PPATHOGENS-D-26-00893

Proteasome-dependent cytoskeleton disruption during Leptospira interrogans infection induces aberrant collective cell migration

PLOS Pathogens

Dear Dr. Toma,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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 within 60 days. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only the individual author can complete the verification step; PLOS staff cannot verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Jon T. Skare

Academic Editor

PLOS Pathogens

Thomas Guillard

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Editor Comments:

While the reviewers were generally supportive of the work presented, they did identify some aspects of the manuscript that require modification. Please pay particular attention to all reviewer suggestions, specifically Reviewer 1, who had substantial comments that will require careful consideration. Some relevant points to address include:

• Issues related to the infection set up used (Reviewer 1).

• The invasion status of Leptospira into cells (Reviewer 1 and 3)

Journal Requirements:

1) We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg ©,  ®, or TM  (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including:- ® on page: 27- TM on page: 27.

2) Some material included in your submission may be copyrighted. According to PLOS’s copyright policy, authors who use figures or other material (e.g., graphics, clipart, maps) from another author or copyright holder must demonstrate or obtain permission to publish this material under the Creative Commons Attribution 4.0 International (CC BY 4.0) License used by PLOS journals. Please closely review the details of PLOS’s copyright requirements here: PLOS Licenses and Copyright. If you need to request permissions from a copyright holder, you may use PLOS's Copyright Content Permission form.

Please respond directly to this email and provide any known details concerning your material's license terms and permissions required for reuse, even if you have not yet obtained copyright permissions or are unsure of your material's copyright compatibility. Once you have responded and addressed all other outstanding technical requirements, you may resubmit your manuscript within Editorial Manager.

Potential Copyright Issues:.

i) Figures 2A, and 3C. Please confirm whether you drew the images / clip-art within the figure panels by hand. If you did not draw the images, please provide (a) a link to the source of the images or icons and their license / terms of use; or (b) written permission from the copyright holder to publish the images or icons under our CC BY 4.0 license. Alternatively, you may replace the images with open source alternatives. See these open source resources you may use to replace images / clip-art: - https://commons.wikimedia.org- https://openclipart.org/. 3) We note that your Data Availability Statement is currently as follows: "All data are in the manuscript and/or supporting information files." Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).For example, authors should submit the following data: 1) The values behind the means, standard deviations and other measures reported;2) The values used to build graphs;3) The points extracted from images for analysis..Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 4) Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.1) State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.". 5) Please revise your current Competing Interest statement to the standard "The authors have declared that no competing interests exist."

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Barbee et al. aim to understand how Leptospira induces kidney disease in the host. Using a wide range of technologies, they look at the ability of bacteria to cause disruption of epithelial cells, identify host pathways of importance, and show that inhibitors can modulate these effects in vitro. This study is novel for many reasons, including being one of the first mechanistic looks into kidney epithelial cell damage and repair processes. Generally, the execution of this project was good (using multiple strategies to measure effects), but there are some major and minor weaknesses that need to be addressed. Overall, once addressed, this paper will be highly relevant to the field and provide a solid foundation for understanding disease processes.

Reviewer #2: t is well known that the kidney possesses the ability to self-repair after acute injury, primarily through collective cell migration. In this manuscript, the authors analyzed collective cell migration in renal proximal tubular epithelial cells (RPTECs) infected with Leptospira, and demonstrated that proteasome-dependent disruption of the cytoskeletal network induced by Leptospira leads to delayed collective cell migration and impaired wound healing. They further showed that inhibition of the proteasome preserves this network and restores both collective cell migration and epithelial repair. As the authors note, this study is based on an epithelial cell-only model and does not address the underlying signaling mechanisms; however, linking microtubule disruption to the pathogenic mechanism of Leptospira is novel. Overall, the interpretation of the experimental data and the logical development of the arguments are largely appropriate.

Below are minor comments:

1) For bar graphs and images showing “representative data,” the number of samples (n) analyzed should be clearly indicated.

2) For p-values, the exact values should also be provided. In addition, in Fig. 5 (B) and (C), there is no explanation of the symbol markers.

3) In Fig. 4, quantification of the cell-free area would help to clarify the conclusions.

