Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

The authors study proteins localised to the apical end of the highly polarised parasites causing Toxoplasmosis and malaria. They find new proteins using BioID and examine the localisation of these along with recently identified proteins in the two different parasites. They key question they address is whether there is a conservation of the apical components in these distantly related parasites as well as in some even more distantly related organisms. This is an important question as the apical part comprises many essential proteins of invasion of host cells and shows a unique structure that defines the apicomplexans as a group. The apical structure can be highly elaborate such as in T. gondii and less elaborate as in P. falciparum. The authors now show that there is a large conservation between the species in the protein makeup of the apical end. The experiments are well performed, displayed and discussed and there is no doubt about the validity of the presented results. The text is eloquently written, if at times a bit wordy.
My only main suggestion would be to possibly add data on gene disruption of the two candidates (0310700 and 1216300) that are not detected in blood stage parasites but in the insect stages. A deletion of these should be technically straightforward and would show whether the proteins are important to the parasite. Likely not all of the now many proteins are essential for the parasites but these are good candidates to rapidly investigate. But showing a functional impact might convince editors at certain journals.
Authors' response: The central aim of this study was to ask if the molecular composition of the conoid complex is conserved across Apicomplexa. Functional dissection of proteins is part of an exciting set of subsequent questions and studies that will now follow by us and others. However, careful and thorough phenotyping of gene disruptions is not trivial work, would be most informative to perform in both Toxoplasma and Plasmodium, and is therefore beyond the scope of this project. Regarding the two proteins suggested by this reviewer for follow-up work and the question of 'essentiality', given that these proteins have not been lost during parasite selection through evolution, this is clear evidence of their relevance to the biology of Plasmodium.
Other suggestions in chronological order (line numbers would have helped) title: maybe write 'conoid complex proteome' Authors' response: while we initially thought that this change would be suitable, given that the subsequent part of the title is 'reveals a cryptic conoid feature' we think it is clearer and more logical to leave this title in its original form. The conoid complex includes the apical polar rings, and these are not considered to be cryptic or previously unrecognised, only the conoid. While our study confirms that there is conservation across all proteome components of the conoid complex, this is secondary to the primary question of this study.
abstract: not sure about the use of the words instrument and substructures Authors' response: we believe that the use of 'instrument' is an appropriate analogy of a tool and not different from the use of 'machine' and 'machinery' that is widely used in molecular and cellular biology. Similarly, 'substructure' acknowledges that within recognised structures, such as the conoid, there is further specific organisation such as the conoid base or apex. page 2 last lines: is tubulin monomeric or polymerized?
Authors' response: to specify the polymerized state of tubulin as mentioned here the text has been changed to 'the presence of tubulin polymers'.

page 3 name protein talked about in 9th line
Authors' response: we have now named this protein (RNG2) as suggested.
third paragraph: mention previous proteomics studies e.g. from Ke Hu (mentioned later in discussion) Authors' response: We feel that it is more appropriate to leave the discussion of the Hu et al (2006) proteomics study, along with various subsequent approaches used in pursuit of discovering conoid-associated proteins, to the discussion as currently occurs. In the introduction we seek to efficiently inform the reader of the current state of knowledge that makes the value and nature of the questions that we have asked in this study apparent. But we do give full credit and evaluation of previous studies in the discussion which we think is the most appropriate place for this.

first paragraph or results could go into introduction
Authors' response: The first paragraph of the Results contains specific detail of just one aspect of this study, the use of hyperLOPIT. This is relevant to the new analysis that we have made of the hyperLOPIT data in this study. We, therefore, believe that it is most appropriately presented here in the Results in association with the new analyses we described. Our aim is that the Introduction is succinct and serves the entire study. As requested, we have complied available expression data for the Plasmodium proteins throughout the different zoite stages and will include these data as supplemental material in our subsequent revision.

Discussion:
Maybe discuss that the conoid complex is a cytoskeletal structure and that the other cytoskeletons (actin, microtubules, subpellicular network) also differ between the species investigated in their composition and overall architecture Authors' response: These are reasonable suggested analogies and we can introduce them in the subsequent revision.
page 9: at least two proteins could be deleted as they seem to not confer any growth defect on blood stages (see main comment) Authors' response: This reviewer has not linked this comment to a specific statement on page 9, however, we are cautious not to interpret lack of observed growth defects in experimental scenarios with unimportant or irrelevant proteins. Maintenance, through natural selection and evolution, of proteins of a structure indicate that they are selectively advantageous and of functional relevance. The two proteins in question are not expressed in the blood stage, so one wouldn't expect their deletion to have consequence in this stage.
Apart from classic TEM images also Cryo EM data is available for apex of merozoite and sporozoite. Worth to discuss?

