RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5323
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0407400; Gene model (P.falciparum): PF3D7_0309200; Gene product: serine/threonine protein kinase ARK2, putative (ARK2)
Name tag: mCherry
TaggedGene model (rodent): PBANKA_1115700; Gene model (P.falciparum): PF3D7_0616200; Gene product: kinetochore protein NDC80, putative (NDC80)
Name tag: GFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 14 September 2023, 13:05
  *RMgm-5323
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37704606
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 R
Name Group/DepartmentSchool of Life Sciences
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-5323
Principal nameNDC80-GFP/ARK2-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteTo investigate the association of ARK2 and the mitotic spindle during male gametogony, its location was compared with that of the kinetochore marker NDC80 and cytoplasmic axonemal protein kinesin-8B. Parasite lines expressing ARK2-mCherry and NDC80-GFP were crossed (RMgm-5323), and the progeny were analysed by live-cell imaging to establish the spatiotemporal relationship of the two tagged proteins. The location of both ARK2-mCherry and NDC80-GFP was next to the stained DNA, and with a partial overlap, although NDC80-GFP was always closer to the DNA.
Fertilization and ookineteThe location of ARK2 relative to that of the kinetochore marker, NDC80, was examined during ookinete development in parasite lines expressing ARK2-mCherry and NDC80-GFP (RMgm-5323). ARK2-mCherry was located on spindles radiating from the poles and NDC80-GFP was detected along the metaphase plate during stages I to III. By stage IV both ARK2 and NDC80 had accumulated at spindle poles.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of ARK2 and a GFP tagged version of NDC80. The mutant has been obtained by crossing ARK2-mCherry (RMgm-5322) with NDC80-GFP (RMgm-4843).
(see mutant RMgm-5321 for a mutant expressing a C-terminal GFP-tagged version of ARK2)

Protein (function)
ARK2: Aurora kinases (AKs) are a conserved family of spindle-associated protein kinases, with critical roles in four aspects of cell division: (I) driving mitotic/meiotic spindle assembly and disassembly, (II) regulating spindle pole structure and dynamics, (III) promoting accurate chromosome segregation, and (IV) orchestrating cellular fission at cytokinesis.  Plasmodium spp. have three divergent aurora-related kinases (ARK1-3) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. Functional studies with both the human parasite Plasmodium falciparum and rodent parasite Plasmodium berghei have suggested that all three Plasmodium ARKs are likely essential for proliferation in asexual blood stage schizogony.

NDC80: NDC80 is a kinetochore marker. Chromosome attachment to spindle microtubules (MTs) is mediated by kinetochores, which are large multiprotein complexes assembled on centromeres located at the constriction point of sister chromatids. Each sister chromatid has its own kinetochore, oriented to facilitate movement to opposite poles of the spindle apparatus. During anaphase, the spindle elongates and the sister chromatids separate, resulting in segregation of the two genomes during telophase. The NDC80 complex is the major component of the kinetochore and mediates its attachment to spindle MTs. In most model organisms, it is a member of the network of conserved KNL1, MIS12 and NDC80 complexes (KMN)
The ∼170–190 kDa NDC80 complex has two globular domains at either end of a ∼57 nm elongated coiled-coil, forming a dumb-bell shape. It is a heterotetramer comprising a 1:1:1:1 ratio of NDC80 (also known as HEC1 in humans), NUF2, SPC24 and SPC25 sub-complexed as two heterodimers: NDC80 with NUF2, and SPC24 with SPC25

Phenotype
See mutant RMgm-5321 for a mutant expressing a C-terminal GFP-tagged version of ARK2. This mutant has been analysed in more detail for expression and location of ARK2 and showed the following:

ARK2 is expressed in the nucleus throughout the P. berghei life cycle.
ARK2-GFP showed a punctate nuclear pattern with one or two focal points during blood schizogony and sporogony. It was present at a single focal point with an additional more diffuse nuclear location during early stages of male gamete formation and in the early zygote, but in later zygote stages it had a more dynamic location on the spindle and spindle pole. ARK2-GFP was not detected in mature asexual (merozoites and sporozoites) and sexual (male gametes and ookinetes) stages of development (see below for more information).

