Conservation of the Toxoplasma conoid proteome in Plasmodium reveals a cryptic conoid feature that differentiates between blood- and vector-stage zoites

Background

Apicomplexan parasites are able to actively seek, bind to, and invade the cellular milieu of suitable animal hosts. From there, they manipulate and exploit these host cells to promote their own growth, and their onward transmission. In apicomplexan parasites, the apical complex is key for invasion. The inner membrane complex (IMC) provides shape and protection to the cell, as well as other functions such as gliding motility in apicomplexans. The apical complex has evolved together with the IMC, to provide a location for cellular functions including exocytosis and endocytosis.

The apical complex has been largely studied in ultrastructural studies, and apical rings are the basis of this structure. An apical polar ring (APR) acts as a microtubule organising centre (MTOC) for the subpellicular microtubules, and a second APR coordinates the apical margin of the IMC. Within this opening created by the APRs are further rings, a notable one being the ‘conoid’. The conoidinteracts intimately with secretory organelles including micronemes, rhoptries and other vesicles. While the APRs appear to play key structural organising roles, the conoid is closely associated with the events and routes of vesicular trafficking, delivery and in some cases uptake. While the conoid and its tubulin-based composition have been well described in Toxoplasma, Aconoidasida are considered to have either completely lost the conoid (e.g. Babesia, Theileria), or lost it from multiple zoite stages, e.g. Plasmodium spp. stages other than the ookinete (although controversy exists on whether the conoid was fully lost in Plasmodium spp.). The difficulties that have affected solving this controversy include that relatively few conoid protein and molecular signatures have been identified, making it difficult to conclude whether it is genuinely missing from some major groups, and making it difficult to objectively test for the presence of a homologous structure.

In their work, Koreny et al have carefully explored the Toxoplasma gondii conoid using multiple proteomic approaches, and then explored if these conoid-specific proteins are present in similar locations within the zoite forms (ookinetes, sporozoites and merozoites) of Plasmodium berghei (Fig.1).

Key findings and developments

The authors began by using multiple spatial proteomic methods to identify new candidate conoid proteins. This included the use of the recently published method by the same group, called hyperplexed Localization of Organelle Proteins by Isotope Tagging (hyperLOPIT) (2) over three separate datasets. This allowed assigning 76 proteins to one of the two apical protein clusters, apical 1 and apical 2. These two clusters were verified as comprising proteins specific to structures associated with the conoid, the apical polar ring, and apical cap of the IMC. A further 1013 proteins were quantified in either one or two datasets (instead of the three discussed above), and allowed distinguishing a further 16 proteins assigned to the apical clusters. From all analyses, 92 proteins were assigned as putative apical proteins across the hyperLOPIT samples. While 57 of these proteins had been validated as being located to specific structures (other than the conoid), a remaining 35 protein candidates had had no independent validation on their location.

A second proteomic strategy was used for these candidates, namely, BioID (proximity-dependent biotinylating and pulldown). Two baits used were known conoid markers in Toxoplasma, SAS6-like protein and RNG2. A third bait protein used as negative control, was MORN3, as its location is in high abundance in the apical cap, although excluded in the apex, where the conoid is located. Biotinylated proteins were purified in a streptavidin matrix and analysed by mass spectrometry. Altogether, of the hyperLOPIT-assigned apical proteins, 25 were also detected by BioID with SAS6L and RNG2 but not MORN3. These data indicate that the BioID spatial proteomics indeed enrich for apical proteins, with the differences between SAS6L/RNG2 and MORN3 labelling offering a level of discrimination for conoid-associated proteins when compared to apical cap proteins.

Further validation was performed using fluorescence microscopy. Widefield microscopy showed 13 proteins to be confined in a single small punctum in the extreme apex of the cell. However, higher resolution imaging was required to show specific localization. For this, the authors used 3D-SIM super-resolution to first determine the localizations of SAS6L and RNG2, as these two markers provide definition of the relative positions of the new proteins. Three of the newly identified proteins co-localized to the SAS6L pattern of the full body of the conoid. A further three proteins, with this same pattern of BioID detection, all formed a ring at the posterior end, or base, of the conoid. A further protein was found to co-localize with the RNG2 marker. Altogether, these 3D-SIM data confirm the identities and specific localization of various proteins across various parts of the conoid.

Having validated conoid proteins by various methods in T. gondii, the next step the authors did was to determine how conserved was the conoid proteome in other apicomplexans and related groups within the Myzozoa, and whether there was indeed evidence for conoid loss in the Aconoidasida. They first used two separate bioinformatic/genomic data analysis methods for identification of orthologues. The orthology analysis showed a high degree of conservation (88%) in other coccidia. In other apicomplexan groups (including known members of the Aconoidasida) and in the nearest apicomplexan relatives, the chromerids, approximately half of the conoid proteins are found. The conserved proteins include those associated with all structural components of the T. gondii conoid- conoid body, conoid base, and preconoidal rings.

To test orthologue localization of known T. gondii conoid-associated proteins in Plasmodium, nine selected conoid proteins (representing the conoid’s body and base, and the preconoidal rings) were tagged in P. berghei. This parasite strain provided access to all invasive zoite forms of the parasite: ookinetes, sporozoites, and merozoites. In ookinetes, an apical location was seen for all 9 proteins. In all cases examined, the location and structures formed by the Plasmodium orthologues phenocopied those of T. gondii, strongly suggestive of conservation of function.

 With the new markers for components of an apparent conoid in P. berghei, the authors tested for presence of these proteins in the other zoite stages: sporozoites and merozoites. In sporozoites all proteins tested for, were detected in the cell apex. In merozoites, only 6 of the 9 proteins tested were detected, and each formed an apical punctum juxtaposed to the nucleus, consistent with apical location.

 

What I like about this preprint

Already before I had liked a lot the development of hyperLOPIT that the authors presented. I was wondering on applications, and I was very excited to see it used in this work now. Moreover, I like that they approach questions previously not asked-and challenge assumptions previously made, with new tools, reaching new conclusions. I think this is the exciting part of science! I now look forward to knowing more about the conoid function in the different organisms studied.

References

1. Koreny, L., et al Conservation of the Toxoplasma conoid proteome in Plasmodium reveals a cryptic conoid feature that differentiates between blood and vector-stage zoites, bioRxiv, 2020
2. Barylyuk, K., A subcellular atlas of Toxoplasma reveals the functional context of the proteome, bioRxiv, 2020.

 

 

doi: https://doi.org/10.1242/prelights.23365

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