The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1

Luis Fernando Montelongo-Martínez; Miguel Díaz-Guerrero; Verónica Roxana Flores-Vega; Martín Paolo Soto-Aceves; Roberto Rosales-Reyes; Sara Elizabeth Quiroz-Morales; Bertha González-Pedrajo; Gloria Soberón-Chávez; Miguel Cocotl-Yañez

doi:10.1371/journal.pone.0307174

Peer Review History

Original SubmissionJanuary 8, 2024
20 Feb 2024 Decision Letter - Rajesh P. Shastry, Editor PONE-D-24-00964The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1.PLOS ONE Dear Dr. Cocotl-Yanez, 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 05 2024 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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GS-Ch and BG-P research were supported by DGAPA, PAPIIT UNAM grant IN201222 and IN229023, respectively. We thank Dr. Annia Rodríguez Hernández from the Cellular and Molecular Pharmacology Department of the University of California San Francisco (UCSF), for critical reading of the manuscript, and Abigail González Valdez and Norma Espinosa for technical support.” We note that you have provided funding information that is 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. 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[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: 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: 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: The current study investigates the QS essential proteins namely Las, Rhl, and Pqs QS receptors, and inducers' role in the T3SS activation in P. aureginosa. I find these researches that correlate the QS activation to other bacterial activity is an essential area and unfortunately, there are no sufficient studies. However, I have a lot of concerns regarding this study as it requires further deep investigations, I still recommend its publication as it could shed the light in this direction. 1- What is the 1ry antibody used in the western blot 2- I think it would be nice if you can quantify the expresed proteins (use flourescent 2ry antibody and quantify the flourecence, for instance) to obtain statistical significance 3- Line 52, please re-write "Mutant strains defective in this system are severely attenuated in their virulence" with clarification 4- Please clarify the methodology section to be more clear to non-experts, as clarifying the importance of Groel protein as reference one 5- The discussion is well understood, however I recommend writing more detailed conclusion 6- I may recommend the authors to draw a represenative graph illustrating their findings 7- Furthermore, I may advise the authors to extend their findings showing the impact on the bacterial virulence in vitro by quantification of pyocyanin as an example to QS or immunstining to the exoS proteins in macrophages or HeLa cells, as an example Reviewer #2: This paper examines the role of the global regulator RhlR in the regulation of Pseudomonas aeruginosa type 3 secretion system (T3SS). The transcriptional regulator RhlR was initially characterised at the end of the last century as the receptor for the quorum-sensing signal molecule: N-butanoyl-L-homoserine lactone (C4). However, it is now known that this protein has multiple mechanisms of action, both C4-dependent and C4-independent. The authors re-examine what the role of QS might be in the regulation of T3SS in light of today's knowledge. Briefly, by means of transcriptional fusions and western-blot experiments, evidence is brought to support the hypothesis that RhlR positively regulates the expression of ExoS (the main effector of T3SS) in a C4-independent manner. Preliminary evidence is also brought to support the hypothesis that RhlR can regulate exoS transcription by positively controlling the promoter of the exsCEBA operon, which in turn encodes for the complex regulatory system that controls T3SS gene expression. Overall, the authors show that: i) RhlR positively regulates the expression of exoS regardless of the presence of the C4 inducer; ii) this mechanism involves activation of the exsCEBA operon promoter; iii) constitutive transcription in trans of exsA, the main activator of T3SS, restores the expression of exoS in a rhlR mutant. This is interesting work because it disproves the common belief that QS negatively regulates T3SS expression and draws attention to the multiple mechanisms by which RhlR can control P. aeruginosa virulence. Unfortunately, the work remains somewhat superficial and does not go deeper into the mechanism underlying the RhlR-dependent regulation of exoS and exCEBA genes. Overall, the work is a bit preliminary and should be enriched with further experiments. Below there are some major issues that the authors need to address in order to get the work published: 1) The regulation of T3SS is very complex, the authors should explain it a little better in the introduction, possibly by including a figure in supplementary showing the exsCEBA operon and the exoS and spcS genes, also indicating the promoters cloned into transcriptional fusions with the lux genes. Concerning T3SS, it would be useful to cite Horna & Ruiz's review (doi:10.1016/j.micres.2021.126719). 2) In Figures 1, 2 and 3, it is not clear whether the experiments were conducted under inducing or not-inducing conditions. In any case, the authors should conduct the experiments shown in Figures 1, 2 and 3 under both conditions. 3) The names given to transcriptional fusions (pS, pP, pC, pA) are too contracted and do not help understanding. Authors should name the promoters as per convention (es. promoter of exoS gene = PexoS. Fusion between PexoS and luxABCDE = PexoS::luxABCDE, the latter could be abbreviated as PexoS::lux). 4) The PexsCEBA promoter is activated by RhlR in a C4- and PqsE-independent manner. Thus, this regulation appears to be indirect. The authors speculate that the regulation might depend on the spermidine binding protein PA2592 (lines 457-463). The authors should construct this mutant and test this hypothesis. 5) The PexsCEBA promoter is regulated by other regulators such as PrsA/RpoS, ArtR, RtsM (Horna & Ruiz of 2021 and references therein). Is there a relationship between these regulators and RhlR? The authors should answer this question referring to literature data or by conducting experiments. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Wael A. H. Hegazy 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.0307174.r001
