RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4058
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1137800; Gene model (P.falciparum): PF3D7_1361800; Gene product: glideosome-associated connector (GAC)
Name tag: GFP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 21 December 2016, 22:28
  *RMgm-4058
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 27978434
MR4 number
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).
The mutant parasite was generated by
Name PI/ResearcherJacot D, Tewari R, Soldati-Favre D
Name Group/DepartmentDepartment of Microbiology & Molecular Medicine
Name InstituteUniversity of Geneva
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4058
Principal namePbGAC-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageExpression of GAC::GFP in cytoplasm. Distinct accumulation at the extreme apical end of the invasive merozoites
Gametocyte/GameteExpression of GAC::GFP in cytoplasm
Fertilization and ookineteExpression of GAC::GFP in cytoplasm. Distinct accumulation at the extreme apical end of the invasive motile ookinetes.
OocystExpression of GAC::GFP in cytoplasm.
SporozoiteExpression of GAC::GFP in cytoplasm. Distinct accumulation at the extreme apical end of the invasive sporozoites.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of GAC

Protein (function)
The paper provides evidence for the following (mainly based on analyses of GAC in T. gondii):

Apicomplexa exhibit a unique form of substrate dependent gliding motility central for host cell invasion and parasite dissemination. Gliding is powered by rearward translocation of apically secreted transmembrane adhesins via their interaction with the parasite actomyosin system. We report a conserved armadillo and pleckstrin homology (PH) domain containing protein, termed glideosome-associated connector (GAC), that mediates apicomplexan gliding motility, invasion, and egress by connecting the micronemal adhesins with the actomyosin system. TgGAC binds to and stabilizes filamentous actin and specifically associates with the transmembrane adhesin TgMIC2. GAC localizes to the apical pole in invasive stages of Toxoplasma gondii and Plasmodium berghei, and apical positioning of TgGAC depends on an apical lysine methyltransferase, TgAKMT. GAC PH domain also binds to phosphatidic acid, a lipid mediator associated with microneme exocytosis. Collectively, these findings indicate a central role for GAC in spatially and temporally coordinating gliding motility and invasion.

Phenotype
P. berghei, endogenous PbGAC fused to GFP was soluble and expressed at all life cycle stages examined. GAC::GFP locates to the cytosol at all stages and shows a distinct accumulation at the extreme apical end of the invasive blood stage merozoites, motile ookinetes, and sporozoites. Refined investigation by 3Dstructured illumination microscopy (SIM) revealed that GAC::GFP forms a ring-like structure at the apical end of both merozoite and ookinete

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1137800
Gene Model P. falciparum ortholog PF3D7_1361800
Gene productglideosome-associated connector
Gene product: Alternative nameGAC
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 modificationTo generate PbGAC-GFP-hDHFR, a PCR on P. berghei ANKA line 2.34 gDNA was performed using primers T135f-T135r. PCR products was digested with KpnI and ApaI and cloned in p277-hDHFR (Guttery et al., 2012) digested with the same enzymes.
PbGAC-GFPhDHFR (linearized EcoRV) was transfected in P. berghei ANKA line 2.34 as previously described (Guttery et al., 2014).
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