Back to search resultsSummaryRMgm-5181
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 35775739 |
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 | RMgm-4945 |
Other information parent line | The mutant expresses the plant auxin receptor transport inhibitor response 1 (Oryza sativa TIR1) under control of the constitutive eef1 promoter. The TIR1 is (C-terminal) tagged with FLAG |
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The mutant parasite was generated by | |
Name PI/Researcher | Quian P, Yuan J |
Name Group/Department | State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o |
Name Institute | Xiamen University |
City | Xiamen |
Country | China |
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Name of the mutant parasite | |
RMgm number | RMgm-5181 |
Principal name | see below |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Treatment with the plant hormone auxin (Indole-3-acetic acid, IAA; 1 mM for 3 h) of the mAID::dhhc2 schizonts efficiently depleted the mAID::DHHC2 protein and affected growth/multiplication of parasites (see below for details) |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Phenotype
This parasite displayed normal proliferation during asexual blood stages and the fusion protein mAID::DHHC2 exhibited IMC localization in the schizonts, indicating no detrimental effect of mAID tagging on DHHC2 localization and function. Treatment with the plant hormone auxin (Indole-3-acetic acid, IAA; 1 mM for 3 h) of the mAID::dhhc2 schizonts efficiently depleted the mAID::DHHC2 protein. To determine whether IAA itself affects parasites development in vivo, mice infected with the 17XNL parasite were injected intraperitoneally with 200 mg/kg/day IAA or vehicle (DMSO) for 3 consecutive days. The in vivo parasitemia increased at an indistinguishable rate in both groups, indicating no notable effect of IAA on parasite proliferation in mice. Next, we tested whether the parasite mAID::DHHC2 protein could be depleted in mice. The mice with ~10% parasitemia of the mAID::dhhc2 parasite were injected intraperitoneally with IAA once and the parasite-infected red blood cells were collected for immunoblot at 1 and 3 h after IAA injection.
The mAID::DHHC2 protein was significantly reduced in the parasites from IAA treated mice, indicating successful mAID::DHHC2 degradation by IAA. As a control, the IAA treatment had little effect on the 6HA::DHHC2 protein in the 6HA::dhhc2 parasite. To dissect the DHHC2 function in vivo, mice were infected with the Tir1 or mAID::dhhc2 schizonts which were pretreated with IAA or vehicle for 3 h in vitro to 364 deplete DHHC2. From 12 h post infection, the parasitemia in mice infected with Tir1 and mAID::dhhc2 was monitored in parallel every 12 h. The parasitemia of Tir1 increased at an equal rate after either IAA or vehicle pretreatment. However, the IAA-pretreated mAID::dhhc2 parasite displayed delayed proliferation compared to the parasite pretreated with vehicle. The parasite with IAA-pretreatment emerged in the mouse blood at 96 h post infection while the parasite with vehicle-pretreatment emerged at 36 h. Notably, continuation of DHHC2 depletion by another IAA injection (IAA+) at time of parasite infection resulted in complete suppression of mAID::dhhc2 in mice, while this treatment had no effect on the proliferation of Tir1. These results provided a direct evidence that DHHC2 is essential for the asexual blood stage development in mice.
Additional information Out of the 11 PATs, only DHHC1 and DHHC2 displayed clear IMC localization in the schizonts with a stronger IFA signal for DHHC2.The IMC localization of DHHC2 was confirmed in two independent strains 6HA::dhhc2 and dhhc2::4Myc, whose endogenous DHHC2 was tagged with a N-terminal 6HA and C-terminal 4Myc tag, respectively.
Immunoblot showed that DHHC2 is highly expressed in the late trophozoites and schizonts, but not in the rings or early trophozoites. These observations were independently confirmed in another strain gfp::dhhc2, in which endogenous DHHC2 was tagged with a N-terminal GFP.
This parasite displayed normal proliferation during asexual blood stages and the fusion protein mAID::DHHC2 exhibited IMC localization in the schizonts, indicating no detrimental effect of mAID tagging on DHHC2 localization and function. Treatment with the plant hormone auxin (Indole-3-acetic acid, IAA; 1 mM for 3 h) of the mAID::dhhc2 schizonts efficiently depleted the mAID::DHHC2 protein. To determine whether IAA itself affects parasites development in vivo, mice infected with the 17XNL parasite were injected intraperitoneally with 200 mg/kg/day IAA or vehicle (DMSO) for 3 consecutive days. The in vivo parasitemia increased at an indistinguishable rate in both groups, indicating no notable effect of IAA on parasite proliferation in mice. Next, we tested whether the parasite mAID::DHHC2 protein could be depleted in mice. The mice with ~10% parasitemia of the mAID::dhhc2 parasite were injected intraperitoneally with IAA once and the parasite-infected red blood cells were collected for immunoblot at 1 and 3 h after IAA injection. The mAID::DHHC2 protein was significantly reduced in the parasites from IAA treated mice, indicating successful mAID::DHHC2 degradation by IAA. As a control, the IAA treatment had little effect on the 6HA::DHHC2 protein in the 6HA::dhhc2 parasite. To dissect the DHHC2 function in vivo, mice were infected with the Tir1 or mAID::dhhc2 schizonts which were pretreated with IAA or vehicle for 3 h in vitro to deplete DHHC2. From 12 h post infection, the parasitemia in mice infected with Tir1 and mAID::dhhc2 was monitored in parallel every 12 h. The parasitemia of Tir1 increased at an equal rate after either IAA or vehicle pretreatment. However, the IAA-pretreated mAID::dhhc2 parasite displayed delayed proliferation compared to the parasite pretreated with vehicle. The parasite with IAA-pretreatment emerged in the mouse blood at 96 h post infection while the parasite with vehicle-pretreatment emerged at 36 h. Notably, continuation of DHHC2 depletion by another IAA injection (IAA+) at time of parasite infection resulted in complete suppression of mAID::dhhc2 in mice, while this treatment had no effect on the proliferation of Tir1. These results provided a direct evidence that DHHC2 is essential for the asexual blood stage development in mice.
