Asexual blood stage | 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 remarks phenotype | Mutant/mutation
In this mutant the pbsip2 gene contains two loxP sequences that were inserted in the 5' and 3' UTR regions. This mutant will lose the entire open reading frame of pbsip2 in the presence of rapamycin.
The loxP sequences in pbsip2 were introduced in a mutant (RMgm-5590) that expresses a dimerizable Cre recombinase (DiCre), constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB and it expresses CAS9. In this mutant the DiCre expression cassette is introduced into the silent 230p locus into mutant RMgm-4870 that contains the cas9 in the silent c/d-ssu-rRNA gene unit and does not contain a drug-selectable marker that has been removed by negative selection.
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
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
It was first assessed whether DiCre-mediated recombination in pbsip2-cKO occurs quickly enough to disrupt pbsip2 before PbSIP2 expression begins. The whole blood from mice infected with synchronized pbsip2-cKO was split into two cultures at 6 hpi, and the one was treated with 10 nM rapamycin (pbsip2-cKORapa+) and the other with dimethyl sulfoxide as a control (pbsip2-cKORapa−). At 18 hpi (12 h of culture), when no parasites had started nuclear division, recombination at the pbsip2 locus was assessed by genotyping PCR. The assay showed that the pbsip2 locus was almost completely excised from the genome in pbsip2-cKORapa+ while no recombination was detected in pbsip2-cKORapa−. Notably, using the parental PbDiCre parasite, it was confirmed that schizont development was not affected by rapamycin.
Evidence is presented for the following:
- PbSIP2 binds to the upstream of genes related to merozoite formation.
- PbSIP2 functions as a transcriptional activator
- PbSIP2 binds to the GTGCA motif through its tandem AP2 domains.
- Th domain RGTGCA is essential for PbSIP2 binding and transcription of the downstream gene
To assess whether RGTGCA is essential for the DNA binding of PbSIP2 and functions as a cis-regulatory element for the downstream gene, we introduced a mutation into the motif within the ChIP peak upstream of rop14 (PBANKA_0111600; PF3D7_0613300). We developed parasites expressing GFP-fused PbSIP2 using PbCas9 (PbSIP2::GFPCas9) and then introduced a mutation upstream of rop14, altering GGTGCA to ttTGCA (PbSIP2::GFPCas9_cismut). ROP14 is a functionally uncharacterized rhoptry protein that localizes to the rhoptry bulbs of merozoites. Rop14 was selected as the target in this experiment because it is not essential for the asexual blood-stage development in P. berghei. Using these parasite lines, we examined whether the disruption of the RGTGCA motif affected the binding of PbSIP2 to the genome by ChIP-qPCR analysis.
- PbSIP2 recognize the bipartite motif upstream of rhoptry genes.
Analysis of a mutant expressing a C-terminal GFP-tagged version of SIP2 (RMgm-5592) showed the following: 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.
Other mutants |