Summary

RMgm-1348
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1007700; Gene model (P.falciparum): PF3D7_0408700; Gene product: perforin-like protein 1 | sporozoite micronemal protein essential for cell traversal (PLP1; PPLP1, SPECT2)
TaggedGene model (rodent): PY17X_0934000; Gene model (P.falciparum): PF3D7_1116000; Gene product: rhoptry neck protein 4 (RON4)
Name tag: mCherry
Phenotype Sporozoite; Liver stage;
Last modified: 27 November 2015, 09:57
  *RMgm-1348
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherRisco-Castillo V; Silvie, O
Name Group/DepartmentCentre d’Immunologie et des Maladies Infectieuses
Name InstituteINSERM
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-1348
Principal namePyΔplp1/RON4::mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
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
SporozoiteMutant 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 stageMutant 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


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1007700
Gene Model P. falciparum ortholog PF3D7_0408700
Gene productperforin-like protein 1 | sporozoite micronemal protein essential for cell traversal
Gene product: Alternative namePLP1; PPLP1, SPECT2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe 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)
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0934000
Gene Model P. falciparum ortholog PF3D7_1116000
Gene productrhoptry neck protein 4
Gene product: Alternative nameRON4
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 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)
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