Authors' response: According to this review's subsequent suggestion (below), we have included a schematic of the zoite stages of Plasmodium and these draw on Cryo EM tomography data.
Add and discuss the recent work from Curr Biol and EMBO J of the Yuan lab on ookinete formation?
Authors' response: These two reports are excellent studies of the polarised development of the cell pellicle during ookinete formation and control of gliding initiation, but don't specifically related to the conoid complex structures that are the subject of our study. We, therefore, do not see a logical place to include discussion of these works.
Reviewer #2 (Significance (Required)): The paper provides a conceptual advance over previous data as it shows clearly a high level of conservation of the protein components of the conoid complex. It could introduce a new terminology for these important apical structure of Apicomplexan parasites and provides a good basis to dissect the molecular functions.
Authors' response: We appreciate this reviewer recognising this opportune point in time to more clearly define the terminology applied to these apical structures so that they can be more clearly and easily compared between taxa. This has now been done in the revision along with clear schematics that show the equivalent structures between taxa and zoite forms. This also makes this important aspect of apicomplexan biology more accessible to general readership. We have used terminology based on precedent in the original ultrastructural literature.
As it stands all scientists investigating Plasmodium and Toxoplasma invasion of host cells will be highly interested in this study, most scientists researching apicomplexan organisms should be and some evolutionary scientists will be interested in this study.
Key papers in the field are the discovery of the Toxoplasma conoid as a highly twisted microtubulelike structure (Hu et al., JCB 2002;doi: 10.1083/jcb.200112086) the first description of an apical proteome (Hu et al., PLoS Path 2006;10.1371/journal.ppat.0020013), the description of a tilted arrangement of the rings in Plasmodium versus Toxoplasma (Kudryashev et al., Cell Microbiol 2012;doi: 10.1111/j.1462-5822.2012 and the discovery of apical located proteins that are essential for conoid formation (Tosetti et al., eLife 2020; 10.7554/eLife.56635) to name a few.
If intended for a broader audience, a cartoon of a conoid complex across the different species investigated and discussed here would help for visual guidance highlighting the similarities and differences

Authors' response: This is a good suggestion and we have acted on this in new figures 1 and 10. Furthermore, we now include transmission electron microscopy to support these cartoon and that further define the apical structures of each of the cell stages examined.
Reviewer #3 (Evidence, reproducibility and clarity (Required)): In this work, Koreny et al. characterized the localization of a new collection of conoid proteins in Toxoplasma gondii as well as in several different stages of Plasmodium berghei. The authors discovered that these proteins are located in several distinct substructures in Plasmodium and are expressed in a stage-specific manner. The data are of high quality, well-organized, and well presented. The paper is well written. The introduction, in particular, was a pleasure to read. This reviewer (Ke Hu) does not have any new experiments to suggest.
However, while the authors present LOPIT+BIOID as a powerful approach to identify conoid proteins, implying that it is more reliable than previously published approaches (see below), the manuscript includes no data to show what the false positive or false negative rate is with the current approach, nor any estimate of how many conoid proteins were missed entirely.
Authors' response: In our validation of putative conoid-associated proteins identified by the hyperLOPIT+BioID approach we reporter-tagged 18 proteins to resolve their cellular location by microscopy. All 18 were verified as being located at the site of the conoid. So, by this measure there were no false positives. The veracity of the hyperLOPIT data was also confirmed across other cell compartments in our report where 62 proteins were reporter-tagged from which there were no false positive assignments of cell location (Barylyuk et al., 2020, Cell Host & Microbe, in press:doi:10.1016/j.chom.2020 Estimating false negatives is more difficult, but we know that these would occur as for any mass spectrometry-based detection technique. However, we have not claimed to have been exhaustive, nor was this required to answer our central question of are there conserved conoid-associated proteins throughout Apicomplexa? To address this question, we required a good sample of proteins, and the methods that we have employed provided this. Page 7: "Previous identification of conoid complex proteins used methods including subcellular enrichment, correlation of mRNA expression, and proximity tagging (BioID) (Hu et al. 2006;Long, Anthony, et al. 2017;Long, Brown, et al. 2017). Amongst these datasets many components have been identified, although often with a high false positive rate. We have found the hyperLOPIT strategy to be a powerful approach for enriching in proteins specific to the apex of the cell, and BioID has further refined identification of proteins specific to the conoid complex region." The authors should state whether the candidate proteins were chosen in an unbiased way or not.
Authors' response: Candidate proteins selected for validation by microscopy were not biased for any known likelihood of being associated with the conoid, other than our proteomics data what we were seeking to test. However, we did preference proteins with the following traits, 1) proteins with strong corresponding gene knockout fitness phenotypes from published studies, 2) proteins with some evidence of conserved functional domains, and 3) genes with orthologues found in Plasmodium spp. and other apicomplexans. These traits were chosen with future functional studies in mind where proteins might be more informative of conoid-related functions and relevance in other apicomplexans. All validated proteins, however, were otherwise uncharacterised and, therefore, were not knowingly biased for more likely conoid-association over others discovered by our proteomics approach. We now include the following statement.
"All proteins selected for validation were previously uncharacterised and with no a priori reason to be identified as conoid-associated other than our proteomics data." If so, how many proteins were localized to the conoid and how many were not?
Authors' response: as stated above, we observed no false positives from the sample of 18 protein locations verified by microscopy.
Related to this, the majority (14 out of 20) of the conoid proteins identified by LOPIT+BIOID in this paper were previously identified as conoid candidate proteins in Hu et al's 2006 paper, based on the number of peptides retrieved from the conoid enriched vs depleted fractions. Those data (see below) have been available from ToxoDB for many years and should be acknowledged.