RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5534
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1443000; Gene model (P.falciparum): PF3D7_1228300; Gene product: NIMA related kinase 1 (NEK1)
Name tag: triple-HA
Phenotype Gametocyte/Gamete;
Last modified: 11 September 2024, 10:11
  *RMgm-5534
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) : 39255311
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/ResearcherZeeshan M, Tewari T
Name Group/DepartmentUniversity of Nottingham
Name InstituteSchool of Life Sciences
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-5534
Principal nameNEK1-HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteSee below
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal 3xHA-tagged version of NEK1

Protein (function)
Mitosis has a key role in the eukaryotic cell cycle when the cell divides and produces daughter cells. During mitosis, centrosomes act as microtubule organising centres (MTOCs) coordinating spindle dynamics with chromosome congression and segregation. They also act as signalling hubs for regulators of mitosis, including cyclin-dependent kinases (CDKs), and Aurora, Polo and NIMA-related (NEK) kinases.
Budding and fission yeast express a single NEK kinase, but the family is expanded to 11 members in mammals, which may reflect the increased complexity of microtubule-dependent processes. Several NEKs are essential to form functional cilia and flagella in mammals and other organisms, including Chlamydomonas, Trypanosoma and Tetrahymena. Other NEKs are more directly implicated in mitotic cell division, with defects in NEK expression likely to contribute to aberrant chromosome segregation in cancer cells. NEKs are located at MTOCs in many organisms, including Aspergillus, yeast and human cells. Of the eleven mammalian NEKs, NEK2, NEK6, NEK7 and NEK9 localise to centrosomes, playing roles in mitotic spindle assembly.
Plasmodium contains a bipartite MTOC, that consists of a cytoplasmic part (outer MTOC) and a nuclear component (inner MTOC). Four NIMA-like kinases were identified in Plasmodium, and all are expressed most highly in gametocytes. These kinases have been named NEK1 to NEK4, but they are not directly analogous to mammalian NEK1 to NEK4.
Previous gene knockout studies showed that Plasmodium NEK2 and NEK4 are required for zygote differentiation and meiosis, but not for mitotic division of parasite cells in mammalian red blood cells, whereas NEK1 is (likely) essential for asexual blood stage schizogony and proliferation.

Phenotype

See also mutant RMgm-5533 for a mutant expressing a C-terminal GFP-tagged version of NEK1.
Ultrastructure Expansion Microscopy (U-ExM) was used to further resolve the location of NEK1 during male gametocyte development. NEK1 was tagged at the C-terminus with a 3xHA tag. In non-activated microgametocytes, the bipartite MTOC, as highlighted by the NHS-ester (amino reactive dye used for fluorescent labelling of protein density) and centrin staining, was visible spanning the nuclear membrane and linking the amorphous cytoplasmic part to the intranuclear body. At this stage, only a few sparse NEK1-HA punctuate signals were visible in both cytoplasmic and nuclear part of MTOC. Thirty seconds to one-minute post-activation, axonemes started to nucleate on the cytoplasmic side, as revealed by tubulin staining. At this stage, NEK1-HA localised next to the centrin positive cluster of basal body and partially to the intranuclear spindle poles. At one to two minutes post-activation, the cluster of basal bodies and spindle poles had separated and migrated towards the opposite side of the nucleus. At this stage, NEK1-HA form a link between the cluster of basal bodies and the spindle poles. From six to eight minutes post-activation, NEK1-HA was located around the basal bodies and spindle poles. The location of NEK1 at the connection between the basal bodies and spindle poles was also more marked. By ten to twelve minutes, the NEK1-HA pattern at the MTOC overlapped that of the NHS-ester suggesting a pronounced connection between the basal body and the spindle pole. The U-ExM approach was also used with γ-tubulin to resolve further the location of NEK1-HA at the MTOC. As described previously, γ-tubulin re-locates from the cytoplasmic centriolar MTOC to spindle pole post-gametocyte activation. In the period from one to ten minutes post-activation, NEK1-HA was located at the MTOC, as described above, enriched towards the cytoplasmic side of the acentriolar MTOC, while γ-tubulin was enriched towards the intranuclear side. Interestingly, a discrete region, apparent as a fine line across the acentriolar MTOC remained free both of NEK1-HA and γ-tubulin.

See RMgm-5533 for a mutant expressing NEK1-GFP. Analysis of these parasites showed that NEK1-GFP was undetectable in ring stages but was visible with a diffuse cytoplasmic location during later stages (trophozoites). At the start of schizogony, NEK1-GFP re-located In the cytoplasm initially to single focal points near individual nuclei, which later divided into two tightly associated punctae. NEK1-GFP signal disappeared at the end of nuclear division and was undetectable in free merozoites.The location of NEK1-GFP was also studied during other asexual stages like liver schizogony and mosquito gut sporogony. The live cell images of the proliferative liver schizogony and sporogony stages showed similar patterns of NEK1-GFP foci formation during proliferative stages.

