RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: SYIPSAEKI amino acids replaced by SIINFEKL (H-2-Kb-restricted CD8+ T-cell epitope of ovalbumin)
PhenotypeNo phenotype has been described
Last modified: 16 May 2021, 21:22
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) : 33709544
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherMüller K, Hafalla JCR
Name Group/DepartmentDepartment of Infection Biology, Faculty of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene and Tropical Medicine
Name of the mutant parasite
RMgm numberRMgm-4990
Principal nameCSP(SIINFEKL)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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 stageNot different from wild type
Additional remarks phenotype

In the mutant the csp gene has been replaced with a mutated csp gene encoding CSP in which the amino acids SYIPSAEKI were replaced by the H-2-Kb-restricted CD8+ T-cell epitope of ovalbumin (SIINFEKL). 
SYIPSAEKI of CSP is the immunodominant H-2-Kd-restricted CD8+ T-cell epitope of CSP. The replacement with SIINFEKL allowed for recognition in H-2-Kb-carrying C57Bl6 mice.

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.

The resulting parasites showed a phenotype comparable to WT parasites, with comparable mosquito infectivity and number of salivary gland sporozoites, functional sporozoite motility and normal invasive capacity and development inside hepatocytes. Thus, the introduced mutations to generate CSPSIINFEKL parasites did not interfere with the completion of the life cycle, in either mosquito vector or mouse. All C57BL/6 mice that received 800 sporozoites of CSPSIINFKEL intravenously developed a detectable (patent) blood stage infection by day 4, comparable to infection with WT sporozoites

Additional information
From the Abstract:
'We engineered Plasmodium berghei parasites that harbour a well-characterised epitope for stimulation of CD8+ T cells, either as an antigen in the sporozoite surface-expressed circumsporozoite protein or the parasitophorous vacuole membrane associated protein upregulated in sporozoites 4 (UIS4) expressed in exo-erythrocytic forms (EEFs). We show that the antigen origin results in profound differences in immunogenicity with a sporozoite antigen eliciting robust, superior antigen-specific CD8+ T-cell responses, whilst an EEF antigen evokes poor responses. Despite their contrasting immunogenic properties, both sporozoite and EEF antigens gain access to antigen presentation pathways in hepatocytes, as recognition and targeting by vaccine-induced effector CD8+T-cells results in high levels of protection when targeting either antigen. Our study is the first demonstration that poorly immunogenic EEF antigens do not preclude their susceptibility to antigen-specific CD8+ T-cell killing.'

Other mutants

  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 mutationSYIPSAEKI amino acids replaced by SIINFEKL (H-2-Kb-restricted CD8+ T-cell epitope of ovalbumin)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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