RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5112
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1362400; Gene model (P.falciparum): PF3D7_1349600; Gene product: conserved Plasmodium protein, unknown function (SKA3)
Name tag: HA, mScarlet or pCP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 26 August 2022, 14:28
  *RMgm-5112
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) : 36006241
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/ResearcherBrusini L, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine, Faculty of Medicine
Name InstituteUniversity of Geneva
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-5112
Principal namePBANKA_1362400-tag
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageSee below
Gametocyte/GameteSee below
Fertilization and ookinetesee below
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal HA, mScarlet or pCP-tagged version of PBANKA_1362400

Protein (function)
From the Abstract: 
Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify six divergent eukaryotic components, in addition to a set of eight apicomplexan kinetochore proteins (AKiTs) that bear no detectable sequence similarity to known proteins. The nanoscale  organization of the apicomplexan kinetochore includes four subdomains, each displaying different evolutionary rates across the phylum. Functional analyses confirm  AKiTs are essential for mitosis and reveal architectures parallel to biorientation at  metaphase.

PBANKA_0414300 - NUF2; PBANKA_0405800 - SKA2
PBANKA_0812300 and PBANKA_1362400: in complex with SKA2 (SKA1; SKA3)
PBANKA_0621300 - Apicomplexan Kinetochore protein 1 (AKiT1)
AKit2 - AKiT6: PBANKA_1202800 (AKiT2); PBANKA_0612200  (AKiT3), PBANKA_1310500 (AKiT4), PBANKA_1243900  (AKiT5), PBANKA_0522000  (AKiT6)
AKiT7-9: PBANKA_0612300 (AKiT7), PBANKA_0213200 (AKiT8), PBANKA_1307000 (AKit9)
AKiT10-11: PBANKA_0406000 (AKiT10), PBANKA_0803900 (AKiT11)

Phenotype

See below

Additional information
From the Abstract: 
Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify six divergent eukaryotic components, in addition to a set of eight apicomplexan kinetochore proteins (AKiTs) that bear no detectable sequence similarity to known proteins. The nanoscale  organization of the apicomplexan kinetochore includes four subdomains, each displaying different evolutionary rates across the phylum. Functional analyses confirm  AKiTs are essential for mitosis and reveal architectures parallel to biorientation at  metaphase.

PBANKA_0414300 - NUF2; PBANKA_0405800 - SKA2
In agreement with previously localized Plasmodium centromeres and kinetochores, location and movement of NUF2 and SKA2 fusion proteins was restricted to the nuclear periphery during progression of asexual blood-stage divisions. SKA2 and NUF2 fusion proteins showed very different localizations during sexual development. Whilst “rod-like bridges”, as previously described for components of the NDC80/NUF2 complex, were visible for NUF2 during the three rounds of DNA replication and mitosis that occur at microgametogenesis – SKA2 signal at kinetochore foci was not clearly detectable. A similar dichotomy was seen post-activation of the macrogametocyte, NUF2 spreading sparingly across the cytoplasm compared to SKA2 residing primarily in the nucleus. However, 24-hours post-fertilization and meiosis, both fusion proteins united as four  distinct nuclear foci in fully developed banana-shaped ookinetes. 

PBANKA_0812300 and PBANKA_1362400: in complex with SKA2
Both tagged PBANKA_0812300 and PBANKA_1362400 proteins showed localization patterns highly similar to that of SKA2 in the malaria parasite, colocalizing with NUF2 during asexual blood stage divisions, not detected during microgametogenesis and present as four nuclear foci in fully developed ookinetes.

PBANKA_0621300 - Apicomplexan Kinetochore protein 1 (AKiT1)
the tagged protein showed clear co-localization with NUF2 throughout asexual blood-stage divisions. In contrast to previously localized SKA components, foci were also seen  across spindle bundles during microgametogenesis. Furthermore, the protein accumulated as foci in the nuclei of female gametes prior to fertilization, suggesting  kinetochore recruitment prior to outer kinetochore NDC80/NUF2 complex assembly. Given biochemical affinities, kinetochore localization and conservation across apicomplexan organisms, the proteinwas named  Apicomplexan Kinetochore protein 1 (AKiT1). During the first round of microgametocyte mitosis, PbAKiT1 localized along the spindle as pairs of kinetochore foci.

