Summary

RMgm-4461
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0711400; Gene model (P.falciparum): PF3D7_0819400; Gene product: perforin-like protein 4 (PPLP4)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 14 August 2018, 18:20
  *RMgm-4461
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) : 30102727
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherDeligianni E, Siden-Kiamos I
Name Group/DepartmentInstitute of Molecular Biology and Biotechnology
Name InstituteFoundation for Research and Technology-Hellas
CityHeraklion
CountryGreece
Name of the mutant parasite
RMgm numberRMgm-4461
Principal namepplp4(-)
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
OocystNormal numbers of (motile) ookinetes (with wild type morphology) are produced. Evidence is presented that ookinetes were unable to invade and traverse the midgut epithelium resulting in severely impaired oocyst formation and no transmission to naïve mice
SporozoiteNormal numbers of (motile) ookinetes (with wild type morphology) are produced. Evidence is presented that ookinetes were unable to invade and traverse the midgut epithelium resulting in severely impaired oocyst formation and no transmission to naïve mice
Liver stageNormal numbers of (motile) ookinetes (with wild type morphology) are produced. Evidence is presented that ookinetes were unable to invade and traverse the midgut epithelium resulting in severely impaired oocyst formation and no transmission to naïve mice
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PPLP4 (perforin-like protein 4)

Protein (function)
PPLP4 encodes one of the five PPLP genes (PPLP1–PPLP5) identified in the P. berghei genome that contains a membrane attack complex/perforin (MACPF) domain (see also the link PPLP for other mutants with disrupted or tagged pplp genes).

Phenotype
Normal numbers of (motile) ookinetes (with wild type morphology) are produced. Evidence is presented that ookinetes were unable to invade and traverse the midgut epithelium resulting in severely impaired oocyst formation and no transmission to naïve mice.

When in vitro cultured ookinetes were injected into the thorax of the mosquito, thus by-passing midgut passage, sporozoites were formed and the mutant parasites were able to infect naïve mice.

Additional information
RT-PCR analyses showed: the pplp4 gene is not transcribed in blood stages, with the transcript first detected at 3 h after the seeding of an ookinete culture, a time corresponding to zygote formation, and being continuously present until the mature ookinete stage. No expression was detected in oocysts or sporozoites.

Using a different antibody developed against PfPPLP4 revealed that the labelling pattern in P. berghei in vitro cultured ookinetes was consistent with our previous finding, showing a dotted peripheral pattern.

To further investigate the trafficking and cellular localization of PPLP4, we generated a parasite mutant (RMgm-4462) expressing PPLP4 fused C-terminally to mCherry. Surprisingly, in live ookinetes the chimeric protein was detected in the punctate structures in the parasite cytoplasm both in dots and diffusely towards the apical end. The pplp4::mCherry mutant formed ookinetes with similar conversion rate to WT; oocysts were also formed and sporozoites transmitted to a naïve mouse in two subsequent experiments.

Dual immunolabeling with antibodies directed against mcherry and the micronemal protein CTRP showed that the punctate structure are distinct from the micronemes.

In order to explain the absence of signal in the pplp4::mCherry ookinete periphery, we hypothesized that during protein export to the ookinete pellicle the mCherry tag at the C-terminal of PPLP4 may be processed leading to cleavage of the tag. To test this hypothesis we performed a Western blot analysis of pplp4::mCherry retorts and ookinetes using the anti-mCherry antibody. The results showed that two bands were recognized of molecular weight ~110 kDA (doublet) and 30 kDA that corresponds to the size of the chimeric protein and mCherry respectively. This supports the hypothesis that the C-terminus tag is cleaved during trafficking of PPLP4 to the periphery of ookinetes.

Taken together, these data indicate that PPLP4 is packaged into vesicular structures that are trafficked to the parasite periphery.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0711400
Gene Model P. falciparum ortholog PF3D7_0819400
Gene productperforin-like protein 4
Gene product: Alternative namePPLP4
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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