RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5196
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1429300; Gene model (P.falciparum): PF3D7_1213500; Gene product: integral membrane protein GPR180, putative (GPR180)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 26 April 2022, 17:36
  *RMgm-5196
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) : 35404079
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherWang PP, Zhu Z
Name Group/DepartmentDepartment of Immunology, College of Basic Medical Sciences
Name InstituteChina Medical University
CityShenyang
CountryChina
Name of the mutant parasite
RMgm numberRMgm-5196
Principal nameΔpbgpr180(del)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal (wild type) numbers of gametocytes are produced; male gamete formation ~2fold reduced (as determined by counting exflagellation); female gamete formation reduced by ~ 30%. Crossing experiments indicate a more severe defect in female gametes compared to males.
Fertilization and ookineteReduced ookinete formation (Whereas >93% of wild-type zygotes were transformed into mature ookinetes after 24 h of culture, Δpbgpr180 parasites had a ~50% conversion rate.
Oocyst~50% reduction in the percentage of infected mosquitoes and ~80% decrease in oocyst density.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of GPR180

Protein (function)
Heptahelical serpentine receptors are the largest group of membrane receptors responsible for transducing extracellular signals to various downstream effectors. The serpentine receptors coupled to heterotrimeric guanine nucleotide-binding proteins belong to G-protein-coupled receptors (GPCRs) with a salient feature of seven transmembrane domains, each consisting of 25–35 residues. Although there is little conservation in amino acid (aa) sequences across the entire GPCR superfamily, they share similar structures, which are used to classify the GPCRs into six main classes (A – F). Rhodopsin-like Class A is the largest class, accounting for around 90% of GPCRs. Structurally, Rhodopsin-like GPCRs have a GpcrRhopsn4 domain, an eighth helix, and a palmitoylated cysteine at the C-terminal tail. Bioinformatic analysis identified a gpr180-like gene in all Plasmodium species, with the GPCR-like transmembrane domain located in the C terminus, as predicted using the HMMER program. The ~250 aa GPCR domain contains residues that are highly conserved in the Rhodopsin-like GPCR transmembrane domain, which classifies the GPR180 proteins as Class A (Rhodopsin-like) family members of GPCRs

Phenotype
Normal (wild type) numbers of gametocytes are produced;  male gamete formation ~2fold reduced (as determined by counting exflagellation); female gamete formation reduced by ~ 30%. Crossing experiments indicate a more severe defect in female gametes compared to males.
Reduced ookinete formation (Whereas >93% of wild-type zygotes were transformed into mature ookinetes after 24 h of culture, Δpbgpr180 parasites had a ~50% conversion rate.
~50% reduction in the percentage of infected mosquitoes and ~80% decrease in oocyst density.

Additional information
Expression of GPR180 in schizonts, gametocytes and ookinetes with highest expression in gametocytes (see mutant RMgm-5197 that expresses HA-tagged GPR180). Evidence is presented that gametocyte activation stimulates GPR180 redistribution from the cytoplasm to the plasma membrane.

To investigate the function of GPR180i, we generated two pbgpr180 knockout (KO) lines Δpbgpr180(Del) (with the PbGEM-340051 plasmid) and Δpbgpr180KO (with the pL0034-PbGPR180-KO plasmid) using a double-crossover homologous recombination strategy.

Crossing experiments indicate a more severe defect in female gametes compared to males.

Evidence is presented that:
- Pbgpr180 deletion leads to dysregulated expression of genes involved in the signaling pathway during sexual development. 
- Pbgpr180 deletion impairs the PKG-mediated signaling
- PbGPR180 is associated with proteins of transporter functions

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1429300
Gene Model P. falciparum ortholog PF3D7_1213500
Gene productintegral membrane protein GPR180, putative
Gene product: Alternative nameGPR180
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-340051
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 For pbgpr180 gene disruption, two plasmids were used. The PbGEM-340051 plasmid (to delete nt: 392 – 2080 bp of pbgpr180 open reading frame–ORF) was obtained from PlasmoGEM. To delete the entire pbgpr180 ORF, an 1157 bp fragment containing the 5’UTR (nt: 21157 – 21 bp) and the 871 bp 3R described above were amplified and cloned into the HindIII/ApaI and XhoI/NotI sites, respectively, of the pL0034 vector to generate the plasmid pL0034-PbGPR180-KO. For transfection, the pL0034-PbGPR180-KO plasmid was linearized with BglI and NotI, while the PbGEM-340051 plasmid was linearized with NotI, followed by ethanol precipitation
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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