FlgV forms a flagellar motor ring that is required for optimal motility of Helicobacter pylori

Jack M. Botting; Shoichi Tachiyama; Katherine H. Gibson; Jun Liu; Vincent J. Starai; Timothy R. Hoover

doi:10.1371/journal.pone.0287514

Peer Review History

Original SubmissionMarch 28, 2023
17 Apr 2023 Decision Letter - Eric Cascales, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-23-09244Novel FlgV ring is required for optimal flagellar motility of Helicobacter pyloriPLOS ONE Dear Dr. Hoover, 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 01 2023 11:59PM. 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We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This work was supported by National Institutes of Health grants AI140444 (TRH), AI1469077 (TRH), AI087946 (JL), and AI132828 (JI). The URL for NIH is https://www.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. 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. 4. Please include a copy of Table 4 which you refer to in your text on page 18. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 6. 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: It has been sent to two external reviewers. As you will see in their comments pasted below, the two reviewers have a very positive appraisal of your work and recommend publication pending minor revisions. I encourage you to carefully modify your manuscript in light of the reviewer's comments. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 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: No ****** 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 ****** 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 manuscript describes the identification of protein that forms a previously uncharacterised ring structure on the flagellar motor of Helicobacter pylori of the Campylobacterota. The authors found that FlgV is required for full flagellation and motility in H. pylori, and proceeded to demonstrate using cryoelectron tomography that FlgV forms a cytoplasmic ring. The paper is overall reasonably well-written and essentially scientifically sound, and presents a nice advance in putting the pieces of the puzzle together that at the structures of the Campylobacterota flagella motors. - The title is grammatically poorly formed, and requires an article ("A novel ring of FlgV"?) - although "novel" is over-used and essentially meaningless. How about "A cytoplasmic ring of FlgV is required for optimal..."? - The abstract is misleading, hinting that this is the first time FlgV has been shown to be required for motility: "Here, we provide the first evidence that deletion of flgV in H. pylori B128 and a highly motile variant of H. pylori G27 (G27M) results in reduced motility in soft agar medium.". This fails to capture the fact that FlgV has already been characterised. Gao 2014 identified FlgV using a screen that essentially selected for less motile variants, and as they stated: "Although some mutations could indirectly lead to lack of motility, through a variety of functional, biochemical, and in vivo imaging analyses we have provided strong evidence that the genes we have identified encode factors directly involved in the assembly and/or function of the flagellar apparatus." - Line 44: MotA has four transmembrane helices, not domains. - Line 48: It is speculation than MotB rotates within MotA - don't present as fact. - Line 50: "Campylobacterota", not "H. pylori"; worth citing https://mbio.asm.org/content/11/1/e02286-19 - Line 91: worth reporting identity/similarity percentages here. - Line 99: "strongly suggest" is more appropriate than "strong evidence". - Line 128: Worth highlighting to non-expert readers that Arcobacter has a mystery, "weird" outlier motor, so this result not expected, but this protein is likely to be FlgV. - Line 131: I think G27M is a new result? Phrase it as such by writing this sentence in present tense (or provide citation if it's a previous result). - Entire paragraph starting Line 131: I don't understand why you did this work in non-standard strain G27M instead of G27? Was it because you can more sensitively assay motility using G27M? Spell it out to your readers. I'd have preferred you to have mentioned the WT B128 result first, THEN move into a non-standard strain, although don't understand why you used B128 instead of the correct comparison, G27? - Line 185: Glu 71,72 come out of the blue - need introduction. - Paragraph commencing line 196: this is currently fantasy/speculation. I'd prefer the authors either support this hypothesis with relevant fliF mutant results, or remove or greatly tone-down this paragraph. Use words like "suggestive" and "speculative" to help guide your readers to understand the underlying science. - Line 221: worth acknowledging that this density was previously highlighted in Campylobacter jejuni by the yellow arrows in Fig. 2 of http://www.pnas.org/content/early/2016/03/09/1518952113 and that your work is nicely