RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5191
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0701900; Gene model (P.falciparum): PF3D7_0828800; Gene product: GPI-anchored micronemal antigen (GAMA, Putative Secreted Ookinete Protein 9, PSOP9)
Details mutation: the promoter of the gama gene replaced by the promoter of the ctrp gene (PBANKA_0412900)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 15 June 2023, 15:58
  *RMgm-5191
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) Reference 1 (PMID number) : 37314965
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-5188
Other information parent lineIn the mutant the gama gene has been replaced by the selectable marker cassette containing the positive/negative selectable marker hdhfr/yfcu. This enables to introduce additional genes in the disrupted gama locus by the method of GIMO transfection
The mutant parasite was generated by
Name PI/ResearcherJennison C, Vaughan AM
Name Group/DepartmentCenter for Global Infectious Disease Research
Name InstituteSeattle Children’s Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-5191
Principal name(CTRP)GAMA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystWild type numbers of oocyst. Reduced numbers of salivary gland sporozoites
SporozoiteWild type numbers of oocyst. Reduced numbers of salivary gland sporozoites.
Liver stage(Midgut) sporozoites showed a strongly reduced capacity to establish an infection in mice (after intravenous injection).
Additional remarks phenotype

Mutant/mutation
In the 'promoter swap' mutant the promoter of the gama gene has been replaced by the promoter of ctrp gene (PBANKA_0412900). In addition, the mutant expresses the reporter GFP-Luciferase under the constitutive eef1a promoter.

In the manuscript two additional promoter swap mutants are described. In these mutants the promoter of the gama gene has been replaced by the promoter of cap380 gene (PBANKA_1218100) and the trap gene (PBANKA_1349800), respectively.

Selected promoters were CTRP, expressed only in ookinetes; CAP380, which is expressed throughout ookinete-oocyst development; and TRAP, which is expressed primarily in mature midgut sporozoites and salivary gland sporozoites

Protein (function)
GAMA is a micronemal protein with a C-terminal GPI-anchor. P. berghei GAMA consists of 625 aa with a predicted molecular mass of ~71 kDa, (signal peptide cleavage results in a 69 kDa mass). In P. falciparum, two additional asparagine rich repeat regions result in a larger 738 aa protein (predicted molecular weight ~85 kDa, 83 kDa after cleavage of the signal peptide). For P. berghei, SignalP-5.0 predicts a signal peptide with a cleavage site at S21/L22. For P.falciparum, cleavage is predicted at the same locus, A21/L22.
P. berghei and P. falciparum share 44.8% identity across the full-length sequence, with areas of higher conservation found towards the N-terminus (residues 64-347 = 60% identity) and C-terminus (residues 501-700 = 60% identity) split by a central repeat region of low shared identity (residues 348-500, with predicted disorderly structure. The C-terminal transmembrane domain is predicted to be cleaved and appended with a GPI anchor. Downstream of signal peptide cleavage, P. falciparum contains seven cysteine residues prior to the central repeat region and one downstream which are conserved across all Plasmodium species investigated. P. berghei and other rodent infective species of Plasmodium bear an additional cysteine at position 152 in the alignment. GAMA is present in all Plasmodium species and indeed all other intracellular apicomplexans.

Phenotype

A mutant lacking GAMA (
∆GAMA; RMgm-5188) showed the following phenotype:
Wild type-like blood stage development. Wild type numbers of ookinetes are produced. (Strongly) reduced oocyst production and strongly reduced numbers of midgut- and salivary gland sporozoites. Evidence is provided for a defect in the egress of sporozoites from oocysts. Sporozoites show strongly reduced motility. (Midgut) sporozoites are unable to establish an infection in mice (after intravenous injection). 

The promoter swap mutants, (ctrp)gama, (cap380)gama and (trap)gama showed the following phenotype:
1. (ctrp)gama
Wild type numbers of oocyst. Reduced numbers of salivary gland sporozoites. (Midgut) sporozoites showed a strongly reduced capacity to establish an infection in mice (after intravenous injection).

2. (cap380)gama
Oocysts exhibited a striking developmental defect, whereby oocysts exhibited lower level GFP expression that wild type and did not grow beyond ~day-five size. No sporozoite formation

3.(trap)gama
Reduced oocyst production; no (strongly reduced) egress of sporozoites from oocysts; however, motility of midgut sporozoites was not affected. Reduced capacity to establish an infection in mice by midgut sporozoites (after intravenous injection). However, salivary gland sporozoites showed wild type like infectivity

In conclusion: in none of the promoter-swap mutants, the promoter swap could rescue (completely) the phenotype(s) observed in the mutant lacking expression of GAMA (∆GAMA; RMgm-5188)

Additional information

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0701900
Gene Model P. falciparum ortholog PF3D7_0828800
Gene productGPI-anchored micronemal antigen
Gene product: Alternative nameGAMA, Putative Secreted Ookinete Protein 9, PSOP9
Details of the genetic modification
Short description of the mutationthe promoter of the gama gene replaced by the promoter of the ctrp gene (PBANKA_0412900)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA CRISPR/Cas9 methodology was used to generate promoter switch parasites. To generate promoter switch parasites, guides were identified in the 188 bp region just upstream of the GAMA ORF. Homology arms corresponding to the 758 bp upstream of this region (5’ flank), or the first 768 bp of the GAMA gene (3’ flank) were amplified from genomic DNA and ligated into the pYC-L2 plasmid (PMID: 24987097) via restriction cloning.
Stage-specific promoters were amplified from genomic DNA and ligated between the homology arms; CTRP = 781bp, CAP380 = 1065 , TRAP = 946.
Single stranded guide primers were annealed in a 50 μL volume (10 μL of each 100 μM primer with 25 μL water and 5 μL cutsmart buffer), by heating to 95 ºC for 5 minutes and allowing to cool to room temperature. Annealed guides were inserted into the pYC-L2 plasmid, via restriction cloning with the enzyme Esp3I. Guide integration was confirmed via sanger sequencing with the primer CJ059.
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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