Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene mutation
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 21445239 |
MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone |
Not applicable
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Other information parent line | |
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The mutant parasite was generated by |
Name PI/Researcher | I.A. Cockburn; F. Zavala |
Name Group/Department | Johns Hopkins Malaria Research Institute and Department of Molecular Microbiology and Immunology |
Name Institute | Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University |
City | Baltimore |
Country | USA |
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Name of the mutant parasite |
RMgm number | RMgm-613 |
Principal name | CS5M |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | The mutant produces normal numbers of salivary gland sporozoites. See also additional remarks phenotype. |
Liver stage | The mutant produces normal numbers of salivary gland sporozoites. Salivary gland sporozoites show normal infectivity to mice. See also additional remarks phenotype. |
Additional remarks phenotype | Mutant/mutation
In the mutant (CS5M) the wild type cs gene is replaced with a mutated cs gene carrying 5 mutations that changed the natural H-2Kd restricted epitope SYIPSAEKI to SIINFEKL, an H-2Kb restricted epitope
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
See additional information below
Additional information
Phenotype analyses of sporozoites of the mutant (CS5M), expressing a mutated CS protein with the H-2Kb epitope SIINFEKL, provide evidence that both dendritic cells (DCs) and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER.
In addition evidence is presented that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes the CS protein must be secreted directly into the cytosol. These observations suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol.
Secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein (see also mutant RMgm-614).
In the CS5M mutant the epitope SIINFEKL is inserted in place of a well-defined natural epitope, leaving intact the neighboring residues to ensure correct proteasomal processing, thus the model epitope is presented exactly as the natural CS epitope. This makes this mutant an useful system in which to study antigen processing and presentation. Therefore, the authors anticipate that the CS5M mutant will be a powerful tool for use in future studies of antigen specific immune responses to malaria sporozoites.
Other mutants
RMgm-614: A mutant expressing a mutated CS containing the model SIINFEKL H-2Kb restricted epitope with additional mutations of the Pexel/VTS motifs |