Dear Dr. Persat,

Thank you for submitting your manuscript entitled "Pseudomonas aeruginosa contracts mucus to form biofilms in tissue-engineered human airways" for consideration as a Research Article by PLOS Biology.

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Paula Jauregui, PhD,

Senior Editor

PLOS Biology

pjaureguionieva@plos.org

Dear Dr. Persat,

Please allow me to first apologize for the delay in the processing of your manuscript. This delay is caused by my difficulty in recruiting reviewers for your manuscript, and is further compounded by one referee promising an overdue report but failing to deliver after long delay and multiple chases. I am sorry for this unexpected event, and I thank you for your patience while your manuscript "Pseudomonas aeruginosa contracts mucus to form biofilms in tissue-engineered human airways" was peer-reviewed at PLOS Biology. It has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports.

As you will see below, the reviewers find the manuscript interesting but raise some issues that should be solved before publication. Please address all the reviewers' issues including the control experiments with the natural isolate of P. aeruginosa from the CF patient, as requested by the reviewer #2.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

We expect to receive your revised manuscript within 3 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 withdraw it.

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

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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,

Paula

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Paula Jauregui, PhD,

Senior Editor

PLOS Biology

pjaureguionieva@plos.org

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REVIEWS:

Reviewer #1: Biophysics.

Reviewer #2: Airway mucus, cystic fibrosis and Pseudomonas aeruginosa.

Reviewer #1: Rossy et al establish a novel lung model system for studying Pseudomonas aeruginosa infection. This model system enables 3D live imaging of bacteria. The authors confirm that after differentiation, epithelia are protected from damage caused by P. aeruginosa as expected for mucus producing epithelia. They show that within this model system, P. aeruginosa forms biofilms at a time scale of hours, mostly driven by fusion of colonies. Importantly, the time scale of biofilm formation is much faster than has been observed in vitro so far. The authors identify type IV pilus activity as the mechanism for mucus contraction and colony fusion. They conclude that mucus formation comes with a trade-off; while it protects the epithelia from bacterial invasion, it facilitates biofilm formation.

I am no expert on infection models, but the model developed in this study looks intriguing, since it reveals bacterial behavior that is not reproducible in vitro. Overall, I believe the authors established a highly promising infection model that will serve the community to address follow up questions about biofilm formation by an important model organism. The result that type IV activity is required for rapid biofilm formation in the close to natural infection environment are both convincing and interesting. I have only a few minor issues that hopefully help improving the manuscript.

1. While the model shows previously unseen behavior, it remains unclear whether mucus contraction affects pathogenicity of P. aeruginosa. It would be good to discuss this point.

2. Line 230: "After this initial biofilm seeding, P. aeruginosa may initiate the secretion of matrix components to strengthen the cohesion of the biofilm." On which experimental hint or evidence is this speculation based? Since this speculation is not required for further discussion I suggest omitting it.

3. The dynamics of colony formation and fusion are astonishingly similar to colony formation of Neisseria species in vitro even in the absence of mucus. It would be interesting to discuss this analogy. (doi.org/10.1016/j.cell.2018.04.010 and doi.org/10.1103/PhysRevLett.121.118102)

4. There is no reference to Supplementary Fig. 2a. Why is its shown? Is it part of the control for jacalin labeling?

5. Supplementary Fig. 3: It would be good to show the fluorescence signals of bacteria (orange) and membrane? (pink) in different panels. It is difficult to distinguish these colors. Also, please explain what the colors refer to the Figure legend.

6. Supplementary Fig. 8: I understand that the total fluorescence intensity is used as a measure for total cell number. If this is correct then the there seems to be no net growth. How can you claim that the growth rate is not influenced by mutations? What means N = 9? There are only three curves per strain. Please correct to N = 3 if there are only three curves per strain.

Reviewer #2: This paper connects important airway biology and microbiology processes in a way that is novel, fills a gap in the field, and addresses previously unknown mechanisms of bacterial function and disease. There are, however, some gaps in the description of the methods and in the results that should be addressed before publication.