consistent/explanatory with previous work. - Line 237: better phrased, "To further assess whether FlgV forms..." (in science we don't try to obtain evidence for a specific hypothesis, as this biases results - rather, we put hypotheses to the test). - Figure 4: labels need to be larger. Reviewer #2: This paper aimed to characterise the role of the protein FlgV, a component of the bacterial flagellum, in the human pathogen H. pylori. This protein had previously been identified in C. Jejuni, but its role if flagellum biogenesis and/or function had not been determined. Here, the authors demonstrate that FlgV plays an important role in motility, by promoting flagellum assembly, but also contributes to the flagellum rotation mechanism. Modelling of this protein suggests that it is likely an integral membrane protein, that interacts with the the flagellum MS-ring protein FliF. Finally, very elegant sub-tomogram averaging experiments link FlgV to unattributed density between the MS-ring, and the C-ring, in the cytoplasm. Collectively, this is a short study, that fills a important gap in our understanding of high-torque-generating flagella such as those of C. Jejune and H. pylori. The manuscript is clear and well-written, and I only have a few minor comments, that would help with clarity and completeness of the reported work. - Lines 64-66: Some of the mentioned symmetries were shown to be artefacts of FliF in isolation, and were not not found in FliF within the full flagellum HBB structure (Tan et al, Cell, 2021). The true oligomeric state of FliF is 34, with the inner ring (RBM2) having 23-fold symmetry. - Figure 1b: What does the last operon correspond to? - Lines 96-97: I think it would be relevant to show the predicted TM topology, in the supplementary material. - Figure 2C: For AlphaFold modelling, it's essential to show the pLDDT plot, and/or the model Color-coded by confidence, to get a sense of how accurate the prediction is. - Figure 2: For completeness, a photo of agar plate, a selection of electron micrograph of cells used for counting the flagella, and a table including the number of flagella for each mutant, should be added in the supplementary material. - Lines 179-183: Showing a multiple alignment of the FlgV sequences, with the GxxxG motif indicated and the predicted tomopogy, would be really helpful. - Figure 3I, J: These would gain to have additional labels for the FliL ring, which is only mentioned in the legend. - Figure 3: Could the authors do a map subtraction, between WT and DflgV, and also between WT and flgV-YFP, to better highlight the regions of density that have changed? - Line 250: This could gain from additional labels, an/or a self-rotation analysis to better illustrate the proposed stoichiometry. - Figure 4: The model suggests that FlgV is in partly in the IM - should be shown in 4G. Does the non-TM region of the protein fits in the density? ****** 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: No Reviewer #2: Yes: Julien Bergeron ******** [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.0287514.r001
Revision 1
10 May 2023 Author Response Dear Dr. Cascales, Thank you for handling the review of the manuscript. We have modified the manuscript based on your comments and the comments of the two reviewers. Below is a point-by-point description of how we addressed the comments, starting with your comments. Our responses follow each specific comment and are in red. We thank both you and the reviewers for the constructive comments as we feel that the suggested changes have significantly improved the manuscript. Sincerely, Tim Hoover 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 We have reformatted the manuscript to meet the PLOS ONE style requirements. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "We thank Jennifer Aronson for critical reading of the manuscript. This work was supported by NIH grants AI140444 and AI146907 to T.R.H. and AI087946 and AI132818 to J.L" We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This work was supported by National Institutes of Health grants AI140444 (TRH), AI1469077 (TRH), AI087946 (JL), and AI132828 (JI). The URL for NIH is https://www.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. The funding statement has been removed from the Acknowledgements Section. The Funding Statement indicated above is correct and does not require any amendment. 3. 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. We removed the information that was referred to by “data not shown” – see lines 233 and 370 on Revised Manuscript with Track Changes. We feel that deleting these statements has no impact on the data presented in the manuscript or interpretation of the results of the study. 4. Please include a copy of Table 4 which you refer to in your text on page 18. “Table 4” was a typographical error in the original manuscript. This is Table 1. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. The information has been added. 6. 