RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4685
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0805900; Gene model (P.falciparum): PF3D7_0319400; Gene product: kinesin-8, putative (kinesin-8X)
Name tag: GFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 27 October 2019, 15:54
  *RMgm-4685
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) : 31600347
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, Queens Medical Centre
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4685
Principal namekinesin-8X-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteKinesin-8X was not detectable by microscopy in asexual blood stages but exhibited a diffuse nuclear localization in both male and female gametocytes.
Fertilization and ookineteFollowing activation of gametogenesis in vitro, kinesin-8X began to accumulate in male gametocytes at one end of the nucleus, presumably at the putative MTOC. Within one-minute of activation we observed an arc-like distribution of kinesin-8X across the nucleus, later forming two distinct foci that is consistent with the formation of two MTOCs. As DNA replication and endomitosis continued, six to eight distinct foci were seen to form 8 to 10 min after activation. These kinesin-8X foci may be associated with the MTOCs of the 8N nucleus that precedes exflagellation to produce eight male gametes. However, there was no detectable expression of Pbkinesin-8X in these male gametes. To examine further the location of kinesin-8X, we investigated its co-localization with MTs (using α-tubulin as a marker) by indirect immunofluorescence assay (IFA) in fixed cells. Kinesin-8X was localized on MTs during the early stages of male gametogenesis but in later stages it was distributed diffusely within the nucleus. To improve visualisation, we used deconvolution microscopy and confirmed that kinesin-8X is localized on mitotic spindles in early stages of male gametogenesis. In female gametocytes there was no major change in kinesin-8X distribution and it remained nuclear even 15 min post-activation. During this period the nucleus, together with the appearance of kinesin-8X staining, became more condensed and centrally located within the female gamete. Next, we examined the location and dynamics of kinesin-8X during zygote differentiation into the motile ookinete over 24 h. Two hours after fertilisation, kinesin-8X began accumulating at one end of the nucleus, as determined by live cell imaging. Following the initial protrusion of the apical prominence during stage I and II of ookinete development, kinesin-8X was observed on spindles. In later stages (stage V), it accumulated at two distinct foci, probably at two spindle poles, and remained there in the mature stages of ookinete development.
OocystDuring oocyst development at about 10 to 14-days post-mosquito feeding, kinesin-8X formed punctate dots located near putative MTOCs (Fig 4C). Many arc or bridge-like structures were also observed that may represent the redistribution of kinesin-8X on spindles during this endomitosis in oocysts.
SporozoiteKinesin-8X was also present in sporozoites, located as a focal point next to the nucleus
Liver stageTo study the expression and location of kinesin-8X during the vertebrate pre-erythrocytic stage, we infected HeLa cells with sporozoites. The pattern of protein location during liver stage development was similar to that in other mitotic stages, with a spindle pattern in cytomere stages and an MTOC-like location in schizont stages.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expresses a C-terminal GFP-tagged version of kinesin-8X

Protein (function)
Kinesins are molecular motors that use ATP to translocate along microtubules (MTs) or control MT-end dynamics. There are 14 to 16 classes of kinesins in eukaryotes which are defined by their conserved motor domain. This domain contains both ATP and MT binding sites, is located in different contexts within the protein primary sequence, and is required by these motor proteins to undertake a wide range of cellular functions. Many kinesins, together with dynein, have important roles in mitosis, including spindle pole separation, kinetochore attachment to spindles, chromosome alignment and segregation, and cytokinesis. Some of these kinesins have also been shown to play an essential role in meiosis, mainly during meiosis I, where recombination takes place.
Kinesin-8s are conserved across eukaryotes. During mitosis, kinesin-8 proteins in many eukaryotes localise to spindles and control spindle length and chromosome positioning at the cell equator. In the absence of functional kinesin-8, mitotic spindle length increases and chromosome alignment at the metaphase plate is perturbed. In addition, kinesin-8 proteins may have a role in maintenance of cell polarity and nuclear positioning in the centre of a fission yeast. At the molecular level, kinesin-8s are plus end directed MT motors that play a key role in controlling MT length, with some exhibiting MT depolymerisation activity.
Phylogenetic analyses have identified 9 kinesin genes in the Plasmodium genome though not much is known overall about the roles of these motors in Apicomplexa. Evidence in this study support earlier studies in identifying two kinesins classified as kinesin-8s which are members of distinct kinesin-8 subgroups, kinesin-8B and kinesin-8X

