SummaryRMgm-4952
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Successful modification | The 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) : 33705380 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Koreny, L, Tewari R, Waller RF |
Name Group/Department | Department of Biochemistry |
Name Institute | University of Cambridge |
City | Cambridge |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-4952 |
Principal name | PBANKA_0907700-GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | No |
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Phenotype | |
Asexual blood stage | Location of the tagged protein at the apical end of merozoites (see below) |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Location of the tagged protein at the apical end of ookinetes (see below) |
Oocyst | Not tested |
Sporozoite | Location of the tagged protein at the apical end of sporozoites (see below) |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation In most known conoids, the walls of the conoid have a spiralling fibrous presentation by electron microscopy, a trait that is chiefly attributed to the presence of tubulin polymers. In the Toxoplasma conoid, tubulin forms unusual open fibres with a comma-shaped profile. The ancestral state of conoid tubulin, however, is likely canonical microtubules as seen in gregarines, Chromera, and other apicomplexan relatives. It is unclear if the modified tubulin fibres of the Toxoplasma conoid arose specifically within coccidians or are more widespread in apicomplexans due to the limits of resolution or preservation of this dense structural feature. Nevertheless, this tubulin component demonstrates a degree of plasticity of the conoid structure. Electron microscopy shows that the tubulin fibres are embedded in electron dense material, evidence of further conoid proteins. This matrix extends to an open apical cover described as a ‘delicate osmophilic . . . canopy’ by Scholtzseck et al (1970) within which two conoidal rings are often seen. These rings are now frequently referred to as ‘preconoidal rings’, however, in recognition of the continuity of conoid ultrastructure from spiral reinforced walls to canopy rings, this entire structure was designated as the conoid and the rings as ‘conoidal rings’. The apical conoid canopy is in closest contact, and probably interacts, with the cell plasma membrane. Electron microscopy does not reveal any direct attachment fibres or structures from the conoid to the plasma membrane at its apex, or to the IMC at its base. However, in Toxoplasma it is known that at least one protein (RNG2) links the conoid directly to the APR2, thus, there is evidence of molecular architecture beyond that observed by ultrastructure. To test if a homologous conoid cell feature is present in Aconoidasida, but cryptic by traditional microscopy techniques, fuller knowledge of the molecules that characterise this feature in a ‘classic’ conoid model is needed. In our study we have sought such knowledge for the Toxoplasma gondii conoid using multiple proteomic approaches. We then asked if these conoid-associated proteins are present in similar locations within Aconoidasida using the model Plasmodium berghei to investigate each of its zoite forms: ookinetes, sporozoites and merozoites. Phenotype
To test if orthologues of T. gondii conoid-associated proteins occur in equivalent apical structures in Plasmodium, nine orthologues were selected for reporter tagging in P. berghei (PBANKA_1025300, PBANKA_1313300, PBANKA_1347000, PBANKA_1419000, PBANKA_0310700, PBANKA_0109800, PBANKA_1216300, PBANKA_0907700, PBANKA_1334800).The nine proteins represented the three sites associated with the conoid (base, walls and canopy) as well as APR1 and APR2 (PBANKA_0907700 and PBANKA_1334800, respectively). GFP fusions of these proteins were initially observed in the large ookinete form by live cell widefield fluorescence imaging, and an apical location was seen for all. Eight of these proteins were resolved only as a dot or short bar at the extreme apical end of the cell, whereas the APR2 orthologue (PBANKA_1334800) presented as an apical cap. To further resolve the location of the P. berghei apical proteins, 3D-SIM was performed on fixed ookinetes for eight proteins representing the different presentations found in T. gondii. The P. berghei orthologue of the conoid wall protein (PBANKA_0310700) was resolved as a ring at the cell apex, and this structural presentation was also seen for orthologues of the conoid base (PBANKA_1216300) and canopy rings (PBANKA_1347000, PBANKA_1419000). Further, two orthologues that are unresolved conoid canopy puncta in T. gondii are seen in P. berghei to mimic this presentation either as an apical punctum (PBANKA_1025300) or a barely resolved small ring (PBANKA_1313300). The APR2 orthologue (PBANKA_1334800) that showed a broader cap signal by widefield imaging was revealed as a ring of much larger diameter than the rings of the conoid orthologues. Furthermore, short spines radiate from this ring in a posterior direction that account for the cap-like signal at lower resolution. The location of this protein is consistent with an APR2 function, although more elaborate in structure than what is seen in T. gondii. Finally, the APR1 orthologue (PBANKA_0907700) also resolved as a ring of larger diameter than the conoid orthologues, and apparently closer to the apical cell surface than APR2 orthologue PBANKA_1334800. In all cases examined, the locations and structures formed by the Plasmodium orthologues were equivalent to those of T. gondii, strongly suggestive of conservation of function.
In merozoites, of the nine proteins tested for, only six were detected in this alternative zoite form of the parasite, and this is generally consistent with differential transcript expression profiles of these nine genes. The conoid wall (PBANKA_0310700) and base (PBANKA_1216300) orthologues were not detected in this cell form, nor was the APR2 protein (PBANKA_1334800). However, all five of the other conoid orthologues are present in merozoites as well as the APR1 protein (PBANKA_0907700), each forming an apical punctum juxtaposed to the nucleus consistent with apical location. These data support conservation of conoid constituents in the apical complex of both sporozoites and merozoites, but either a reduction in the complexity of this structure in merozoites or the possible substitution for other proteins that are yet to be identified. Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0907700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1141300 | ||||||||||||||||||||||||||
Gene product | conserved protein, unknown function | ||||||||||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | GFP | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid single cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | Unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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