Dear Dr Qi,

Thank you for submitting your manuscript entitled "Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGF β -dependent Smad/YAP/TAZ signaling" for consideration as a Research Article by PLOS Biology.

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PLOS Biology

Dear Dr Qi,

Thank you very much for submitting your manuscript "Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGF β -dependent Smad/YAP/TAZ signaling" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers.

In light of the reviews (below), we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a much-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 for further evaluation by the reviewers.

We expect to receive your revised manuscript within 2 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

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*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

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*Published Peer Review*

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https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any 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

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

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

Sincerely,

Di Jiang

PLOS Biology

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

REVIEWS:

Reviewer #1: This paper provided a mechanistic explanation by which Porphyromonas gingivalis may mediate the pro-tumor effects of esophageal cancers. Through the combination of molecular biology and clinical samples, the authors have provided evidence for the role of TGF-b-YAP/TAZ in the tumor promoting effects of the intracellular invasion of Porphyromonas gingivalis. The mechniastic investigation on the host sides are extensive and rigorous. However, there are some significant concerns that I hope the authors can address.

1. In the mechanistic control, only PBS was used as negative control. More appropriate control is other intracellular bacteria without the pro-tumor effects associated with Porphyromonas gingivalis.

2. While there are extensive mechanistic investigations of the host cells, what are the bacterial determinants of the TGF-b pathways. Without such understanding of the molecular interface between host and pathogens, the studies appear to be more descriptive.

3. The gene signature of Porphyromonas gingivalis need to be unbiased defined based on the RNA-Seq data (line 307-309) instead of hand-picked genes noted to be involved in the oncogenesis.

4. I cannot find the dataset of GSE532655 in GEO relevant for the study. There is also no cited reference for this data. It is not clear what the source of data used in the gene expression analysis.

5. Overexpression of Last1/2 or S518A Merlin in ESCC 209 cells affect the migration phenotypes. Does this effect mediate through the mitigation of TAZ/YAP activation ?

6. How does the YAP/TAZ regulate the SMAD2/3 activities in the absence of altered phosphorylation ?

Reviewer #2: This is a very interesting experimental study dealing with the interaction of P. gingivalis and cancer cells. The authors provide a huge number of data and demonstrate the effect of P.g. on oesophageal cancer cells.

This reviewer inserted a number of comments in the manuscript and asks the authors to take these comments into consideration for the improvement of their documents.

Some aspects seem to be important:

1. there was no non-malignant cell line/primary cells involved and used as controls to be able to compare the effects of P.g.

2. DNA-probes used should be described and the sequence should be given together with the appropriate reference(s)

3. The statistical methods and algorithms used should be described in a more detailed form.

4. Antibodies used should be characterised and described in a detailed form (together with the sources/References).

5. A number of abbreviations are used in the manuscript, which should be explained, if they are used for the first time (especially if included in the abstract)

Some minor things have been marked in the ms.

Reviewer #3: In this manuscript, authors explore how P. gingivalis affects esophageal squamous cancer cells (ESCC). Results show that P. gingivalis promotes proliferation, migration and invasion. Analysis of the mechanisms driving aggresive progression lead them to establish a TGF-beta-dependent Smads/YAP/TAZ pathway.

This is a work of undoubted interest and includes a large amount of data. Nevertheless, there are some issues that need to be clarified and/or solved.

Major issues:

1. Authors provide evidence that P. gingivalis increases TGF-beta bioactivity in tumor cells, which results in activation of TGF-beta signaling pathway, ultimately leading to EMT activation.

Data evidence a regulation of GARP as the potential mechanism involved in TGF-beta activation. However, in figure 2 authors show that P. gingivalis indeed increases not only active TGF-beta but also total TGF-beta. Could authors please provide some comment on that?.

2. Have the authors tested some representative TGF-beta target genes related with its tumor supressor activity (cell growth inhibition- and/or apoptosis-related genes)?. It is conceivable that ESSC cells are likely resistant to the tumor supressor activity while being responsive to the tumor promoter activity (associated with the EMT response) but nevertheless it would be nice to show it.

3. Authors show that infection with P. gingivalis in ESCC tumor cells promotes a more aggresive phenotype, and it is correlated with a poor prognosis, so P. gingivalis seems to be important for progression. But, authors state that P. gingivalis is a causative agent during development and progression of ESCC (lanes 332-333). In order to test the causative relationship between P. gingivalis and ESCC development, infection of normal or premalignant cells would be neccesary, to see if it is really capable of malignant conversion by itself. So, authors should be more careful when proposing such effect.