AKit2 - AKiT6: PBANKA_1202800 (AKiT2); PBANKA_0612200  (AKiT3), PBANKA_1310500 (AKiT4), PBANKA_1243900  (AKiT5), PBANKA_0522000  (AKiT6)
Along with AKiT1, 5 proteins of unknown function were most abundant across PbAKiT1 immunoprecipitates. Each taggedhypothetical protein showed PbAKiT1-like localization patterns, colocalizing with NUF2 during blood-stage and microgametocyte mitosis, also present as foci in activated female macrogametes and accumulating as four puncta in fully developed ookinetes.

AKiT7-9: PBANKA_0612300 (AKiT7), PBANKA_0213200 (AKiT8), PBANKA_1307000 (AKiT9)  
Three proteins of unknown function formed a distinct enrichment profile compared to AKiTs 1 - 6. Each tagged protein showed characteristic kinetochore localizations, with  that encoded by PBANKA_0612300 displaying an additional localization reminiscent of  the nuclear membrane during sexual development.

AKiT10-11: PBANKA_0406000 (AKiT10), PBANKA_0803900 (AKiT11)
Principle components 2 and 3 further resolved AKiT clustering, with the distinction of AKiTs 1 - 6 relative to AKiT9, itself clustering with histones H3 and H4, the histone modifier SPT16, and two additional proteins of unknown function (Gene IDs: PBANKA_0406000 and PBANKA_0803900) that we named AKiT10 and AKiT11.

From the paper:
'Taken together, our immuno-affinity purification strategy has identified 4 biochemically stable compartments at the Plasmodium kinetochore (Table S4). Between compartments, more labile interactions were also detected between the outer kinetochore NDC80/NUF2 complex and PbAKiT1, itself closely associated with 5 additional AKiT proteins. AKiT1 - 6 also interact with AKiT9 - 11, which, given their association with proteins known to interact with DNA, likely form the most centromere proximal kinetochore sub-domain identified in this study.'

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1362400
Gene Model P. falciparum ortholog PF3D7_1349600
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameSKA3
Details of the genetic modification
Name of the tagHA, mScarlet or pCP
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 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 modificationFor C-terminal tagging of P. berghei proteins by PbGEM technology, 3xHA and mScarlet-I tagging
constructs were generated using phage recombineering in E. coli TSA strain with PlasmoGEM vectors
(http://plasmogem.sanger.ac.uk/) using sequential recombineering and gateway steps as described
previously (Pfander et al., 2011). For genes SKA2 and NUF2, the Zeocin-resistance/Phe-sensitivity
cassette was introduced using oligonucleotides goi-recR1 x goi-recR2 for 3xHA tagging and goi mSc-F x
goi mSc-R for mSc tagging vectors. Insertion of the GW cassette following gateway reaction was
confirmed using primer pairs GW1 x goi-QCR1 and GW2 x goi-QCR2.
For C-terminal tagging of P. berghei proteins by pCP, constructs were newly derived from pOB277
(Patzewitz et al., 2013) in order to target endogenous loci by allele replacement instead of insertion.
Briefly a 588 bp fragment encompassing the coding sequence of 3xHA and DHFR flanked by KpnI and
EcoRI was amplified from PbGEM plasmid GW-R6K-3xHA using primers MB1033 and MB1034 and
replaced the corresponding fragment in pOB277 to generate pCP-3xHA. The coding sequence for mNG
flanked by AvrII and SacII sites was purchased from GeneArt and inserted upstream to 3xHA to generate
pCP-mNG-3xHA. Sequences comprising 500 ∼500 bp from the C-terminus of the coding sequence and
~500 bp from the immediate 3’ UTR for genes SKA1, SKA3 and AKiTs 1 - 9 were cloned into KpnI and
AvrII sites upstream to the mNG coding sequence, along with a NotI linearisation site between the
targeting sequences.
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