Summary

RMgm-68
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: Region II plus (substituted with alanines)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 4 March 2010, 23:39
  *RMgm-68
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) : 16201021
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, 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-68
Principal nameCS-RIImut
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 and oocyst-sporozoites at day 14 after infection of A. stephensi mosquitoes. After day 14 oocyst-sporozoite numbers remain high compared to wild type, whereas in the hemolymph only minimal numbers of free sporozoites were detected, indicating a defect in release of sporozoites in the hemolymph. Mutant oocysts were more resistant to trypsin treatment (see further additional remarks phenotype).
SporozoiteSee also the description of the phenotype of the oocyst.
Mutant sporozoites display normal morphology and motility.
Oocyst-sporozoites obtained by disruption of oocysts are not infective to young Sprague/Dawley rats and they show a reduced binding rate (~40%) to HepG2 cells in vitro.
Liver stageOocyst-sporozoites obtained by disruption of oocysts are not infective to young Sprague/Dawley rats and they show a reduced binding rate (~40%) to HepG2 cells in vitro.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of the circumsporozoite protein (CS).
The mutant expresses CS in which region II-plus of the protein has been substituted with alanines (R290A, K291A, R292A, K293A). Region II-plus is located at the 5' end of the thrombospondin type I repeat (TSR) domain.

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 analysis shows that CS is involved in the exit of the sporozoites from the oocysts and that mutation in region II-plus of CS prevents the exit of sporozoites from oocysts. The trypsin-resistant phenotype of the mutant oocysts indicate that sporozoite egress from oocysts is a protease-dependent process. CS is present on the inner surface of the oocyst capsule and it is possible that proteolysis (involving cysteine and/or serine proteases) of the CS protein layer underneath the capsule is required for sporozoite egress. See also mutant RMgm-102 which lacks expression of a papain-like cysteine protease (Egress Cysteine Protease 1; ECP1) which is required for sporozoite egress from sporozoites.
The reduced binding of mutant sporozoites to HepG2 cells in vitro and the lack of infectivity to rats indicate that the Region II-plus is involved in hepatocyte invasion (possibly through binding to heparan sulfate proteoglycans (HSPGs).
A. P. berghei mutant has been described expressing a mutated from of CS of P. falciparum lacking Region II (RMgm-71). Sporozoites of this mutant show no gliding motility, do not invade salivary glands and are not infective to the mammalian host.. A difference between the mutated CS genes is that the Region II-plus deletion in the mutant described here does not affect the thrombospondin (TSR) domain whereas in the RMgm-71 mutant two of the four cysteines of the TSR domain are deleted.

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).
P. berghei mutants have been generated with a mutated GPI-anchor addition sequence (RMgm-73).
A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. falciparum CS (RMgm-69).
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 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).
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 mutationRegion II plus (substituted with alanines)
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 modificationThe mutant expresses CS in which region II-plus of the protein has been substituted with alanines (R290A, K291A, R292A, K293A). Region II-plus is located at the 5' end of the thrombospondin type I repeat (TSR) domain.

Mutations were introduced into the CS coding region by using QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, California, United States). Primer1 (sense, 5′-GTATAAGAGTTGCTGCAGCAGCAGGTTCAAATAAGAAAGC-3′) and its reverse and complement primer2 were used to mutate R290, K291, R292, and K293 to alanines.
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