Dear Professor Cui,

Thank you very much for submitting your manuscript "Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion" 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.

The reviewers commented that this is an interesting study, however, editing and potentially other experiments are required before the manuscript is suitable for publication. Please ensure that sufficient information is provided for the methods and address all the comments raised by the reviewers.

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,

Krystyna Cwiklinski, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

Section Editor

PLOS Neglected Tropical Diseases

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

The reviewers commented that this is an interesting study, however, editing and potentially other experiments are required before the manuscript is suitable for publication. Please ensure that sufficient information is provided for the methods and address all the comments raised by the reviewers.

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: Methods, line 136. What is the anti-rTsASP2 serum mentioned? Define this and how it was produced.

For Western blotting, how much soluble lysate was added per well? How was this quantified?

Figure 1 – how many biological replicates of the RNAi experiment have been performed? This should be clearly stated in the text/legend.

How was the % protein expression (Figure 1) quantified? More detail should be added to line 175 in the methods section.

Reviewer #2: The methods used are appropriate and the results support the conclusions.

1. Line 191,how to obtain "anti-MBP serum" should be describe clearly ;

2. Line 209~211, “rTsCatL2, MBP or PBS were ...... added to the surface of Caco-2 cells”，MBP here is protein-tag, how to obtain MBP? also expressed in E.coli and purified？ or Purchased from the company?

3. The abbreviation that first appears should indicated，such as NBL, EBSS.

Reviewer #3: The methods are well described.

Study design could include addition of specific inhibitor such as E64 to rTsCL2 to show that inhibitor-bound cysteine protease does not affect Caco2 cells. Also, why did authors only test one concentration of enzyme - was the effect of the protease titratable?

In experiments where anti-TsCL2 was used to block parasite entry into cells - reduced at low significance - specificity could have been shown by adding rTsCL2 to the medium and demonstrating that this reversed the effect of the enzyme

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

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: What time point post-RNAi is shown in Figure 2? Please add.

It would be of interest to see if the cellular damage shown in Figure 3 is due only to the mechanical action of worms migrating through the monolayer as whether secreted products contribute as well. Could this assay be performed with the addition of dead worms as well as T. spiralis secreted products and recombinant TsCatL2?

Additionally, experiments with heat-denatured enzyme (or enzyme inhibited with E-64) would show whether these effects are due to active enzyme (same for later experiments; e.g. Fig 7 and the pulldown in Fig. 9).

Figure 4 – I don’t think it is necessary to plot the data as both & % invasion and % inhibition. Pick one readout for this figure.

How many individual works were measured in each group (Figure 5)?

Results describing Figure 6. It is stated in text that “Immunofluorescence with anti-rTsCatL2 antibody showed that rTsCatL2 bound mainly to the tight junctions of Caco-2 cells…”. It is not possible to say for certain what the TsCatL2 is binding to at this resolution. At best you can say green fluorescence was seen around the cell periphery. This section should be tempered to reflect this.

Collagen is misspelt as “collage” in several places throughout the manuscript.

The labelling of the micrographs shown in Figure 10 are too small/unclear. How many cells/field of view were observed/counted to quantify autophagy at the ultrastructural level?

Reviewer #2: The results are clearly and believable，and several minor problems should be noted.

1. Line 321~324，“rTsCatL2 bound mainly to the tight junctions of Caco-2 cells..., no binding of MBP to Caco-2 cells was detected.” actually, Fig 6, MBP-tag showed faint fluorescenec，you should describe more accurately.

2. Fig 8 and Fig 9 seem to be contradictory. Fig 8, the rTsCatL2 was added to Caco-2 cells, western blot showed no change in laminin and collagen I expression; and Fig 9, showed rTsCatL2 did not interact with claudin 1, e-cadherin, occludin and fibronectin but with collagen I and laminin. How to explain?

Reviewer #3: The amount of knockdown by iRNA is shown to be specific and significant but is nevertheless low - less than 50% with all iRNAs and for transcripts and protein -and it does not add confidence that parasites with only this reduction would be necessarily damaged and lack viability.

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

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: Since the silencing/antibody blocking of TsCatL2 was only partially successful at blocking entry of the worms into the epithelial cells, could the authors please speculate on other enzymes that may be compensating and the implications of this for parasite control.

