Dear Dr. Siqueira-Neto,

Thank you very much for submitting your manuscript "Genome-scale metabolic models highlight stage-specific differences in essential metabolic pathways in Trypanosoma cruzi" for consideration at PLOS Neglected Tropical Diseases. 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.

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).

Important additional instructions are given below your reviewer comments.

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,

Igor C. Almeida

Associate Editor

PLOS Neglected Tropical Diseases

Joseph Vinetz

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: see summary and general comments section

Reviewer #2: The objectives are clear. and methodology is in teh direction of objectives. Authors develop a computational approach, and confrim their results through biochemical validation.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: see summary and general comments section

Reviewer #2: Results are complete, original, and Figures, Tables and Supplementary information allow to follow them clearly.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: see summary and general comments section

Reviewer #2: The conclusions are supported by the results, and authors discuss how these data can be helpful in the identification of targets and development of drugs against T. cruzi, causative agent of Chagas disease, a neglected disease of relevance in public health.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: see summary and general comments section

Reviewer #2: Minor Revision

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The manuscript by Shiratsubaki et al. describes the reconstruction of an updated genome scale metabolic model of Trypanosoma cruzi, iIS3142. The authors used the expansion of the genome annotation of T. cruzi to extend a previous model of core metabolism iSR215, while using also information from the Leishmania model iAC560. Using transcriptome data for the different lifecycle stages of T. cruzi, they made lifestage specific models. This allowed the authors to compare the metabolic networks in the lifestages and how metabolism sustains growth. They validated some of the network differences by measuring enzyme activities in the different lifestages.

This is a very relevant study as this extends the knowledge on the metabolic capabilities of T. cruzi. Metabolic enzymes could be potential drug targets and models can help in identifying the most promising drug targets in metabolism.

While it is an interesting paper, there are a few major points that I would like to see addressed – also to make it more accessible to a non-GEM/FBA readership

Major points

1. Although there is an intrinsic value in extending the previous model, it is not clear to me whether the new reconstruction improved predictions or not. The authors should make a comparison of the prediction capabilities between both models and comment why the new model is better or worse. For example, for the genes which are common to both models, how many of them are accurately predicted to be essential using both models.

2. I wonder why the authors considered the biomass objective as the function to optimize for all the stages. This make sense for epimastigotes and amastigotes that replicate. However, it does not make sense for trypomastigotes which do not replicate. Non-replicating cells need to produce considerably less compounds, mainly for maintenance and pathogenicity and it seems very unlikely that the cell is trying to optimize biomass yield as they don’t replicate. Although the authors modified the biomass equations as stated in the supplementary material methods, it seems that this change is insufficient. For example, the new biomass equation still considers the same coefficients for amino acids. It is highly unlikely that replicating and non-replicating cell need the same amount of amino acids for protein synthesis. Please comment on this.

3. Despite that the model was validated regarding by-products and reaction essentiality, the model should also be validated in other aspects too to ensure a basic functioning and usability. Recently, Memote, a suite to assist in the assessment of genome-scale models, was published. We strongly suggest the authors to use this suite to further validate the model and include the results in the manuscript (see: https://doi.org/10.1038/s41587-020-0446-y)

4. It is not clear to me how big is the non-metabolic effect regarding inactivation of genes. I would like to have a bigger picture regarding transcriptomics and the genome-scale model. For example, how many genes overlap between the transcriptomics and the genome-scale model? How many genes are present in the transcriptomics and missing in the model? How many genes were deactivated according to the transcriptomics but they cannot be taken into account because they are not in the model? Do the authors expect only metabolic (and not regulatory) changes among the different stages?

5. The authors at several points in the manuscript state that metabolism could yield potential drug targets. While I agree with this, many metabolic pathways and enzymes are also operational in the human host. Therefore, the authors should discuss how side-effects could be prevented (e.g. because some pathways or enzymes may be less important in host metabolism)(see e.g. https://doi.org/10.1038/msb.2010.115 and https://doi.org/10.1038/srep40406)

6. It seems that the authors calculated the results of Fig 2C using parsimonious FBA. This is not fully correct to explore the potential of a metabolic network regarding products. Flux variability analysis should be performed to explore which metabolic products can be produced at optimal state. Also, it would be interesting to see what products must be produced at optimal state. For a reference regarding studying the potential for metabolic products, see https://doi.org/10.1038/nbt.3703

7. Why is the Pan-model part of Fig 2C? This model is not biologically relevant as this is not a lifecycle stage that exists. It could, theoretically, be interesting to show the full metabolic potential of T. cruzi. But a flux analysis is a functional analysis and should be restricted to the actual lifestages. This comment is also relevant for the analysis of PAN for deletions.

In addition, it would be more informative if Fig 2C could compare the FBA outcome per lifestage to what is known of the excreted products for the different lifestages from literature.

