Summary

RMgm-4509
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1138200; Gene model (P.falciparum): PF3D7_1360500; Gene product: guanylyl cyclase beta (GCbeta)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 4 September 2018, 13:11
  *RMgm-4509
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) : 30146157
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/ResearcherGao H, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network
Name InstituteSchool of Life Sciences, Xiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4509
Principal nameΔgcβ
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 ookineteOokinetes are formed in wild type numbers and with wild type morphology. Ookinetes lost gliding motility.
OocystNo oocysts
SporozoiteNo sporozoites
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of gcβ (deleted the first three exons of gcβ causing frame shift mutation).
In the paper several additional mutants are described expressing mutated forms of gcβ (see also below)

Protein (function)

Phenotype
Wild type gametocyte production, ookinete production and morphology, Ookinetes lost gliding motility. No oocyst production and sporozoite production

Additional information
This study describes a large number of mutants expressing tagged (mutated) versions of different proteins, gene-deletion mutants and mutants with mutated genes (see link).

genes tagged:
gcβ (PY17X_1138200), gcα (PY17X_0911700), pdeδ (PY17X_1338400), pkg (PY17X_1009800), cdc50a (PY17X_0619700), cdc50b (PY17X_0916600); imc1 (f,g,i,j,k,l,m), alvX (PY17X_1240600), isp1 (PY17X_1212600), isp3 (PY17X_1328100),

mtip (PY17X_1462100); ara1 (PY17X_1412750); myosinb (PY17X_0931400); soap (PY17X_1040200); dhhc10 (PY17X_0513100), mpodd (PY17X_1225400)

genes deleted:
gcβ (PY17X_1138200), cdc50a (PY17X_0619700), cdc50b (PY17X_0916600); cdpk3 (PY17X_0410700), pdeδ (PY17X_1338400), Δisp1 (PY17X_1212600)

P28M/Δcdc50a, Δcdc50a/Δgcβ, Δcdpk3/Δgcβ, Δcdpk3/Δcdc50a, Δpdeδ/Δgcβ, Δpdeδ/Δcdc50a,

Mutated GCβ
5 mutants

Analyses of these mutants showed the following (from the Abstract):
'Using real-time imaging to visualize Plasmodium yoelii guanylate cyclase β (GCβ), we show that cytoplasmic GCβ translocates and polarizes to the parasite plasma membrane at ‘‘ookinete extrados site’’ (OES) during zygote-to-ookinete differentiation. The polarization of enzymatic active GCβ at OES initiates gliding of matured ookinete. Both the P4-ATPase-like domain and guanylate cyclase domain are required for GCβ polarization and ookinete gliding. CDC50A, a co-factor of P4-ATPase, binds to and stabilizes GCβ during ookinete development. Screening of inner membrane complex proteins identifies ISP1 as a key molecule that anchors GCβ/CDC50A complex at the OES of mature ookinetes.'

From the paper:
'3'–5'-cyclic guanosine monophosphate (cGMP), cGMP-dependent protein kinase G (PKG), phosphodiesterase delta (PDEδ), and guanylate cyclase beta (GCβ) have been shown to be crucial for ookinete motility in the rodent malaria parasite Plasmodium berghei. Coordinated activities of GCβ (synthesizes cGMP) and PDEδ (hydrolyzes cGMP) regulate cGMP levels that activate PKG, leading to phospholipase C (PLC)/inositol triphosphate (IP3)-mediated Ca2+ release, phosphorylation of multiple proteins in the glideosome, and initiation of ookinete gliding.
The Plasmodium yoelii parasite encodes two large guanylate cyclases (GCα, 3,850 amino acids [aas] and GCβ, 3,015 aas that contain 22 transmembrane (TM) helixes spanning an N-terminal P4-ATPase-like domain (ALD) and a C-terminal guanylate cyclase domain (GCD). The GC enzymes possessing this ALD/GCD structure are observed in many protozoan species.'

From the paper:
'To test whether cGMP synthesis activity of polarized GCb at OES is required for ookinete gliding, we generated GCb mutant parasites that maintained GCb OES polarization but lost the ability to synthesize cGMP. Sequences analysis reveal the conserved residues Asn-Thr-Ala-Ser-Arg (NTASR) in the a4 helix of catalytic domain 1 (C1) of GC, which are likely critical for the cyclase to bind its substrate guanosine triphosphate (GTP), and mutations in these residues may reduce or abolish the cyclase activity (Figure S3H). Accordingly, we introduced mutations by replacing ‘‘NTASR’’ with ‘‘NKASR’’ or ‘‘AKASA’’ in the gcβ::6HA parasite, generating GCDm1 and GCDm2 parasites, respectively. Both mutants showed normal GCβ polarization and expression levels similar to that of gcβ::6HA parasite but had severely impaired ookinete motility, resembling the phenotype of gcβ disruption.'

Other mutants
This study describes a large number of mutants expressing tagged (mutated) versions of different proteins, gene-deletion mutants and mutants with mutated genes (see link)..

In the paper several additional mutants are described expressing mutated forms of gcβ (see also above).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1138200
Gene Model P. falciparum ortholog PF3D7_1360500
Gene productguanylyl cyclase beta
Gene product: Alternative nameGCbeta
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 genePartial
Additional remarks partial/complete disruption Deleted the first three exons of gcβ causing frame shift mutation
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for parasite genomic modification. To construct the vectors for gene deleting, we amplified the 5'- and 3'- genomic sequence (400 to 700 bp) of target genes as left and right homologous arms using specific primers and inserted into the restriction sites in pYCm. Oligonucleotides for guide RNAs (sgRNAs) were annealed and ligated into pYCm. For each gene, two sgRNAs were designed to target the coding region of gene using the online program ZiFit. To construct the vectors for gene tagging and T2A insertion, we first amplified the C- or N-terminal segments (400 to 800 bp) of the coding regions as left or right arm and 400 to 800 bp from 5' UTR or 3' UTR following the translation stop codon as left and right arm, respectively. A DNA fragment (encoding mCherry, mScarlet, 6HA, 4Myc, or 3V5 tag) was inserted between the left and right arms in frame with the gene of interest. For each gene, two sgRNAs were designed to target sites close to the C- or N-terminal part of the coding region. To construct vectors for site-directed nucleotide mutations, the substitution sites were designed with a restriction site for modification detection and placed in the middle of the homologous arms. Parasite-infected red blood cells (RBC) were electroporated with 5 mg purified circular plasmid DNA using the Lonza Nucleotector.
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