Dear Dr. Parkinson,

Thank you very much for submitting your manuscript "ToxoNet: A high confidence map of protein-protein interactions in Toxoplasma gondii" for consideration at PLOS Computational Biology.

As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

All three reviewers raised similar issues but agree that the work is of potential interest to the field. Please respond productively to all reviewers' comments. Please pay close attention to comments regarding the issue of the apparent low correlation between hyperLOPIT predicted localization in identified complexes (i.e. many complexes have components that are apparently in different compartments).

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael L Reese, PhD

Guest Editor

PLOS Computational Biology

Stacey Finley

Section Editor

PLOS Computational Biology

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All three reviewers raised similar issues but agree that the work is of potential interest to the field. Please respond productively to all reviewers' comments. Please pay close attention to comments regarding the issue of the apparent low correlation between hyperLOPIT predicted localization in identified complexes (i.e. many complexes have components that are apparently in different compartments).

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Toxonet is an excellent resource for the toxoplasma community, combined with some interesting analysis I think this is a great paper. It includes new data, new appproaches and follow up analysis in several cases.

I have some minor comments that I hope that authors can address.

1) Could the authors, where possible, show the pointers underlying the data - i.e. when they present error bars?

2) The authors show results improve with adding more data quite convincingly but how SNF compares with other approaches for data integration is not clearly presented in the manuscript.

3) 38% percentage of interacting proteins being in the same compartment appears quite low - is there any explaination for this? Did the authors think about integrating this data also?

4) It is unclear whether the experimental mass-spectrometry was replicated - as far a I understand the 480 fraction were generated once but the replicate to replicate variation wasn't examined?

5) The FDR of ~0 doesnt really quite make sense - do the authors really believe there are no false positives with there >2,000 interactions. I think some naunce is need here too, since proteins could co-elute but not necessarily form an interaction (and visa-versa if they come apart during lysis)

Reviewer #2: review is uploaded as an attachment (240210_Review.doc)

Reviewer #3: The manuscript from Swapna et al seeks to address the knowledge gap of protein-protein interactions (PPIs) and complex formation within Toxoplasma gondii. The manuscript is generally well written, with significant amount of experimental and computational work carried out to refine and validate the interactions identified. There are a few issues that need to be addressed before publication:

Major:

1. Page 7: The study reports 1423 unique proteins identified (supp table 2), however, over 100 proteins have been identified with only a single spectrum across 420 fractions. These are not reliable identifications and should be removed from the results.

2. Page 8: Gold standard negative interactions were generated based on differences in cellular localization. However, in page 15 and table 1, only 38% of interacting pairs shared the same localization according to hyperLOPIT. Were the rest of 62% of interacting pairs based in different localization or not identified in hyperLOPIT dataset? hyperLOPIT information should be added when generating negative controls.

3. Page 14: “Pearson correlation values were calculated for protein pairs across eleven time-points encompassing different lifecycle stages”. Since tachyzoite was the only lifecycle stages investigated in the study, adding expression data from other lifecycle stages would add noise to the analysis and protein interactions may not sustain across lifecycle stages, such as invasion related ones.

Minor:

1. Page 9 and figure 1C: “the ribosome, and a snRNP complex, demonstrating its ability to capture features in the training datasets (Figure 1C…” figure 1C appear to be a wrong reference to the sentence.

2. Page 12 and figure 2D: Essential proteins are highlighted as mediating more central roles in the network; however, the associated Figure 2D shows a lower betweenness centrality score for essential proteins.

3. Page 28: The 93 clusters identified should not be referred to as complexes. Comparing to experimentally validated complexes (page 16), there are many additional proteins grouped into the same cluster.

4. Page 28: Please provide more details on how the data was made freely available on ToxoDB, which was not easily searchable.

5. Page 31: Please provide parameters used in SEQUEST searching.

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: None

Reviewer #3: Yes

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Reviewer #1: Yes: Oliver Crook

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: 240210_Review.docx

Dear Dr. Parkinson,

We are pleased to inform you that your manuscript 'ToxoNet: A high confidence map of protein-protein interactions in Toxoplasma gondii' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Michael L Reese, PhD

Guest Editor

PLOS Computational Biology

Stacey Finley

Section Editor

PLOS Computational Biology

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Nice work on the revision. Everything looks good to me.

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have answered my questions and I appreciate they're remarks on hyperLOPIT data. I'm convinced that the data and analysis will be extremely useful for the toxo community.

Reviewer #2: Thank you for addressing my comments, the revised manuscript is very nice.

Reviewer #3: This revision has addressed my previous comments. I recommend it to be accepted for publication.

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Oliver M. Crook

Reviewer #2: No

Reviewer #3: No