Summary

RMgm-286
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: Mutation of two putative pexel/VTS motifs by mutation of conserved arginine and leucine to alanine
Phenotype Liver stage;
Last modified: 25 April 2011, 11:01
  *RMgm-286
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) : 17981117
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/ResearcherA.P. Sing, V. Nussenzweig
Name Group/DepartmentDepartment of Pathology
Name InstituteNew York University School of Medicine
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-286
Principal nameCS pexel/VTS
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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
SporozoiteNot different from wild type
Liver stageInvasion of mutant sporozoites of HepG2 cells was comparable to wild type sporozoites. Release of CS in the cytoplasm of hepatocytes was strongly reduced (>90%) at 1-3 hours after invasion. Parasites were affected in development within hepatocytes both in vivo and in vitro in HepG2 cells. Mature stages in HepG2 cells were reduced in number (2.5 times) and reduced in size (3 times). In vivo, a 10-14 times reduction in growth of mature liver stages was observed.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of CS in which the two putative pexel/VTS motifs are mutated. The conserved arginine and leucine were mutated to alanine by site directed mutagenesis. The endogenous cs gene was replaced with the mutated form.

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 phenotype analyses indicate that mutation of the pexel/VTS motifs in the CS protein had no effect on the in vitro invasion of hepatocytes  by mutant sporozoites. However, development within hepatocytes was strongly reduced. Evidence is presented that expression of the mutated CS protein resulted in abortion of transport of CS into the cytoplasm of the host hepatocyte. The lack of CS release into the cytoplasm/nucleus of the hepatocyte affected the development of the liver stages.

Additional information
In wild type sporozoites, the CS-protein is cleaved at the N-terminal by a parasite cysteine protease, and this processing is required for infectivity. Although several substitutions were introduced in the amino terminus of the pexel/VTS of the CS mutants, the protein was processed correctly.

See also the mutant RMgm-614 which expresses the CS-protein with similar mutated Pexel/VTS motifs. For this mutant it was shown the CS was exported into the cytoplasm of the infected hepatocyte and no evidence was reported for affected CS export.

Other mutants
RMgm-285: Mutants expressing a CS-GFP fusion protein in blood stages. Mutants express the fusion protein in which the putative pexel/VTS motifs of the CS gene are mutated.


  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 mutationMutation of two putative pexel/VTS motifs by mutation of conserved arginine and leucine to alanine
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate the mutant, both pexel motifs were mutated in the context of full-length CS and the final construct was named pRCS pexel1-2. Conserved arginine and leucine were mutated to alanine by site directed mutagenesis. A Pst I restriction site next to pexel2 and a Sac I next to pexel1 mutation site were introduced, without changing amino acid sequence, for restriction analysis.
The endogenous cs gene was replaced with the mutated form of cs by double cross-over integration, using 5'and 3'UTR regions of cs as target regions. No information is provided on the sequence of the target regions or the primers used to amplify the target regions.
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