RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5139
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0808100; Gene model (P.falciparum): PF3D7_0317100; Gene product: 6-cysteine protein (B9)
TaggedGene model (rodent): PBANKA_0932000; Gene model (P.falciparum): PF3D7_1116000; Gene product: rhoptry neck protein 4 (RON4)
Name tag: mCherry
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0808100; Gene product: 6-cysteine protein (B9)
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 6 January 2022, 17:30
  *RMgm-5139
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-5137
Other information parent lineThe mutant lacks expression of B9 and expresses GFP under the control of the HSP70 promoter. The parasites have been generated and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method and the construct used)
The mutant parasite was generated by
Name PI/ResearcherFernandes P, Silvie O
Name Group/DepartmentSorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses
Name InstituteCIMI-Paris
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-5139
Principal namePbΔb9/RON4-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageExpression of RON4 in merozoites and sporozoites
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteExpression of RON4 in merozoites and sporozoites
Liver stageIn vitro in HepG2 cells RON4-mCherry signal was lost in a vast majority of intracellular wild-type PbGFP/RON4-mCherry sporozoites, reflecting rhoptry discharge during productive invasion.
In sharp contrast, RON4-mCherry was detected in all examined PbΔb9/RON4-mCherry intracellular sporozoites, indicating that sporozoites lacking B9 invade cells without secreting their rhoptries, i.e. through traversal mode only.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of B9 and expresses GFP under the control of the HSP70 promoter. In addition, it expresses a C-terminal, mCherry-tagged version of RON4. The tagged version of ron4 has been introduced into mutant RMgm-5137. This mutant lacks expression of B9 and expresses GFP under the control of the HSP70 promoter.  

The PbΔb9 parasites have been generated and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method and the construct used)

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.10.25.465731

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. 

RON4 is a rhoptry protein. While microneme proteins are typically involved in gliding motility, adhesion to substrates and host cell recognition, rhoptry proteins have been implicated in MJ (moving junction) and PV formation as well as host cell modifications.

Phenotype
Phenotype analyses of a mutant lacking expression of B9 (RMgm-5137) shows the following: Normal/wild-type numbers of sporozoites. C57BL/6 mice injected with 10,000 PbΔb9 did not develop a patent blood infection. Dramatic reduction in the number of PbΔb9 exoerythrocytic forms (EEFs) in vitro cultured HepG2 cells. Evidence presented that PbΔb9 were not able to form productive vacuoles in hepatocytes.
A cell wound-repair assay indicated that the cell traversal activity of PbΔb9 sporozoites is not different to wild-type sporozoites, in both HepG2 and HepG2/CD81 cells.

Analysis of PbΔb9/RON4-mCherry parasites showed the following:
In vitro in HepG2 cells RON4-mCherry signal was lost in a vast majority of intracellular wild-type PbGFP/RON4-mCherry sporozoites, reflecting rhoptry discharge during productive invasion.
In sharp contrast, RON4-mCherry was detected in all examined PbΔb9/RON4-mCherry intracellular sporozoites, indicating that sporozoites lacking B9 invade cells without secreting their rhoptries, i.e. through traversal mode only.

Additional information
Evidence is presented that: 
- B9 is required for sporozoite invasion
- B9 is secreted from the sporozoite micronemes
- B9 contains a CyRPA-like beta propeller domain, required for B9 function
- The propeller domain of B9 interacts with P36 and P52

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0808100
Gene Model P. falciparum ortholog PF3D7_0317100
Gene product6-cysteine protein
Gene product: Alternative nameB9
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 parasites have been generated and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method).
For each target gene, a 5’ fragment and a 3’ fragment were amplified by PCR from P. berghei (ANKA) or P. yoelii (17XNL) WT genomic DNA and inserted into SacII/NotI and XhoI/KpnI restriction sites, respectively, of the GOMO-GFP vector, using the In-Fusion HD Cloning Kit (Clontech). The resulting targeting constructs were linearized with SacII and KpnI before transfection.
Additional remarks selection procedureThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP+mCherry- and marker-free parasites are selected by FAC sorting and used to infect a mouse
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_0932000
Gene Model P. falciparum ortholog PF3D7_1116000
Gene productrhoptry neck protein 4
Gene product: Alternative nameRON4
Details of the genetic modification
Name of the tagmCherry
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse
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 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 PBANKA_0808100
Gene product6-cysteine protein
Gene product: Alternative nameB9
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