Summary

RMgm-1346
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)
Phenotype Sporozoite; Liver stage;
Last modified: 27 November 2015, 09:56
  *RMgm-1346
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
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-1346
Principal namePyΔplp1
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.
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.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PLP1 (SPECT2). and expresses GFP under the control of the HSP70 promoter.  The parasites have been generated and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method and the construct used).

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

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.

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.

The mutant parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting (GOMO transfection; 'Gene Out Marker Out'; see mutant RMgm-1026 for more details for this transfection method and the construct used).

Other mutants
See the link PPLP for other mutants with disrupted or tagged pplp genes


  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 and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method)
Additional remarks selection procedureThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP+mCherry- and marker free parasites are selected by FACS sorting and used to infect a mouse
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 15’-TCCCCGCGGTCTCTACCTACATATTCAGAAAGAATCC-3’
Additional information primer 1PyPLP1rep1for
Sequence Primer 25’-ATAAGAATGCGGCCGCATATTCCACAATTCAATATGCATAAATGCC-3’
Additional information primer 2PyPLP1rep2rev
Sequence Primer 35’-CCGCTCGAGTTGATTATACTACCTTTTTAAGTTGAACC-3’
Additional information primer 3PyPLP1rep3for
Sequence Primer 45’-CGGGGTACCAGCAAATAACGATTTATAATTTGTATATCC-3’
Additional information primer 4PyPLP1rep4rev
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6