Reviewer #3: Pathogenic Leptospiral spirochetes cause severe renal failure. It has been demonstrated that pathogenic Leptospira can attach to kidney epithelial cells. Some studies showed that Leptospira can be found in the kidney cells cultivated ex vivo. In this study, Barbee et al. attempted to identify the mechanisms that Leptospira used to modulate cells ex vivo and found these bacteria can cause proteosome-dependent cytoskeleton disruption and aberrant collective cell migration. The strength of this study is that the authors have spent significant efforts in using cell biology based approaches to study mechanisms underlying Leptospira caused damages and cell repairs. The paper was also well written and clear. I only have few comments that require the authors to clarify.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. Cell infection method disparity and inconsistency:

a. In the methods section, the authors state that live bacteria were added to the basolateral side whereas heat killed control bacteria were added to the apical side (Lines 486-488). In their diagram of the mass spectrometry workflow (Figure 2C), it is suggested from the location of the arrows that bacteria were added to the apical side. From their reference (#21), bacteria were added to the basolateral side, and only pathogenic L. interrogans crossed to the apical side (model in that paper also shows this). This disparity causes significant issues in interpreting results:

i. Lines 140-142: A comparison between live bacteria added to basolateral and heat-killed added to apical are not comparable. Although live pathogenic bacteria may cross to the apical side, the effects of crossing and virulence factors and cell interactions during that cannot be ruled out as causing effects seen. Thus, the control would need to be added at the same location as the living bacteria to allow valid interpretation. Ideally, cells would be infected with live or dead bacteria in both apical and basolateral locations to determine cause and requirements for their effects, as right now there is no valid control.

b. Apical vs. basolateral study two different processes: Bacteria are transiently spread in the blood stream (<8 days post infection in humans). Symptoms start 5-14 days after infection, during which the bacteria likely have reached and colonized the proximal tubules. Kidney disease typically starts 3 days after onset of systemic symptoms, suggesting the bacteria are causing disease after being on the apical side of the epithelial barrier. Thus, the model used and inconsistencies, make the data hard to interpret.

c. Thus, by infecting on the basolateral side, and suggesting through addition of heat killed on the apical side that effects are through the apical side, there is a likely possibility of virulence mechanisms and host effects occur through the transmigration process, and this topic is not discussed within the current manuscript.

d. Similarly, the proteomics was based upon differences between L. biflexa and L. interrogans, which according to methods, were infected on basolateral side. L. biflexa does not cross the epithelial barrier (according to reference #21). An interpretable proteomics experiment would include a set infected on both apical and basolateral, with both L. biflexa and L. interrogans so that this data can be compared adequately.

e. Then with the scratch assays, these are not performed on membranes, and thus, the bacteria are infected on the apical side. This is a requirement of the assay, but for consistency, the previous experiments need to be confirmed with apical infection, with valid controls.

2. Figure 1C and D:

a. In this figure, comparisons are made that serve as a foundation for future experiments. Specifically, they claim that actin is disrupted after 5 hours post infection. However, no uninfected control in IF, or L. biflexa infected control for IF and TEER is shown. Thus, one cannot rule out based upon this figure that Actin changes over the course of 24 hours in uninfected cells, or cells infected with a non-pathogen. They may therefore not be specific to pathogenesis.

b. Further, in these images, please identify the sites being referred to regarding actin disorganization, as from the images seen and way it will show up in publication, the difference is not clear.

3. Line 350-352: The authors draw the conclusion that Leptospira “invades” epithelial cells. This is unfounded in the data. What is shown is that Leptospira colocalize with LAMP1 signal by IF, suggesting the Leptospira are within lysozomes. The authors also state that these bacteria do not survive. An alternate interpretation of this data is that the bacteria are engulfed by host cells, and killed as part of the immune response as opposed to bacteria “invading” the host cells. “Invasion” implies its to the bacteria’s advantage, but even the data presented show that once inside they are quickly inactivated (thus not invasion but rather an immune response to the bacteria). In keeping with this, it is then no surprise that the engulfed bacteria do not cause an actin disruption, as they are likely sequestered to be destroyed by the host and therefore not viable and/or able to cause disease (further evidence its not invasion, but rather a response by the host).

Reviewer #2: (No Response)

Reviewer #3: N.A.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Line 19 and 45: “Leptospira interrogans is the…” and “caused by Leptospira interrogans” – L. interrogans is not the only cause. This is addressed in the introduction, but please correct to “a”, or “a major cause”, or something to that effect.

Line 23: “possess an intrinsic”, not “and”

Line 33-34: Did the authors identify this protein for the first time? Or identify it in the context of it’s the analysis and its meant to say “identified as a cause of…”? Change wording.

Line 68: “EGFR induces”, should read “EGFR activation induces”, as it’s the activation of the receptor not just the receptor alone?

Line 147: missing period at end of sentence

Figure 2A: is DM mean DMSO? This abbreviation is not specified.

Line 200: “host protein(s) to late-stage” � is this supposed to read “lead to” late-stage….

Line 204-206: Addition fo BF+MG pre-infection also stopped TEER reduction, but results only discuss 5h.p.i.

