Translational gene expression control in Chlamydia trachomatis

Nicole A. Grieshaber; Travis J. Chiarelli; Cody R. Appa; Grace Neiswanger; Kristina Peretti; Scott S. Grieshaber

doi:10.1371/journal.pone.0257259

Peer Review History

Original SubmissionAugust 25, 2021
5 Oct 2021 Decision Letter - Jorn Coers, Editor PONE-D-21-27589Translational gene expression control in Chlamydia trachomatis.PLOS ONE Dear Dr. Grieshaber, 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 Nov 19 2021 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. 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See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 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. Additional Editor Comments: Both reviewers asked for a Chlamydia loading control. These concerns need to be addressed with the relevant data provided in a revised MS [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: Partly 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: 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: In this manuscript by Grieshaber et al., the authors describe a new method of translational control, riboswitch E, for use in Chlamydia trachomatis. The ability to control translation of chlamydial factors will enable functional characterization of regulatory factors and effectors. Overall the manuscript is well written and easy to follow, however I have some concerns related to data presentation. Major comments: Fig 1B, Fig. 3B, Fig. 4B, etc.: A loading control (Ctr HSP) should be included. In my opinion, Fig. 2A should be part of Fig. 1 (maybe Fig. 1D?). The bar graph should also have N.S. to indicate no significant difference following Theo treatment. The decimal seems to be missing in the legend in Fig. 2B. Should also include mM in the legend. Fig. 7 should have N.S. on graph to indicate no significant difference. Scale bars are missing from many of the microscopy images. Details on the number of experimental replicates and/or technical replicates is missing. This should be included in the figure legends or materials and methods section. Minor comments: In many places, Chlamydia is italicized but trachomatis is not. Both should be italicized. In some instances, the authors use Chlamydia trachomatis whereas in others they use Ctr. Consistent use of one or the other should be used. Line 237, 454 should be 434 Reviewer #2: Greishaber et al. describe the development of a theophylline-inducible riboswitch E that allows for regulatable translation in Chlamydia trachomatis L2 from plasmid-encoded genes. Riboswitch-regulation was assessed with both native (pgp4) and synthetic promoters (T5-lac and tet). The tet-promoter system allowed for transcriptional control (tet operator/repressor) in addition to translational control via the riboswitch. In general, the data are convincing and regulation appears to be very tight and dose-responsive. The riboswitch approach should have significant utility for the chlamydial field as highlighted by the construction of hctB-plasmid transformants using the dual tet-riboswitch E vector to negate HctB-toxicity owing to leaky expression. In addition, the authors document that the T5-lac/tet promoters appear to show developmental-stage specific expression which has relevance for many of the chlamydial expression-vectors in use. Suggestions for improving the manuscript are listed below as major and minor criticisms. Major criticisms 1) The Flag-tag western blots showing induction of the protein of interest in figures 1, 3, 4, and 6 would greatly benefit from loading controls to demonstrate that chlamydial proteins were present in the Flag-tag negative lanes. Inclusion of sample-matched anti-MOMP western blots (or another prominent chlamydial protein) would address this weakness. 2) The group has already reported the use of the riboswitch E in a recent publication. The study is referenced in the methods regarding how transformations were performed (ref 38, line 132), but nowhere else. It is clear that the current manuscript is an in-depth analysis of the riboswitch and it provides significant new details that merit sharing with the field. A brief discussion of the use of the riboswitch in the prior study would further support the utility of the system and it is recommended that the authors highlight the prior usage with dbdA, hctA, and scc1. Minor criticisms 1) The RNAseq data under SRA 10220676 (https://www.ncbi.nlm.nih.gov/sra/?term=10220676, accessed on 9-30-21) do not appear to match the current study. Please confirm the submission number. 2) The methods mention using pen G or spec as appropriate for the plasmids (lines 133-135). All of the plasmids reported appear to be p2TK2-SW2 based (ref 20), which uses the bla marker. Please indicate which vector required spec-selection. 3) Please state in the methods the vehicle used to dissolve theophylline. 4) Line 265 – “two” should be too 5) The methods state that glycogen staining was performed at 40 hpi (line 197), while the results indicate 36 hpi (328). Please clarify. 6) When discussing the toxicity of HctB leakiness in the discussion (lines 586-593) it is perhaps worth noting that HctA (ref 38) did not require a dual transcription/translation regulated system to obtain transformants. Is this due to less leakiness for the hctA riboswitch construct or differences in the impact of these proteins on development? 7) Lines 588-589: “Additionally, ectopic expression of HctB early in infection (16 hpi) inhibited the formation of infectious progeny.” Recommend replacing “inhibited” with significantly reduced. At least in my mind, inhibited implies a complete block in IFU production which is not the case (figure 6D). 8) Recommend unifying the Y-axis formats in Fig_S1. ****** 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.] 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.0257259.r001