Evidence is presented that:
- DHHC2 regulates schizont segmentation
- DHHC2 controls merozoite invasion
- DHHC2 palmitoylates GAP45 and CDPK1
(To investigate whether DHHC1 also contributes to the palmitoylation of GAP45 and CDPK1, we generated the dhhc1::mAID parasite clone in which the C-terminus of endogenous DHHC1 was tagged with the mAID::HA module in the Tir1 strain. IAA treatment depleted the DHHC1::mAID protein in the dhhc1::mAID schizonts, but had little impact on the palmitoylation level of GAP45 and CDPK1. These results indicated that DHHC2, but not DHHC1, contributes to the palmitoylation of GAP45 and CDPK1 in the schizonts).
Residues for palmitoylation in GAP45 and CDPK1 In CDPK1, two cysteines (C3 and C252) were predicted as the potential residues for palmitoylation. Using the same approach, we found that only the C3A mutation caused a complete loss in both protein palmitoylation and IMC targeting of CDPK1 in the schizonts while the C252A mutation had no effect (Figure 7E-F), suggesting C3 as the critical residue for protein palmitoylation and IMC targeting of CDPK1 in schizonts. Interestingly, the cysteine residues C5, C156, C158, C169, and C172 of GAP45 and C3 of CDPK1 are evolutionarily conserved among different Plasmodium species. Together, these results suggest that C5, C156, C158, C169, and C172 of GAP45 and C3 of CDPK1 are residues for palmitoylation which direct IMC targeting of the proteins in schizonts. Lastly we asked whether the palmitoylation in GAP45 and CDPK1 is essential for protein function and thus parasite viability. The above cysteine to alanine mutation experiments indicated that the palmitoylation of the N-terminal cysteine (C5 in GAP45 and C3 in CDPK1) is required for the correct IMC targeting of proteins. We attempted to replace the C5 wi 517 th alanine in the endogenous GAP45 of 17XNL parasite. A 742 bp DNA donor template containing the nucleotide substitution was used for homologous replacement. Seven sgRNAs were designed for guiding the Cas9 complex to the target DNA. After three independent transfections with each of these seven Cas9/sgRNA plasmids, we failed to obtain the GAP45 C5A mutant parasites. In contrast, a control mutant parasite clone GAP45 C5C was generated with a silent mutation still encoding C5. Using the same approach, we attempted to replace the C3 with A in the endogenous CDPK1 of 17XNL parasite (Fig S9C). Similarly, only mutant parasite clones with CDPK1 C3C, but not CDPK1 C3A, were generated. Together, these results suggest that palmitoylation of C5 in GAP45 and C3 in CDPK1 is essential for protein function and parasite viability in the asexual blood stage development.
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0108300 | ||||||||||||||||||||||||||
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 | AID degron motif (mAID-2HA) | ||||||||||||||||||||||||||
Details of tagging | N-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 | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
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 | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | To construct the plasmids for gene tagging, the DNA fragment (encoding 6HA, 4Myc, GFP, or mAID-2HA) was inserted between the left and right arms in frame with the gene of interest. For each gene tagging, two sgRNAs were designed to target sites close to the N- or C-terminal part of the coding region. To construct the plasmid for amino acid substitution, the donor template (700-800 bp) for homologous recombination was introduced with the targeted mutations for amino acid substitution and extra shield mutations via mutagenesis. These shield mutations in or adjacent to the protospacer-adjacent motif (PAM) were used to prevent the recognition and cleavage of the replaced locus by the gRNA/Cas9 complex. Seven sgRNAs were designed to target sites close to the desired mutation sites. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | the plant Oryza sativa auxin receptor transport inhibitor response 1 (TIR1) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||
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 | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | The CRISPR/Cas9 plasmid pYCm was used to edit the parasite genome. To generate the plasmid for deleting the gene p230p (PY17X_0306600), a 973 bp of the 5’ untranslational region (UTR) upstream the initiation codon and an 857 bp of the 3’UTR following the translation stop codon were amplified as the homologous left and right arm, respectively. The left and right arms were inserted into the pYCm plasmid. Eight single guide RNAs (sgRNAs) were designed to target the coding region of the p230p gene. To generate the plasmid for replacing the coding region of p230p gene with the Tir1 expression cassette, the coding sequence of Tir1 was amplified from the Oryza sativa genome, tagged with a Flag epitope sequence, and put under the control of both the 5’UTR (promoter) of eef1a (551 bp) and the 3’UTR of the dhfr (456 bp). The Tir1 expression cassette was inserted between the left and right homologous arms in the pYCm plasmid. | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PY17X_1134900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PY17X_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | DHFR-TS | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PY17X_0306600 | ||||||||||||||||||
Gene product | 6-cysteine protein P230p | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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