PONE-D-22-33243A novel  in vitro  model to study prolonged  Pseudomonas aeruginosa  infection in the cystic fibrosis bronchial epitheliumPLOS ONE

Dear Dr. Stefanie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Harish Chandra, PhD

Academic Editor

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The manuscript entitled “A novel in vitro model to study prolonged Pseudomonas aeruginosa infection in the cystic fibrosis bronchial epithelium” by Meghan et al. to explore novel invitro infection model is very interesting. The manuscript is well written however, the results lack crucial data points and are not well represented. A comparison with 1-6 hours infection model needs to be shown in all data points.

Therefore, I feel the current version is not suitable for publication in PlosOne until the following major concerns are addressed.

Figure 1: Bright field image of the cells is not clear.

Figure2: CFUs for both inoculum used and endpoint should be converted to Log CFUs for better representation. Further, the inoculum CFUs and endpoint CFUs may be plotted together for better comparision.

The authors should also include 0 hours CFUs Post infection after washing of the unbound bacteria.

Figure 3A: mRNA fold change for Il-8, IL-6, IL-1B, for 6 HR data is shown only for PA01. At what dilution was this data obtained? Why the authors did not include other dilutions (1:100, 1: 500, 1:1000) and the mucoid infections as shown for the 24 HR infection? Since the manuscript compares the 6 hours infection model therefore, authors must replicate the 6 HR data points as shown for 24 HR model.

Similar problem is associated with Fig 3B, C and D. Authors should include all the data points.

Fig 3C, the control cells have high levels of Il-8 while we do not see significant mRNA increase?

Fig 4: The western blot do not show the loading control band (GAPDH or Actin). The phosphorylated and the total proteins should be compared with the loading control in each well for densitometric analysis.

It is difficult to conclude that the there is increase in phosphorylation. In fact, it looks other way round if you compare it with the untreated control data points!

Fig 4E is not clear!

Reviewer #2: Hirsch et al. has provided a well-written manuscript on Pseudomonas aeruginosa in people with CF with nice translational potential with their in vitro model. Their science is sound, and their research contribution is valuable.

I’ve attached some minor corrections, below. Other than that, I would highlight the aim of the study and the impact in the discussion.

Unfortunately, I can’t seem to see the figures so I cannot comment on that.

Well don, Hirsch et al!

L36: comma after tissue

L37: comma after lungs

L39: remove space between 25-34

L40: capital G for Gram

L47,49: ‘therapy’ following HEMT seems redundant?

L80: phospholipase C gamma (PLCy)

L92: remove ‘-’ (i.e., 1 h)

L107,112: 4 °C

L113: 60× magnification (use symbol not the letter x)

L130-131: Roche cOmplete™ Protease Inhibitor Cocktail

L132: elsewhere you’ve not cited location with company, i.e. Millipore Sigma (L131), L124: ThermoFischer. Please keep it consistent throughout.

L146: were (?) performed

L147: standard error of mean (SEM)

L148: P is always capitalised and italicised

L150: use hour in subheading

L166: Air Liquid Interface (ALI)

L170-171: use hours, hour in subheading

L187: tdTomato

L216: italicise ‘post hoc’

L244: 6 to 24

Throughout: hrs is an incorrect abbreviation of hours. Instead of hr / hrs, use h.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-33243R1A novel  in vitro  model to study prolonged  Pseudomonas aeruginosa  infection in the cystic fibrosis bronchial epitheliumPLOS ONE

Dear Dr. Krick,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 10 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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.

We look forward to receiving your revised manuscript.

Kind regards,

Harish Chandra, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

The manuscript entitled “A novel in vitro model to study prolonged

Pseudomonas aeruginosa infection in the cystic fibrosis bronchial epithelium” by Meghan

et al. to explore novel invitro infection model was revised. However, there are still some concerns that needs to be resolved.

Therefore, I feel the current version is not suitable for publication in PlosOne until the

following major concerns are addressed.

Line 130: gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference in QRT PCR, while the authors dispute the use of GAPDH and B actin as controls in the western blot. Please use another internal control for fold change calculations. How was the fold change calculated? Please describe in methods.

Did the authors quantify the total proteins in the lysates before loading? The authors should use equal amount of proteins in each well for normalization. How the results were compared in densitometer analysis?

The authors used various dilutions for infections (1: 100, 1:500, 1:1000),. For better comparison, the authors should have optimized it with real numbers of CFUs.

Figures provided are very blurred and not clear. Therefore, not suitable for publication at this stage.

Please provide clear Images in fig 1A and 2B.

All figures provided should be clear and distinct. Error bar font sizes are too big and placed irregularly.

Thanks

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-22-33243R2A novel  in vitro  model to study prolonged  Pseudomonas aeruginosa  infection in the cystic fibrosis bronchial epitheliumPLOS ONE

Dear Dr. Krick,

Thank you for submitting your revised manuscript to PLOS ONE. After careful consideration, we feel that it has merit  and is very close to publication. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 02 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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.

We look forward to receiving your revised manuscript.

Kind regards,

Harish Chandra, PhD

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

Minor Comments:

Description of Fig 2 legends is superficial. Fig 2A and its description is not clear. Please improve the bar diagram and the error bars specifically the round filled dots (Reduce the size).

Is the inoculum same to infect at different time points (1 hr, 6h and 24 hr)?

If yes then it should not be repeated for every time points instead it should be mentioned as zero time point (zero hours, 1 hours, 6 hours 24 hours). The term endpoints should be then removed!

The inoculum and the endpoint nomenclature is not clear in the experimental description.

(Dilutions of the initial inoculum may be mention as 102, 0.5 x 103 , 103 etc.

All figure legends should be described in detail including the statistical analysis.

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

A novel  in vitro  model to study prolonged  Pseudomonas aeruginosa  infection in the cystic fibrosis bronchial epithelium

PONE-D-22-33243R3

Dear Dr. Stefanie Krick,

Thanks you for submiiting your revised manuscript. After careful reviewing, we found that the all the major concerns raised during reviewe process have been addressed and the manuscript has been improved significantly. Therefore, we’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Harish Chandra, PhD

Academic Editor

PLOS ONE