Differential expression of groEL-1, incB, pyk-F, tal, hctA and omcB genes during Chlamydia trachomatis developmental cycle

Gugulethu F. Mzobe; Sinaye Ngcapu; Bronwyn C. Joubert; Willem A. Sturm

doi:10.1371/journal.pone.0249358

Peer Review History

Original SubmissionJanuary 22, 2021
24 Feb 2021 Decision Letter - Michael F Minnick, Editor PONE-D-21-02174 Differential gene expression by Chlamydia trachomatis in keratinocytes at different temperatures PLOS ONE Dear Dr. Mzobe, 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 Apr 03 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. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Michael F Minnick, PhD 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. Thank you for stating the following in the Acknowledgments Section of your manuscript: '..This study was funded by grants from the National Research Foundation (SFH14061869970) and The University of Kwa-Zulu Natal College of Health Sciences. GFM is funded with a postdoctoral fellowship from the National Research Foundation (SFP180507326699).' 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. a. 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: 'Author: G.F Mzobe Funder: National Research Foundation (www.nrf.ac.za) Grant number: SFH14061869970 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.' b. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical. 4. Please include a separate caption for each figure in your manuscript. 5. Please include your table 1 as part of your main manuscript and remove the individual file. Please note that supplementary tables should remain as separate "supporting information" files. 6. 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 Additional Editor Comments: While there is certainly value in examining alternate host cells and various serovars of Chlamydia for analysis of expression patterns, the manuscript contains a number of major issues that must be addressed before it is suitable for publication. The two reviewers have disparate evaluations of your manuscript and both are experts in the field. Unfortunately, my analysis of the paper falls more in line with reviewer 1. My major concerns are as follows: 1) There are missing data in both figures without any explanations, 2) lack of statistical analyses for comparing data, and 3) inconsistencies between the data in your study and to previously-published results on temporal gene expression in Chlamydia (see citation [10]). For example, the late-cycle genes examined (hctA, omcB) are only modestly expressed at all the time points, and a mid-cycle gene (pykF) peaked at 2 hpi. [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: No 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 examines developmental gene regulation of 4 Chlamydia trachomatis strains in an immortalized human keratinocyte cell line. Six chlamydial genes shown in multiple studies to be differentially expressed throughout the developmental cycle are used to analyze temporal gene expression at 5 different time points. There may be some utility in this study for those studying chlamydial strains infected keratinocytes. However, the results really don't follow any patterns established in the literature and do not appear to be consistent between Figs 1 and 2. I have only a few comments for consideration. 1. line 87. The HaCaT cell line may be useful in specific instances but I doubt that it is "the most appropriate cell line" to use. Statements of significance or priority should be minimized. 2. There are no statistics for Figures 1 or 2. The legend of Fig 1 states that the standard deviations were incorporated into the fold-difference calculations. Perhaps a more qualified statistician might be needed to evaluate this but the statement on line 163 that " could not produce any p-values due to limited data points" 3. There are missing data points in both figures with no explanation. There are no discernable patterns in Fig. 1. Indeed, in the bottom 3 panels some genes did not change at all. The differences do not really seem congruent with Fig. 2. 4. Caution should be exercised in interpreting results from the earliest time points. Carryover mRNA is known to be present in EBs and not due to early transcription. 5. line 200. "G (E) strain" What is this? Does G represent genital. If so, please spell it out and identify serovars as such. 6. Urogenital strains are generally slower growing than LGV strains. Some of the LGV isolates seem to have different patterns of expression. A better way to get at this would be one step growth curves plotting numbers of progeny IFUs over time. 7. Fig 3. Scale bars are unreadable and it appears that different magnifications are shown. It would be easier to evaluate if all images were at a similar magnification. 8. The manuscript would benefit from editing for English grammar. Reviewer #2: The manuscript by Mzobe and colleagues addresses chlamydial gene expression in keratinocytes. The work is thorough and conducted well. The conclusions, though only modestly incremental, are sufficiently significant to warrant publication. The electron microscopy images will be particularly useful to readers. I have no significant criticisms of the study, the following are minor changes that should be addressed by the authors: Line 35: keratinocytes should be plural Line 45: Use Chlamydia spp., and italicize. Line 49: “can occur either as…” should be changed. Line 62: indicate which inc Line 104: “strains” should be used in place of “serovars”. Same with line 120 Line 198: Why use biovar here instead of serovar? Figure 2: The color for US151 should be changed. The distinction will not be visible to most readers. There is some funniness in the references that should be worked out. ****** 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.0249358.r001
Revision 1
9 Mar 2021 Author Response Reviewer #1: This manuscript examines developmental gene regulation of 4 Chlamydia trachomatis strains in an immortalized human keratinocyte cell line. Six chlamydial genes shown in multiple studies to be differentially expressed throughout the developmental cycle are used to analyze temporal gene expression at 5 different time points. There may be some utility in this study for those studying chlamydial strains infected keratinocytes. However, the results really don't follow any patterns established in the literature and do not appear to be consistent between Figs 1 and 2. I have only a few comments for consideration. 