Summary

RMgm-4813
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1116300; Gene model (P.falciparum): PF3D7_0515500; Gene product: gametogenesis essential protein 1, putative (GEP1)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 19 June 2020, 20:48
  *RMgm-4813
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) : 32273496
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/ResearcherJiang Y, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network
Name InstituteSchool of Life Sciences, Xiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4813
Principal nameΔ1116300
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal gametocyte production; no exflagellation; no fertile males and male gametes are produced;
Fertilization and ookineteNo gamete formation, absence of ookinete formation
OocystNo gamete formation, absence of ookinete formation; no oocyst formation
SporozoiteNo gamete formation, absence of ookinete formation; no oocyst formation, no sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PY17X_1116300 (GEP1)

Protein (function)
To identify membrane proteins critical in sensing XA or transducing XA-induced signal during gametogenesis, we identified 59 P. yoelii genes that are expressed in gametocytes and encode proteins with 1 to 22 predicted transmembrane domains (TMs) from the PlasmoDB database. We designed single guide RNA (sgRNA) to disrupt each of these genes using CRISPR/Cas9 methods16,17 and were able to successfully knockout (KO) 45 (76%) of the genes in the P. yoelii 17XNL strain, obtaining at least two cloned lines for each mutant . The remaining 14 genes (24%) were refractory to repeated deletion attempts using three independent sgRNA sequences, suggesting their essential roles for asexual blood-stage growth. The 45 gene deletion mutants proliferated asexually in mouse blood normally and were able to produce both male and female gametocytes although the gametocytemia level varied among these mutants.
Next we measured the gametogenesis of male gametocyte by counting exflagellation centers (ECs) formed in vitro after stimulation with 50 μMXAat 22 °C.Only one mutant (PY17X_1116300; GEP1) showed complete deficiency in EC formation and male gamete release and this mutant was analysed in greater detail.

The PY17X_1116300 (GEP1) gene contains four exons encoding a putative amino acid transporter protein with 14 predicted transmembrane domains.

Phenotype

Normal asexual blood stage growth/multiplication;

Normal gametocyte production; no exflagellation; no fertile males and male gametes are produced; absence of ookinete formation; no oocyst formation, no sporozoite formation

Additional information
From the Abstract:
GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption.

To further confirm the phenotype of Δgep1, we generated three additional gep1 mutant parasites (Δgep1n, Δgep1fl, and Δgep1mScarlet). The Δgep1n parasite had a 464 bp deletion at the 5’ coding region, causing a frameshift for the remaining coding region. The Δgep1fl parasite had the whole gep1 coding region deleted, and the Δgep1mScarlet parasite had its gep1 coding regions replaced with a gene encoding red florescent protein mScarlet. These mutant parasites displayed developmental phenotypes similar to those of Δgep1 in both mouse and mosquito stages. 
We also reintroduced the 558 bp deleted segment plus a sextuple HA epitope (6HA) into the Δgep1 parasite to rescue the gene function using Cas9-mediated homologous replacement. Two clones of the rescued parasite (Δgep1/gep1::6HAc1 and Δgep1/gep1::6HAc2) showed expression of the GEP1::6HA protein in both Western blotting and immunofluorescence analysis (IFA). Importantly, both clones produced wild type (WT) levels of EC in vitro and midgut oocyst in mosquitoes.

To investigate protein expression and localization, we tagged the endogenous GEP1 with 6HA at N-terminus, generating 6HA::gep1 parasite that had normal development throughout the life cycle. The GEP1 protein is expressed in gametocytes and ookinetes, but not in asexual blood stages and other mosquito stages of the 6HA::gep1 parasite. We also tagged the GEP1 protein with quadruple Myc (4Myc)  and observed similar expression pattern in the 4Myc::gep1 parasite. In addition, mScarlet fluorescent signals driven by the endogenous gep1 promoter were detected only in gametocytes, but not in asexual blood stages of the Δgep1mScarlet parasite. Co-staining 6HA::gep1 gametocytes with anti-α-Tubulin (male gametocyte specific) and anti-HA antibody showed that GEP1 was expressed in both male and female gametocytes. Interestingly, GEP1 is not expressed in plasma membrane, but in punctate dots in the cytoplasm of gametocytes and ookinetes.

Deletion of P. berghei gep1 gene (PBANKA_1115100) resulted in parasite clones that failed to form XA-stimulated ECs 9exflagellation centers) in vitro and midgut oocyst in mosquitoes.

We next performed genetic crosses between Δgep1 and Δmap2 (male gamete-deficient) or Δnek4 (female gamete-deficient) parasites. No midgut oocyst was observed in mosquitoes from the Δgep1 × Δmap2 or Δgep1 × Δnek4 cross day 7 post infection (pi), whereas the Δmap2 × Δnek4 cross produced slightly fewer oocysts than the WT parasite, suggesting no functional male and female gametes in the Δgep1 parasite. Together, these results demonstrate that GEP1 is essential for both male and female gametogenesis.

Evidence is presented that:
- GEP1-depleted gametocytes are viable, but lost the ability to produce functional male and female gametes.
- GEP1 depletion blocks PKG-mediated signaling
- axoneme formation was not observed in the Δgep1 parasite
- no axoneme assembly or mitotic division in the stimulated Δgep1 male gametocytes.
- GEP1 depletion affects erythrocyte membrane lysis in activated gametocytes.
- no PVM lysis in stimulated Δgep1 gametocytes
- Impaired cGMP synthesis in GEP1 deficient parasite
- GEP1 interacts and co-localizes with GCα
- GCα depletion causes defect in XA-stimulated gametogenesis
- XA stimulation likely enhances the GEP1/GCα interaction


Other mutants
See other mutants generated in this study


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1116300
Gene Model P. falciparum ortholog PF3D7_0515500
Gene productgametogenesis essential protein 1, putative
Gene product: Alternative nameGEP1
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for all the genetic modifications. For gene deleting, 5’-genomic and 3’-genomic segments (400 to 700 bp) of the target genes were amplified as left and right homologous arms, respectively, using gene specific primers. The PCR products were digested with appropriate restriction enzymes and the digested products were inserted into matched restriction sites of pYCm.
Oligonucleotides for sgRNAs were annealed and ligated into pYCm17. For each deletion modification, two sgRNAs were designed to disrupt the coding region of a target gene using the online program ZiFit47. For gene tagging, a 400 to 800 bp segment from N-terminal or C-terminal of the coding region and 400 to 800 bp sequences from 5’UTR or 3’UTR of a target gene were amplified and fused with a DNA fragment encoding 6HA or 4Myc in frame at N-terminal or C-terminal of the gene. For each tagging modification, two sgRNAs were designed to target sites close to the C-terminal or N-terminal of the gene coding region.
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