Extensible membrane nanotubules mediate attachment of Trypanosoma cruzi epimastigotes under flow

Cristhian David Perdomo-Gómez; Nancy E. Ruiz-Uribe; John Mario González; Manu Forero-Shelton

doi:10.1371/journal.pone.0283182

Peer Review History

Original SubmissionDecember 19, 2022
19 Dec 2022 Author Response Attachments Attachment Submitted filename: 20221212 Rebuttal Plos Pathogens.pdf https://doi.org/10.1371/journal.pone.0283182.r001
11 Jan 2023 Decision Letter - Vyacheslav Yurchenko, Editor PONE-D-22-32336Extensible membrane nanotubules mediate attachment of Trypanosoma cruzi epimastigotes under flowPLOS ONE Dear Dr. Forero-Shelton, 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 Feb 25 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, Vyacheslav Yurchenko, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. In your revised cover letter, please address the following prompts: a) 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. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. We will update your Data Availability statement on your behalf to reflect the information you provide. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 5. 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: The work of Perdomo-Gómez et al. on membrane nanotubules-mediated attachment of Trypanosoma cruzi epimastigotes under flow was reviewed by three independent scientists and they all were positive and supportive of its publication in PLoS One. Please address their comments and submit a revised manuscript, which (likely) will not go through another round of peer-review. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes Reviewer #3: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is a very interesting and original manuscript focusing on how epimastigote forms may attach through their flagelum. Those forms are usually found in the triatomine vector where parasites should attach during their life cyclo to avoid passage with the blood meal.The authors showed using microscopic (SEM and Immunofluorescence) and videos a mechanism of in vitro attachment that open perspective to undertande flagellar extension using nanotubules. The paper is very well-written and I found only some misspellings. I therefore recommend acceptance of the manuscript. Major comments I think the text (lines 1-55) could be shortened or even removed since it includes basic information on T. cruzi biology. Introduction could start at the 3rd paragraph since the flagellum is the main focus of the paper. Line 425 - I do not think is the GPI-mucins, it is probably another major glycoconjugate, the GIPLs. The best reference to cite here is: Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus. Nogueira NF, Gonzalez MS, Gomes JE, de Souza W, Garcia ES, Azambuja P, Nohara LL, Almeida IC, Zingales B, Colli W. Exp Parasitol. 2007 Jun;116(2):120-8. doi: 10.1016/j.exppara.2006.12.014. Minor comments. line 195 - nanotubules lines 325, 336/337 and 370/399 - 1,55/1,24 or 1.55/1.24? Lines 361-362/396 and several parts in figure legends - 0.31 –– 0.62 – 0.93 – 1.24 line 460 - total? line 473 - brucei not Brucei Reviewer #2: The submitted manuscript has been transferred from Plos Pathogens following revisions to address the main comments of the reviewers. The revised manuscript is well written and it adequately addresses the concerns regarding the relevance of the filaments to attachment in vivo, and the reproducibility of the observations regarding filament elongation under varying levels of shear stress. Despite the absence of in vivo studies, the data on on the biophysical properties of the filaments are sound, they are novel with respect to T. cruzi, and provide a meaningful advance to the study of microbial adhesion under conditions of shear stress. Reviewer #3: I enjoyed so much reading this manuscript. It is shown that epimastigotes submitted to shear force after attachment to a surface release nanotubes that is formed by membranes of the parasite. I have some comments that could help to clarify some issues I found. 1- it should be discussed that attachment only occurs in parasite washed in PBS (why?) and extended in PBS. 2- The nanotubes can be seen after addition of medium and shear force (I know that they remain attached)? 3- The authors should clarify what surface they used? Only glass? 4- The text should include and discuss the formation of similar structures in T. cruzi amastigotes (J . Euk. Microbioi.,43(4)1996, 275-28 and references in). 5- The author suggest that T. cruzi mucins could be in the membrane of the nanotubes. It would be interesting if they test it using monoclonal antibodies. There are also antibodies that labeled the flagellum membrane (FCBP for example) that could be used, if possible. 6- Recheck orthography. I found a few typos. ****** 6. 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: Yes: Rodrigo Pedro Soares Reviewer #2: Yes: David Sacks Reviewer #3: Yes: Sergio Schenkman ******** [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. https://doi.org/10.1371/journal.pone.0283182.r002
