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

Ludek Koreny, Mohammad Zeeshan, Konstantin Barylyuk, Declan Brady, Huiling Ke, Rita Tewari, Ross F. Waller

Posted on: 23 July 2020

Preprint posted on 13 July 2020

Article now published in PLOS Biology at http://dx.doi.org/10.1371/journal.pbio.3001081

Understanding the conoid in Plasmodium parasites

Selected by Mariana De Niz

Categories: cell biology

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).

Figure 1. Super-resolution imaging of P. berghei conoid complex orthologues in ookinetes (From ref.1, fig.5).

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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Author's response

Ross Waller and Ludey Koreny shared

Open questions

1.Out of curiosity, did you find conoid orthologues in Plasmodium berghei stages other than the zoites? Including the gametocytes (and its various maturation stages). Following directly from this question, when does the conoid begin to form, and how? Is it also present in liver-derived merozoites? And is it present equally in all the merozoite population?

To date we focused on the zoite stages for these new proteins, but the next step will be to look more thoroughly at the developmental behavior of these proteins. We know from Toxoplasma that the conoid is one of the first structures assembled during the formation of new cells. Similarly, in Plasmodium ookinete development from the zygote, the emergence of the apex of this newly polarized cell appears to lead with these apical proteins. In our previous study of SAS6L we saw an initial cytosolic location in the post-fertilized spherical female gamete then form a peripheral SAS6L spot at the point of ookinete emergence (PMID: 27339728). So, we anticipate that other conoid-associated proteins will be recruited during these developmental stages. However, we have also shown that an apical myosin is recruited late to the apex in ookinetes, so there is clearly a developmental sequence (PMID: 31283102). Regarding liver-derived merozoites, this is a stage that we haven’t currently examined, and it will be fascinating to see if these are the same or different from the blood-stage merozoites. We would anticipate these structures to be equally present in all blood merozoites, but another question that needs careful examination.

2.Plasmodium species were believed not to have a conoid, largely due to lack of fibrous tubulin that characterizes the conoid in parasites such as Toxoplasma. Do you envisage that functionally the conoid in Plasmodium vs. Toxoplasma would be very different from each other given the apparent difference in tubulin?

The function and requirement for the more elongate tubulin-reinforced conoid of Toxoplasma is actually relatively poorly understood. A mechanical role in helping to penetrate host extracellular matrices has been suggested, and this seems logical, but so far is difficult to test. But if this is the case then it would seem that a Plasmodium conoid would have less potential to act in this way. However, the presence of proteins associated with signal cascades relevant to controlling the secretory and motility events of invasion are common to both Toxoplasma and Plasmodium conoids. Hence, we hypothesis that there will be common functions also. It might be similar to the patella in humans versus frogs. We have different mechanical requirements for these joints, and their composition has some differences, but they share common developmental and core functional features also.

3.Regarding the three very defined structures in the conoid, do you envisage they have different functions? Have you tried generating mutants to determine their survival and overall the impact of loss of proteins in any of these structures?

Our knowledge of the organization of ‘substructures’ of the conoid is still in its infancy, some based on ultrastructure (e.g. preconoidal rings) and some by specific protein recruitment. We have little functional insight for the preconoidal rings at present, but where the tubulin part of the conoid is reduced these structures and their proteins tend to persist. So, this might be a core part of the conoid’s function. Proteins at the base of the conoid might be involved in its interaction with other parts of the apical complex, such as we’ve shown for the protein RNG2 (PMID: 24743791). We, and others, have started examining mutation phenotypes for these proteins (PMID: 32487504) and this is work that will keep us occupied now for some time I suspect.

4.Why did you choose Plasmodium berghei as a model? Given so, did you find orthologues in other Plasmodium spp. for the same conoid proteins, including Plasmodium falciparum?

Plasmodium berghei has served as an amenable model for some time to access all stages of the Plasmodium life cycle. Curiously, this taxon is probably primarily a bat pathogen, but it can be propagated in mice for the vertebrate portion of its cycle, and in mosquitoes for the insect vector stage — both of which can be maintained in the laboratory. As evidence of the utility of this model, all conoid proteins we found in P. berghei are also present in human-infective taxa such as P. falciparum. It is true that we can’t always assume that what we find in one Plasmodium species will be the case for another, however fundamental aspects of their biology are often conserved. Strong mutant phenotypes for many of these conoid proteins in P. falciparum supports that this will be the case for this structure.

5.Beyond genetic modification, are there known pharmacological treatments that would help study the function of the conoid in various different organisms?

A previous screen of small molecules against Toxoplasma has shown that some can specifically inhibit conoid activities such as conoid protrusion during invasion (PMID: 15123807). Similarly, we know that conoid protrusion is activated by Ca2+– and cGMP-sensitive kinases and signaling cascades, and drugs that perturb these kinases or their secondary messenger balance can also affect conoid function. Drugs that modulate tubulin polymerization also have potential to be used to functionally dissect these structures. However, secondary effects of perturbing other cell pathways and structures need to be factored into these approaches.

6.If the conoid is not specific to invasion phenotypes, what other functions do you predict it could have developed in other organisms?

Our data provides strong evidence that similar looking ultrastructures found in non-parasitic relatives of apicomplexans are genuine homologues of the conoid and associated apical structures. In most cases these organisms are microbial predators that attach to and feed directly on their prey — a heterotrophic behavior called myzocytosis, or more imaginatively ‘cellular vampirism’. The apical complex and conoid serve as feeding structures in these examples. However, other relatives are thought to be symbionts of animals, and it is less clear what the role of the conoid might be. Nevertheless, in all cases the conoid-bearing organism engages in some form of interaction with an unrelated organism, and we believe that the conoid helps to mediate this interaction through controlled secretion of proteins and other molecules. These interactions start with a kiss, but the relationship can then go in several directions!

7.Since Babesia and Theileria are thought to have fully lost the conoid, have you checked for orthologues in those parasites too?

Yes, our orthology analysis included Babesia and Theileria, and in both cases similar representation of conoid protein orthologues was found to that of Plasmodium. This suggests that, again, microscopy has overlooked cryptic cell structures that compositionally, and likely functionally, are homologous and equivalent to the conoid.

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