RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4193
Malaria parasiteP. yoelii
Genotype
MutatedGene model (rodent): PY17X_1337400; Gene model (P.falciparum): PF3D7_1301600; Gene product: erythrocyte binding antigen-140 (EBL; erythrocyte binding antigen-140; EBA140; BAEBL, EBP2)
Details mutation: an amino acid substitution (Tyr >Cys) at position 351
Phenotype Asexual bloodstage;
Last modified: 19 July 2017, 19:09
  *RMgm-4193
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28704525
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/line17X1.1pp
Name parent line/clone Not applicable
Other information parent line17X1.1pp (with intermediate growth rate phenotype)
The mutant parasite was generated by
Name PI/ResearcherAbkallo HM, Culleton R
Name Group/DepartmentMalaria Unit, Department of Pathology
Name InstituteInstitute of Tropical Medicine, Nagasaki University
CityNagasaki
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-4193
Principal name17x1.1pp-EBL-351Y>C
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageReduced growth rate of blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of Pyebl with an amino acid substitution (Tyr >Cys) at position 351(17x1.1pp-EBL-351Y>C)

Protein (function)
Pyebl is a gene encoding a type I integral transmembrane protein encoded by the ebl (erythrocyte-binding-like) gene family. Upon release from the micronemes, EBL proteins recognize erythrocyte receptors and initiate the formation of the tight junction. Plasmodium vivax uses an EBL orthologue, PvDBP (Pv110810), to recognize the Duffy antigen on the erythrocyte surface. P. falciparum has an expanded family of EBL proteins, for example EBA175 (PF07_0128), EBA181/JESEBL (PFA0125c), EBA140/BAEBL (MAL13P1.60), EBL1 (PFD1145c).

EBL proteins possess 2 Cys-rich regions conserved among EBL orthologues. The N-terminal Cys-rich region named the DBL (Duffy-binding-like) domain or region 2 recognizes a specific erythrocyte surface receptor. The C-terminal Cys-rich region named the C-cys domain or region 6 is located adjacent to the transmembrane domain, and the number and location of Cys residues are well conserved among  Plasmodium species. Region 6 exhibits structural similarity to the KIX-binding domain of the coactivator CREB-binding protein and has been proposed to be a protein trafficking signal for transportation to the micronemes.

Phenotype

Reduced growth rate of blood stages of the mutant (17x1.1pp-EBL-351Y>C) compared to wild type (17x1.1pp)

Additional information
Examining the Pyebl gene, Sanger capillary sequencing re-confirmed the existence in 17X1.1pp of an amino acid substitution (Cys >Tyr) at position 351 within region 2 of the encoded protein. When aligned against other P. yoelii strains and other Plasmodium species, this cysteine residue is highly conserved, and the substitution observed in 17X1.1pp was novel. Crucially, no other polymorphisms were detected in the coding sequence of the gene, including in region 6, the location of the SNP previously implicated in parasite virulence in other strains of P. yoelii.
Structural modeling of the EBL protein in both wild-type and 17x1.1pp (C351Y) mutants predicted the abolition of a a disulphide bond between C351 and C420 in the 17x1.1pp parasites that alters the tertiary structure of the receptor binding region of the ligand in these parasites.

Other mutants
see PY17X_1337400


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1337400
Gene Model P. falciparum ortholog PF3D7_1301600
Gene producterythrocyte binding antigen-140
Gene product: Alternative nameEBL; erythrocyte binding antigen-140; EBA140; BAEBL, EBP2
Details of the genetic modification
Short description of the mutationan amino acid substitution (Tyr >Cys) at position 351
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 parasitepyrimethamine resistant gene
Promoter of the selectable markernot available
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