RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
TaggedGene model (rodent): PY17X_0109900; Gene model (P.falciparum): PF3D7_0609800; Gene product: palmitoyltransferase DHHC2, putative (DHHC2)
Name tag: sextuple HA epitope (6HA)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 30 October 2020, 13:23
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) 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 Not applicable
Other information parent line
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
Name of the mutant parasite
RMgm numberRMgm-4896
Principal nameDHCC2::6HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Asexual blood stageNot tested
Gametocyte/GameteDHHC2 is expressed in the cytoplasm (likely membrane-bound
vesicles) of both female gametocytes and female gametes
Fertilization and ookineteDHHC2 showed polarized expression with minor cytoplasmic distribution in the dhhc2::6HA parasite zygotes
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal 6HA-tagged version of DHCC2

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. We tagged all 11 P. yoelii endogenous PATs with 6HA and analyzed their localization in zygotes. Out of the 11 PATs, only DHHC2 showed polarized expression with minor cytoplasmic distribution in the dhhc2::6HA parasite zygotes. The polarized localization of DHHC2 was confirmed in 3D imaging of the dhhc2::6HA zygotes and observed in two other independent parasites, 6HA::dhhc2 (DHHC2 tagged with N-terminal 6HA) and dhhc2::4Myc (DHHC2 tagged with C-terminal 4Myc). To confirm co-localization of DHHC2 and ISP1/ISP3, we tagged the endogenous DHHC2 with 4Myc in the DTS parasite (RMgm-4895), generating the triple-tagged parasite isp1::3V5/isp3::6HA/dhhc2::4Myc (TTS). DHHC2 was indeed co-localized with ISP1 and ISP3 at a polarity patch of zygote. Similar to ISP1/ISP3, DHHC2 was expressed only at periphery of the protrusion and elongation where IMC is assembled, but not at zygote remnant during zygote-to-ookinete differentiation, suggesting that DHHC2, ISP1, and ISP3 are localized at IMC, but not at PPM. DHHC2 possesses typical four transmembrane domains surrounding the DHHC motif, with both N- and C-termini facing the cytoplasm. 

Additional information

DHHC2 polarization at cell periphery occurred in P28-positive parasites in a time- dependent manner, starting at 30 min and peaking at 2 h post-XA stimulation. This lag in time correlates with the time required for gamete fertilization and zygote formation after gametogenesis. In addition, no DHHC2 polarization was detected in parasites stimulated with only either XA or 22°C due to the failure of gametogenesis and lack of fertilization.

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

  Tagged: Mutant parasite with a tagged 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
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 markerpbdhfr
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