RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4899
Malaria parasiteP. yoelii
Genotype
MutatedGene model (rodent): PY17X_0109900; Gene model (P.falciparum): PF3D7_0609800; Gene product: palmitoyltransferase DHHC2, putative (DHHC2)
Details mutation: Glycine 128 changed to alanine
TaggedGene model (rodent): PY17X_1212600; Gene model (P.falciparum): PF3D7_1011000; Gene product: inner membrane complex sub-compartment protein 1 (ISP1)
Name tag: sextuple HA epitope (6HA)
Phenotype Fertilization and ookinete;
Last modified: 30 October 2020, 14:37
  *RMgm-4899
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32395856
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-4893
Other information parent lineThis background line expresses C-terminal tagged form of ISP1 (6xHA tag)
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
Name of the mutant parasite
RMgm numberRMgm-4899
Principal nameDHHC2::6HA/C128A
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteCompared to WT DHHC2, the catalytic-deficient DHHC2C128A failed to restore zygote polarization of ISP1 as well as ookinete differentiation of the dhhc2kd (RMgm-4898), indicating that the PAT activity is essential for DHHC2 function. Surprisingly, this C128A mutation in the DHHC motif led to cytoplasmic distribution of DHHC2, indicating that its own PAT activity is also required for the proper localization of DHHC2.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of DHHC2 (glycine 128 changed to alanine).  In addition, line expresses C-terminal tagged forms of ISP1 (6xHA tag)

Protein (function)
Many proteins are post-translationally modified by the addition of lipids. Palmitoylation results in the addition of a C-16 fatty acid to a cysteine residue within a given protein. Palmitoylation is reversible and thus can dynamically regulate a protein’s subcellular localization, gene expression and activity. There are 11 predicted palmitoyl-S-acyl-transferases (PATs; named DHHC1–11) in the genomes of P. yoelii and P. berghei parasites.

Phenotype
Replacing cysteine to alanine within DHHC motif impairs the catalytic activity of PATs. Compared to WT DHHC2, the catalytic-deficient DHHC2C128A failed to restore zygote polarization of ISP1 as well as ookinete differentiation of the dhhc2kd (RMgm-4898), indicating that the PAT activity is essential for DHHC2 function. Surprisingly, this C128A mutation in the DHHC motif led to cytoplasmic distribution of DHHC2, indicating that its own PAT  activity is also required for the proper localization of DHHC2. Together, these results demonstrate that the palmitoylation of ISP1/ISP3 by DHHC2 mediates their IMC targeting, which is essential for zygote-to-ookinete differentiation.

Additional information
DHHC2 possesses a 75 aa (residue 210–284) cytosolic C-terminus conserved among Plasmodium spp. Hydrophilicity analysis revealed high hydrophobicity in a segment of residue 251–261. To test whether this C-terminus regulates DHHC2 localization at zygote, we episomally expressed three 6HA-tagged DHHC2, each with a truncated C- terminus of different lengths (ΔC1: missing residues 210–232, ΔC2: missing 233–262, and ΔC3 missing: 263–284). The expression of all three mutants was comparable to that of WT in zygotes. However, only ΔC2 lost IMC targeting and polarized expression, suggesting that the C2 segment is required for proper DHHC2 trafficking. Sequence alignment revealed four conserved cysteine residues (C255, C258, C260, and C262) in C2 segment, and palmitoylation of these cysteines likely plays a role in IMC targeting of DHHC2. Replacement of all four cysteine with alanine (C255A/C258A/C260A/C262A:C4A) completely abolished the palmitoylation of DHHC2, leading to cytoplasmic distribution of the DHHC2 in zygotes and retorts.

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
Details of the target gene
Gene Model of Rodent Parasite PY17X_0109900
Gene Model P. falciparum ortholog PF3D7_0609800
Gene productpalmitoyltransferase DHHC2, putative
Gene product: Alternative nameDHHC2
Details of the genetic modification
Short description of the mutationGlycine 128 changed to alanine
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

  Tagged: Mutant parasite with a tagged gene
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
Details of the genetic modification
Name of the tagsextuple HA epitope (6HA)
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructCRISPR/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
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