Summary

RMgm-58
Malaria parasiteP. berghei
Genotype
MutatedGene 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)
Details mutation: The endogenous P. berghei gene replaced with the ortholog of P. falciparum
Phenotype Sporozoite; Liver stage;
Last modified: 26 January 2011, 14:21
  *RMgm-58
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) : 10508153
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 line
The mutant parasite was generated by
Name PI/ResearcherK. Wengelnik, A. Crisanti
Name Group/DepartmentDepartment of Biology
Name InstituteImperial College of Science
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-58
Principal namePfTRAP (clone 87-11)
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
SporozoiteSporozoites showed (reduced) gliding motility. Sporozoites invaded salivary glands (in comparison with wild type sporozoites relatively low numbers of salivary gland sporozoites were observed with a mean of 3750 per salivary gland). Sporozoites were infectious to mice (however, infectivity was reduced as shown by a longer prepatent period).
Liver stageSporozoites were infectious to mice (however, infectivity was reduced as shown by a longer prepatent period).
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei TRAP is replaced by P. falciparum 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 as has been shown by analysis of mutant parasites lacking expression of TRAP (RMgm-47; RMgm-53).

Phenotype
Analysis of the phenotype of the mutant show that P. falciparum TRAP can complement the function of P. berghei TRAP. However, the phenotype analyses indicate that the replacement impairs both the gliding motility and infectivity of the sporozoites as is discussed by Menard and Nussenzweig (2000, Parasitol. Today, 16, 222-24). In the same study mutants has been generated that contain mutated forms of the trap gene of P. falciparum carrying amino acid substitutions or deletions in the adhesive domains. Mutations in the A-domain affect invasion of salivary glands but has no effect on gliding motility or invasion of hepatocytes. Mutations in the TSP (TSR) domain affects both gliding motility and invasion of salivary glands but not invasion of hepatocytes. These results are (partly) in contradiction with results obtained with other mutants that contain mutated A-domains and TSR domains (A-domain: RMgm-49; TSR domain: RMgm-50; RMgm-51; A-domain and TSR domain: RMgm-52). As discussed by Menard and Nussenzweig (2000, Parasitol. Today, 16, 222-24) several methodological problems of heterologous expression of TRAP may limit the validity of the conclusions based on expression of mutated P. falciparum genes in P. berghei. The role of the different domains of the protein in motility and invasion has been analysed in other mutant parasites expressing mutated forms of TRAP (see below).

Other mutants
P. berghei mutants have been generated with mutated cytoplasmic tails of TRAP (RMgm-54; RMgm-55; RMgm-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).


  Mutated: Mutant parasite with a mutated 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
Short description of the mutationThe endogenous P. berghei gene replaced with the ortholog of P. falciparum
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct integrates by double cross-over, homologous recombination resulting in the (i) the deletion of endogenous PbTRAP; (ii) its replacement by PfTRAP at a position identical to that of the wild-type gene and under the control of endogenous 3'UTR and 5'UTR regulatory sequences.
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