Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene disruption,
Gene tagging
|
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 26607162 |
MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone |
Not applicable
|
Other information parent line | |
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The mutant parasite was generated by |
Name PI/Researcher | Risco-Castillo V; Silvie, O |
Name Group/Department | Centre d’Immunologie et des Maladies Infectieuses |
Name Institute | INSERM |
City | Paris |
Country | France |
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Name of the mutant parasite |
RMgm number | RMgm-1348 |
Principal name | PyΔplp1/RON4::mCherry |
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 | Mutant sporozoites were motile and developed into EEFs in vitro (in HepG2/CD81 cells) as efficiently as control parasites, but were poorly infective to mice in vivo, especially when administered through mosquito bites, the natural transmission route.
RON4-mCherry is expressed in both oocyst-derived and salivary gland sporozoites, with an apical distribution consistent with rhoptry localization. |
Liver stage | Mutant sporozoites were motile and developed into EEFs in vitro (in HepG2/CD81 cells) as efficiently as control parasites, but were poorly infective to mice in vivo, especially when administered through mosquito bites, the natural transmission route.
RON4-mCherry is expressed in both oocyst-derived and salivary gland sporozoites, with an apical distribution consistent with rhoptry localization. See additional information about rhoptry discharge and release of RON4-mCherry in transient vacuoles (TVs) and in the PV of infected hepatocytes. |
Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of PLP1 (SPECT2), expresses GFP under the control of the HSP70 promoter and expresses a C-terminal mCherry-tagged version of RON4. The parasites have been generated by tagging RON4 in mutant PyΔplp1 (RMgm-1346) using the same strategy and construct as described for mutant RON4::mCherry (RMgm-1034)
Protein (function)
The spect2/pplp1 gene is a member of a small, conserved family of proteins encoding perforin-like proteins containing membrane-attack complex/perforin domains (MACPF). The P. berghei genome contains 5 PLP proteins.
RON4 is a rhoptry protein and plays a critical role during hepatocyte infection (RMgm-685).
Phenotype
Mutant sporozoites were motile and developed into EEFs in vitro (in HepG2/CD81 cells) as efficiently as control parasites, but were poorly infective to mice in vivo, especially when administered through mosquito bites, the natural transmission route. Evidence is presented that sporozoites form transient vacuoles (TVs) in traversed hepatocytes and that mutant sporozoites fail to egress from TVs, indicating that PLP1 is required for sporozoite egress from nonreplicative TVs, but not for entry into cells. Evidence is presented that mCherry::RON4 is not released from the rhoptries in TVs but only in the PV (see below), indicating that the formation of TVs, unlike PVs, occurs without rhoptry discharge.
Additional information
Sporozoites migrate from the dermis to the liver, where they invade hepatocytes through a moving junction (MJ) to form a replicative parasitophorous vacuole (PV). Sporozoites need to traverse cells during progression through host tissues, a process requiring parasite perforin-like protein 1 (PLP1). In the paper evidence is presented that i) sporozoites traverse cells inside transient vacuoles that precede PV formation; ii) sporozoites initially invade cells inside transient vacuoles by an active MJ-independent process that does not require vacuole membrane remodeling or release of parasite secretory organelles typically involved in invasion; iii) sporozoites use pH sensing and PLP1 to exit these vacuoles and avoid degradation by host lysosomes; iv parasites enter the MJ-dependent PV, which has a different membrane composition, precluding lysosome fusion.
Other mutants
See the link PPLP for other mutants with disrupted or tagged pplp genes.
See the link RON4 for other RON4 mutants |