Summary

RMgm-4519
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0512800; Gene model (P.falciparum): PF3D7_1028700; Gene product: merozoite TRAP-like protein (MTRAP)
Transgene
Transgene not Plasmodium: Nanoluciferase
Promoter: Gene model: PBANKA_0412900; Gene model (P.falciparum): PF3D7_0315200; Gene product: circumsporozoite- and TRAP-related protein (CTRP)
3'UTR: Gene model: PBANKA_1349800; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (sporozoite surface protein 2; SSP2; SSP-2; TRAP)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Fertilization and ookinete;
Last modified: 7 September 2018, 14:22
  *RMgm-4519
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30181368
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4518
Other information parent lineA reporter line expressing nanoluciferase under control of the (ookinete specific) ctrp promoter. The nanoluciferase expression cassette has been integrated into the neutral 230p locus.
The mutant parasite was generated by
Name PI/ResearcherCalit J, Bargieri DY
Name Group/DepartmentDepartment of Parasitology, Institute of Biomedical Sciences
Name InstituteUniversity of São Paulo
CitySão Paulo
CountryBrazil
Name of the mutant parasite
RMgm numberRMgm-4519
Principal nameOokluc-MTRAP-KO
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 ookineteTo verify if the increase in nanoluciferase (nLuc) activity in (ookinete) conversion assays with Ookluc was dependent on fertilization, we deleted the mtrap gene from the Ookluc genome. MTRAP-knockout (MTRAP-KO) gametocytes can activate, but the gametes remain trapped inside the host cell. There was no increase of nLuc activity in Ookluc-MTRAP-KO parasites after 24 hr of conversion assay – the absence of ookinetes was confirmed by blood smears – showing that gametocyte activation per se is not sufficient for activation of the ctrp promoter, hence nLuc expression and activity in Ookluc parasites depend on gamete egress and fertilization.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MTRAP and expresses nanoluciferase under control of the (ookinete specific) ctrp promoter. The nanoluciferase expression cassette has been integrated into the neutral 230p locus.

Protein (function)
The thrombospondin-related anonymous protein (TRAP) family of proteins are type I transmembrane proteins that share a functionally conserved cytoplasmic tail and an ectodomain exposing various ligand-binding modules including a thrombospondin type I repeat (TSR). In Plasmodium, the sporozoite stage expresses three members of the family—TRAP; TRAP-related protein (TREP), also called S6; and TRAP-ike protein (TLP)—which all play a role in sporozoite gliding on substrates and within tissues. The ookinete stage expresses a single member, called circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP), which is essential for ookinete gliding motility.
Merozoite TRAP (MTRAP) is a TRAP family member that was reported as expressed in the merozoite. The mtrap gene is conserved and syntenic among Plasmodium species.

Analyses of P. berghei mutants lacking expression of MTRAP (RMgm-1493, RMgm-4056) showed a role of MTRAP in rupture of the parasitophorous membrane of gametocytes, resulting in escape of gametes resulting in absence of fertilisation and ookinete formation.

Phenotype
To verify if the increase in nanoluciferase (nLuc) activity in (ookinete) conversion assays with Ookluc (RMgm-4518) was  dependent on fertilization, we deleted the mtrap gene from the Ookluc genome. MTRAP-knockout (MTRAP-KO) gametocytes can activate, but the gametes remain trapped inside the host cell. There was no increase of nLuc activity in Ookluc-MTRAP-KO parasites after 24 hr of conversion assay – the absence of ookinetes was confirmed by blood smears – showing that gametocyte activation per se is not sufficient for activation of the ctrp promoter, hence nLuc expression and activity in Ookluc parasites depend on gamete egress and fertilization.

Additional information
From the Abstract:
'Here we demonstrate the generation of a Plasmodium model for drug screens against  gametes and fertilization. The new P. berghei line, named Ookluc, was genetically and pharmacologically validated and scalable for HTS. Screening the Pathogen Box from the Medicines for Malaria Venture (MMV) using the new model identified promising TB compounds. The use of Ookluc in different libraries of compounds may aid in the identification of transmission-blocking drugs not assessed in screens against asexual stages or gametocytes.'

Other mutants

P. berghei mutants lacking expression of MTRAP (RMgm-1493, RMgm-4056)
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0512800
Gene Model P. falciparum ortholog PF3D7_1028700
Gene productmerozoite TRAP-like protein
Gene product: Alternative nameMTRAP
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 usedNo
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
Promoter of the selectable markerunknown
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameNanoluciferase
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 parasiteNo selectable marker
Promoter of the selectable markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0412900
Gene Model P. falciparum ortholog PF3D7_0315200
Gene productcircumsporozoite- and TRAP-related protein
Gene product: Alternative nameCTRP
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_1349800
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2; TRAP
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