Summary

RMgm-4513
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1212600; Gene model (P.falciparum): PF3D7_1011000; Gene product: phosphodiesterase delta, putative (isp1)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 5 September 2018, 13:04
  *RMgm-4513
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-4513
Principal nameΔisp1
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. Many ookinetes with aberrant morphology.
OocystStrongly reduced oocyst formation (mean of 8 oocysts per mosquitoe compared to 100 oocyst in wild type infected mosquitoes).
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of isp1.
 
Protein (function)

Phenotype
Ookinetes are formed in wild type numbers. Many ookinetes with aberrant morphology.
Strongly reduced oocyst formation (mean of 8 oocysts per mosquitoe compared to 100 oocyst in wild type infected mosquitoes).

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:
'GCβ is likely anchored by the molecules at the IMC of mature ookinetes because (1) GCβ polarizes at a curved region of the ookinete that is mostly maintained by the IMC and (2) PPM-residing GCb remains polarized at OES even after trypsin digestion. Therefore, we searched putative IMC proteins expressed in ookinetes identified previously and selected 10 genes for protein localization analyses by tagging the protein with 6HA or 4Myc. Out of 10 proteins, only the ISP1 displayed OES polarization as well as some distribution along the cell periphery in the isp1::6HA ookinete. We observed the same location of ISP1 in the ookinetes of another tagged parasite—isp1::3V5. ISP3, another member of the ISP proteins, distributes along the periphery of ookinete. We generated doubly tagged gcβ::6HA/isp1::3V5 parasites by tagging endogenous isp1 with 3V5 in the gcβ::6HA parasite to investigate GCβ and ISP1 expression in the same parasite. ISP1 was expressed and polarized as an elongated dot in early zygotes, became two branches lining the future apical in the retort, and polarized at the OES in mature ookinete. ISP1 was reported as an essential gene refractory to deletion in P. berghei asexual blood stages. However, we were able to disrupt the isp1 gene in P. yoelii 17XNL using the Cas9 method and obtained three mutant clones from two independent transfections. Δisp1 parasites showed normal asexual blood stages and gametocyte development in mouse, male gametocyte activation, and mature ookinetes with normal morphology. However, isp1 disruption caused a slight decrease in conversion rate to mature ookinete (25% in Δisp1; 51% in WT;. Importantly, the Δisp1 ookinetes with normal morphology showed significantly reduced ookinete gliding and oocyst counts in mosquito.
Evidence is presented that ISP1, with signal for tracking to OES and residing at the IMC, could anchor GCβ at the OES of mature ookinetes

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)..


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1212600
Gene Model P. falciparum ortholog PF3D7_1011000
Gene productphosphodiesterase delta, putative
Gene product: Alternative nameisp1
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) 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