| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene mutation,
Introduction of a transgene
|
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 28137845
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| 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 |
P. berghei ANKA 676m1cl1 (RMgm-29)
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| Other information parent line | 676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). |
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| The mutant parasite was generated by |
| Name PI/Researcher | Sá E Cunha C, Mota MM, Prudencio M, Mueller AK |
| Name Group/Department | Parasitology Unit, Centre for Infectious Diseases |
| Name Institute | Heidelberg University Hospital |
| City | Heidelberg |
| Country | Germany |
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| Name of the mutant parasite |
| RMgm number | RMgm-4564 |
| Principal name | PbEXP-1ΔC2 |
| 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 | Not different from wild type |
| Liver stage | The characteristic movement of infectious sporozoites and their invasion capacity were also not impaired in the PbEXP-1ΔC2 parasites. Additionally, immunofluorescence microscopy analysis of Huh7 cells infected with clonal PbEXP-1ΔC2 lines and stained with antibodies against the PVM proteins UIS4 and EXP-1 indicated that neither the integrity of the vacuolar membrane nor the localization of EXP-1 were compromised in transgenic parasites.
Analyses of infected Huh7 cells 48 h after sporozoite addition showed that infection with any of the three PbEXP-1ΔC2 clones led to lower EEF numbers and sizes than those yielded by their counterparts containing full-length EXP-1. Merosome formation is clearly delayed in transgenic PbEXP-1ΔC2 parasites compared with their WT parasite controls.
A decrease in the liver parasite load was observed of mice infected with PbEXP-1ΔC2 at 42 hpi, compared with their respective controls. Late intrahepatic development (72–96 hpi) was significantly reduced in PbEXP-1ΔC2 compared with WT infections. |
| Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated form of exp1/hep17 where a part of the C-terminal region (C2; 93bp) of exp1/hep17 is deleted.
Protein (function)
Exp1 (HEP17; circumsporozoite-related antigen) is a protein that localizes to the parasitophorous vacuole membrane (PVM) of blood stages and liver stages.
Exported protein 1 (EXP-1) was the first protein described to associate with the PVM. It harbors a classical N-terminal signal peptide, and, upon trafficking via the endoplasmic reticulum–Golgi transport route, is inserted with its transmembrane domain into the PVM of both blood and liver parasite stages. It was shown later that the protein’s N-terminal region faces the PV lumen, whereas its C terminus (CT) extends into the host-cell cytosol.
Phenotype
Normal blood stage development. This indicates that the carboxyl-terminal C2 part of EXP-1 is not essential for intraerythrocytic parasite development because we were able to generate deletion mutants lacking this particular portion of the PVM-resident protein.
Analyses of infected Huh7 cells 48 h after sporozoite addition showed that infection with any of the three PbEXP-1ΔC2 clones led to lower EEF numbers and sizes than those yielded by their counterparts containing full-length EXP-1. Merosome formation is clearly delayed in transgenic PbEXP-1ΔC2 parasites compared with their WT parasite controls.
A decrease in the liver parasite load was observed of mice infected with PbEXP-1ΔC2 at 42 hpi, compared with their respective controls. Late intrahepatic development (72–96 hpi) was significantly reduced in PbEXP-1ΔC2 compared with WT infections.
From the paper:
'Here, we show that, during the parasite’s hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency.'
Additional information
A significantly decreased colocalization of EXP-1 and ApoH was observed in additional immunofluorescence experiments using PbEXP-1ΔC2–infected Huh7 cells, compared with WT Pb-infected cells. Furthermore, quantification of accumulated ApoH in liver stages of PbWT and PbEXP-1ΔC2 parasites showed a significantly reduced uptake of ApoH in intrahepatic stages of parasites expressing the C-terminally truncated version of EXP-1. This finding clearly suggests that the interaction between the two proteins is severely compromised when the C2 region of EXP-1 is truncated.
Other mutants |
*RMgm-4564
