Summary

RMgm-140
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0412900; Gene model (P.falciparum): PF3D7_0315200; Gene product: circumsporozoite- and TRAP-related protein (CTRP)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 18 February 2009, 14:20
  *RMgm-140
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) : 10587361
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/ResearcherM. Yuda, Y. Chinzei
Name Group/DepartmentDepartment of Medical Zoology
Name InstituteSchool of Medicine, Mie University
CityTsu
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-140
Principal nameCTRP(-)1; CTRP(-)2
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 numbers of ookinetes are produced (in vitro and in vivo in A. stephensi). Ookinetes do not show any morphological differences from wild-type parasites with Giemsa staining under microscopic observation. Ookinetes do not develop into oocysts.
OocystNormal numbers of ookinetes are produced. Ookinetes do not develop into oocysts.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CTRP (circumsporozoite protein [CSP] and thrombospondin-related adhesive protein [TRAP]-related protein; CSP and TRAP-related protein (CTRP).

Protein (function)
CTRP 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). CTRP has six tandemly arrayed A domains followed by seven TSP type 1-like domains. CTRP is located in the micronemes of ookinetes. This structure is similar to that of TRAP (PF13_0201; PB000374.03.0), a malaria sporozoite protein critical for sporozoite motility and invasion into host cells.
CTRP plays a role in the motility of ookinetes and invasion of midgut epithelial cells.

Phenotype
The phenotype analyses indicate a role in the transformation of the ookinete into the oocyst. This and other studies indicate a role in motility and invasion of midgut epithelial cells.

Additional information
Ookinetes lacking expression of CTRP that are directly injected into the hemocoel, thereby by-passing the midgut wall, develop into oocysts and these ookinetes can transform in vitro into oocysts, indicating that CTRP is not essential for transformation of ookinetes into oocysts (Nacer, A. et al., Malar J.  19;7:82).

A P. falciparum mutant lacking CTRP (PFC0640w) has been generated which show a comparable defect in the formation of oocysts (Templeton, T.J. 2000. Mol. Microbiol. 36, 1-9).

A mutant has been generated (RMgm-150) that expresses a mutated form of TRAP (thrombospondin-related anonymous protein; PB000374.03.0; PF13_0201), in which the cytoplasmic tail domain (CTD) of TRAP is replaced with the CTD domain of CTRP. The CTD of CTRP can complement the function of the CTD of TRAP, albeit not as well as TRAP as shown by the reduced invasion of salivary glands and hepatocytes. 

Other mutants
RMgm-141: an independent mutant lacking expression of CTRP.
RMgm-150: A mutant that expresses a mutated form of TRAP (thrombospondin-related anonymous protein; PB000374.03.0; PF13_0201), in which the cytoplasmic tail domain (CTD) of TRAP is replaced with the CTD domain of CTRP.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0412900
Gene Model P. falciparum ortholog PF3D7_0315200
Gene productcircumsporozoite- and TRAP-related protein
Gene product: Alternative nameCTRP
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 genePartial
Additional remarks partial/complete disruption The DNA fragment containing the 5' portion of ctrp (2.05 kb) was subcloned into pBluescript II. The selectable marker gene pbdhfr was inserted into the MunI site of this fragment after ligation of EcoRI linkers to both ends. Integration of the construct results in disruption of the 5'-UTR region of ctrp.
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe DNA fragment containing the 5' portion of ctrp (2.05 kb) was subcloned into pBluescript II. The selectable marker gene pbdhfr was inserted into the MunI site of this fragment after ligation of EcoRI linkers to both ends. Integration of the construct results in disruption of the 5'-UTR region of ctrp.
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