RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5541
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0416300; Gene model (P.falciparum): PF3D7_0905100; Gene product: nucleoporin NUP221, putative (nucleoporin NUP100)
Name tag: 3xHA/GFP
Phenotype Asexual bloodstage;
Last modified: 29 December 2024, 18:41
  *RMgm-5541
Successful modificationThe 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) : 39526784
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherBlauwkamp J, Absalon S
Name Group/DepartmentIndiana University School of Medicine, Department of Pharmacology and Toxicology
Name InstituteIndiana University School of Medicine
CityIndianapolis
CountryUS
Name of the mutant parasite
RMgm numberRMgm-5541
Principal nameNup221-3xHA/GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageU-ExM visualization of Nups around the nucleus. Nuclear pore complexes (NPCs) are primarily produced before mitosis. NPCs are organized around the centriolar plaque. The RITE system reveals NPC assembly and maintenance. P. berghei replaces Nups in post-mitotic NPCs.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal 3xHA/GFP-tagged version of NUP221

Protein (function)
Unlike closed mitosis in yeast, P. berghei parasites undergo multiple rounds of asynchronous nuclear divisions in a shared cytoplasm which results in a multinucleated organism prior to formation of daughter cells within an infected red blood cell. During this replication process, intact nuclear pore complexes (NPCs) and their component nucleoporins play critical roles in parasite growth, facilitating selective bi-directional nucleocytoplasmic transport and genome organiza on. Here we ulize ultrastructure expansion microscopy (U-ExM) to investigate tagged P. berghei nucleoporins NUP138 (PBANKA_0417900), NUP313 (PBANKA_1310200) and NUP221 (PBANKA_0416300) at the single nucleus level throughout the 24-hour blood-stage replication cycle. To visualize nucleoporins (Nups) at single NPC resolution in P. berghei, we introduced C-terminal endogenous epitope tags on three previously identified P. berghei FG Nups: spagheti monster Hemagglutinin (smHA) tags on Nup138 and Nup313 and a 3xHA/GFP tag on Nup221.
Our findings reveal that these Nups are evenly distributed around the nuclei and organized in a rosette structure previously undescribed around the centriolar plaque, which is responsible for intranuclear microtubule nucleation during mitosis.
By adapting the recombinaton-induced tag exchange (RITE) system to P. berghei, we provide evidence of NPC maintenance, demonstrating Nup221 turnover during parasite asexual replication. Our data shed light on the distribution of NPCs and their homeostasis during the blood-stage replica on of P. berghei parasites

Phenotype
In this study, we employed U-ExM to visualize individual NPCs using tagged versions of P. berghei Nup138, Nup221, and Nup313. We successfully localized these Nups to the nuclear envelope by employing NHS ester or BodipyFL Ceramide staining throughout the asexual life cycle of the P. berghei parasite. Consistent with prior findings using “serial surface” imaging in  focused ion beam – scanning electron microscopy (FIB-SEM), we observed an increase in NPC number until the parasite's first mitosis, during which the NPCs were then distributed between the newly developing nuclei.  We proceeded to assess NPC assembly and protein turnover using the Recombination Induced Tag Exchange (RITE) system (using NUP221) marking the first time this has been accomplished in Plasmodium. The RITE system uses a conditional, hormone activated Cre-recombinase to generate permanent epitope tag switch in the target coding sequence NUP221. This method has been previously utilized in human cells to observe the dynamic replacement of NPC components in human neurons.  
Leveraging the enhanced resolution of U-ExM coupled with the RITE system, we were able to monitor NPC assembly and maintenance dynamics within a single nucleus during Plasmodium replication. In line with previous observations in mammalian cells, we found that NPCs in P. berghei undergo protein maintenance after the NPCs have been produced. Remarkably, we also confirmed the presence of Nup221 proteins of different ages in a single NPC by colocalization of original and induced Nup221 signal.

Additional information
In the early stages of the parasite's life cycle, before the parasites begin their mitotic phase (1n, Ring), we observed Nup138 being uniformly distributed around the nucleus, while the Nup313 signal is concentrated at either end of the nucleus . At this stage, we noticed a higher number of Nup313 foci (approximately 40-50 NPCs) compared to Nup138 (around 25-35 NPCs) (Fig. 3b). As the parasites began mitosis (1n, Trophozoite premitotic and mitotic), there was a significant 5-fold increase in both Nup138 and Nup313 numbers. The parasites produce over 250 NPCs containing Nup313 and over 150 NPCs containing Nup138, distributed around the single nucleus. As more nuclei are formed (2-4n Schizont, 5-9n Schizont, 10+n Cytokinesis), the number of NPCs around each nucleus decreased. Initially, approximately 250 NPCs surrounded a single nucleus, which then reduced to around 90-100 NPCs around each of the 2 or 3 nuclei. Eventually, the count further decreases to approximately 25-30 NPCs around 10-12 nuclei. Our data suggests that NPC production peaks before mitosis and NPCs are then distributed during each karyokinesis.

Quantification of NPCs around the centriolar plaque reveals a higher count of Nup313 compared to the other two FG Nups

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0416300
Gene Model P. falciparum ortholog PF3D7_0905100
Gene productnucleoporin NUP221, putative
Gene product: Alternative namenucleoporin NUP100
Details of the genetic modification
Name of the tag3xHA/GFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmid cloning was carried out using NEBuilder HIFI DNA assembly (NEB). To generate a smHA fusion at the endogenous C terminus of P. berghei Nup138 the smHA coding sequence (spagheti monster Hemagglutinin (smHA)) was amplified from plasmid pCAG_smFP_HA using primers P4121/4122 and inserted between BamHI/BsiWI immediately downstream of the Pbnup138 homology region in the plasmid pLIS0654 resulting in the plasmid pSA026. To generate a smHA fusion at the endogenous C terminus of P. berghei Nup313, a homology region corresponding to the 3’ coding sequence of Pbnup313 up to but not including the stop codon was amplified with primers P4140/4126 from P. berghei genomic DNA and inserted between AvrII/BamHI in pSA026, replacing the Pbnup138 homology region and resulting in the plasmid pSA028.
To generate parasites expressing Cre-EBD with the RITE cassette fused to the endogenous C terminus of P. berghei Nup221, the Cre-EBD coding sequence was amplified from the plasmid pTWO40 (Addgene #64769) and the RITE cassette was amplified from the plasmid pKV016, which includes a 3xHA-GFP fusion in the pre-excised state and a 3xMYC-mRFP fusion in the post-excised state (Addgene #64767). These sequences were assembled together with a Pbnup221 homology region, placing Cre-EBD under the control of P. berghei EF1 alpha promoter and the hDHFR selectable marker under the control of the P. berghei EF1 delta promoter and positioning the hDHFR cassette between the two loxP-containing RITE tags to facilitate marker removed upon excision. The mRFP sequence in the post-excision RITE fusion was subsequently replaced with mRuby3 to yield the final plasmid pL857. Plasmids pSA026, pSA028 and pL857 were linearized within the Pbnup138, Pbnup313 or Pbnup221 homology region using HindIII, PacI or ScaI, respectively, and transfected into P. berghei
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