Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus

Connor L. Brown; Emily Garner; Guillaume Jospin; David A. Coil; David O. Schwake; Jonathan A. Eisen; Biswarup Mukhopadhyay; Amy J. Pruden

doi:10.1371/journal.pone.0223033

Peer Review History

Original SubmissionSeptember 9, 2019
14 Oct 2019 Decision Letter - Daniel E. Voth, Editor PONE-D-19-25375 Whole genome sequence analysis reveals broad distribution of the RtxA type 1 secretion system and four novel type 1 secretion systems throughout the Legionella genus PLOS ONE Dear Dr. Pruden, 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. We would appreciate receiving your revised manuscript by Nov 28 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Daniel E. Voth, 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. 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. Additional Editor Comments (if provided): [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: 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 ****** 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 well organized report on the prevalence of T1SS in isolates of Legionella. Genome sequence was generated from specimen isolated in Flint, Michigan groundwater. Four isolates were described with one novel genome revealed. The emphasis of the study examines distribution of RtxA-like type 1 secretion systems, with the important conclusion that this system is not limited to distribution among human pathogenic strains of Legionella as previously proposed. The majority of the paper reads like a comprehensive review of classes of T1SS, focusing on classifying new secretion systems in L.taurinensis. The use of 'virulence factor' throughout to describe the RtxA locus in L.pneumophila is somewhat generous. When one examines the papers that this study cites, nearly all phenotypes said to be associated with the rtxA locus present as less than two-fold differences in most experiments. of the three enhanced entry loci described in reference 15, the rtxA-containing locus has not been pursued since these publications. Other enhanced entry (enh) have been explored. The wording in the abstract (lines 41-42) 'conserved virulence factor in L.pneumophila' doesn't convey the message correctly. It should be changed to 'virulence factor exclusive to L.pneumophila'. Reviewer #2: This manuscript describes an in silico analysis of predicted type I secretion system (T1SS) distribution in multiple Legionella species. Brown et al. describe isolation of four new strains of Legionella taurinensis from Genesee county Michigan. Upon genome sequencing of these strains, the authors identified four putative T1SSs, one with homology to the RtxA T1SS and three putative novel T1SSs. They also probed the genomes of 45 additional Legionella species for the presence of T1SSs using amino acid sequence homology as an indicator. They found predicted RtxA and novel LB2SS T1SS in previously sequenced Legionella species and determined that distribution of T1SS is not a predictor of virulence. This is an interesting study that re-evaluates the distribution of T1SS within Legionella. Comments: The title should be changed to ..”four putative novel type 1 secretion…”. There is no experimental evidence demonstrating that these are functional type 1 secretion systems. Table 1 is missing from the submission files. As there is a virulence defect observed for loss of the rtxA T1SS in L. pneumophila (Cirillo et al, 2001), the authors should rephrase the sentence in lines 56-57. The RtxA system could be a virulence determinant, it’s just not sufficient if other virulence factors are not present/active, etc. Another example is that the Dot/Icm secretion system is present in all sequences strains of Legionella but they're not all pathogenic to humans. Line 96: add the word “potential” or “putative” in front of “novel T1SS” Line 103: Remove “(Garner et al. in review)” unless the study has been published. Figure 2: B and C are switched in the figure legend and text. Out of curiosity, any idea what the hypothetical genes in the L. taurinensis lssXYZABD locus could be doing? Line 130: How did you decide on a 40% identity cut-off? In your predictions, was the rtxA substrate homolog also identified? Line 147: The blastp algorithm was used here, correct? If so, please specify. Line 200: Please reference the sentence starting “Of these 19,…”. Line 216: Please reference the statement ending in “…Legionella tested (n = 10)”. Line 229: please change the language in the sentence starting with “In contrast,…” to emphasize that presence if these systems is predicted based on homology. Figure 4 is of poor quality (pixelated), please improve the quality of this image to increase its readability. Also, what does the light pink color indicate? Please also keep in mind that just because a Legionella species has not been isolated from a patient, it does not have potential to cause disease. It is likely that some species may be less able to colonize sources of human infection. ****** 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: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0223033.r001
