Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene mutation,
Introduction of a transgene
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 36761022 |
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 |
RMgm-5137
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Other information parent line | The mutant, PbΔb9, lacks expression of B9 and expresses GFP under the control of the HSP70 promoter. It is drug selectable marker-free |
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The mutant parasite was generated by |
Name PI/Researcher | Fernandes P, Silvie O |
Name Group/Department | Sorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses |
Name Institute | CIMI-Paris |
City | Paris |
Country | France |
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Name of the mutant parasite |
RMgm number | RMgm-5141 |
Principal name | Pb-b9(mut) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Complementation of PbΔb9 sporozoites with a construct encoding the entire PbB9 fully restored sporozoite infectivity in HepG2 cell cultures, validating the genetic complementation approach. In contrast, parasites complemented with a truncated B9 lacking the propeller domain, alone or in combination with the first 6-cys domain, were not infectious, phenocopying the parental B9-deficient parasites. These results show that the propeller domain is required for B9 function during sporozoite entry. Chimeric B9 versions where the propeller domain of PbB9 was replaced by the equivalent sequence from PyB9 (Pyprop, Pyprop6cys1) restored sporozoite infectivity. In contrast, substitution of the PfB9 propeller domain for the PbB9 propeller (Pfprop) did not restore infectivity in complemented parasites. Complementation with the PyB9 propeller domain restored infection in both HepG2, which express SR-B1 but not CD81, and HepG2/CD81 cells, which express both receptors, suggesting that the B9 propeller domain does not restrict host receptor usage. |
Liver stage | Complementation of PbΔb9 sporozoites with a construct encoding the entire PbB9 fully restored sporozoite infectivity in HepG2 cell cultures, validating the genetic complementation approach. In contrast, parasites complemented with a truncated B9 lacking the propeller domain, alone or in combination with the first 6-cys domain, were not infectious, phenocopying the parental B9-deficient parasites. These results show that the propeller domain is required for B9 function during sporozoite entry. Chimeric B9 versions where the propeller domain of PbB9 was replaced by the equivalent sequence from PyB9 (Pyprop, Pyprop6cys1) restored sporozoite infectivity. In contrast, substitution of the PfB9 propeller domain for the PbB9 propeller (Pfprop) did not restore infectivity in complemented parasites. Complementation with the PyB9 propeller domain restored infection in both HepG2, which express SR-B1 but not CD81, and HepG2/CD81 cells, which express both receptors, suggesting that the B9 propeller domain does not restrict host receptor usage. |
Additional remarks phenotype | Mutant/mutation
Several mutants are described that express mutated forms of B9 (see below for details for the different mutants/mutated versions). These mutante lacks express GFP under the control of the HSP70 promoter.
The mutants have been generated by introducing mutated forms of the b9 gene in the mutant, PbΔb9, that lacks expression of B9 and is drug selectable marker-free
Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored.
From the paper:
Three domains were predicted at the structural level using HHpred: an N-terminus propeller domain similar to that of CyRPA encoded by the first exon, and two putative but poorly supported 6-cys domains encoded by the second exon. CyRPA is a cysteine-rich protein expressed in P. falciparum merozoites, where it forms a protein complex that is essential for invasion of erythrocytes. B9 is enriched in cysteines, nine being located in the predicted propeller domain that we suppose are involved in the formation of disulphide bonds in a similar manner to CyRPA, to stabilize the protein structure.
Phenotype
To define the functional importance of the predicted propeller domain (see above), various DNA constructs were generated encoding the entire or partially deleted B9, all containing an intact signal peptide and C-terminus sequences to ensure correct secretion and GPI-anchoring of the protein. Constructs were used for transfection of the drug selectable marker-free PbΔb9 mutant line (RMgm-5137).
Complementation of PbΔb9 sporozoites with a construct encoding the entire PbB9 fully restored sporozoite infectivity in HepG2 cell cultures, validating the genetic complementation approach. In contrast, parasites complemented with a truncated B9 lacking the propeller domain, alone or in combination with the first 6-cys domain, were not infectious, phenocopying the parental B9-deficient parasites. These results show that the propeller domain is required for B9 function during sporozoite entry. Chimeric B9 versions where the propeller domain of PbB9 was replaced by the equivalent sequence from PyB9 (Pyprop, Pyprop6cys1) restored sporozoite infectivity. In contrast, substitution of the PfB9 propeller domain for the PbB9 propeller (Pfprop) did not restore infectivity in complemented parasites. Complementation with the PyB9 propeller domain restored infection in both HepG2, which express SR-B1 but not CD81, and HepG2/CD81 cells, which express both receptors, suggesting that the B9 propeller domain does not restrict host receptor usage.
Additional information
Evidence is presented in the paper that:
- B9 is required for sporozoite invasion
- B9 is secreted from the sporozoite micronemes
- B9 contains a CyRPA-like beta propeller domain, required for B9 function
- The propeller domain of B9 interacts with P36 and P52
Other mutants
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