4. Authors claim that inactivation of Merlin is involved in the P. gingivalis-induced mechanism driving malignant progression of ESSC cells. However, the downregulation of Merlin provoked by P. gingivalis (fig. 3D) seems quite modest.

5. It is proved that GARP is needed for TGF-beta bioactivation and activation of TGF-beta signaling cascade in P. gingivalis-treated ESSC cells, but how is being GARP upregulated by P. gingivalis? In other words, which is the mechanisms driving GARP upregulation?. Is GARP itself a target of the Hippo pathway?

6. A number of figures contain images whose quality and/or size is not optimal to allow a good visualization. Examples: Figure 1E, S1F, 3B, 5C.

Authors should consider the possibility to include an inset with a zoomed up area to help visualization, at least in those cases in which it is important to see the staining pattern or subcellular distribution. Alternatively, higher amplification images might also help.

7. Figure S1: Representative Ki67 staining images should be accompanied by quantitative graphs for a proper analysis of tumor cell proliferation rate.

8. Figure S2: In Fig. S2D no significant differences in Smad7 between P. gingivalis alone or with SB and/or Tinidazole are seen. How do authors interpret this?

Please, revisit figure labeling to confirm that authors actually did a combined treatment with SB and tinidazole or the fourth experimental group corresponds only to Tinidazole treatment (w/o SB).

9. Figure 3: In Fig. 3C, differences between different experimental conditions are in general clear but not in all cases, for example changes in PAI-1 are not that obvious. It is recommended to include quantitative graphs of the blots performed (in both F3C and F3D) to reinforce the analysis of changes in protein levels. This analysis could be included as supplementary information.

This quantitative reinforcement applies to other analysis, particularly:

- the effect of siYAP/TAZ on pSMAD2/3 and SMAD2/3 in Fig. 4A (since it is not fully clear that phosphorylation is actually unaffected by deleting YAP/TAZ). This result is important to be certain on the connection between these two pathways and the mechanims mediating P. gingivalis effect.

- the effect of Verteporfin on Smad2/3 binding to CTGF promoter (Figure 4D)

- Co-immunoprecipitation results also need to be improved. The increase in Smad2/3 binding to TAZ upon P. gingivalis is a bit poor (Fig 4E).

10. Figure S3: Controls for the overexpression of Lats1/2 and S518A Merlin should be included.

11. Figure S4I: increase in lung metastasis in animals inoculated with P. gingivalis-treated KYSE30 cells is not seen in this experiment.

12. Figure S5: In Fig. S5B, migration in P. gingivalis-treated KYSE30 cells decreases instead of increasing.

13. Figure 6:

- Fig. 6B: Authors claim that there is also a correlation between TGF-beta and YAP/TAZ, but I do not see such correlation.

- Fig. 6E: The overlap between the poor prognosis group (I presume represented as High risk group in the figure) and the differentiation, or invasion depth is not as clear as in other parameters, in fact, statistical significance is not indicated. Authors statements should be somehow softened.

- Fig. 6F: the proposed model of P. gingivalis-induced aggressive progression of ESCC should be briefly summarized in figure legend. Figure 6F should be also cited in the text.

Minor comments:

14. Scale bars in photographs from Figures F1A and F1D are not easily seen.

15. Figure 1G, right panel: I suggest to label the metastatic cells in the histological section image to help identification/visualization.

16. Figure S3: revisit text (S518A Merlin) and figure (S51A Merlin).

17. Lane 281: S5A should be located inside the parenthesis.

18. Lanes 306-309, GEO database analysis. Authors include Slug and Nanog as molecules substantiated in the present study, but these two genes are not within the target genes analyzed in previous experiments, although they are definitely related to those analyzed. Please, rewrite text to fit more precisely to the approach done.

19. Assay to detect P. gingivalis: IHC or in situ hybridization? Authors mention the use of specific probes in hybridization buffer. Please, clarify.

20. Authors should provide a few important details about siRNA approach, such as the siRNA concentration used, siRNA combination approach, timings and culture conditions.

Attachments

Attachment

Submitted filename: PBIOLOGY-D-20-00010_R1_reviewer Kopie-Jm01_SG Kopie 2.pdf

Dear Dr Qi,

Thank you very much for submitting a revised version of your manuscript "Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGF β -dependent Smad/YAP/TAZ signaling" for consideration as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

In light of the reviews (below) and based on our discussion, we would like to offer you the opportunity to address the remaining concerns from reviewer 1 and us in a revised version that we anticipate should not take you very long. Below I have outlined several points to aid you in revision.

1 You will need to include the DH5α data currently shown in Response to Reviewers in the manuscript. Please make sure to add a description of the materials and methods used in these experiments and include the statistical analysis of the results. You will need to add a text in the manuscript to describe and discuss the data.

2 Please add the data for heat-killed P. gingivalis and LPS which are currently textually described in Response to Reviewers and please remove the PBS control. Please make sure to add a description of the materials and methods used in these experiments and include the statistical analysis of the results. You will need to add a text in the manuscript to describe and discuss the data.

3 The data showing that long-term P. gingivalis-infected esophageal immortal cell line NE6 failed to form colony and the xenograte experiment using NE6 cells will also need to be added to the manuscript and discussed. Please make sure to add a description of the materials and methods used in these experiments and include the statistical analysis of the results. We also encourage you to discuss the differences between cell lines and about to discuss choosing appropriate models for the cancer of interest.

4 We ask you to temper the conclusions about the direct involvement of fimA in the phenotype.

5 We would like to also ask you to discuss the limitations of modelling ESCC through subcutaneous injection into the flank of nude mice.

6 For publication of this study, we will not ask for additional data that will increase the mechanistic insight into how YAP/TAZ regulates SMAD2/3 activities.

We will assess your revised manuscript and your response to the reviewers' comments and we may consult the reviewers again. We expect to receive your revised manuscript within 1 month.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

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. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any 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

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

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

Sincerely,

Di Jiang, PhD

Senior Editor

PLOS Biology

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

REVIEWS:

Reviewer #1:

I appreciate that authors have performed additional experiments and responded to my previous concerns. However, I am a bit disappointed that the authors did not effectively deal with my concerns and fill in some mechanistic gaps in their studies.

First, if the DH5a similarly augmented the migration and invasion of ESCC cells, then I am somewhat skeptical of using this culture ESCC cells systems to examine the mechanism by which Porphyromonas gingivalis promotes the esophageal cancers.

Other two concerns are about the request for more mechanistic insights. First, I hope that the idea to identify the bacterial determinants of YAP/TAZ regulation as the relevant bacterial virulence factor that may mediate the relevant molecular pathways. While fimA mutant P. gingivalis led to markedly decreased Smad2/3, this may be due to the simple reason of poor adherence.

Another concern is the authors failed to provide more mechanistic insights into how YAP/TAZ regulate the SMAD2/3 activities. The authors provide some hand-waving statements without gaining more mechanistic insights.

Because of the concern for proper control and lack of enough mechanistic insights, I feel that this manuscript may not be appropriate for the top journal, such as PLoS Biology.

Reviewer #2:

Reviewer #3: Authors have adequately/reasonably addressed most of the issues raised during reviewing process. The very few pending issues can be disregarded at this stage.

Dear Dr Qi,

Thank you for submitting your revised Research Article entitled "Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGF β -dependent Smad/YAP/TAZ signaling" for publication in PLOS Biology.

Based on our evaluation, we will accept this manuscript for publication only after you modify the manuscript to address the remaining points (3-5) listed below.

1 The DH5a data are added with statistics in Fig. S1E & S1F and described and discussed. This is fine.

2 The data for heat-killed P. gingivalis and LPS are added with statistics, discussion added in the text. We are fine that you keep the PBS results.

3 The data of an esophageal immortal cell line NE6 have been incorporated in the revised manuscript and the pictures are presented in Fig S1H & S1I. However, these are photographs of cells and mice without quantitation. Also the added text doesn't mention the figure panels. You will need to quantify the results and perform statistics on the results. You will also need to tell our readers where to find the data.

4 You have tempered down the conclusions about the direct involvement of fimA in the phenotype. However, you don't say that fimA mutant P. gingivalis has poor adherence but only say there are other mechanisms imparting the oncogenic effect of P. gingivalis. You will need to discuss the significant limitation of fimA mutant data in supporting your conclusion because it impairs adherence.

5 You didn't discuss the limitations of modelling ESCC through subcutaneous injection into the flank of nude mice but let us know about an review on this and that "this issue is beyond the object of this study". You will need to add a concise discussion on this point.

Please also make sure to address the data and other policy-related requests noted at the end of this email.

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Dear Dr Qi,

On behalf of my colleagues and the Academic Editor, Christina L Stallings, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

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on behalf of

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