Revision 1
2 Mar 2023 Author Response 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. Response: We have revised our manuscript in order to fulfill the journal’s requirements 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Response: The funding we received was not in the form of formal grants, and there are no grant numbers. We included the (intramural) financial support in the acknowledgements. 3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.te Response: we have uploaded the data which we analyzed as a dataset and the movies to Figshare. You can find them in: supporting information: https://doi.org/10.6084/m9.figshare.22178795 S1 Dataset: https://doi.org/10.6084/m9.figshare.22178789.v1 S1 Movie: https://doi.org/10.6084/m9.figshare.22177418.v1 S2 Movie: https://doi.org/10.6084/m9.figshare.22177583.v1 S3 Movie: https://doi.org/10.6084/m9.figshare.22177586.v1 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Response: In that particular section we are describing the biophysical properties of adhesion nanotubules in T. cruzi epimastigotes after they were exposed to shear stress. We observed that some epimastigotes did not produce nanotubules, so we did not consider those parasites for the analysis of nanotubule properties, even if we observed them, so the phrase “data not shown” was inaccurate. We have rewritten lines 379 – 385 to clarify that. 5. 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. Response: Concerning the articles we referenced in our manuscript, we could not find retracted articles. We did find one paper that has a corrected section that is not related to our study or the data we cited. Nevertheless, taking into consideration a comment given by one of the reviewers (below), we have shortened the first part of the introduction, the section in which we used the mentioned article, and we have removed that reference from our manuscript. On the other hand, we have added references 8 and 9 to broaden the description of the flagellum functionalities in the introduction. (Removed) Ballesteros-Rodea G, Santillán M, Martínez-Calvillo S, Manning-Cela R. Flagellar Motility of Trypanosoma cruzi Epimastigotes. Journal of Biomedicine and Biotechnology. 2012 8. Vaughan S. Assembly of the flagellum and its role in cell morphogenesis in Trypanosoma brucei. Curr Opin Microbiol. 2010 Aug;13(4):453–8. 9. Sharma R, Peacock L, Gluenz E, Gull K, Gibson W, Carrington M. Asymmetric Cell Division as a Route to Reduction in Cell Length and Change in Cell Morphology in Trypanosomes. Protist. 2008 Jan;159(1):137–51. Response to de reviewers • Reviewer #1, Dr. Rodrigo Pedro Soares 1. I think the text (lines 1-55) could be shortened or even removed since it includes basic information on T. cruzi biology. Introduction could start at the 3rd paragraph since the flagellum is the main focus of the paper. Response: We thank the reviewer for the comment, and agree the introduction was a bit lengthy. We have rewritten the introduction to improve its focus and clarity. 2. Line 425 - I do not think is the GPI-mucins, it is probably another major glycoconjugate, the GIPLs. The best reference to cite here is: Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus. Nogueira NF, Gonzalez MS, Gomes JE, de Souza W, Garcia ES, Azambuja P, Nohara LL, Almeida IC, Zingales B, Colli W. Exp Parasitol. 