The ARK2-mCherry mutant (RMgm-5322) has been used to cross with a mutant expressing GFP-tagged NDC80 (see mutant RMgm-4843) resulting in mutant ARK2-mCherry/NDC80-GFP (RMgm-5323). Analysis of this mutant showed the following:

'To investigate the association of ARK2 and the mitotic spindle during male gametogony, its location was compared with that of the kinetochore marker NDC80 and cytoplasmic axonemal protein kinesin-8B. Parasite lines expressing ARK2-mCherry and NDC80-GFP were crossed (RMgm-5323), and the progeny were analysed by live-cell imaging to establish the spatiotemporal relationship of the two tagged proteins. The location of both ARK2-mCherry and NDC80-GFP was next to the stained DNA, and with a partial overlap, although NDC80-GFP was always closer to the DNA.
Parasite lines expressing ARK2-GFP (RMgm-5321) and kinesin-8B-mCherry (RMgm-5324; PBANKA_0202700; PF3D7_0111000; kinesin-8B, putative) were crossed resulting in mutant ARK2-GFP/Kinesin8b::mCherry  (RMgm-5325) and examined by live cell imaging of both markers. One to two minutes after gametocyte activation, ARK2-GFP was observed close to the DNA and adjacent to, but not overlapping, the kinesin-8B-mCherry tetrad. ARK2-GFP remained distributed on spindles, while there was duplication of kinesin-8B-mCherry labelled tetrads. In later stages of male gametogony, ARK2-GFP remained associated with spindles and spindle poles, while kinesin-8B-mCherry showed  a distinct cytoplasmic axonemal location.
The location of ARK2 relative to that of the kinetochore marker, NDC80, was examined during ookinete development in parasite lines expressing ARK2-mCherry and NDC80-GFP. ARK2-mCherry was located on spindles radiating from the poles and NDC80-GFP was detected along the metaphase plate during stages I to III. By stage IV both ARK2 and NDC80 had accumulated at spindle poles.'

In addition the ARK2-mCherry mutant has been used to cross with a mutant expressing GFP-tagged EB1 (see mutant RMgm-5319) resulting in mutant EB1-GFP/ARK2-mCherry (RMgm-5326). Analysis of this mutant showed the following:

'To further resolve the location of EB1 with respect to the kinetochore and basal body at higher resolution, 3D-SIM was performed on EB1-GFP/NDC80-mCherry (RMgm-RMgm-5327), EB1-GFP/ARK2-mCherry (RMgm-RMgm-5326) and EB1-mCherry/SAS4-GFP (RMgm-RMgm-5328) fixed gametocytes. The 3D-SIM images of gametocytes expressing EB1-GFP/NDC80-mCherry showed EB1 bridge(s) across the nucleus with NDC80 distributed like beads on the bridge, each bead presenting a kinetochore. The 3D-SIM images of gametocytes expressing EB1-GFP/ARK2-mCherry showed EB1 bridge(s) across the nucleus with ARK2, overlapping each other. The bridged pattern of spindles for EB1 were restricted to the nucleus as shown by 3D-SIM images of gametocytes expressing EB1-mCherry/SAS4-GFP; whereby SAS4 was located in the cytoplasm but aligned with the EB1 bridge in the nucleus'.

Analysis of a 'promoter swap mutant' (RMgm-5320) in which the promoter of ark2  gene has been replaced with the the promoter of clag9 (PBANKA_0836300) that is active in asexual blood stages but not in gametocytes, showed the following:
- Normal growth/multiplication of asexual blood stages. and normal formation of mature ookinetes with wild-type-like motility.
- A significant reduction (up to 70%) in the number of oocysts per mosquito midgut, detectable from day 7 post-infection, and remaining significantly lower through to day 21. The few oocysts present were smaller than those of wild-type parasites after day 7 and absence of sporozoite formation inside oocysts
- No midgut and salivary gland sporozoites.

Additional information
In the paper evidence is presented for a unique scaffold of an aurora-related kinase (ARK2; PBANKA_0407400; PF3D7_0309200; serine/threonine protein kinase ARK2, putative) at the spindle including the microtubule plus end-binding protein EB1 (PBANKA_0405600; PF3D7_0307300; end-binding protein 1), lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission.

To examine the role of ARK2 during sexual stages we first tagged the endogenous ARK2 locus with sequence encoding an auxin-inducible degron (AID) and an HA epitope tag to degrade the fusion protein in the presence of auxin in a parasite line expressing the TIR1 protein (Philip and Waters, 2015). Although the genetic modification was confirmed by diagnostic PCR, addition of auxin to gametocytes did not lead to ARK2-AID/HA degradation and there was no detectable phenotype in male gamete formation. Since the AID system was unsuccessful, we used a promoter trap strategy, replacing the ark2 promoter with that of cytoadherence-linked asexual protein (CLAG – PBANKA_1400600), which is not transcribed in gametocytes.

Since ARK2 is expressed in male gametocytes and parasite development is affected after fertilization, we investigated whether the defect is due to inheritance from the male gamete. We performed genetic crosses between Pclag-ark2 parasites and other mutants deficient in production of either male (Δhap2) or female (Δdozi) gametocytes. Crosses between Pclag-ark2 and Δdozi mutants produced some normal-sized oocysts that were able to sporulate, showing a partial rescue of the Pclag-ark2 phenotype. In contrast, crosses between Pclag-ark2 and Δhap2 did not rescue the Pclag-ark2 phenotype. These results reveal that a functional ark2 gene copy from a male gamete is required for subsequent oocyst development.

Analysis of a mutant expressing GFP-tagged ARK2 (RMgm-5321) showed the following:
- ARK2 is expressed in the nucleus throughout the P. berghei life cycle.
ARK2-GFP showed a punctate nuclear pattern with one or two focal points during blood schizogony and sporogony. It was present at a single focal point with an additional more diffuse nuclear location during early stages of male gamete formation and in the early zygote, but in later zygote stages it had a more dynamic location on the spindle and spindle pole. ARK2-GFP was not detected in mature asexual (merozoites and sporozoites) and sexual (male gametes and ookinetes) stages of development 
- Spatiotemporal dynamics of ARK2-GFP during male gamete formation demonstrates its association with rapid spindle dynamics.
The location of ARK2 was investigated by indirect immunofluorescence assay (IFA) and its co-localization with microtubules (MTs) in activated gametocytes was analysed by labelling MTs with an α-tubulin antibody.  Alpha-tubulin antibody detected both nuclear mitotic spindles and developing cytoplasmic axonemes, but ARK2 colocalized only with the mitotic spindles at all stages of male gamete formation. This result provides evidence that ARK2 is involved in mitosis within the male gametocyte. Deconvolution microscopy confirmed that ARK2 is located on mitotic spindles during male gamete formation. 
- ARK2 and kinetochore dynamics are associated, but cytoplasmic axonemal microtubule dynamics are not, male gamete formation. 
To investigate further the association of ARK2 and the mitotic spindle during male gametogony, we compared its location with that of the kinetochore marker NDC80 and cytoplasmic axonemal protein kinesin-8B. Parasite lines expressing ARK2-mCherry and NDC80-GFP were crossed, and the progeny were analysed by live-cell imaging to establish the spatiotemporal relationship of the two tagged proteins. The location of both ARK2-mCherry and NDC80-GFP was next to the stained DNA, and with a partial overlap, although NDC80-GFP was always closer to the DNA. Parasite lines expressing ARK2-GFP and kinesin-8B-mCherry were crossed and examined by live cell imaging of both markers. One to two minutes after gametocyte activation, ARK2-GFP was observed close to the DNA and adjacent to, but not overlapping, the kinesin-8B-mCherry tetrad. ARK2-GFP remained distributed on spindles, while there was duplication of kinesin-8B-mCherry labelled tetrads. In later stages of male gametogony, ARK2-GFP remained associated with spindles and spindle poles, while kinesin-8B-mCherry showed  a distinct cytoplasmic axonemal location 
- Tracing ARK2-GFP location during the zygote to ookinete transition indicates a role at the meiotic spindle.
In zygotes (2h after gametocyte activation and fertilisation), ARK2-GFP was detected at one or two foci. These foci migrated away from each other over the next 8-10h through development into stage IV ookinetes, to opposite sides of the nucleus. During this time, the ARK2-GFP signal appeared to radiate into the centre of the nucleus, typical of a classic metaphase spindle arrangement. These two foci then divided again to form four foci, before the signal faded into a diffuse distribution within nuclei of mature ookinetes. The location of ARK2 relative to that of the kinetochore marker, NDC80, was examined during ookinete development in parasite lines expressing ARK2-mCherry and NDC80-GFP. ARK2-mCherry was located on spindles radiating from the poles and NDC80-GFP was detected along the metaphase plate during stages I to III. By stage IV both ARK2 and NDC80 had accumulated at spindle poles.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0407400
Gene Model P. falciparum ortholog PF3D7_0309200
Gene productserine/threonine protein kinase ARK2, putative
Gene product: Alternative nameARK2
Details of the genetic modification
Name of the tagmCherry
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate the mCherry-tag line, a region of the ark2 gene downstream of the ATG start codon was amplified, ligated to p277 vector, and transfected. The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine.
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1115700
Gene Model P. falciparum ortholog PF3D7_0616200
Gene productkinetochore protein NDC80, putative
Gene product: Alternative nameNDC80
Details of the genetic modification
Name of the tagGFP
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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