Revision 1
8 Nov 2021 Author Response We have addressed the reviews comments as follows: Both reviewers asked for a Chlamydia loading control. These concerns need to be addressed with the relevant data provided in a revised MS We have provided a tubulin control as loading controls for the westerns. We chose to not use a chlamydial antigen as induction of both Pgp4 and HctB affect the developmental cycle of chlamydial making any chlamydial protein a poor choice for a loading control. Comments to the Author Reviewer #1: In this manuscript by Grieshaber et al., the authors describe a new method of translational control, riboswitch E, for use in Chlamydia trachomatis. The ability to control translation of chlamydial factors will enable functional characterization of regulatory factors and effectors. Overall the manuscript is well written and easy to follow, however I have some concerns related to data presentation. Major comments: Fig 1B, Fig. 3B, Fig. 4B, etc.: A loading control (Ctr HSP) should be included. We have added beta tubulin control as loading controls for the westerns. We chose not to use a chlamydial antigen as induction of both Pgp4 and HctB affect the developmental cycle of chlamydial making any chlamydial protein a poor choice for a loading control. In my opinion, Fig. 2A should be part of Fig. 1 (maybe Fig. 1D?). The bar graph should also have N.S. to indicate no significant difference following Theo treatment. We agreed and updated the figure and text. We have added N. S. Where appropriate The decimal seems to be missing in the legend in Fig. 2B. Should also include mM in the legend. Fixed Fig. 7 should have N.S. on graphs to indicate no significant difference. This is now addressed Scale bars are missing from many of the microscopy images. This is now addressed Details on the number of experimental replicates and/or technical replicates is missing. This should be included in the figure legends or materials and methods section. This is now addressed Minor comments: In many places, Chlamydia is italicized but trachomatis is not. Both should be italicized. This is now addressed In some instances, the authors use Chlamydia trachomatis whereas in others they use Ctr. Consistent use of one or the other should be used. We are now consistently using Ctr Line 237, 454 should be 434 fixed Reviewer #2: Greishaber et al. describe the development of a theophylline-inducible riboswitch E that allows for regulatable translation in Chlamydia trachomatis L2 from plasmid-encoded genes. Riboswitch-regulation was assessed with both native (pgp4) and synthetic promoters (T5-lac and tet). The tet-promoter system allowed for transcriptional control (tet operator/repressor) in addition to translational control via the riboswitch. In general, the data are convincing and regulation appears to be very tight and dose-responsive. The riboswitch approach should have significant utility for the chlamydial field as highlighted by the construction of hctB-plasmid transformants using the dual tet-riboswitch E vector to negate HctB-toxicity owing to leaky expression. In addition, the authors document that the T5-lac/tet promoters appear to show developmental-stage specific expression which has relevance for many of the chlamydial expression-vectors in use. Suggestions for improving the manuscript are listed below as major and minor criticisms. Major criticisms 1) The Flag-tag western blots showing induction of the protein of interest in figures 1, 3, 4, and 6 would greatly benefit from loading controls to demonstrate that chlamydial proteins were present in the Flag-tag negative lanes. Inclusion of sample-matched anti-MOMP western blots (or another prominent chlamydial protein) would address this weakness. We have added a beta tubulin control as loading controls for the westerns. We chose not to use a chlamydial antigen as induction of both Pgp4 and HctB affect the developmental cycle of chlamydial making any chlamydial protein a poor choice for a loading control. 2) The group has already reported the use of the riboswitch E in a recent publication. The study is referenced in the methods regarding how transformations were performed (ref 38, line 132), but nowhere else. It is clear that the current manuscript is an in-depth analysis of the riboswitch and it provides significant new details that merit sharing with the field. A brief discussion of the use of the riboswitch in the prior study would further support the utility of the system and it is recommended that the authors highlight the prior usage with dbdA, hctA, and scc1. We have added this to the discussion Minor criticisms 1) The RNAseq data under SRA 10220676 (https://www.ncbi.nlm.nih.gov/sra/?term=10220676, accessed on 9-30-21) do not appear to match the current study. Please confirm the submission number. Our mistake, this was the temporary accession number the correct one is PRJNA756251 