RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5254

Malaria parasite	P. berghei
Genotype
Disrupted	Gene 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: 14 February 2023, 15:00

*RMgm-5254

Successful modification	The 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) : 36780562
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Nishi T, Yuda M
Name Group/Department	1Laboratory of Medical Zoology, Department of Medicine
Name Institute	Mie University
City	Mie, Tsu
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-5254
Principal name	pbap2-fg2(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	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.
Oocyst	Formation of aberrant ookinetes. No oocyst/sporozoite formation
Sporozoite	Formation of aberrant ookinetes. No oocyst/sporozoite formation
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of PbAP2-FG2 (PbAP2-O3). 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

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1015500

Gene Model P. falciparum ortholog

PF3D7_1429200

Gene product

AP2 domain transcription factor AP2-O3, putative

Gene product: Alternative name

ApiAP2; AP2-O3; PbAP2-FG2

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The 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

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