2007 Jun;116(2):120-8. doi: 10.1016/j.exppara.2006.12.014. Response: We agree that the proteins involved in the process could indeed be glycoinositolphospholipids (GIPL) and the article suggested is very interesting; considering that the focus of the manuscript is on the biomechanical properties of membrane nanotubules, we have removed assumptions regarding the molecules that could mediate attachment. Minor comments. a. line 195 -– nanotubules Corrected b. lines 325, 336/337 and 370/399 - 1,55/1,24 or 1.55/1.24? We corrected all numbers to period separated decimals instead of comma separated decimals. c. Lines 361-362/396 and several parts in figure legends - 0.31 –– 0.62 – 0.93 – 1.24 corrected d. line 460 - total? Corrected e. line 473 - brucei not Brucei ✓ Corrected • Reviewer #2, Dr. David Sacks o The submitted manuscript has been transferred from Plos Pathogens following revisions to address the main comments of the reviewers. The revised manuscript is well written and it adequately addresses the concerns regarding the relevance of the filaments to attachment in vivo, and the reproducibility of the observations regarding filament elongation under varying levels of shear stress. Despite the absence of in vivo studies, the data on on the biophysical properties of the filaments are sound, they are novel with respect to T. cruzi, and provide a meaningful advance to the study of microbial adhesion under conditions of shear stress. Response: We appreciate the time taken to review and comment the paper. • Reviewer #3, Dr. Sergio Schenkman 1. It should be discussed that attachment only occurs in parasite washed in PBS (why?) and extended in PBS. Response: We thank the reviewer for the comment. We used PBS because it has a known and stable viscosity (necessary to calculate shear stress and the resulting forces on the parasite), is isoosmolar; then, we did not disturb the homeostasis or motility of the parasite. Attachment of the parasites was only studied under PBS, but is likely to happen in other buffers. 2. The nanotubes can be seen after addition of medium and shear force (I know that they remain attached)? Response: Parasites were added in the medium, and nanotubules were observed after these attached to the surface and force resulting from shear stress was turned on. We did not change media for the reasons explained above. 3. The authors should clarify what surface they used? Only glass? Response: We used non-functionalized glass, and have clarified this in the methods, we thank the reviewer for pointing this oversight. We also tested poly-l-lysine but found it was not necessary to study the mechanical properties of the nanotubules and resulted in additional complexity. 4. The text should include and discuss the formation of similar structures in T. cruzi amastigotes (J . Euk. Microbioi.,43(4)1996, 275-28 and references in). Response: We thank the reviewer for pointing it out this paper as we had missed it. We now discuss it in the context of our findings in lines 512 - 526. Similarly, we included reference 59 to enrich the discussion. 5. The author suggest that T. cruzi mucins could be in the membrane of the nanotubes. It would be interesting if they test it using monoclonal antibodies. There are also antibodies that labeled the flagellum membrane (FCBP for example) that could be used, if possible. Response: We agree that this would be an interesting experiment to attempt in order to elucidate the molecules involved in this attachment process. However, considering that the scope of our research is to describe the biomechanical properties of the nanotubules in the attachment process, not to identify the molecules that mediate attachment, we removed the part where we discuss the molecules that could mediate the interaction since it is detract from the focus of the paper. 6. Recheck orthography. I found a few typos Response: We corrected a couple typos we found using our text editing software, hopefully they are the same. Again, we thank the reviewers and the editor for taking the time to review the paper and make constructive comments that have improved the quality of the paper. Attachments Attachment Submitted filename: Response to Reviwers 20230225.docx https://doi.org/10.1371/journal.pone.0283182.r003
6 Mar 2023 Decision Letter - Vyacheslav Yurchenko, Editor Extensible membrane nanotubules mediate attachment of Trypanosoma cruzi epimastigotes under flow PONE-D-22-32336R1 Dear Dr. Forero-Shelton, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Vyacheslav Yurchenko, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Kudos to authors for producing an interesting and important paper! Reviewers' comments: https://doi.org/10.1371/journal.pone.0283182.r004
Formally Accepted
14 Mar 2023 Acceptance Letter - Vyacheslav Yurchenko, Editor PONE-D-22-32336R1 Extensible membrane nanotubules mediate attachment of Trypanosoma cruzi epimastigotes under flow Dear Dr. Forero-Shelton: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Vyacheslav Yurchenko Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0283182.r005

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .