Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of SIP2
Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.08.02.606280
Protein (function)
SIP2 is an AP2 transcription factor expressed during the schizont stage. AP2 transcription factors are the major family of transcription factors found in Plasmodium. Members in this family contain sequence-specific DNA binding domains with three anti-parallel β-sheets and one α-helix, called AP2 domain. In P. falciparum, SIP2 (PF3D7_0604100) has been reported to bind to the subtelomeric var promoter element 2 (SPE2) and has been proposed to be involved in chromosome end biology.
PbSIP2 contains two tandem AP2 domains at its N-terminus and a putative nuclear localization signal (NLS) on the C-terminal side of the AP2 domains. Protein-protein BLAST (BLASTP) search using the AA sequence of its AP2 domains detected proteins in Hepatocystis, Babesia, and Theileria species but not from other apicomplexan species. These species belong to the Haemosporida or Piroplasmida, which constitute a group of parasites that infect host RBCs.
Phenotype
No fluorescent signals were observed in the ring or trophozoite stages. Fluorescent signals first appeared in the maturing schizonts with four nuclei (at 22 hpi). The signals continued until the schizonts had eight nuclei and then faded in later stages.
Analysis of a mutant after conditional knockdown of SIP2 (RMgm-5591) showed the following:
To investigate the role of PbSIP2 during schizont development, we performed a conditional knockout of pbsip2 using a dimerizable Cre recombinase (DiCre) system. A parasite mutant (PbDiCre; RMgm-5590) was generated constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB, using the Cas9 expressing parasite, PbCas9 (RMgm-4870). Subsequently, two loxP sequences were inserted on 5’ and 3’ sides of pbsip2, arranged in the same direction (pbsip2-cKO). This parasite will lose the entire open reading frame of pbsip2 in the presence of rapamycin.
In culture, pbsip2-cKORapa+ developed as comparable to pbsip2-cKORapa− until 24 hpi. At 26 hpi, approximately 20% of pbsip2-cKORapa− became mature schizonts (number of nuclei>10), and some have already formed merozoites. In contrast, most schizonts of pbsip2-cKORapa+ had less than ten nuclei at 26 hpi. The number ofpbsip2-cKORapa− schizonts with six–ten nuclei increased at 28 hpi, mature schizonts were barely produced in pbsip2-cKORapa+, and no merozoite formation was observed. These results indicate that PbSIP2 plays an essential role in schizont development.
Additional information
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