Line 261-262: Fix “without combination with”

Line 269: RPTEC/TERT1 cells are not the same as RPTECs (which would imply primary cells). Please adjust the references to RPTECs throughout. RPTECs and their immortalized versions often behave quite differently, and are apt to reverting to other cell types. Therefore, please also include the number of passages of the cells used, and if any verification was performed on the cells prior to use. I do not object to their use as they are a valid model, just more information is needed.

Line 346: Some discussioin should be added here. Although pathogens may counteract repair strategies, one major aspect of Leptospira pathogenesis is colonization of hosts (human and reservoirs) without causing disease. Especially with regards to reservoirs, repair is not bad as this allows the animal to live and sustain colonization and ultimate spread. Further, disrupting the repair does not help “establish infection”, but rather induces disease. “Establish infection” would imply more the long term survival and colonization.

Line 383: “and” should not be italicized

Line 418: clarify that it has not been studied following leptospiral infection, because it was just stated it was studied in other bacteria.

Reviewer #2: (No Response)

Reviewer #3: 1. Leptospira is generally considered as a extracellular infectious bacteria. The fact from previous studies and this study showing Leptospira in the cells were done in cultivated cell lines. Is it possible that this is an artifact that only can be observed ex vivo? Is there any evidence supporting Leptopsira can enter the cells in vivo? This needs to be discussed.

2. In lines 166-167, the authors stated “cell associated leptopsira….at 5.h.i.” Did you mean the cell-associated leptospires grows larger (larger size)? If so, the length or anyway to qualify the size/length needs to be reported and compared statistically if possible.

3. All experiments in this work was performed using cultivated cells ex vivo. Different time points were justified based on the experimental finding. Is there any evidence to correlate these ex vivo time points to the in vivo progression of the Leptospirosis?

4. In line 261, please add the justification of why BZ is used. It would also be helpful to describe the differences of all proteosomal inhibitor used in this study. Is there any difference among those inhibitors?

5. Several editing comments:

(1) Line 53, which cell? Epithelial cells?

(2) Line 135, Specify what BF and MG the first time when these termed were used. (if it has mentioned as full names in abstract, it may be better to be spelled out as full name again in the main text of the manuscript.

(3) Lines 136-137, Which PAMP, please specify it. The full name of PAMPs also needs to be spelled out the first time when this term is used.

(4) Line 141, Please specify TEER the first time when this is used.

(5) Line 158, Please clarify why DMSO is used what was this used for?

(6) Line 267, what is "afadin"?

**********

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

[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.]

Figure resubmission:

While revising your submission, we strongly recommend that you use PLOS’s NAAS tool (https://ngplosjournals.pagemajik.ai/artanalysis) to test your figure files. NAAS can convert your figure files to the TIFF file type and meet basic requirements (such as print size, resolution), or provide you with a report on issues that do not meet our requirements and that NAAS cannot fix.

After uploading your figures to PLOS’s NAAS tool - https://ngplosjournals.pagemajik.ai/artanalysis, NAAS will process the files provided and display the results in the "Uploaded Files" section of the page as the processing is complete. If the uploaded figures meet our requirements (or NAAS is able to fix the files to meet our requirements), the figure will be marked as "fixed" above. If NAAS is unable to fix the files, a red "failed" label will appear above. When NAAS has confirmed that the figure files meet our requirements, please download the file via the download option, and include these NAAS processed figure files when submitting your revised manuscript.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies 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. 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 Dr. Toma,

We are pleased to inform you that your manuscript 'Proteasome-dependent cytoskeleton disruption during Leptospira interrogans infection induces aberrant collective cell migration' has been provisionally accepted for publication in PLOS Pathogens.

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 Pathogens.

Best regards,

Jon T. Skare

Academic Editor

PLOS Pathogens

Thomas Guillard

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Barbee et al. aim to understand how Leptospira induces kidney disease in the host. Using a wide range of technologies, they look at the ability of bacteria to cause disruption of epithelial cells, identify host pathways of importance, and show that inhibitors can modulate these effects in vitro. This study is novel for many reasons, including being one of the first mechanistic looks into kidney epithelial cell damage and repair processes.

Reviewer #2: The authors have satisfactorily addressed all of the reviewer's comments and suggestions, and the revised manuscript has been substantially improved.

Reviewer #3: (No Response)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The Authors did a great job responding to and addressing all reviewer comments, I am endorsing publication of this revision.

Reviewer #2: No additional major experiments or major modifications are required.

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: The Authors did a great job responding to and addressing all reviewer comments, I am endorsing publication of this revision.

Reviewer #2: No further minor issues to address.

Reviewer #3: (No Response)

**********

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