Revision 1
4 Apr 2024 Author Response 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: No. R: We carried out an additional statistical analysis to be more rigorous with our results, this analysis has been included in the revised version. 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: The current study investigates the QS essential proteins namely Las, Rhl, and Pqs QS receptors, and inducers' role in the T3SS activation in P. aureginosa. I find these researches that correlate the QS activation to other bacterial activity is an essential area and unfortunately, there are no sufficient studies. However, I have a lot of concerns regarding this study as it requires further deep investigations, I still recommend its publication as it could shed the light in this direction. 1- What is the 1ry antibody used in the western blot R: The first antibody used was anti-ExoS, the information was included in Materials and methods and in the figure legends. 2- I think it would be nice if you can quantify the expresed proteins (use flourescent 2ry antibody and quantify the flourecence, for instance) to obtain statistical significance R: Thank you for your suggestion. We carried out a densitometry analysis to obtain statistical significance using at least three Western blot assays and it was included in the Western blot figures. Also, the methodology was included (lines 264-268). 3- Line 52, please re-write "Mutant strains defective in this system are severely attenuated in their virulence" with clarification R: Thanks for your suggestion. The sentence was modified for clarification (lines 51-52) 4- Please clarify the methodology section to be more clear to non-experts, as clarifying the importance of Groel protein as reference one R: The methodology section was reviewed and modified to be clear. 5- The discussion is well understood, however I recommend writing more detailed conclusion. R: We have written a more detailed conclusion according to your suggestion (lines 562-565) 6- I may recommend the authors to draw a represenative graph illustrating their findings R: Thank you for your recommendation. A figure summarizing our findings has been included (Fig 9). 7- Furthermore, I may advise the authors to extend their findings showing the impact on the bacterial virulence in vitro by quantification of pyocyanin as an example to QS or immunstining to the exoS proteins in macrophages or HeLa cells, as an example R: As you suggested, we quantified pyocyanin production in T3SS-induction conditions. This was included in the revised manuscript (lines 258-262 and 458-476). Reviewer #2: This paper examines the role of the global regulator RhlR in the regulation of Pseudomonas aeruginosa type 3 secretion system (T3SS). The transcriptional regulator RhlR was initially characterised at the end of the last century as the receptor for the quorum-sensing signal molecule: N-butanoyl-L-homoserine lactone (C4). However, it is now known that this protein has multiple mechanisms of action, both C4-dependent and C4-independent. The authors re-examine what the role of QS might be in the regulation of T3SS in light of today's knowledge. Briefly, by means of transcriptional fusions and western-blot experiments, evidence is brought to support the hypothesis that RhlR positively regulates the expression of ExoS (the main effector of T3SS) in a C4-independent manner. Preliminary evidence is also brought to support the hypothesis that RhlR can regulate exoS transcription by positively controlling the promoter of the exsCEBA operon, which in turn encodes for the complex regulatory system that controls T3SS gene expression. Overall, the authors show that: i) RhlR positively regulates the expression of exoS regardless of the presence of the C4 inducer; ii) this mechanism involves activation of the exsCEBA operon promoter; iii) constitutive transcription in trans of exsA, the main activator of T3SS, restores the expression of exoS in a rhlR mutant. This is interesting work because it disproves the common belief that QS negatively regulates T3SS expression and draws attention to the multiple mechanisms by which RhlR can control P. aeruginosa virulence. Unfortunately, the work remains somewhat superficial and does not go deeper into the mechanism underlying the RhlR-dependent regulation of exoS and exCEBA genes. Overall, the work is a bit preliminary and should be enriched with further experiments. Below there are some major issues that the authors need to address in order to get the work published: 1) The regulation of T3SS is very complex, the authors should explain it a little better in the introduction, possibly by including a figure in supplementary showing the exsCEBA operon and the exoS and spcS genes, also indicating the promoters cloned into transcriptional fusions with the lux genes. Concerning T3SS, it would be useful to cite Horna & Ruiz's review (doi:10.1016/j.micres.2021.126719). R: Thank you for your suggestion. In this revised version we have included two additional supplementary figures related to the regulation of the T3SS and the transcriptional fusions constructed in this work (Fig S1 and S2). Also, the cite Horna and Ruiz was included. 2) In Figures 1, 2 and 3, it is not clear whether the experiments were conducted under inducing or not-inducing conditions. In any case, the authors should conduct the experiments shown in Figures 1, 2 and 3 under both conditions. R: All experiments were conducted under inducing conditions since in LB the T3SS is not active. We have included this information in the revised version and also, we have included a supplementary figure where we showed that ExoS is not detected in non-induction conditions (lines 281-282, 498-500) (Fig S6). 3) The names given to transcriptional fusions (pS, pP, pC, pA) are too contracted and do not help understanding. Authors should name the promoters as per convention (es. promoter of exoS gene = PexoS. Fusion between PexoS and luxABCDE = PexoS::luxABCDE, the latter could be abbreviated as PexoS::lux). R: Names of plasmids were changed in the text according to your suggestion. 