Minor points

a. Line 119 and Fig 2a: Please explain why an extracellular compartment is part of a GEM of a cell. This may be logic to modellers, but a short explanation would make the paper wider accessible.

b. The manuscript often refers to metabolic BYproducts. This suggests that there a more relevant purpose of metabolism (making growth possible!), but the authors never make this explicit in the manuscript

c. At line 128, FBA is first mentioned. While the methods section explains it further and provide useful references, for the less-informed reader we would like to see a short overall explanation of what is the purpose of this method and what type of questions this approach will answer. This should include the objective function that is used (a biomass equation) and the quasi-steady state assumption. The authors can keep it short, but it should convey the essence of the approach.

d. The legend to Fig 2C should be extended with explain what the PAN model is (if it stays in Fig 2C) and why these excreted metabolism are listed in the model (previous experimental data?)

e. Fig 3: There are no reactions that are deactivated in all lifestages. Was this expected?

f. Fig 4: It would be helpful to include substrates, end-products and directions of flux. In the amastigotes, there is no substantial flux from pyruvate to TCA, so the substrate for lower part are the amino acids, but this does not become immediately clear from the figure. Especially as line 269 mentions a gluconeogenic flux.

g. Fig 5: Please make the order of the lifecycle stages the same as Fig 4 for easy reference.

h. Fig 5: How is hexokinase a validation of the models? It has a high flux intensity in all models according to figure 4.

i. Line 391: the medium that was used for growth simulation was based on earlier in silico studies? Why didn’t the authors use the composition of the medium that was used in the validation experiments.

j. Line 421: Does MEM include gap-filling? Or gap-filling was performed after MEM?

k. Lines 447-456: The authors say that some essential genes were deactivated in the model due to low gene-expression levels. Therefore, they removed a few reactions which made the model infeasible. To restore growth, they then do gap-filling using the template model. Why they didn’t used the BiGG database for the gap-filling procedure? In this way they can expand the search for alternative pathways as they suggest

Reviewer #2: In this manuscript, authors a genome scale metabolic model with focus on targets for development of drugs against Chagas disease, a neglected disease, in which currently two drugs are used -Nifurtimox nd Benznidazol- with several side/undesirable effects. They take into account the expression in the different stages, extracted from previously published transcriptomic studies. Particularly, they describe metabolic differences among stages, some of them already validated in previous publications, and by measuring the activity of the enzymes G-6-P dehydrogenase, malic enzyme, hexokinase, glutamate dehydrogenase and citrate synthase, confirming their predicted results.

I consider that this manuscript gives very relevant information about metabolic fluxes in the relevant stages in mammals (amastigotes and trypomastigotes), which gives important clues about valid drug targets in T. cruzi. In my opinion this paper should be accepted after minor changes:

- Figure 1, "epimastigote" (instead of "espimastigote")

- Line: 191: "amastigotes" (instead of "amastigote")

- authors should consider to discuss about the role of malic enzymes in amastigotes, i.e.: anaplerotic reaction vs. NADPH source, taking into acount that PPP is not so active at this stage.

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Reviewer #1: No

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Dear Dr. Siqueira-Neto,

Thank you very much for submitting your manuscript "Genome-scale metabolic models highlight stage-specific differences in essential metabolic pathways in Trypanosoma cruzi" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

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

[1] A letter containing a detailed list of your responses to all 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).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. 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,

Igor C. Almeida

Associate Editor

PLOS Neglected Tropical Diseases

Joseph Vinetz

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: yes

Reviewer #2: (No Response)

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: yes

Reviewer #2: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: yes

Reviewer #2: (No Response)

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Panel 2C may go to Fig3

Reviewer #2: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Many thanks to the authors for the answers to the points. The manuscript has greatly approved in the revision.

There are a few remaining points on the revised version. Where relevant, there is a reference to the lines in the Word document that contained the track changes (with the track changes on):

1. Considering the response to our first point: please indicate in the main text that only 23 genes could be validated (due to limited experimental studies) and that for those genes 79% of the predictions was accurate.

2. Considering the response to our third point: can the authors say something on the MEMOTE results with respect to biomass consistency?

3. Considering the response to our fourth point: A very good comparison – we advise to put the most relevant outcomes in the main text

4. It is confusing that at line 135 and Fig 2C the stage-specific models are already used, while the text on how they are made comes only at line 162. Fig 2C could go to Fig 3 and the text about these results too?

5. The sentence at line 163 is superfluous as lines 165-169 say the same, but are more informative

6. At line 205 there is still a reference to the pan-model. Maybe include the number of the reconstruction as this makes it more clear what the pan model means

7. Line 218 suggests that only one reaction is deactivated in epimastigotes, but according to Fig 3 this should be seven reactions.

8. Line 332/333: a better formulation would be: “…, or enzymes that are more important to sustain glycolytic flux in the pathogen than in the host”.

9. At line 354-357, please write dpi (days post induction??) in full

Reviewer #2: (No Response)

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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: JR Haanstra

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Siqueira-Neto,

We are pleased to inform you that your manuscript 'Genome-scale metabolic models highlight stage-specific differences in essential metabolic pathways in Trypanosoma cruzi' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Igor C. Almeida

Associate Editor

PLOS Neglected Tropical Diseases

Joseph Vinetz

Deputy Editor

PLOS Neglected Tropical Diseases

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