The methods mention using pen G or spec as appropriate for the plasmids (lines 133-135). All of the plasmids reported appear to be p2TK2-SW2 based (ref 20), which uses the bla marker. Please indicate which vector required spec-selection. We have updated the material and methods to reflect that we replaced bla with spec in this construct 3) Please state in the methods the vehicle used to dissolve theophylline. Theophylline was dissolved straight into RPMI media. We have added this to the materials and methods 4) Line 265 – “two” should be too Fixed 5) The methods state that glycogen staining was performed at 40 hpi (line 197), while the results indicate 36 hpi (328). Please clarify. This was done at 36 hours and has been updated in the materials and methods 6) When discussing the toxicity of HctB leakiness in the discussion (lines 586-593) it is perhaps worth noting that HctA (ref 38) did not require a dual transcription/translation regulated system to obtain transformants. Is this due to less leakiness for the hctA riboswitch construct or differences in the impact of these proteins on development? We elaborate on this in the discussion. Basically HctA does not appear to be as toxic even though there is some evidence of leakiness. 7) Lines 588-589: “Additionally, ectopic expression of HctB early in infection (16 hpi) inhibited the formation of infectious progeny.” Recommend replacing “inhibited” with significantly reduced. At least in my mind, inhibited implies a complete block in IFU production which is not the case (figure 6D). We agree and have updated the sentence. 8) Recommend unifying the Y-axis formats in Fig_S1. Agreed and fixed Attachments Attachment Submitted filename: Reviews comments to Fancytet.docx https://doi.org/10.1371/journal.pone.0257259.r002
17 Nov 2021 Decision Letter - Jorn Coers, Editor PONE-D-21-27589R1Translational gene expression control in Chlamydia trachomatisPLOS ONE Dear Dr. Grieshaber, There are some minor points that need to be addressed (missing file for supplementary figure S2, etc.). Please , send in revised version that addresses these minor point and I will make an editorial decision shortly thereafter. Thanks! Please submit your revised manuscript by Jan 01 2022 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, Jorn Coers Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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: (No Response) ******** 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: (No Response) Reviewer #2: The authors did an excellent job responding to the prior criticisms. Two minor points that should be addressed: 1) A file for Figure S2 (RNA-seq results) does not appear to be included with the revised submission. Page 35 is S1 and page 36 is S3. If the figure S2 intended for the revised submission matches the first submission, then I do not have additional comments for this data. 2) The authors indicate that they revised Figure S1 to unify the Y-axis formats in response to the round 1 reviewer comment. The included Figure S1 still uses 3 different numbering formats. Perhaps the incorrect version of the figure was uploaded. Recommend using scientific notation for panels A and B as shown in panels C and D. ****** 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: 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.] 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.0257259.r003
Revision 2
7 Dec 2021 Author Response We have addressed the reviewers comments as follows: Second review Reviewer #2: The authors did an excellent job responding to the prior criticisms. Two minor points that should be addressed: 1) A file for Figure S2 (RNA-seq results) does not appear to be included with the revised submission. Page 35 is S1 and page 36 is S3. If the figure S2 intended for the revised submission matches the first submission, then I do not have additional comments for this data. Yes this was an oversight on resubmission. Fig S2 is unchanged from the original submission and is now included in this submission. 2) The authors indicate that they revised Figure S1 to unify the Y-axis formats in response to the round 1 reviewer comment. The included Figure S1 still uses 3 different numbering formats. Perhaps the incorrect version of the figure was uploaded. Recommend using scientific notation for panels A and B as shown in panels C and D. We have fixed the graphs in fig S1 to all have the same style of Y axis. Attachments Attachment Submitted filename: rev_response.docx https://doi.org/10.1371/journal.pone.0257259.r004
13 Jan 2022 Decision Letter - Jorn Coers, Editor Translational gene expression control in Chlamydia trachomatis PONE-D-21-27589R2 Dear Dr. Grieshaber, 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, Jorn Coers Academic Editor PLOS ONE Additional Editor Comments (optional): the second revision address the very minor outstanding critique points. Congratulations to having your work accepted for publication. Reviewers' comments: https://doi.org/10.1371/journal.pone.0257259.r005
Formally Accepted
18 Jan 2022 Acceptance Letter - Jorn Coers, Editor PONE-D-21-27589R2 Translational gene expression control in Chlamydia trachomatis. Dear Dr. Grieshaber: 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. Jorn Coers Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0257259.r006

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