RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4898

Malaria parasite	P. yoelii
Genotype
Mutated	Gene model (rodent): PY17X_0109900; Gene model (P.falciparum): PF3D7_0609800; Gene product: palmitoyltransferase DHHC2, putative (DHHC2) Details mutation: 'promoter-swap' mutant; the promoter of DHHC2 replaced by the promoter of PY17X_1402200 (clag)
Phenotype	Fertilization and ookinete; Oocyst;

Last modified: 30 October 2020, 14:38

*RMgm-4898

Successful modification	The 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 Parasite	P. yoelii
Parent strain/line	P. y. yoelii 17XNL
Name parent line/clone	isp1::3V5/isp3::6HA/dhhc2::4Myc
Other information parent line	This background line expresses C-terminal tagged forms of ISP1 (3V5 tag); ISP3 (6xHA tag) and DHHC2 (4xMyc tag)
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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-4898
Principal name	dhhc2kd
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	The dhhc2kd parasite displayed a developmental arrest at early stages with no formation of mature ookinetes
Oocyst	No formation of oocysts
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation In the 'promoter-swap' mutant the promoter of DHHC2 replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PY17X_1402200; clag; cytoadherence linked asexual protein). In addition, line expresses C-terminal tagged forms of ISP1 (3V5 tag); ISP3 (6xHA tag) and DHHC2 (4xMyc 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 Attempts to disrupt the dhhc2 gene in P. yoelii were unsuccessful (see RMgm-4897) indicating an essential role of DHCC2 for blood stage development/multiplication. Promoter replacement significantly reduced the level of DHHC2 protein in zygotes of the resulted parasite dhhc2kd. The dhhc2kd parasite proliferated in mouse blood, produced functional male and female gametocytes, expressed ISP1 and ISP3 at levels comparable to those of the TTS parasite, but had significantly reduced palmitoylation on ISP1 and ISP3 in zygotes. Consequently, ISP1 and ISP3 lost polarized localization in the dhhc2kd zygotes. These results indicate that DHHC2 is the major PAT palmitoylating ISP1/ISP3. The dhhc2kd parasite displayed a developmental arrest at early stages with no formation of mature ookinetes, repeating the phenotype of dhhc2kd knockdown in P. berghei. No oocyst was observed in midguts from mosquitoes infected with the dhhc2kd parasites 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_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

Short description of the mutation

'promoter-swap' mutant; the promoter of DHHC2 replaced by the promoter of PY17X_1402200 (clag)

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

CRISPR/Cas9 construct; episomally maintained

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

eef1a

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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