Summary

RMgm-47
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: sporozoite surface protein 2|thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2; TRAP)
Phenotype Sporozoite; Liver stage;
Last modified: 26 February 2010, 20:23
  *RMgm-47
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) : 9267031
MR4 number MRA-266
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherA.A. Sultan, V. Nussenzweig, V. Thathy, R. Menard
Name Group/DepartmentDepartment of Pathology, Kaplan Cancer Center
Name InstituteNew York University Medical Center
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-47
Principal nameTRAP(-); REP1; REP2
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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNormal numbers of midgut sporozoites are formed. Sporozoites do not display gliding motility. Sporozoites are strongly affected in their capacity to invade salivary glands (the number of salivary gland–associated, sporozoites per infected mosquito was 34 times smaller than in wild type). Sporozoites are also affected in their infectivity to Sprague-Dawley rats (~10,000 times less infective than wild type sporozoites).
Liver stageSporozoites are affected in their infectivity to Sprague-Dawley rats (~10,000 times less infective than wild type sporozoites).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of TRAP (thrombospondin-related anonymous protein).

Protein (function)
TRAP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells.

The role of the different domains of the protein in motility and invasion has been analysed in mutant parasites expressing mutated forms of TRAP (see below).

Phenotype
The phenotype analyses show that the lack of expression of TRAP results in the loss of the gliding motility of the sporozoites. The mutant sporozoites show a strongly reduced capacity to invade salivary glands and infectivity to the mammalian host.

Additional information
This mutant that lacks expression of TRAP has been complemented with a wild-type copy of the trap gene, introduced on episomes that contain the trap gene and the human dihydrofolate reductase (hdhfr) as a drug-selectable marker which confers resistance to the antifolate drug WR99210 (Sultan, A.A. et al., 2001, Mol. Biochem. Parasitol. 113, 151-6). Complementation resulted in restoration of the wild type phenotype.

In the same study mutants are described (INT1, INT2) that have been generated using a construct that integrates by single cross-over integration. The disadvantage of using such integration constructs is that the construct can be removed from the genome, thereby restoring the wild type genotype. One mutant has been deposited at MR4 (MRA-265)

Adhesion of sporozoites of this mutant has been analysed in another study (Hegge et al., 2010, FASEB J; PMID: 20159960). Mutant sporozoites showed significant weaker adhesion on glass slides as compared to wild type parasites.

Other mutants
A. P. yoelii mutant has been generated that lacks expression of TRAP (RMgm-53).
P. berghei mutants have been generated with mutated cytoplasmic tails of TRAP (RMgm-54; RMgm-55; RMgm-56; RMgm-57; RMgm-149; RMgm-150; RMgm-151).
Other P. berghei mutants have been generated with mutated extracellular (adhesive) domains (A-domain: RMgm-48, RMgm-49; TSR domain: RMgm-50; RMgm-51; A-domain and TSR domain: RMgm-52).
A P. berghei mutant has been generated in which the endogenous P. berghei trap was replaced with (mutated forms of) P. falciparum trap (RMgm-58).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1349800
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productsporozoite surface protein 2|thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2; TRAP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe mutants have been generated using a construct that disrupt the trap gene by double cross-over integration (replacement vector). In the same study mutants are described (INT1, INT2) that have been generated using a construct that integrates by single cross-over integration. The disadvantage of using such integration constructs is that the construct can be removed from the genome, thereby restoring the wild type genotype.
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