RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4896

Malaria parasite	P. yoelii
Genotype
Tagged	Gene 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

*RMgm-4896

Successful modification	The 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
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. yoelii
Parent strain/line	P. 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/Researcher	Wang X, Yuan J
Name Group/Department	State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network
Name Institute	School of Life Sciences, Xiamen University
City	Xiamen
Country	China
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Name of the mutant parasite
RMgm number	RMgm-4896
Principal name	DHCC2::6HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	Not tested
Gametocyte/Gamete	DHHC2 is expressed in the cytoplasm (likely membrane-bound vesicles) of both female gametocytes and female gametes
Fertilization and ookinete	DHHC2 showed polarized expression with minor cytoplasmic distribution in the dhhc2::6HA parasite zygotes
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation 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. Phenotype 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

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_0109900

Gene Model P. falciparum ortholog

PF3D7_0609800

Gene product

palmitoyltransferase DHHC2, putative

Gene product: Alternative name

DHHC2

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Details of the genetic modification

Name of the tag

sextuple HA epitope (6HA)

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

CRISPR/Cas9 construct: integration through double strand break repair

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite

hdhfr/yfcu

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

CRISPR/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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