RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5254
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1015500; Gene model (P.falciparum): PF3D7_1429200; Gene product: AP2 domain transcription factor AP2-O3, putative (ApiAP2; AP2-O3; PbAP2-FG2)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 28 September 2022, 14:09
  *RMgm-5254
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherNishi T, Yuda M
Name Group/Department1Laboratory of Medical Zoology, Department of Medicine
Name InstituteMie University
CityMie, Tsu
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5254
Principal namepbap2-fg2(-)
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 ookineteThe mutant formed morphologically normal female and male gametocytes, and the male gametocytes showed normal exflagellation. Pbap2-fg2(-) parasites failed to produce banana-shaped ookinetes; more than half of the fertilized population stopped developing at round zygotes, and the others, at retort-form ookinetes.
OocystFormation of aberrant ookinetes. No oocyst/sporozoite formation
SporozoiteFormation of aberrant ookinetes. No oocyst/sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PbAP2-FG2 (PbAP2-O3).

Published in bioRxiv preprint doi: https://doi.org/10.1101/2022.09.22.508975

Protein (function)
In Plasmodium spp., sexual development is triggered by AP2-G. It is an AP2-family transcription factor that is expressed in a subpopulation of blood-stage parasites, with disruption of the gene resulting in complete loss of the parasite’s capability to produce gametocytes. The target genes of AP2-G includes a female-specific transcription factor gene, ap2-fg. AP2-FG activates most female-specific genes, and disruption of this gene results in the formation of abnormal female gametocytes. Ap2-o3 (ap2-fg2) and ap2r-2 are also a target gene of AP2-G.It has been reported that in both P. berghei and P. yoelii, disruption of ap2-o3 results in arrest of parasite development during ookinete development (and therefore the name ap2-o3. AP2-R2, on the other hand, is expressed in female gametocytes, and disruption of this gene impairs ookinete development.

Here, we report that PbAP2-O3 and  AP2R-2 are expressed in female gametocytes and function together as a transcriptional repressor complex in P. berghei. Accordingly, we renamed PbAP2-O3 PbAP2-FG2.

Phenotype

The mutant formed morphologically normal female and male gametocytes, and the male gametocytes showed normal exflagellation. Pbap2-fg2(-) parasites failed to produce banana-shaped ookinetes; more than half of the fertilized population stopped developing at round zygotes, and the others, at retort-form ookinetes. No oocyst/sporozoite formation.

Additional information
Evidence is presented that:
- Crossing experiments showed that Pbap2-fg2(-) parasites produce fertile males; only females are 'infertile' and produce aberrant ookinetes
- Disruption of pbap2-fg2 affected the female transcriptome, causing downregulation of female-enriched genes
- Target genes of PbAP2-FG2 were upregulated in ap2-fg2(-)
- The binding motifs of PbAP2-FG2 functioned as a cis-acting repressive element. PbAP2-FG2 functions as a transcriptional repressor.
- PbAP2-FG2 requires a co-repressor AP2R-2 (PBANKA_1418100; PF3D7_1319600) to repress its target genes.
Previously  two putative transcriptional regulator genes were identified, ap2r-1 and ap2r-2, as a target gene of AP2-G and AP2-FG. Of these, ap2r-1 functions as a transcriptional activator in zygotes and is renamed ap2-z, but the functional role of ap2r-2 remains unknown. AP2R-2 has an ACDC domain at its C-terminus, but no AP2 domain. It is expressed in females, and ap2r-2 knockout parasites [ap2r-2(-)] are not able to form banana-shaped ookinetes.
- PbAP2-FG2 and AP2R-2 repress the target genes of AP2-G
- The function of PyAP2-FG2 is identical to that of PbAP2-FG2.
The analyses revealed that PbAP2-FG2 and PyAP2-FG2 both repress not only male genes but also a wide-variety of genes to support female differentiation.

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1015500
Gene Model P. falciparum ortholog PF3D7_1429200
Gene productAP2 domain transcription factor AP2-O3, putative
Gene product: Alternative nameApiAP2; AP2-O3; PbAP2-FG2
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe DNA constructs for tagging PbAP2-FG2 with GFP and knocking out pbap2-fg2 were prepared as previously reported. Briefly, for gfp-tagging, two homologous regions were cloned into the gfp-fusion vector to fuse pbap2-fg2 in-frame with gfp. This vector possesses a hdhfr expression cassette next to the gfp so that mutants can be selected by treatment with pyrimethamine. The plasmid was linearized by XhoI and NotI digestion before use in transfection experiments. To knock out pbap2-fg2, the targeting construct was prepared using overlap PCR. The construct had two homologous regions around the pbap2-fg2 locus flanking a hdhfr expression cassette.
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