Revision 1
10 Dec 2019 Author Response PONE-D-19-25375 Whole genome sequence analysis reveals broad distribution of the RtxA type 1 secretion system and four novel type 1 secretion systems throughout the Legionella genus PLOS ONE Dear Dr. Pruden, 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. We would appreciate receiving your revised manuscript by Nov 28 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. • A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. • An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Daniel E. Voth, Ph.D. Academic Editor PLOS ONE [Note: Line references refer to the revised version of the manuscript, rather than the version showing tracked changes.] Reviewers’ Comments to Authors: Reviewer #1: This is a well-organized report on the prevalence of T1SS in isolates of Legionella. Genome sequence was generated from specimen isolated in Flint, Michigan groundwater. Four isolates were described with one novel genome revealed. The emphasis of the study examines distribution of RtxA-like type 1 secretion systems, with the important conclusion that this system is not limited to distribution among human pathogenic strains of Legionella as previously proposed. The majority of the paper reads like a comprehensive review of classes of T1SS, focusing on classifying new secretion systems in L.taurinensis. 1. The use of 'virulence factor' throughout to describe the RtxA locus in L. pneumophila is somewhat generous. When one examines the papers that this study cites, nearly all phenotypes said to be associated with the rtxA locus present as less than two-fold differences in most experiments. of the three enhanced entry loci described in reference 15, the rtxA-containing locus has not been pursued since these publications. Other enhanced entry (enh) have been explored. We agree that the nature of RtxA as a virulence factor is uncertain. However, several experimental studies since Cirillo et al. 2001 have supported the role of RtxA and its type 1 secretion system (T1SS) as a possible virulence factor (Cirillo et al. 2002, Fuche et al 2015, Qin et al. 2017) and these observations have been reported in a substantial review (Smith et al. 2019). Of note, Cirillo et al. 2002 examined 20 non-pneumophila Legionella genomes and found that RtxA was restricted to L. pneumophila and L. feeleii; this led the authors to conclude: “This observation, along with previous studies demonstrating that this gene plays an important role in virulence (Cirillo et al., 2001), has helped to fulfill ‘molecular Koch’s postulates’ for rtxA, supporting the conclusion that this gene plays a role in disease.” Therefore, we argue that the use of “virulence factor” is justified to reflect the assumptions of the current available literature. However, as these studies were not conducted in animal models, we have updated the first paragraph of the discussion (lines 234-236) to reflect the spirit of the reviewer’s comment: Thus, the emergent consensus model has been that the RtxA system is an important conserved genetic virulence determinant unique to L. pneumophila, despite the absence of additional experimental investigation of its function in animal models since the discovery of the enhanced entry locus in 2001 [15]. 2. The wording in the abstract (lines 41-42) 'conserved virulence factor in L.pneumophila' doesn't convey the message correctly. It should be changed to 'virulence factor exclusive to L. pneumophila'. The wording has been changed to reflect that it is a virulence factor exclusive to L. pneumophila rather than being conserved (lines 40-41). Reviewer #2: This manuscript describes an in-silico analysis of predicted type I secretion system (T1SS) distribution in multiple Legionella species. Brown et al. describe isolation of four new strains of Legionella taurinensis from Genesee county Michigan. Upon genome sequencing of these strains, the authors identified four putative T1SSs, one with homology to the RtxA T1SS and three putative novel T1SSs. They also probed the genomes of 45 additional Legionella species for the presence of T1SSs using amino acid sequence homology as an indicator. They found predicted RtxA and novel LB2SS T1SS in previously sequenced Legionella species and determined that distribution of T1SS is not a predictor of virulence. This is an interesting study that re-evaluates the distribution of T1SS within Legionella. 1. The title should be changed to ”four putative novel type 1 secretion…”. There is no experimental evidence demonstrating that these are functional type 1 secretion systems. The title has been updated to reflect that these systems are putative. 2. Table 1 is missing from the submission files. This table has been uploaded along with the resubmission. 