Reviewer #2: (No Response)

Reviewer #3: I feel that most experiments do not achieve strong significance in parasite invasion or infectivity and additional experiments as suggested above would strengthen the data

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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: Line 358 – previous research?

Reviewer #2: Minor revision. The quality of English needs improving, I suggest that you obtain assistance from a colleague whose native language is English.

Reviewer #3: (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: In this study the authors aimed to investigate the role of Trichinella spiralis cathepsin L (TsCatL2) during penetration of intestinal epithelial cells. Invasion of cells in vitro, as well as establishment of infection of mice, were both reduced when TsCatL2 was silenced using RNAi. They propose a mechanism for this where TsCatL2 disrupts the barrier function of the intestine by degradation of extracellular matrix/cell junction proteins.

Specific comments:

Introduction, line 106. Replace “substance” with “cysteine peptidase” to describe Giardipain-1 here.

Introduction, line 109. Explain what domains TsCatL2 refers to – was only the mature enzyme domain cloned and expressed as a recombinant?

Do the authors have any information on the transcriptional profile of TsCatL? Is it specifically upregulated to coincide with arrival of the worms in the intestine? This should be included if known.

Line 376 – it should be made clear that Fasciola hepatica secretes a large family of cathepsins L, only some of which (primarily FhCL2 and FhCL3) have collagenolytic activity.

The section of ECM/cell junctions in the discussion was overlong and could be made more concise.

Suggested further experiments are suggested in the results comments.

Reviewer #2: The topic is novel and interesting, and the methods used are appropriate. "MBP" I mentioned above is important, for rTsCatL2 were expressed in Escherichia coli, recombinant protein of Prokaryotic expression may contain endotoxins，it should avoid the effect of endotoxins on Caco-2 cell junctions, when you observe the effect of rTsCatL2 on Caco-2 cell junctions.

Reviewer #3: The data could be improved by better experimental design and control experiments as suggested above e.g. titrating protease, using specific inhibitors, uisng reverse inhibtion of antibodies.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: 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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Professor Cui,

Thank you very much for submitting your manuscript "Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion" 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.

The authors have addressed the reviewers comments, however some minor points still need addressing before the manuscript is suitable for publication.

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,

Krystyna Cwiklinski, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

Section Editor

PLOS Neglected Tropical Diseases

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

The authors have addressed the reviewers comments, however some minor points still need addressing before the manuscript is suitable for publication.

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: (No Response)

Reviewer #2: 1. the sequence of TsCatL2 should have signal peptide, when construct pMAL-c2X/TsCatL2 recombinant plasmid, contained the signal peptide ?

Reviewer #3: Authors have addressed my concerns and improved figures as suggested.

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

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: (No Response)

Reviewer #2: 1. Line 308，“in the TsCatL siRNA-487”， and line 310 "lower in the siRNA-487 group than in the control siRNA...Fig 4B", I'm not find siRNA-487 in the methods, and Fig 4B showed siRNA-302, but the legend is siRNA-487 group, maybe an error?

2. legend of Fig4, "the rate of worm invasion was lower in the siRNA-487 group", the picture showed inhibition%, be consistent.

3. Fig 6 and Fig 7, rTsCatL2,MBP, rTsCatL2+E64, incubation with Caco-2 cells within 60min, your response mentioned "because adding rTsCatL2 to the liquid medium severely damaged the Caco-2 cell monolayer within 1 hour", if co-culture of MBP and rTsCatL2+E64 with Caco-2 cells more than 1h, whether the similar appearance ?

Reviewer #3: Yes

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

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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Yes

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

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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: None

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

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: (No Response)

Reviewer #2: rTsCatL2 downregulated claudin 1 expression, but not interacted with claudin 1, what's the possible mechanisms, should further discussed.

Reviewer #3: Paper is much improved

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

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: No

Reviewer #2: No

Reviewer #3: 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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Professor Cui,

We are pleased to inform you that your manuscript 'Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion' 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.

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 Neglected Tropical Diseases.

Best regards,

Krystyna Cwiklinski, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

Section Editor

PLOS Neglected Tropical Diseases

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

The authors have addressed the comments raised by the reviewers. The manuscript is now suitable for publication in PLoS NTD.