Summary

RMgm-73
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: Mutations of the C-terminal glycosylphosphatidylinositol (GPI)-anchor addition sequence
Phenotype Oocyst; Sporozoite;
Last modified: 4 March 2010, 23:40
  *RMgm-73
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) : 16207248
MR4 number
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/ResearcherQ. Wang, H. Fujioka, V. Nussenzweig
Name Group/DepartmentDepartment of Pathology
Name InstituteMichael Heidelberger Division, New York University School of Medicine
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-73
Principal nameCS-∆GPI, CS-TM1, CS-TM2
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
OocystNormal numbers of of mature oocysts are produced by all three mutants in A. stephensi mosquitoes. However no sporozoites are formed within the oocysts. Up to day eight after feeding of the mosquitoes the morphology of oocysts is normal; after day 8, the oocysts display a highly vacuolated structure (see for further details 'Additional remarks phenotype').
SporozoiteSee phenotype description of the oocyst and for further details 'Additional remarks phenotype'.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The three mutants (CS-∆GPI, CS-TM1, CS-TM2) express mutated forms of the circumsporozoite protein (CS). Mutations have been introduced in the C-terminal glycosylphosphatidylinositol (GPI)-anchor addition sequence (see below).

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
The mutants express CS with mutated C-terminal glycosylphosphatidylinositol (GPI)-anchor addition sequence. This seqence contains two pairs of cysteines in the C-terminal, and the last cysteine is predicted to serve as the site of attachment of the GPI-anchor. In mutant CS-∆GPI the GPI-anchor addition sequence is deleted. In mutants CS-TM1, CS-TM2 the GPI-anchor addition sequence of CS was replaced with the trans-membrane domain (TMD) and part of cytoplasmic tail (truncated to different degree) of thrombospondin-related anonymous protein (TRAP; PB000374.03.0).  See for further details 'Additional information genetic modification'.

The phenotype analysis shows that normal numbers of  mature oocysts are produced by all three mutants in A. stephensi mosquitoes. However no sporozoites are formed, comparable to the phenotype of a mutant lacking CS (RMgm-9) with a comparable (EM) morphology of the aberrant oocysts. These results suggest that CS function during sporozoite development occurs on the plasma membrane of the oocysts and requires the specific presence of its GPI-anchor.
In CS-∆GPI oocysts, CS is never detected on the oocyst plasma membrane, and instead remains in the cytoplasm, associated with the periphery of vesicles of various sizes. In CS-TM1 CS is found both in the cytoplasm and plasma membrane of the oocysts.

Other mutants
A P. berghei mutant has been generated that lacks expression of CS (RMgm-9) .
A. P. berghei mutant has been generated that produces reduced levels of CS (RMgm-72).
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. falciparum CS (RMgm-69).
A P. berghei mutant has been generated that express a hybrid form of CS of P. berghei and  P. falciparum (the P. berghei CS repeat region  is exchanged with the P. falciparum CS repeat region)(RMgm-76).
Two P. berghei mutants have been generated that express mutated forms of P. falciparum CS (RMgm-70 with a mutated Region I; RMgm-71 with a mutated Region II).
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. gallinaceum CS (RMgm-74).
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. yoelii CS (RMgm-75).

 


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
Details of the genetic modification
Short description of the mutationMutations of the C-terminal glycosylphosphatidylinositol (GPI)-anchor addition sequence
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn the mutants the endogenous CS is replaced with the mutated forms using replacement vectors.
In CS-∆GPI, the addition of a GPI-anchor to CS is prevented by deletion of nucleotides encoding the C-terminal stretch of hydrophobic amino acids that contains the GPI-anchor addition sequence (S317 to F340). In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was replaced with two versions of the C-terminal of another sporozoite surface protein, TRAP (PB000374.03.0). CS-TM1 contains only the TRAP Trans Membrane Domain (TMD; I544-G559) followed by five alanines at the C-terminus. CS-TM2 contains a larger portion of TRAP (K534-S569), which includes its TMD, 10 amino acids upstream of the TMD (K534–K543) and 10 amino acids downstream from the TMD (V560-S569). The choice of TM2 replacement was based on the finding that these amino acids are sufficient to direct TRAP to the sporozoite plasma membrane
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