RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_1415700; Gene model (P.falciparum): PF3D7_1317200; Gene product: AP2 domain transcription factor AP2-FG, putative (AP2-FG, ApiAP2)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 6 August 2019, 14:03
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) : 31231888
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/ResearcherYuda M, Kato T
Name Group/DepartmentDepartment of Medical Zoology
Name InstituteMie University School of Medicine
CityTsu, Mie
Name of the mutant parasite
RMgm numberRMgm-4649
Principal nameAP2-FG(−)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of male gametocytes (and exflagellation). No production of mature female gametocytes. Evidence provided that immature female gametocytes are produced.
Fertilization and ookineteStrongly reduced numbers of ookinetes in in vitro cultures. All ookinetes show aberrant morphology.
OocystNo oocyst production
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of AP2-FG

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.

Normal production of male gametocytes (and exflagellation). No production of mature female gametocytes. Evidence provided that immature female gametocytes are produced.Strongly reduced numbers of ookinetes in in vitro cultures. All ookinetes show aberrant morphology. No oocyst production

Additional information
AP2-FG was expressed in the nucleus of female gametocytes, but not in other blood stages, including male gametocytes (see mutant RMgm-4650).

See also other mutants in the databse targeting this gene PF3D7_1317200

Both clones proliferated at rates similar to wild-type parasites and produced mature male gametocytes with normal exflagellation. However, no mature female gametocytes were observed in them; instead, immature female gametocyte-like parasites (i.e., small mononuclear cells with a light violet cytoplasm) were observed on Giemsa-stained blood smears in both clones. Because it was difficult to conclude that they were female gametocytes, we further transfected AP2-FG(−)1 parasites with a centromere plasmid that conferred female-specific expression of the red fluorescent protein mCherry. Here, the small mononuclear parasites displayed red fluorescence, indicating that they were indeed female gametocytes. By flow cytometric analysis, the rate of mCherry-positive cells in AP2-FG(−) parasites was shown to be similar to that in wild-type parasites transfected with the same centromere plasmid, but the fluorescence intensity was significantly weaker than that in the wild-type. In contrast, when AP2-FG(−)1 parasites were transfected with a centromere plasmid that conferred the male-specific expression of mCherry, the rates of mCherry-positive cells and signal intensities were essentially the same as those of wild-type parasites. Collectively, these results suggested that female gametocytes were produced in AP2-FG(−) parasites, but they were unable to develop properly, and the expression of female-specific genes might decrease significantly.

Cross‐fertilization of AP2‐FG(−)1 parasites with p47‐deficient parasites, where females are infertile, did not rescue the abnormal morphology, but cross‐fertilization with p48/45‐deficient parasites, where males are infertile, resulted in the formation of ookinetes with normal morphology (approximately 90% of ookinetes were with normal morphology). These results indicated that the capacity to mediate transmission to the mosquito was lost in the female gametocytes of AP2‐FG(−) parasites.

To detect the weak expression of AP2‐FG more effectively, we used transgenic P. berghei parasites that produce mNeonGreen‐tagged AP2‐FG (see mutant RMgm-4651); mNeonGreen is a green fluorescent protein that produces a more intense signal than GFP. Specific expression of AP2‐FG in female gametocytes was also observed in these parasites. We transfected these AP2‐FG::mNeonGreen parasites with a centromere plasmid, which confers male‐ or female‐specific expression of the mCherry gene, and performed the fluorescent microscopic study. In all 100 females examined in this study, the expression of AP2‐FG was observed in the nucleus. In contrast, in all 100 males examined, the expression of AP2‐FG was not observed in the nucleus or in the cytoplasm. These results again confirmed the female‐specific expression of AP2‐FG and its localization in the nucleus.
To investigate the expression profile of AP2‐FG, rats were synchronously infected with AP2‐FG::mNeonGreen parasites by the intravenous inoculation of mature schizonts and the blood smears were examined at 2‐h intervals under a fluorescent microscope. AP2‐FG expression was not observed in mature schizonts before inoculation into the rats (0 hpi). At 14 hpi, weak expression was first observed in a small number of the parasites. Then, the fluorescent signals gradually increased with their development and reached a steady state at around 22–24 hpi. This result indicated that AP2‐FG expression begins before gametocyte‐specific features appear and then reaches a peak in accordance with their manifestation of sexual dimorphism. This expression profile strongly suggested that AP2‐FG is involved in female development from the early phase of gametocytogenesis.

From the Abstract:
Chromatin immunoprecipitation sequencing analysis suggested that AP2‐FG directly regulates over 700 genes. Its targets include genes for female gametocyte‐specific functions, such as gametogenesis, fertilization and zygote development. AP2‐FG binding to target gene promoters was associated with a 10 bp sequence motif. These results indicate that AP2‐FG plays a role in the differentiation of early gametocytes to mature females by governing a female‐specific gene expression repertoire.  

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1415700
Gene Model P. falciparum ortholog PF3D7_1317200
Gene productAP2 domain transcription factor AP2-FG, putative
Gene product: Alternative nameAP2-FG, ApiAP2
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 genePartial
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 modificationFor the disruption, a construct containing the pyrimethamine-resistant selectable marker gene was inserted into the 5′ side of the coding region of the AP2 domain by double cross-over homologous recombination.
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