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. The references have been checked. Reviewer #1: - The title is grammatically poorly formed, and requires an article ("A novel ring of FlgV"?) - although "novel" is over-used and essentially meaningless. How about "A cytoplasmic ring of FlgV is required for optimal..."? The title has been changed to “FlgV forms a flagellar motor ring that is required for optimal motility of Helicobacter pylori”. We have also removed “novel” throughout the manuscript. - The abstract is misleading, hinting that this is the first time FlgV has been shown to be required for motility: "Here, we provide the first evidence that deletion of flgV in H. pylori B128 and a highly motile variant of H. pylori G27 (G27M) results in reduced motility in soft agar medium.". This fails to capture the fact that FlgV has already been characterised. Gao 2014 identified FlgV using a screen that essentially selected for less motile variants, and as they stated: "Although some mutations could indirectly lead to lack of motility, through a variety of functional, biochemical, and in vivo imaging analyses we have provided strong evidence that the genes we have identified encode factors directly involved in the assembly and/or function of the flagellar apparatus." We changed the wording of the indicated sentence by replacing “provide the first evidence” to “confirm”. (line 26 in copy with track changes) - Line 44: MotA has four transmembrane helices, not domains. The change was made (line 44 of copy with track changes). - Line 48: It is speculation than MotB rotates within MotA - don't present as fact. We are not completely clear about the reviewer’s objection, but we modified the sentence to indicate that the MotA pentamer is thought to rotate around the stationary Mot dimer (line 49 of copy with track changes). - Line 50: "Campylobacterota", not "H. pylori"; worth citing https://mbio.asm.org/content/11/1/e02286-19 We made the change and included the citation (line 51 of copy with track changes). - Line 91: worth reporting identity/similarity percentages here. We included the information on identity/similarity (line 226 of copy with track changes). - Line 99: "strongly suggest" is more appropriate than "strong evidence". The change was made (line 235 of copy with track changes). - Line 128: Worth highlighting to non-expert readers that Arcobacter has a mystery, "weird" outlier motor, so this result not expected, but this protein is likely to be FlgV. We included an additional sentence to indicate that the Arcobacter flagellar proteins are divergent to flagellar proteins of other Campylobacterota and included a reference (lines 263-265 of copy with track changes). - Line 131: I think G27M is a new result? Phrase it as such by writing this sentence in present tense (or provide citation if it's a previous result). See response to next comment. - Entire paragraph starting Line 131: I don't understand why you did this work in non-standard strain G27M instead of G27? Was it because you can more sensitively assay motility using G27M? Spell it out to your readers. I'd have preferred you to have mentioned the WT B128 result first, THEN move into a non-standard strain, although don't understand why you used B128 instead of the correct comparison, G27? We indicate the reason we enriched for a variant of G27 that displayed greater motility in soft agar medium (lines 269-274 of copy with track changes). We do not feel that starting with B128 and then moving to G27M makes as much sense since most of the data presented in the manuscript was obtained with G27M. - Line 185: Glu 71,72 come out of the blue - need introduction. We included an introduction for these amino acid residues (line 336 of the copy with track changes). - Paragraph commencing line 196: this is currently fantasy/speculation. I'd prefer the authors either support this hypothesis with relevant fliF mutant results, or remove or greatly tone-down this paragraph. Use words like "suggestive" and "speculative" to help guide your readers to understand the underlying science. We disagree that the paragraph needs to be toned down as we feel it is clear that this is a model based on predicted protein-protein interactions. We agree with the reviewer that generating mutations in fliF would be a reasonable way to examine the validity of the model, and we hope to do those experiments in the future. To support the confidence of the model, we included a figure showing the predicted aligned error (PAE) score for the model for interactions between FlgV and FliF (Fig 2C), as per Reviewer #2’s recommendation. - Line 221: worth acknowledging that this density was previously highlighted in Campylobacter jejuni by the yellow arrows in Fig. 2 of http://www.pnas.org/content/early/2016/03/09/1518952113 and that your work is nicely consistent/explanatory with previous work. We included the suggested statement (lines 372-373 of the copy with track changes). - Line 