RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1347000; Gene model (P.falciparum): PF3D7_1332200; Gene product: conserved protein, unknown function
Name tag: GFP
Phenotype Asexual bloodstage; Fertilization and ookinete; Sporozoite;
Last modified: 12 March 2021, 10:57
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) : 33705380
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherKoreny, L, Tewari R, Waller RF
Name Group/DepartmentDepartment of Biochemistry
Name InstituteUniversity of Cambridge
Name of the mutant parasite
RMgm numberRMgm-4949
Principal namePBANKA_1347000-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Asexual blood stageLocation of the tagged protein at the apical end of merozoites (see below)
Gametocyte/GameteNot tested
Fertilization and ookineteLocation of the tagged protein at the apical end of ookinetes (see below)
OocystNot tested
SporozoiteLocation of the tagged protein at the apical end of sporozoites (see below)
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal GFP-tagged version of PBANKA_1347000

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2020.06.26.174284; this version posted December 7, 2020.

Protein (function)
Apical rings are the basis of the apical complex. An apical polar ring (APR1) coordinates the apical margin of the IMC, and a second APR (APR2) acts as a microtubule organising centre (MTOC) for the subpellicular microtubules. Within this opening created by the APRs are further rings, a notable one being the ‘conoid’ that is conspicuous throughout much of Apicomplexa. The conoid is a tapered tubular structure of variable length and cone pitch. It interacts intimately with secretory organelles including micronemes, rhoptries and other vesicles that penetrate and occupy its lumen. While the APRs appear to play chiefly structural organising roles, the conoid is closely associated with the events and routes of vesicular trafficking—delivery and in some cases uptake.

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. 

A predicted structural deviation to the apical complex in Apicomplexa is the interpretation of loss of the conoid in some groups, a state enshrined within the class name Aconoidasida. This class contains two important groups: Haemosporida, such as Plasmodium spp., and Piroplasmida. Aconoidasida are considered to have either completely lost the conoid (e.g. Babesia, Theileria), or at least lost it from multiple zoite stages, e.g. Plasmodium spp. stages other than the ookinete. However, while conoids have been attributed to micrographs of ookinete stages in some Plasmodium spp., in other studies these are alternatively labelled as ‘apical polar rings’, and the prevailing understanding of many is that a conoid was lost outright.

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. 

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. 

Additional information 
The presence of a possible conoid in Plasmodium has been previously attributed to the ookinete stage, but the conoid is widely considered to be absent from asexual blood-stage merozoites. With our new markers for components of apparent conoid-associated structures in P. berghei, we tested for presence and location of these proteins in the other zoite stages: sporozoites and merozoites.

In sporozoites all proteins tested for are detected at the cell apex and super-resolution imaging of five of these again showed either a ring or unresolved apical punctum. 

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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1347000
Gene Model P. falciparum ortholog PF3D7_1332200
Gene productconserved protein, unknown function
Gene product: Alternative name
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