Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene tagging
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30102727 |
MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone |
P. berghei ANKA 2.34
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Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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The mutant parasite was generated by |
Name PI/Researcher | Deligianni E, Siden-Kiamos I |
Name Group/Department | Institute of Molecular Biology and Biotechnology |
Name Institute | Foundation for Research and Technology-Hellas |
City | Heraklion |
Country | Greece |
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Name of the mutant parasite |
RMgm number | RMgm-4462 |
Principal name | pplp4::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 | 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. |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version 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
Analysis of a mutant lacking expression of PPLP4 (RMgm-4461) showed the following: 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.
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.
Analysis of the mutant expressing PPLP4 fused C-terminally to mCherry, described here showed the following: 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.
Additional information
Other mutants |