RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4979
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1017000; Gene model (P.falciparum): PF3D7_1429200; Gene product: AP2 domain transcription factor AP2-O3, putative (ApiAP2; AP2-O3)
Phenotype Fertilization and ookinete;
Last modified: 13 May 2021, 15:46
  *RMgm-4979
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) : 33665945
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/ResearcherZhenkui C; Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, Schoo
Name InstituteXiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4979
Principal nameΔap2-o3
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 ookineteNormal numbers of gametocytes are produced. Normal exflagellation. Normal egress of gametes. Males are fertile (shown in crossing-experiments). Fertilization ability of females is impaired. Fertilized zygotes of the Δap2-o3 mutants were developmentally arrested at the early stages (stage II-III), displaying incomplete elongation. Only ookinetes with aberrant morphology are produced.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of AP2-O3
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).

Protein (function)
Most sequence-specific transcription factor families found in other eukaryotes seem to be absent from Plasmodium. Instead, an expansion of a protein family containing one or more apetala2 (AP2) DNA-binding domains was observed across the phylum apicomplexa. In total, 27 members of this family have been found in the human malaria parasite Plasmodium falciparum (although a possible 28th member of the family may be present. In total, 26 of these have syntenic orthologs in rodent malaria species, each with its unique stage-specific expression profile.
From the paper: ' Recently, Modrzynska et al and our group systematically investigated the functions of ApiAP2 TFs in the rodent malaria parasite P. berghei and P. yoelii, respectively (Modrzynska et al, 2017; Zhang et al, 2017b). Both studies revealed that AP2-O3, an ApiAP2 member, is expressed in the gametocytes and essential for the ookinete formation and mosquito transmission (Modrzynska et al, 2017; Zhang et al, 2017b). However, the underlying mechanism is unknown. In this study, we established AP2-O3 as a transcription repressor of the male-associated genes in the female gametocytes, which is essential for maintaining the female specific transcriptome.

Phenotype
Normal numbers of gametocytes are produced. Normal exflagellation. Normal egress of gametes. Males are fertile (shown in crossing-experiments). Fertilization ability of females is impaired. Fertilized zygotes of the Δap2-o3 mutants were developmentally arrested at the early stages (stage II-III), displaying incomplete elongation. Only ookinetes with aberrant morphology are produced (see also Additional information)

Additional information
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).

See mutant RMgm-4978 for expression analyses: Tagged AP2-O3 expressed in the nucleus of female gametocytes (not in males) and in mature oocysts (not in ookinetes, early stage oocysts and sporozoites).

Evidence is presented that:
- AP2-O3 null female gametocytes fail to develop into mature fertile gametes
- AP2-O3 represses male gene transcription in the female gametocytes
- Protein expression of upregulated male genes in the AP2-O3 null female gametocytes
- AP2-O3 binds to the upstream promoter of specific male genes
- Decreased expression of highly expressed female-specific genes in the AP2-O3 null female gametocytes
- Downregulation of AP2-O3 expression after fertilization
- Ectopic expression of AP2-O3 after fertilization impairs the ookinete development
- Sequential expression of AP2-O3 and AP2-O during female gametogenesis.

- Protein expression of upregulated male genes in the AP2-O3 null female gametocytes
From the paper: ' We selected 4: dpod2 (PY17X_0408600, DNA polymerase delta small subunit), dpod1 (PY17X_0502300, DNA polymerase delta catalytic subunit), rpa1 (PY17X_0419400, replication protein A1 small fragment), and mcm7 (PY17X_0805800, DNA replication licensing factor). Each of these 4 genes was endogenously tagged with a gfp-coding sequence at the C-terminus in the female-identity reporter strain ccp2::mCherry, yielding 4 double-tagged strains, including ccp2::
mCherry;dpod2::gfp (DTS1), ccp2::mCherry;dpod1::gfp (DTS2), ccp2::
mCerry;rpa1::gfp (DTS3), and ccp2::mCherry;mcm7::gfp (DTS4).
All the 4 GFP-fusion proteins were expressed and nuclear localized in the male gametocytes , but not were detectable (Dpod2::GFP and Dpod1::GFP) or present at extremely low abundance (Rpa1::GFP and Mcm7::GFP) in the female gametocytes. Next, we removed ap2-o3 gene in each individual DTS strain. As expected, AP2-O3 disruption did not significantly affect the level of any of these 4 proteins in the male gametocytes as revealed by both fluorescence microscopy and flow cytometry In clear contrast, protein levels of Dpod2::GFP and Dpod1::GFP in the female gametocytes were dramatically increased, while Rpa1::GFP and Mcm7::GFP levels in the female gametocytes showed a modest increase compared to the corresponding parental strains.'

- Ectopic expression of AP2-O3 after fertilization impairs the ookinete development
From the paper: ' We speculate that ectopic AP2-O3 expression after fertilization will affect normal zygote to ookinete development. To test it, we attempted to generate a strain ectopically expressing AP2-O3 after the gametogenesis. Specifically, in  the ap2-o3::6HA parasites, the 800 bp promoter sequence of ap2-o3 was replaced with a
1,200 bp promoter of ccp2, a gene that is transcribed in the female gametocytes, female gametes, zygotes, and ookinetes (Liu et al, 2018). Correct replacement in the resulting mutant Pccp2 was confirmed by PCR, which drove AP2-O3 expression in the non-activated gametocytes at a level comparable to that of the parental ap2-o3::6HA parasites. The AP2-O3 expression was maintained in the activated Pccp2 gametocytes even at 2 h post-XA stimulation. IFA analyses also confirmed the AP2-O3 expression in all (93/93) of the activated female gametocytes of the Pccp2 parasites. Compared to the parental strain, the Pccp2 strain produced normal gametocytes in mice, male gametes in vitro, and fertilized  zygotes. However, the ability of the zygotes to develop into ookinetes in vitro and midgut oocysts in the mosquitoes was significantly reduced. The detrimental effect imposed on the ookinete development caused by the ectopic expression of AP2-O3 post-fertilization suggests that AP2-O3 expression is precisely regulated to meet the developmental demands.'

Other mutants
see ApiAP2


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1017000
Gene Model P. falciparum ortholog PF3D7_1429200
Gene productAP2 domain transcription factor AP2-O3, putative
Gene product: Alternative nameApiAP2; AP2-O3
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for genomic modification (Zhang et al, 2014; Zhang et al, 2017a). To construct the plasmids for gene deleting, the 50 - and 30-flanking genomic sequence (400–700 bp) of target genes was PCR-amplified as left and right homologous arms and inserted into the restriction sites of pYCm. Oligonucleotides for small guide RNAs (sgRNAs) were annealed and ligated into pYCm. Two sgRNAs were designed to target the coding region of each gene.
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