RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5181
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PBANKA_0108300; Gene model (P.falciparum): PF3D7_0609800; Gene product: palmitoyltransferase DHHC2, putative (DHHC2)
Name tag: AID degron motif (mAID-2HA)
Transgene
Transgene not Plasmodium: the plant Oryza sativa auxin receptor transport inhibitor response 1 (TIR1)
Promoter: Gene model: PY17X_1134900; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PY17X_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (DHFR-TS)
Replacement locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein P230p
Phenotype Asexual bloodstage;
Last modified: 4 July 2022, 14:07
  *RMgm-5181
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-4945
Other information parent lineThe 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
The mutant parasite was generated by
Name PI/ResearcherQuian P, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o
Name InstituteXiamen University
CityXiamen
CountryChina
Name of the mutant parasite
RMgm numberRMgm-5181
Principal namesee below
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageTreatment 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/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
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. In addition, it expresses DHHC2 N-terminal tagged with AID  

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.01.28.478263

Protein (function)
TIR1 and AID: The plant hormone auxin induces rapid proteasomal degradation of certain proteins by a specific E3 ubiquitin ligase. To implement this system in Plasmodium, only two transgenic components are needed: a plant auxin receptor called transport inhibitor response 1 (TIR1) and a protein of interest (POI) tagged with an AID (degron) motif. In these engineered parasite lines stably expressing the plant TIR1, the auxin functions as a molecular glue promoting specific interaction between the ubiquitin ligase complex and AID-tagged POI, which triggers proteasomal degradation of the latter.

DHHC2: Protein palmitoylation is a reversible lipid modification that facilitates protein attachment to the plasma and organelle membranes. Previous studies have shown that palmitoylation is important for the binding or targeting of proteins to inner membrane complex (IMC) in the Plasmodium. 11 putative palmitoyl-S-acyl-transferasesPATs (named DHHC1–11) were predicted in the genomes of rodent malaria parasites.
Attempts to disrupt the dhhc2 gene in P. berghei were unsuccessful (see RMgm-1350) indicating an essential role of DHCC2 for blood stage development/multiplication.

To explore the functions of DHHC2, we applied an Auxin-inducible degron (AID)-based protein degradation system in the P. yoelii transgenic strain Tir1, which allows depletion of the target protein fused to a miniAID (mAID) motif with the aid of the plant hormone auxin (Indole-3-acetic acid, IAA). The N-terminus of the endogenous dhhc2 locus was tagged with the sequence encoding mAID::2HA in the Tir1 strain, generating the mAID::dhhc2 clone

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
To identify the residue(s) of palmitoylation in GAP45, we used an online software CSS485 Palm (csspalm.biocuckoo.org) for prediction, which generated 6 candidate cysteines (C5, C140, C156, C158, C169, and C172). To test them, we initially generated 4 constructs expressing HA-tagged GAP45, each with a single or double cysteine-to-alanine mutations (C5A, C140A, C156A/C158A, and C169A/C172A). These constructs were episomally expressed in the schizonts. Only the C140A mutant displayed the IMC localization similar to wildtype (WT) GAP45; other mutants (C5A, C156A/C158A, and C169A/C172A) lost the IMC localization. These results suggest that these cysteines (C5, C156 and/or C158, C169 and/or C172) are critical for IMC targeting of GAP45 and might be the residues for modification. Indeed, the Acyl-RAC assay detected significantly decreased palmitoylation of GAP45 in the C5A, C156A/C158A, and C169A/C172A mutants, but not the C140A mutant. Thus, for GAP45, there is an association between IMC localization and palmitoylation. Additionally, the degree of palmitoylation was further reduced in the triple cysteine mutants (C5A/C156A/C158A and C5A/C169A/C172A) relative to the single and double mutants. 

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.

Other mutants

 


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0108300
Gene Model P. falciparum ortholog PF3D7_0609800
Gene productpalmitoyltransferase DHHC2, putative
Gene product: Alternative nameDHHC2
Details of the genetic modification
Name of the tagAID degron motif (mAID-2HA)
Details of taggingN-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 modificationTo 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
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namethe plant Oryza sativa auxin receptor transport inhibitor response 1 (TIR1)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe 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
Other details transgene
Promoter
Gene Model of Parasite PY17X_1134900
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PY17X_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative nameDHFR-TS
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PY17X_0306600
Gene product6-cysteine protein P230p
Gene product: Alternative name
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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