Summary

RMgm-4406
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: 2 January 2018, 14:04
  *RMgm-4406
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) : 29253313
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/ResearcherSteel RWJ, Kappe SHI
Name Group/DepartmentCenter for Infectious Disease Research
Name Instituteformerly Seattle Biomedical Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4406
Principal nameplp1/spect2(-)
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
SporozoiteNormal numbers of salivary gland sporozoites. Injection of 100 sporozoites revealed that Py plp1/spect2¯ sporozoites were significantly less infectious than Py WT sporozoites (1 out of 10 mice developed blood stage parasitemia). When infection was delivered by five mosquito bites, all Py WT infected mice but none of the Py plp1/spect2¯ infected mice developed blood stage infection. Increasing the challenge to 15 infectious bites resulted in 7% of Py plp1/spect2¯ infected mice developing blood stage infection, with a greater than one-day delay to patency observed for infected mice of both groups compared to Py WT. Reduced hepatocyte cell traversal in vitro. Normal/higher hepatocyte invasion rate in vitro compared to wild type.
Liver stageInjection of 100 sporozoites revealed that Py plp1/spect2¯ sporozoites were significantly less infectious than Py WT sporozoites (1 out of 10 mice developed blood stage parasitemia). When infection was delivered by five mosquito bites, all Py WT infected mice but none of the Py plp1/spect2¯ infected mice developed blood stage infection. Increasing the challenge to 15 infectious bites resulted in 7% of Py plp1/spect2¯ infected mice developing blood stage infection, with a greater than one-day delay to patency observed for infected mice of both groups compared to Py WT. Reduced hepatocyte cell traversal in vitro. Normal/higher hepatocyte invasion rate in vitro compared to wild type.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of plp1 (spect2).

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. yoelii genome contains 5 PLP proteins

Phenotype
Normal numbers of salivary gland sporozoites. Injection of 100 sporozoites  revealed that Py plp1/spect2¯ sporozoites were significantly less infectious than Py WT sporozoites (1 out of 10 mice developed blood stage parasitemia). When infection was delivered by five mosquito bites, all Py WT infected mice but none of the Py plp1/spect2¯ infected mice developed blood stage infection. Increasing the challenge to 15 infectious bites resulted in 7% of Py plp1/spect2¯ infected mice developing blood stage infection, with a greater than one-day delay to patency observed for infected mice of both groups compared to Py WT. Reduced hepatocyte cell traversal in vitro. Normal/higher hepatocyte invasion rate in vitro compared to wild type.

Additional information

Other 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 modificationMarker-free Py plp1/spect2¯ parasites were created using a sequential double crossover recombination strategy known as Gene Insertion/Marker Out (GIMO).

Briefly, approximately 1 kb of the 5’ and 3’ UTR of Py PLP1/SPECT2 was amplified from WT Py 17XNL genomic DNA and cloned into the pDEF vector and blood stage schizonts transfected. Following positive selection using pyrimethamine and cloning, the drug selectable marker was removed by a second transfection with only the 5’ and 3’ UTR fragment and negative selection with 5 flurocytosine.
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