237: better phrased, "To further assess whether FlgV forms..." (in science we don't try to obtain evidence for a specific hypothesis, as this biases results - rather, we put hypotheses to the test). The sentence was modified (line 392 of the copy with track changes). - Figure 4: labels need to be larger. The labels on Figure 4 have been enlarged. Reviewer #2: - Lines 64-66: Some of the mentioned symmetries were shown to be artefacts of FliF in isolation, and were not not found in FliF within the full flagellum HBB structure (Tan et al, Cell, 2021). The true oligomeric state of FliF is 34, with the inner ring (RBM2) having 23-fold symmetry. We made the changes in the Introduction (lines 66-68 of the copy with track changes) and Discussion (lines 471 and 475 of the copy with track changes). - Figure 1b: What does the last operon correspond to? The figure has been fixed. - Lines 96-97: I think it would be relevant to show the predicted TM topology, in the supplementary material. A figure showing the predicted membrane topology has been included (S1 Fig). - Figure 2C: For AlphaFold modelling, it's essential to show the pLDDT plot, and/or the model Color-coded by confidence, to get a sense of how accurate the prediction is. A figure showing the PAE score has been added (Fig 2C). The original Figure 2C is not Figure 2D. - Figure 2: For completeness, a photo of agar plate, a selection of electron micrograph of cells used for counting the flagella, and a table including the number of flagella for each mutant, should be added in the supplementary material. A figure showing representative electron micrographs of flagellated cells of G27M and G27M-derived strains, together with a photo of a plate for the soft agar motility assays has been added (S4 Fig). - Lines 179-183: Showing a multiple alignment of the FlgV sequences, with the GxxxG motif indicated and the predicted tomopogy, would be really helpful. A figure showing multiple alignments of FlgV homologs has been included (S3 Fig). - Figure 3I, J: These would gain to have additional labels for the FliL ring, which is only mentioned in the legend. The FliL-rings are indicated in the images. - Figure 3: Could the authors do a map subtraction, between WT and DflgV, and also between WT and flgV-YFP, to better highlight the regions of density that have changed? We used "map subtraction" between WT and the mutant to better highlight the FlgV ring as shown in the NEW Figure 4E, F. The difference between WT and flgV-YFP could be better visualized in Fig. 3D. - Line 250: This could gain from additional labels, an/or a self-rotation analysis to better illustrate the proposed stoichiometry. We included additional labels. The symmetries of the C-ring and the FlgV-ring are 39 and 21, respectively. - Figure 4: The model suggests that FlgV is in partly in the IM - should be shown in 4G. Does the non-TM region of the protein fits in the density? FlgV is inserted in the curved inner membrane. We also resolved 21-fold symmetry of the FlgV ring. However, we do not know how many subunits of FlgV are required for the formation of the FlgV ring or if there are other proteins associated with the density that we attribute to FlgV. Therefore, we do not feel convinced to fit the FlgV model into the density. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0287514.r002
7 Jun 2023 Decision Letter - Eric Cascales, Editor FlgV forms a flagellar motor ring that is required for optimal motility in Helicobacter pylori PONE-D-23-09244R1 Dear Dr. Hoover, Thank you for addressing the reviewer's comments and for the submission of the revised manuscript. It has been sent back to the two original reviewers who noticed that you have appropriately addressed their comments and now recommend publication of your work. I am therefore please to accept your manuscript for publication. Please note that it will be formally accepted 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. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. 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 ****** 5. 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 ****** 6. 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: I am satisfied with the Author's response to my suggestions and am happy with the manuscript being published. Reviewer #2: The authors have addressed all the points from both reviewers, and I am happy to recommend the publication of this manuscript in PLoS One. ****** 7. 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: Yes: Julien Bergeron ******** https://doi.org/10.1371/journal.pone.0287514.r003
Formally Accepted
5 Jul 2023 Acceptance Letter - Eric Cascales, Editor PONE-D-23-09244R1 FlgV forms a flagellar motor ring that is required for optimal motility of Helicobacter pylori Dear Dr. Hoover: 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 Dr. Eric Cascales Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0287514.r004

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