1. Figure 1 presents an interesting and innovative method of culturing airway epithelial cells in a more physiologically relevant way. However, it's not clear how the cells are able to maintain air-liquid interface. The diagram in Figure 1b shows a luminal access port, but how is this port protected from the surrounding media and how is air moved in? Is there a pressure pump moving air across? Perhaps a picture of the actual device rather than a graphic would make the ALI more clear.

2. The methods section indicates that HBEs were obtained from both CF and non-CF sources. Which were used in the data presented here?

3. Figure 2 nicely shows appropriate architecture. However, MUC5AC and MUC5B should be differentially stained, to understand which mucin is primarily expressed. This is particularly important for the later figures, because previous studies have shown that the arrangement of mucins in the airway strands may influence disease.

4. In figure 2 iii, the measured mucociliary clearance is very slow. This is important because the rate of mucus clearance could affect biofilm formation. The references cited to defend this rate as physiologic are a very small subset of many studies measuring MCC ex vivo, (one of the papers cited is a review, which does not present primary data) and one of the papers present MCC data that is ten-fold higher than the data presented here. The authors should include more references to capture a more complete picture of the data found in the field, and a comparison of MCC and ciliary beat frequency measured by these methods in flat monolayers, which have been shown to closely represent ciliary function, would be needed to ensure that the method of culturing is not affecting mucus and cilia movement. This is particularly important given that these host factors may influence mucus contraction in addition to the bacterially-driven mechanisms proposed later in the study.

5. There is some important information missing from the methods regarding the infections. At what CFU are the bacteria inoculated? And at what stage of growth? The formation of biofilms in the mucus may be dependent on if the bacteria are collected in the doubling or stationary phases.

6. The study as presented is convincing, particularly around with the mutants. However, confirmatory studies showing this relationship with mucus in a non-PAO1 strain would be a critical component. Of particular interest would be a lung-adapted strain. This is vital, because patients, especially with CF and other muco-obstructive disorders, have cultured isolates of P. aeruginosa that are phenotypically and mechanistically very different from PAO1.

Dear Dr Persat,

Thank you for your patience while we considered your revised manuscript "Pseudomonas aeruginosa contracts mucus to form biofilms in tissue-engineered human airways" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors and by the Academic Editor. I'm handling your paper temporarily while my colleague Dr Paula Jauregui is out of the office.

Based on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

a) The unusual transitive use of "contracts" in the Title may cause confusion. Please change your Title to "Pseudomonas aeruginosa type IV pili actively induce mucus contraction to form biofilms in tissue-engineered human airways"

b) Please address my Data Policy requests below; specifically, we need you to supply the numerical values underlying Figs 2AB, 3, 4BC, S1, S2, S3, S5ABCD, S6AB, either as a supplementary data file or as a permanent DOI’d deposition.

c) Please cite the location of the data clearly in all relevant main and supplementary Figure legends, e.g. “The data underlying this Figure can be found in S1 Data” or “The data underlying this Figure can be found in https://doi.org/10.5281/zenodo.XXXXX”

d) We note that your Materials and Methods section is currently in the Supplement – please move it to the main manuscript.

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that 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/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*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. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Roli Roberts

Roland G Roberts PhD

Senior Editor

rroberts@plos.org

PLOS Biology

on behalf of

Paula Jauregui, PhD,

Editor,

pjaureguionieva@plos.org,

PLOS Biology

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DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it: Figs 2AB, 3, 4BC, S1, S2, S3, S5ABCD, S6AB. NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

IMPORTANT: Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

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DATA NOT SHOWN?

- Please note that per journal policy, we do not allow the mention of "data not shown", "personal communication", "manuscript in preparation" or other references to data that is not publicly available or contained within this manuscript. Please either remove mention of these data or provide figures presenting the results and the data underlying the figure(s).

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Dear Dr Persat,

Thank you for the submission of your revised Research Article "Pseudomonas aeruginosa type IV pili actively induce mucus contraction to form biofilms in tissue-engineered human airways" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Victor Sourjik, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Paula

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Paula Jauregui, PhD,

Senior Editor

PLOS Biology

pjaureguionieva@plos.org