Summary

RMgm-4710
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0820300; Gene model (P.falciparum): PF3D7_0919400; Gene product: protein disulfide isomerase (PDI9; PDI-Trans)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 23 January 2020, 10:15
  *RMgm-4710
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31797966
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherAngrisano F, Blagborough AM
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College of Science, Technology and Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4710
Principal nameΔPDI-Trans
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal production of gametocytes; normal activation, gamete formation and egress of gametocytes/gametes from host red blood cells. No ookinete formation in vitro. Crossing experiments with mutants defective in either male or female gamete production showed that ΔPDI-Trans female gametes are fertile while ΔPDI-Trans male gamete fertility is strongly reduced.
OocystNormal production of gametocytes; normal activation, gamete formation and egress of gametocytes/gametes from host red blood cells. No ookinete formation in vitro. Crossing experiments with mutants defective in either male or female gamete production showed that ΔPDI-Trans female gametes are fertile while ΔPDI-Trans male gamete fertility is strongly reduced.
Strongly reduced oocyst formation (63-96% inhibition) of oocyst formation
SporozoiteStrongly reduced oocyst formation (63-96% inhibition) of oocyst formation; the small number of oocysts formed are morphologically normal and form normal sporozoites that are infective to mice.
Liver stageStrongly reduced oocyst formation (63-96% inhibition) of oocyst formation; the small number of oocysts formed are morphologically normal and form normal sporozoites that are infective to mice.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PDI-Trans

Protein (function)
Protein Disulphide Isomerase (PDI) (EC: 5.3.4.1) is a multifunctional member of the thioredoxin superfamily of redox proteins, characterized by the presence of the βαβαβαββα fold. PDIs typically have three catalytic activities; disulphide isomerase, thiol-disulphide oxidoreductase, and redox-dependent chaperone. PDI homologues have been identified in multiple species, where they are “classically” located in the endoplasmic reticulum (ER) and facilitate the folding and assembly of secretory and membrane proteins within the lumen. In Plasmodium, a small number of proteins have been putatively identified (by sequence homology) as PDI-like molecules in Plasmodium falciparum, vivax, knowlesi, berghei and yoelii. Conclusive demonstration of PDI activity has currently only been demonstrated with PF3D7_0827900/PDI-8, with transcription and translation demonstrated in asexual blood schizonts, gametocytes and sporozoites. Knowledge regarding the process of disulphide bond-dependent protein folding in Plasmodium is scarce.
Previous proteomic analysis of a P. berghei male gamete proteome  followed by advanced bioinformatics analysis encompassing a suite of functional and localization-based algorithms identified the expression of PDI-Trans (PBANKA_0820300) in the male gamete, and suggested that the resulting transmembrane protein was potentially located on the surface of the plasma membrane of male gametes.

Phenotype
Normal production of gametocytes; normal activation, gamete formation and egress of gametocytes/gametes from host red blood cells. No ookinete formation in vitro. Crossing experiments with mutants defective in either male or female gamete production showed that ΔPDI-Trans female gametes are fertile while ΔPDI-Trans male gamete fertility is strongly reduced.
Strongly reduced oocyst formation (63-96% inhibition) of oocyst formation; the small number of oocysts formed are morphologically normal and form normal sporozoites that are infective to mice.

Additional information
Analysis of a mutant expressing a C-terminal GFP-tagged version of PDI-Trans (RMgm-4711) showed:
- Immunofluorescence microscopy on non-permeabilized parasites confirmed PDI-Trans-GFP expression at the surface of activated male gametes and ookinetes. Live microscopy of mixed blood stages, female gametes and fixed immunofluorescence of sporozoites demonstrated that PDI-Trans-GFP is expressed across the entire parasitic lifecycle. 

Evidence is presented that:

- PDI shows classical PDI activity
- fertilization is inhibited reversibly by the PDI inhibitor Bacitracin in vitro
- transmission is inhibited by Bacitracin in P. berghei and P. falciparum
- PDI-Trans can be targeted specifically with antibodies to block transmission in vitro and ex vivo

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0820300
Gene Model P. falciparum ortholog PF3D7_0919400
Gene productprotein disulfide isomerase
Gene product: Alternative namePDI9; PDI-Trans
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-239637
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
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