Summary

RMgm-74
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: The endogenous P. berghei gene replaced with the homolog of P. gallinaceum
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 12 September 2012, 11:17
  *RMgm-74
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) : 15839899
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 lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherR. Tewari, D. Rathore, A. Crisanti
Name Group/DepartmentImperial College of Science, Technology and Medicine
Name InstituteImperial College of Science
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-74
Principal namePgCS-14
Alternative namePgCS-14 (replacement with PgCS)
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. Oocysts and oocyst-sporozoite numbers in A. gambiae were comparable to that of wild type. Both wild type and mutant parasites did not infect Aedes aegypti.
A proportion (12%) of PgCS-14 infected A. stephensi mosquito, showed melanotic encapsulation of oocysts at
late (18–21 days p.i.) developmental stages.
SporozoiteNormal numbers of oocyst-sporozoites are formed but sporozoites fail to invade salivary glands. The frequency of motile oocyst-derived sporozoites was comparable to that observed for oocyst-derived sporozoites of wild type.
Sporozoites were not infective to mice (C57Bl/6).
Liver stageSporozoites were not infective to mice (C57Bl/6).
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei CS is replaced by P. gallinaceum CS (circumsporozoite protein) .

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 analyzes show that the CS protein of P. gallinaceum (PgCS) can only partly complement the activity of the endogenous P. berghei CS protein. P. berghei mutant parasites lacking expression of CS (RMgm-9) do not form sporozoites within the oocysts. The oocysts-derived sporozoites expressing PgCS showed a normal surface localization of CS as shown by immunofluorescence assays.
Parasites expressing PgCS do form sporozoites, however they fail to invade salivary glands and are not infective to the mammalian host. P. berghei mutants expressing CS of P. falciparum (PfCS; RMgm-69) and P. yoelii (RMgm-75) are able to invade salivary glands and are able to infect the mammalian host, although the invasion of salivary glands of mutants expressing PfCS is reduced. Combined, these results suggest that P. berghei sporozoites expressing CS of P. gallinaceum lack the correct receptor-ligand interaction for invasion of salivary glands of Anopheles mosquitoes.

Other mutants
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 in which the endogenous P. berghei CS was replaced with  P. yoelii (RMgm-75)
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 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 with a mutated Region II-plus (RMgm-68).
P. berghei mutants have been generated with a mutated GPI-anchor addition sequence (RMgm-73).

 


  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 mutationThe endogenous P. berghei gene replaced with the homolog of P. gallinaceum
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe complete CS coding sequence from P. gallinaceum was introduced into the genome, thereby replacing the endogenous P. berghei CS gene. The pgCS gene is under control of the PbCS regulatory sequences. In the paper details are missing (not provided!) on the sequence of the CS gene of P. gallinaceum and no details are given of the primers used for the target sequences to introduce the construct into the genome
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