1. line 87. The HaCaT cell line may be useful in specific instances but I doubt that it is "the most appropriate cell line" to use. Statements of significance or priority should be minimized. Response: We have since downplayed the statement and changed to “HaCaT cells are one of the cell lines than can be used to interrogate cell differentiation, growth kinetics, and gene expression of C. trachomatis infection” line 98 of the manuscript 2. There are no statistics for Figures 1 or 2. The legend of Fig 1 states that the standard deviations were incorporated into the fold-difference calculations. Perhaps a more qualified statistician might be needed to evaluate this but the statement on line 163 that " could not produce any p-values due to limited data points" Response: We have consulted with a qualified senior statistician in our group and she shared the same sentiments that p values can only be produced when there is a hypothesis to be tested. She added that, statistically, p value gives us the probability of obtaining data as extreme as or more extreme than observed given the null hypothesis. On these basis, it mean that the p value and null hypothesis are tied together. Our study is descriptive with limited data points and we don’t intend to produce any inferential statistics. 3. There are missing data points in both figures with no explanation. There are no discernable patterns in Fig. 1. Indeed, in the bottom 3 panels some genes did not change at all. The differences do not really seem congruent with Fig. 2. Response: We have since provided detailed explanation on missing data points in the text and figure legends in the manuscript. For Fig. 1: Some of the genes in response to serovar infections could not be expressed at 2 (strain L2: tal), 12 (strain L2: inc B, tal, omcB; serovar E: hctA; US151: tal, omcB), 24 (serovar E: omcB; US151: tal, omcB), 36 (serovar E: incB, pyk-F, tal, hctA, omcB; US151: tal, omcB), 48 hours post-infection (strain L2: tal, hctA, omcB; serovar E: tal; US151: htcA) using qPCR. For figure 2: Gene expression profile of certain genes in HeLa cells could not be detect at 2 (strain L2: pyk-F, hctA; US151: pyk-F, htcA) and 12 hours (L2: hctA; US151:hctA) post infection using qPCR, which is in keeping with published data. Fig. 1 shows gene expression in HaCaT cells only, whereas Fig.2 compares expression of 3 genes (one for each chlamydial developmental stage) in HaCaT (L2: maroon; US151: blue) Vs HeLa (L2: orange; US151: purple) cells. Patterns for gene expression levels in HaCaT cells (Fig. 2) are in agreement with the patterns shown in Fig.1. However, the patterns observed in HeLa cells are in keeping with previously published data and not our HaCaT cell derived findings. 4. Caution should be exercised in interpreting results from the earliest time points. Carryover mRNA is known to be present in EBs and not due to early transcription. Response: we note the reviewer’s valuable comment and we have added this to our limitation section in the discussion and reads as follows: “Taken together, the observed differences in transcriptional expression of chlamydial genes between reference strain (L2 434) and clinical isolates (US151 and serovar E) and inconsistency with published data may not be attributed exclusively to the type of model or the MOI used, but to several other factors. These include carryover mRNA that may be present in EBs, high-passage numbers, different regulatory systems for gene expression in HaCaT compared to HeLa cell lines, and the origin (site of infection) of the isolated strain.” 5. line 200. "G (E) strain" What is this? Does G represent genital. If so, please spell it out and identify serovars as such. Response: "G (E) strain" has been changed to “the genital biovar (serovar E)”. This has also been corrected throughout the manuscript. 6. Urogenital strains are generally slower growing than LGV strains. Some of the LGV isolates seem to have different patterns of expression. A better way to get at this would be one step growth curves plotting numbers of progeny IFUs over time. Response: this study is an ancillary of a parent study (Joubert and Sturm, 2011) that has done the growth curves plotting numbers of progeny IFUs over time Joubert, B. C., and Sturm, A. W. (2011). Differences in Chlamydia trachomatis growth rates in human keratinocytes among lymphogranuloma venereum reference strains and clinical isolates. J. Med. Microbiol. 60, 1565–1569. doi: 10.1099/jmm.0.032169-0 7. Fig 3. Scale bars are unreadable and it appears that different magnifications are shown. It would be easier to evaluate if all images were at a similar magnification. Response: Scale bars have been made visible. Magnification is similar across each time point. 8. The manuscript would benefit from editing for English grammar. Response: this has been resolve throughout the manuscript Reviewer #2: The manuscript by Mzobe and colleagues addresses chlamydial gene expression in keratinocytes. The work is thorough and conducted well. The conclusions, though only modestly incremental, are sufficiently significant to warrant publication. The electron microscopy images will be particularly useful to readers. I have no significant criticisms of the study, the following are minor changes that should be addressed by the authors: 1. Line 35: keratinocytes should be plural Response: “Keratinocyte” has been changed to “keratinocytes” 2. Line 45: Use Chlamydia spp., and italicize. Response: “Chlamydia” was changed to “Chlamydia spp.” 3. Line 49: “can occur either as…” should be changed. Response: “can occur either as” to “In the extracellular phase, the organism manifests as the elementary body (EB)” 4. Line 62: indicate which inc Response: Specified inc protein as incB 5. Line 104: “strains” should be used in place of “serovars”. Same with line 120 Response: “serovars” changed to “strains” 6. Line 198: Why use biovar here instead of serovar? Response: a combination of serovar can be termed biovar 7. Figure 2: The color for US151 should be changed. The distinction will not be visible to most readers. Response: The colour for US151 in figure 2 has been changed from red to purple. 8. There is some funniness in the references that should be worked out. Response: References have been sorted out Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0249358.r002
17 Mar 2021 Decision Letter - Michael F Minnick, Editor Differential expression of groEL-1, incB, pyk-F, tal, hctA and omcB genes during Chlamydia trachomatis developmental cycle PONE-D-21-02174R1 Dear Dr. Mzobe, 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, Michael F Minnick, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): 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: 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: Partly Reviewer #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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 https://doi.org/10.1371/journal.pone.0249358.r003
Formally Accepted
7 Apr 2021 Acceptance Letter - Michael F Minnick, Editor PONE-D-21-02174R1 Differential expression of groEL-1, incB, pyk-F, tal, hctA and omcB genes during Chlamydia trachomatis developmental cycle Dear Dr. Mzobe: 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. Michael F Minnick Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0249358.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 .