RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5219
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1409100; Gene model (P.falciparum): PF3D7_1310600; Gene product: ras-related protein Rab-5B (RAB5b; secretory complex protein 61 alpha; Sec61-alpha; Rab GTPase 5b)
Details mutation: Rab5b with the mutation Q91L (GTPase deficient)
Transgene
 Transgene not Plasmodium: GFP (gfp-mu3)
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 Liver stage;
Last modified: 12 July 2022, 13:53
  *RMgm-5219
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) : 35649358
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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The mutant parasite was generated by
Name PI/ResearcherLahree A, Mota MM
Name Group/DepartmentInstituto de Medicina Molecular- João Lobo Antunes (iMM-JLA), Faculdade de Medicina
Name InstituteUniversidade de Lisboa
CityLisboa
CountryPortugal
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Name of the mutant parasite
RMgm numberRMgm-5219
Principal namePbRab5b_Q91L_HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stagesee below
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated Rab5b with the Q91L mutation (Rab5b is C-terminally tagged with Azami Green; see RMgm-1460).
The Q91L mutation in PbRab5b is similar to the Q79L mutation in the human Rab5 (hRab5), which renders it GTPase deficient, leading to a prolonged GTP-bound state.

Protein (function)
P. falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport.
This family contains three Rab5 isoforms (Rab5a,b and c)

Phenotype
Evidence is presented for the following: 
Hepatocyte expressed endosomal; protein APPL1 deposition at the P. berghei PVM occurs independently of host cell (hepatocyte) Rab5 but depends on a parasite Rab5 isoform. Ectopic expression of a host Rab5 mutant (Q79L, lacking GTPase activity) results in the retention of APPL1 on host endosomes, leading to a stripping of its signal at the PVM together with a reduction in parasite size, both of which were rescued in parasites expressing the Plasmodium Rab5b Q91L isoform (also lacking its GTPase function; RMgm-5219).

Upon infecting hRab5_Q79L-expressing hepatocytes with P. berghei parasites expressing the GTPase-deficient mutant of PbRab5b (PbRab5b_Q91L), the PVM APPL1 signal and the EEF size were rescued relative to WT parasites. 

Additional information

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1409100
Gene Model P. falciparum ortholog PF3D7_1310600
Gene productras-related protein Rab-5B
Gene product: Alternative nameRAB5b; secretory complex protein 61 alpha; Sec61-alpha; Rab GTPase 5b
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Details of the genetic modification
Short description of the mutationRab5b with the mutation Q91L (GTPase deficient)
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationConstructs for the generation of rab5b (Q91L) mutant (Pbrab5b _Q91L-mAG) were a kind gift from the laboratory of Yumiko Saito-Nakano (National Institute of Health, Tokyo, Japan). Prior to transfection, the Pbrab5b-mAG construct was linearized with HindIII and EcoRI and the Pbrab5b (Q91L)-mAG construct was linearized with HindIII
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
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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 gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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Other details transgene
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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
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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
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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren