/development of sporozoites in hepatocytes (see PF3D7_0404400 and PF3D7_0404500.

Phenotype
Three knockout parasite lines were created: ΔP36, ΔP52 and ΔP52ΔP36.
None of the 17 mice injected with ΔP52 sporozoites became patent, and only 2 in 17 mice injected with the ΔP36 line became patent, the latter exhibited a patency delay of 1 day. In addition, only 2 in 45 mice injected with ΔP52ΔP36 parasites showed a blood infection, and patency in these animals was further delayed by an extra day.
In all 3 mutants no PVM was observed in infected Hepa 1–6 hepatoma cells. 

From the paper:  
'Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.'

Additional information


Other mutants
see PF3D7_0404400 and PF3D7_0404500
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1003600
Gene Model P. falciparum ortholog PF3D7_0404500
Gene product6-cysteine protein
Gene product: Alternative nameP36p; Pb36p; P52
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 usedNo
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 ΔP36, ΔP52 and ΔP52ΔP36 knockout parasite lines were created by gene replacement through double crossover homologous recombination as previously described (Mikolajczak et al., 2008), where a red fluorescent protein (RFP) cassette was inserted in place of each gene, or both genes simultaneously; RFP is expressed in the parasite cytoplasm.
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 nameRFP
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe ΔP36, ΔP52 and ΔP52ΔP36 knockout parasite lines were created by gene replacement through double crossover homologous recombination as previously described (Mikolajczak et al., 2008), where a red fluorescent protein (RFP) cassette was inserted in place of each gene, or both genes simultaneously; RFP is expressed in the parasite cytoplasm.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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 PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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 / InsertionReplacement locus
Gene Model of Parasite PY17X_1003500
Gene product6-cysteine protein
Gene product: Alternative nameP36
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