3. As there is a virulence defect observed for loss of the rtxA T1SS in L. pneumophila (Cirillo et al, 2001), the authors should rephrase the sentence in lines 56-57. The RtxA system could be a virulence determinant, it’s just not sufficient if other virulence factors are not present/active, etc. Another example is that the Dot/Icm secretion system is present in all sequences strains of Legionella but they're not all pathogenic to humans. We have updated the final line of the abstract (lines 56-57): “These results draw into question the nature of RtxA and its T1SS as a singular genetic virulence determinant. Future investigations should focus on mechanistic explanations for the association of RtxA with virulence.” 4. Line 96: add the word “potential” or “putative” in front of “novel T1SS” This line has been updated (line 96). 5. Line 103: Remove “(Garner et al. in review)” unless the study has been published. This reference has been updated (lines 103). 6. Figure 2: B and C are switched in the figure legend and text. This has been fixed (lines 111-117). 7. Out of curiosity, any idea what the hypothetical genes in the L. taurinensis lssXYZABD locus could be doing? These proteins appear to be part of a zeta toxin/antitoxin system. The only homologs of these genes we could find were in Legionella species, and the colocalization of the two genes was preserved in all Legionella genomes we looked at. Analyzing the amino acid sequence of the larger protein, we found evidence of an NTP-ase p-loop domain associated with UDP-kinase/zeta toxin activity (domain IPR010488). On the Integrated Microbial Genomes repository, many of the homologs of the larger protein were annotated as putative zeta toxins. 8. Line 130: How did you decide on a 40% identity cut-off? In your predictions, was the rtxA substrate homolog also identified? The 40% identity cut-off is used by the Enzyme Commission and, when comparing domains with the same fold, reflects specific functional conservation well (Wilson et al. 2000). Further, Qin et al. 2017 used this cut-off value to identify homologs of secretion system components in Legionella spp. This text (and appropriate citations) was added to reflect this (lines 298-301): “The 40% identity cut-off is used by the Enzyme Commission to establish functional conservation between similar proteins (33, 34) Further, Qin et al. 2017 used the 40% identity cut-off previously to investigate the distribution of secretion systems in Legionella species (23).” 9. Line 147: The blastp algorithm was used here, correct? If so, please specify. This line was updated with the search algorithm used (tblastn) (lines 146-147). 10. Line 200: Please reference the sentence starting “Of these 19,…”. A reference was added to this line (lines 202). 11. Line 216: Please reference the statement ending in “…Legionella tested (n = 10)”. A reference was added to this line (line 222). 12. Line 229: please change the language in the sentence starting with “In contrast,…” to emphasize that presence if these systems is predicted based on homology. This sentence was changed to (lines 236-238): “In contrast, we found bioinformatic evidence that homologs of the RtxA T1SS and the four novel T1SSs are prevalent throughout the genus, even among species not presently known to cause disease.” 13. Figure 4 is of poor quality (pixelated), please improve the quality of this image to increase its readability. Also, what does the light pink color indicate? The resolution of Figure 4 has been improved and a sentence was added to the figure legend of Figure 4 (line 197-198): “Pink color indicates species that have been associated with human infections (27)” Please note: the version of the figure within the PLOS submission PDF has less resolution than the actual TIF file. To see the actual figure, please use the download link in the top right corner of the submission document. 14. Please also keep in mind that just because a Legionella species has not been isolated from a patient, it does not have potential to cause disease. It is likely that some species may be less able to colonize sources of human infection. We agree with the reviewer’s comment and have introduced the following changes to the end of the results section (lines 212-217): “However, our observation brings up the possibility that some of the Legionella species that thus far have not been isolated from patients are inefficient colonizers of built environments that are common exposure routes and, therefore, remain classified as non-pathogenic.” Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0223033.r002
18 Dec 2019 Decision Letter - Daniel E. Voth, Editor Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus PONE-D-19-25375R1 Dear Dr. Pruden, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Daniel E. Voth, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0223033.r003
Formally Accepted
27 Dec 2019 Acceptance Letter - Daniel E. Voth, Editor PONE-D-19-25375R1 Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus Dear Dr. Pruden: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Daniel E. Voth Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0223033.r004

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 .