Additional information
The following mutants were generated to analyse the location and function of NEK1 in more detail:
- A mutant expressing a C-terminal 3xHA tagged version of NEK1 (RMgm-5534)
- (Conditional) knockdown mutants of NEK1: i) a mutant expressing a AID/HA tagged version of NEK1 to degrade NEK1 in gametocytes in the presence of auxin and ii) a mutant in which the promoter region of NEK1 was replaced with the promoter of clag, a gene that is specifically expressed in asexual blood stages (and not in gametocytes) (RMgm-5535).

By (mosquito-)crossing NEK1-GFP parasites (RMgm-5533) with other (existing) transgenic lines expressing mCherry tagged proteins (NDC80 RMgm-4844; EB1 RMgm-5329; ARK2 RMgm-5322; kinesin-8b RMgm-5324) 'dual tagged' parasites were generated that expresses:
- GFP-tagged NEK1 and mCherry tagged kinetochore marker NDC80
- GFP-tagged NEK1 and mCherry tagged spindle microtubule plus-end binding protein EB1
- GFP-tagged NEK1 and mCherry tagged spindle-associated aurora kinase 2 ARK2
- GFP-tagged NEK1 and mCherry tagged cytoplasmic axonemal protein kinesin 8B

Generation of dual tagged parasite lines
The NEK1-GFP parasites were mixed with either NDC80-cherry or EB1-mCherry or ARK2-mCherry or kinesin-8B-mCherry parasites in equal numbers and injected into mice. Mosquitoes were fed on mice 4 to 5 days after infection when gametocyte parasitaemia was high. These mosquitoes were checked for oocyst development and sporozoite formation at day 14 and day 21 after feeding. Infected mosquitoes were then allowed to feed on naïve mice and after 4 - 746 5 days, and the mice were examined for blood stage parasitaemia by microscopy with Giemsa-stained blood smears. In this way, some parasites expressed both NEK1-GFP and NDC80-mCherry; or EB1-mCherry; or ARK2-mCherry; or kinesin-8B-mCherry in the resultant gametocytes, and these were purified, and fluorescence microscopy images were collected as described above.

The analyses of these mutants revealed the following (from the Abstract):
'We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organizing centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation and kinetochore attachment. These data suggest that Plasmodium NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation'
NEK1 is (likely) essential for asexual blood stage schizogony and proliferation.

Evidence is presented that:
- Plasmodium NEK1 has a punctate location, partially overlapping with the kinetochore and MTOC during blood stage schizogony
- NEK1-GFP associates with MTOC formation and spindle dynamics during male gamete formation
- The spatiotemporal location of NEK1 with respect to kinetochore, spindle and axoneme markers during male gametogenesis
- NEK1-GFP interacting proteins are components of the axoneme and flagellum
- Conditional knockdown/depletion of NEK1 reveals an essential role during male gametogenesis
- Abnormal MTOC organization and kinetochore attachment following of conditional knockdown of NEK1 level

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1443000
Gene Model P. falciparum ortholog PF3D7_1228300
Gene productNIMA related kinase 1
Gene product: Alternative nameNEK1
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
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 modification- GFP-tagging vectors were designed using the p277 plasmid vector. For GFP-tagging of NEK1 by single crossover homologous recombination, a region of nek1 downstream of the start codon was used to generate the construct.

- NEK1 was tagged at the C-terminus with a 3xHA tag generated using PlasmoGEM vectors and a recombineering strategy (https://plasmogem.umu.se/pbgem/).

- To study the function of NEK1, we used two conditional knock down systems: an auxin inducible degron (PP1AID) and a promoter exchange/trap using double homologous recombination (PP1PTD). The NEK1AID construct was derived from the p277 plasmid, where the GFP sequence was excised following digestion with AgeI and NotI restriction enzymes and replaced with an AID/HA coding sequence. The AID-HA sequence was PCR amplified (using primers: 5’-CCCCAGACGTCGGATCCAATGATGGGCAGTGTCGAGCT-3’ and 5’- ATATAAGTAAGAAAAACGGCTTAAGCGTAATCTGGA-3’) from the GW-AID/HA plasmid (http://plasmogem.sanger.ac.uk/). Fragments were assembled following the Gibson assembly protocol to generate the NEK1-AID/HA transfection plasmid that was transfected in the line.
The conditional knockdown construct NEK1clag was derived from Pclag (pSS368) where nek1 was placed under the control of the clag promoter, as described previously (Sebastian et al., 2012).

- Generation of dual tagged parasite lines
The NEK1-GFP parasites were mixed with either NDC80-cherry or EB1-mCherry or ARK2-mCherry or kinesin-8B-mCherry parasites in equal numbers and injected into mice. Mosquitoes were fed on mice 4 to 5 days after infection when gametocyte parasitaemia was high. These mosquitoes were checked for oocyst development and sporozoite formation at day 14 and day 21 after feeding. Infected mosquitoes were then allowed to feed on naïve mice and after 4 - 746 5 days, and the mice were examined for blood stage parasitaemia by microscopy with Giemsa-stained blood smears. In this way, some parasites expressed both NEK1-GFP and NDC80-mCherry; or EB1-mCherry; or ARK2-mCherry; or kinesin-8B-mCherry in the resultant gametocytes, and these were purified, and fluorescence microscopy images were collected as described above.
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