Phenotype

Analyses of a mutant lacking expression of kinesin-8X (RMgm-4684) showed the following: No significant difference in the number of Δkinesin-8X oocysts compared to wild-type controls at 7 dpi (days post-infection), but by 10 dpi we observed a significant reduction in mutant oocysts, which became even more significant at 14 dpi. By 21 dpi the number of GFP-positive Δkinesin-8X oocysts had decreased further to only 8–10% of the WT-GFP number. We also detected a significant decrease in the size of the Δkinesin-8X oocysts at 10, 14 and 21 dpi. At 21 dpi most of the Δkinesin-8X oocysts were dead, with diminished GFP expression and the presence of disintegrated nuclei. We also observed no viable sporozoites in these oocysts, and were unable to detect any salivary gland sporozoites. No infection of mice by bite of infected mosquitoes. Ultrastructure of Δkinesin-8X oocysts shows defects in growth and sporozoite budding.

By anlysing the mutant line expressing a GFP tagged version of kinesin-8X the following was shown:
- Live cell imaging of P. berghei showed that kinesin-8X is located on the spindle during mitotic and meiotic division at various stages of the parasite life cycle.
Kinesin-8X was not detectable by microscopy in asexual blood stages but exhibited a diffuse nuclear localization in both male and female gametocytes. 
Following activation of gametogenesis in vitro, kinesin-8X began to accumulate in male gametocytes at one end of the nucleus, presumably at the putative MTOC. Within one-minute of activation we observed an arc-like distribution of kinesin-8X across the nucleus, later forming two distinct foci that is consistent with the formation of two MTOCs. As DNA replication and endomitosis continued, six to eight distinct foci were seen to form 8 to 10 min after activation. These kinesin-8X foci may be associated with the MTOCs of the 8N nucleus that precedes exflagellation to produce eight male gametes. However, there was no detectable expression of Pbkinesin-8X in these male gametes. To examine further the location of kinesin-8X, we investigated its co-localization with MTs (using α-tubulin as a marker) by indirect immunofluorescence assay (IFA) in fixed cells. Kinesin-8X was localized on MTs during the early stages of male gametogenesis but in later stages it was distributed diffusely within the nucleus. To improve visualisation, we used deconvolution microscopy and confirmed that kinesin-8X is localized on mitotic spindles in early stages of male gametogenesis. In female gametocytes there was no major change in kinesin-8X distribution and it remained nuclear even 15 min post-activation. During this period the nucleus, together with the appearance of kinesin-8X staining, became more condensed and centrally located within the female gamete. Next, we examined the location and dynamics of kinesin-8X during zygote differentiation into the motile ookinete over 24 h. Two hours after fertilisation, kinesin-8X began accumulating at one end of the nucleus, as determined by live cell imaging. Following the initial protrusion of the apical prominence during stage I and II of ookinete development, kinesin-8X was observed on spindles. In later stages (stage V), it accumulated at two distinct foci, probably at two spindle poles, and remained there in the mature stages of ookinete development.
During oocyst development at about 10 to 14-days post-mosquito feeding, kinesin-8X formed punctate dots located near putative MTOCs (Fig 4C). Many arc or bridge-like structures were also observed that may represent the redistribution of kinesin-8X on spindles during this endomitosis in oocysts. Kinesin-8X was also present in sporozoites, located as a focal point next to the nucleus.
To study the expression and location of kinesin-8X during the vertebrate pre-erythrocytic stage, we infected HeLa cells with sporozoites. The pattern of protein location during liver stage development was similar to that in other mitotic stages, with a spindle pattern in cytomere stages and an MTOC-like location in schizont stages.


Additional information
Evidence is presented that:
- the motor domain of
kinesin-8X is an microtubule(MT)-stimulated ATPase that drives MT gliding and has MT depolymerization activity.

Other mutants 
a mutant lacking expression of kinesin-8X (RMgm-4684)
 


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0805900
Gene Model P. falciparum ortholog PF3D7_0319400
Gene productkinesin-8, putative
Gene product: Alternative namekinesin-8X
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
Click to view information
Click to hide information
The C-terminus of kinesin-8X was tagged with GFP by single crossover homologous recombination in the parasite. To generate the kinesin-8X-GFP line, a region of the kinesin-8 gene downstream of the ATG start codon was amplified using primers T1931 and T1932, ligated to p277 vector, and transfected as described previously. The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine
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 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