RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5249

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0609700; Gene model (P.falciparum): PF3D7_1211200; Gene product: conserved Plasmodium protein, unknown function Name tag: GFP
Phenotype	Asexual bloodstage;

Last modified: 22 September 2022, 16:43

*RMgm-5249

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) : 36040030
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 2.34
Other information parent line	P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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The mutant parasite was generated by
Name PI/Researcher	Ambekar, SV, Mair GR
Name Group/Department	Biomedical Sciences,
Name Institute	Iowa State University
City	Ames
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-5249
Principal name	PBANKA_0609700
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Diffuse cytoplasmic location in blood stages
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation In the paper different mutants are described that express putative nucleoporins (NUPs) C-terminally tagged with different TurboID of GFP. These putative NUPs are: NUP390 (PBANKA_0309200; TurboID-HA tagging); NUP434 (PBANKA_0309400), NUP 335 (PBANKA_0807900), NUP 176 (PBANKA_1365100) and NUP 269 (PBANKA_1454600). These all show a nuclear, perhipheral localization in blood stages, typical for FG-repeat nucleoporins in P. berghei. In addition, a mutant expressing GFP-tagged PBANKA_0609700 was analysed, which showed a diffuse cytoplasmic GFP signal (localization) in blood stages. Protein (function) and Phenotype With an aim to identifying novel Nups, we focused on 8 proteins within our data set that were conserved among Plasmodium spp. but lacked functional annotation: PBANKA_0309200, PBANKA_0309400, PBANKA_0609700, PBANKA_0807900, PBANKA_1211700, PBANKA_1365100, PBANKA_1404700, and PBANKA_1454600. Of those, PBANKA_1211700 and PBANKA_1404700 contained predicted signal peptides and were not pursued any further. Among the remaining candidates, PBANKA_0309200, PBANKA_0309400, and PBANKA_1365100 were similar in spectral abundance (NSAF) to the bait, Nup313. As a first step toward their characterization, we performed C-terminal GFP-tagging with genomic integration to establish their subcellular localization; turboID-HA tagging was used for PBANKA_0309400 as GFP-tagging failed repeatedly (n = 5). Five of the 6 candidate proteins localized to the nuclear periphery typical for FG-repeat nucleoporins in P. berghei (Fig. 3B). In contrast, PBANKA_0609700 presented a diffuse cytoplasmic localization despite the presence of four predicted transmembrane domains. Apart from the localization to the nuclear periphery, the transcriptome profiles of the new nucleoporins were similar to those of the 5 annotated FG-Nups with the exception of Nup335 Additional information Mass-spectrometric analysis of nup313::turboid proximity-labeled parasite lysates identifies known and novel Plasmodium berghei NPC components. proteins identified included the bait protein Nup313 as well as all 5 FG-Nups 138, 205, 221, 313, and 637 but not Sec13 (PBANKA_1445400). Of the 88 detected proteins, 41 contained a predicted nuclear localization signal; this included all known FG-Nups apart from Nup138. Of the 88 proteins 38 had been detected in the nuclear core proteome of P. falciparum, including all FG-Nups. Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0609700

Gene Model P. falciparum ortholog

PF3D7_1211200

Gene product

conserved Plasmodium protein, unknown function

Gene product: Alternative name

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Details of the genetic modification

Name of the tag

GFP

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

(Linear) plasmid single cross-over

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

tgdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

For comparison of various biotin ligases, nucleoporin 138 (Nup138) was used as the bait protein. The C-terminal region of the gene was PCR-amplified from P. berghei genomic DNA using Q5 High-Fidelity DNA polymerase. BioID2-3XHA was obtained from Addgene (#74224) and was fused in-frame to the C-terminal end of the Nup138 resulting in plasmid pLIS0574 which also contains the Toxoplasma gondii DHFR selection marker. Each Nup138 tagging construct includes a 1,848 bp fragment encoding the C-terminal region of the 3,714 bp long gene to promote homologous recombination. Similarly, another fusion protein plasmid was made with the same bait protein but with a 13X GGGGS linker between the C-terminus of the protein and BioID2. TurboID-3XHA (Addgene #107171) was fused in-frame to the C-terminal end of the Nup138 using the plasmid pLIS0574; likewise mini-TurboID-3XHA (Addgene #107172) and BirA* (12). As a wild-type control, we used the 2.34 P. berghei clone containing none of the biotin ligases. To ensure that the nuclear periphery-specific signal was from the biotin ligase fused to Nup138, we constructed a plasmid with the promoter sequence of Nup138 and TurboID as a negative control. All cloning was carried out either with T4 DNA ligase or NEBuilder HiFi (NEB).

To tag all additional FG-Nups with TurboID, we replaced the Nup138 flank in pLIS0654 with flanks for the respective genes by amplifying the C-terminal regions from the genomic locus. For visualization of candidate-Nups identified via mass spectrometry, we used pLIS0010 to tag each protein with GFP. For nup434::turboID-ha, we replaced the gene flank using pLIS0654.

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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