RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4892
Malaria parasiteP. yoelii
Genotype
MutatedGene model (rodent): PY17X_1212600; Gene model (P.falciparum): PF3D7_1011000; Gene product: inner membrane complex sub-compartment protein 1 (ISP1)
Details mutation: N-terminal cysteines (C7/C8) replaced with A7/A8 (alanine) and C-terminal BFP::3V5 tag
Phenotype Fertilization and ookinete;
Last modified: 29 October 2020, 18:24
  *RMgm-4892
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32395856
MR4 number
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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
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The mutant parasite was generated by
Name PI/ResearcherWang X, 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
CountryChina
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Name of the mutant parasite
RMgm numberRMgm-4892
Principal nameisp1 (C7/C8-A7/A8)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteLocalization of the BFP::3V5 tagged and mutated ISP1 in the cytoplasm of zygotes/ookinetes
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated ISP1 with N-terminal cysteines (C7/C8) replaced with A7/A8 (alanine). in addition ISP is tagged with the BFP::3V5 reporter

Protein (function)
The inner membrane complex (IMC)-residing protein ISP (IMC subcompartment protein) is restricted to the phylum apicomplexa and plays key roles in parasite biology. Two ISP members, ISP1 and ISP3, are found in Plasmodium parasites, and display clear apical polarity of expression in zygotes, suggesting possible role in zygote protrusion or elongation. However, parasites with disruption of ISP1 display a modest decrease in zygote-to-ookinete differentiation, while ISP3 depletion has no significant effect on this process.

Phenotype
Both ISP1 and ISP3 have two N-terminal cysteines (C7/C8 in ISP1 and C6/C7 in ISP3) that can potentially be palmitoylated for IMC targeting as shown in schizonts of P. falciparum . Using resin-assisted capture (Acyl-RAC) method, we detected palmitoylation of ISP1 and ISP3 in the zygotes. To study the effect of C7/C8 palmitoylation on protein IMC localization, we generated an ISP1 mutant by replacing C7/C8 with A7/A8 (alanine) and fused the mutant ISP1 with BFP::3V5 reporter. The C7A/C8A mutant protein lost palmitoylation and was localized in the cytoplasm when episomally expressed in WT parasites. In addition, glycine-to-alanine substitution at position 2 (G2A) abolished ISP1 palmitoylation and IMC localization, supporting the observations that glycine myristoylation enhances palmitoylation of the adjacent cysteine.

In wild type zygotes ookinetes ISP1 and ISP3 are concentrated at an apical dot in zygotes (stage I), with residual ISP3 distributed at cytoplasm. In stage III, both proteins are expressed at the periphery of the protrusion, but not zygote remnant. These results support that ISP1/ISP3 is localized in IMC, but not PPM during this differentiation. The localization patterns of ISP1/ISP3 suggest critical roles of these two proteins in zygote protrusion and/or elongation.

Additional information
From the Abstract:
'Here, we show that palmitoylation of N-terminal cysteines of two inner membrane complex (IMC) proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote-to-ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an the IMC-residing palmitoyl-S-acyl-transferase (PAT) DHHC2. IMC-anchored ISP1 and ISP3 interact with microtubule component b-tubulin, serving as tethers to maintain the proper structure of subpellicular microtubules (SPMs) during zygote elongation.

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_1212600
Gene Model P. falciparum ortholog PF3D7_1011000
Gene productinner membrane complex sub-compartment protein 1
Gene product: Alternative nameISP1
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Details of the genetic modification
Short description of the mutationN-terminal cysteines (C7/C8) replaced with A7/A8 (alanine) and C-terminal BFP::3V5 tag
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructCRISPR/Cas9 construct; episomally maintained
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
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 parasite genomic modification. To construct plasmid vectors for gene editing, we amplified 50 and 30 genomic sequence (400–500 bp) of target genes as homologous arms using specific primers and inserted the sequences into specific restriction sites in pYCm. The sgRNAs were designed to target the coding region of a gene using the online program EuPaGDT. Oligonucleotides for guide RNAs (sgRNAs) were mixed in pairs, denatured at 95°C for 3 min, annealed at room temperature for 5 min, and ligated into pYCm. DNA fragments encoding 6HA, 4Myc, 3V5, and Flag tags or BFP were inserted between the left and right arms in frame with 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. Infected red blood cells (iRBCs) were electroporated with 5–10 lg plasmid DNA using Lonza Nucleofector described previously (Zhang et al, 2014). Transfected parasites were immediately intravenously injected into a naı¨ve mouse and were exposed to pyrimethamine (7 mg/ml) 24 h post-transfection. Parasites with transfected plasmids usually appear after 5–6 days under drug pressure. Some modified parasites subjected for sequential modification were negatively selected to remove pYCm plasmid.
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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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren