RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5141
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0808100; Gene model (P.falciparum): PF3D7_0317100; Gene product: 6-cysteine protein (B9)
Details mutation: Mutations in the 'propeller domain' of b9
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 Sporozoite; Liver stage;
Last modified: 7 January 2022, 17:50
  *RMgm-5141
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, 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, PbΔb9, lacks expression of B9 and expresses GFP under the control of the HSP70 promoter. It is drug selectable marker-free
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-5141
Principal namePb-b9(mut)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteComplementation of PbΔb9 sporozoites with a construct encoding the entire PbB9 fully restored sporozoite infectivity in HepG2 cell cultures, validating the genetic complementation approach. In contrast, parasites complemented with a truncated B9 lacking the propeller domain, alone or in combination with the first 6-cys domain, were not infectious, phenocopying the parental B9-deficient parasites. These results show that the propeller domain is required for B9 function during sporozoite entry. Chimeric B9 versions where the propeller domain of PbB9 was replaced by the equivalent sequence from PyB9 (Pyprop, Pyprop6cys1) restored sporozoite infectivity. In contrast, substitution of the PfB9 propeller domain for the PbB9 propeller (Pfprop) did not restore infectivity in complemented parasites. Complementation with the PyB9 propeller domain restored infection in both HepG2, which express SR-B1 but not CD81, and HepG2/CD81 cells, which express both receptors, suggesting that the B9 propeller domain does not restrict host receptor usage.
Liver stageComplementation of PbΔb9 sporozoites with a construct encoding the entire PbB9 fully restored sporozoite infectivity in HepG2 cell cultures, validating the genetic complementation approach. In contrast, parasites complemented with a truncated B9 lacking the propeller domain, alone or in combination with the first 6-cys domain, were not infectious, phenocopying the parental B9-deficient parasites. These results show that the propeller domain is required for B9 function during sporozoite entry. Chimeric B9 versions where the propeller domain of PbB9 was replaced by the equivalent sequence from PyB9 (Pyprop, Pyprop6cys1) restored sporozoite infectivity. In contrast, substitution of the PfB9 propeller domain for the PbB9 propeller (Pfprop) did not restore infectivity in complemented parasites. Complementation with the PyB9 propeller domain restored infection in both HepG2, which express SR-B1 but not CD81, and HepG2/CD81 cells, which express both receptors, suggesting that the B9 propeller domain does not restrict host receptor usage.
Additional remarks phenotype

Mutant/mutation
Several mutants are described that express mutated forms of B9 (see below for details for the different mutants/mutated versions). These mutante lacks  express GFP under the control of the HSP70 promoter.

The mutants have been generated by introducing mutated forms of the b9 gene in the mutant, PbΔb9, that lacks expression of B9 and is drug selectable marker-free 

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. 

From the paper:
Three domains were predicted at the structural level using HHpred: an N-terminus propeller domain similar to that of CyRPA encoded by the first exon, and two putative but poorly supported 6-cys domains encoded by the second exon. CyRPA is a cysteine-rich protein expressed in P. falciparum merozoites, where it forms a protein complex that is essential for invasion of erythrocytes. B9 is enriched in cysteines, nine being located in the predicted propeller domain that we suppose are involved in the formation of disulphide bonds in a similar manner to CyRPA, to stabilize the protein structure.

Phenotype
To define the functional importance of the predicted propeller domain (see above), various DNA constructs were generated encoding the entire or partially deleted B9, all containing an intact signal peptide and C-terminus sequences to ensure correct secretion and GPI-anchoring of the protein. Constructs were used for transfection of the drug selectable marker-free PbΔb9 mutant line (RMgm-5137).
Complementation of PbΔb9 sporozoites with a construct encoding the entire PbB9 fully restored sporozoite infectivity in HepG2 cell cultures, validating the genetic complementation approach. In contrast, parasites complemented with a truncated B9 lacking the propeller domain, alone or in combination with the first 6-cys domain, were not infectious, phenocopying the parental B9-deficient parasites. These results show that the propeller domain is required for B9 function during sporozoite entry. Chimeric B9 versions where the propeller domain of PbB9 was replaced by the equivalent sequence from PyB9 (Pyprop, Pyprop6cys1) restored sporozoite infectivity. In contrast, substitution of the PfB9 propeller domain for the PbB9 propeller (Pfprop) did not restore infectivity in complemented parasites. Complementation with the PyB9 propeller domain restored infection in both HepG2, which express SR-B1 but not CD81, and HepG2/CD81 cells, which express both receptors, suggesting that the B9 propeller domain does not restrict host receptor usage.

Additional information
Evidence is presented in the paper 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


  Mutated: Mutant parasite with a mutated 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
Short description of the mutationMutations in the 'propeller domain' of b9
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 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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationGenetic complementation of PbΔb9 parasites was achieved by double crossover homologous recombination using a vector containing a hDHFR cassette and a 3’ homology arm corresponding to the 5’ sequence of the HSP70 promoter of the GFP cassette in the PbDb9 line. First, an 840-bp fragment including the coding sequence for PbB9 N-terminus (amino acids 1-29), and a 1096-bp fragment encoding the C627 terminus (amino acids 647-852) followed by the 3’ UTR of PbB9 were sequentially inserted into the plasmid, in KpnI/EcoRI sites, resulting in the ΔpropΔ6cys1 construct. Cloning of a 1950-bp fragment of PbB9 gene (including the coding sequence for amino acids 30-646) into XhoI/KpnI sites of the ΔpropΔ6cys1 plasmid resulted in the PbB9 construct, encoding the full-length PbB9 protein. Cloning of a 912-bp fragment of PbB9 gene (including the coding sequence for amino acids 344-646) into XhoI/KpnI sites of the ΔpropΔ6cys1 plasmid resulted in the Δprop construct. Cloning of a 1992-bp fragment from PyB9 gene (including the coding sequence for amino acids 30-653 of PyB9) into XhoI/KpnI sites of the ΔpropΔ6cys1 plasmid resulted in the PyProp6cys1 construct. Cloning of a 948-bp fragment from PyB9 gene (encoding 636 amino acids 30-342 of PyB9) and a 903-bp fragment from PbB9 gene (encoding amino acids 346-646 of PbB9) into XhoI/KpnI sites of the ΔpropΔ6cys1 plasmid resulted in the PyProp construct. Cloning of a 1071-bp fragment from PfB9 gene (encoding amino acids 25-379 of PfB9) and a 903-bp fragment from PbB9 gene (encoding amino acids 346-646 of PbB9) into XhoI/KpnI sites of the ΔpropΔ6cys1 plasmid resulted in the PfProp construct. The constructs were linearized with NheI before transfection of PbΔb9 purified schizonts.
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