4) The PexsCEBA promoter is activated by RhlR in a C4- and PqsE-independent manner. Thus, this regulation appears to be indirect. The authors speculate that the regulation might depend on the spermidine binding protein PA2592 (lines 457-463). The authors should construct this mutant and test this hypothesis. R: In this work, our main objective was to define the role of the quorum sensing systems on the T3SS expression. Therefore, we did not determine the target of RhlR to control the T3SS expression. However, according to your suggestion, in this revised version, we explored whether PA2595 has a role in the T3SS control. Our approach was to evaluate whether the expression of PA2592 under a constitutive promoter was able to restore ExoS secretion in the rhlR mutant strain. These results were included as a new section (lines 432-456). Also, the discussion was modified according to these results (lines 551-560). 5) The PexsCEBA promoter is regulated by other regulators such as PrsA/RpoS, ArtR, RtsM (Horna & Ruiz of 2021 and references therein). Is there a relationship between these regulators and RhlR? The authors should answer this question referring to literature data or by conducting experiments. R: Thank you for your comment. There is not a relationship previously reported between RhlR and PsrA, ArtR, or RetS (RtsM). Also, we searched for a las-box in these and other regulators of the T3SS reported by Horna & Ruiz but we could not find any consensus sequence for RhlR. We discussed these findings in the discussion section (lines 547-551). 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Wael A. H. Hegazy Reviewer #2: No Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0307174.r002
14 May 2024 Decision Letter - Rajesh P. Shastry, Editor PONE-D-24-00964R1The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1.PLOS ONE Dear Dr. Cocotl-Yanez, 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 28 2024 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, Rajesh P. Shastry, Ph.D 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 #3: (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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: 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 #3: 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 #3: 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 #3: In this MS Cocotl-Yanez et al. delve into the role of QS in regulating T3SS in Pseudomonas aeruginosa. Since QS has branching global effects on virulence in this pathogen, this is a very relevant topic and the results provided by this group shed some light on T3SS regulation and provide highly needed clarifications to some experimental discrepancies found regarding this topic. The authors make a sound analysis on the regulation of the T3SS and provide solid data that support most of their conclusions. Although I consider it a high quality work and I would support this MS for its acceptance, there are a few concerns that I think need to be addressed regarding some of their conclusions: Major concerns: The authors conclude that, as overexpressing exsA restores ExoS production in a rhlR mutant, RhlR controls the T3SS expression via ExsA. This is not necessarily the case, they can both act independently, and overexpressing one of the two independent activators may still correct the lack of the other. Their data proves that RhlR does not act downstream of ExsA, but there is still the question if RhlR acts upstream of ExsA as they claim or if they act in different signaling pathways. This would be clarified if they introduced their rhlR overexpression plasmid in a exsA mutant background. If overproducing RhlR can also restore ExoS production they might be independent activators. On the other hand, if ExoS production is not restored, it may indicate that both regulators act hierarchically as they describe. I have trouble relating ExoS quantifications in figure 5b with their associated western blot data. What I see in the graphs is not an increase in ExoS protein levels in the rhlI mutant background as claimed in the main text (L399) and glimpsed in the western blot (in any case average levels in the graphs seem to be higher in the pqsE mutant although not significant). Also the statistical analysis does not seem to indicate that any of the mutants besides rhlR is significantly different from the wild type to make any of these statements. Please clarify these discrepancies. Minor concerns: Also regarding ExsA and RhlR relationship, it would be interesting to see if the internal PexsA promoter is also affected by the rhlR mutation. L118: I fail to understand why are there discrepancies on T3SS regulation by the Las system. It is clear why Rhl role in T3SS needs further clarification, but all evidence discussed up to this point about Las system seems to agree that it is not involved. I would suggest changing this to “there are some discrepancies in the virulence and regulation of the T3SS by QS systems” so it is more accurate. L302: The first section of the results jumps from figure 1 to figure 3. If the authors consider that discussing this mutant at this point of the MS is important I would suggest to rename it as figure 2. Otherwise they can try to introduce this result at a later point. L395: As they also mentioned in the introduction, RhlR is known to be able to act independently of C4. I would rephrase this to “can be dependent” or other formulation that better reflects that this is not an absolute dependence. L401-404: It is a bit hard to understand why this result is discussed here, and it also introduces a bit of difficulties in following the reasoning of the experimental procedure. I would move this phrase to a different point, maybe after discussing the results on Fig 6, so it is easier to follow and shows in a clear way why there are differences between exsC promoter activities in Fig 6 and S5. L438, 442, 551, and 553: The authors probably mean a rhl box or a las-rhl box? L461: The authors should clarify how their results show dependence on C4 and PqsE. By looking at figure 8 one can see PqsE dependence, but this is not discussed in the text, and C4 dependence is equally not properly described in this section despite being shown in the picture. Please reformulate this whole section so it is clear to the reader why your data supports your claims (which does). Minor errata and things the authors may want to check before submitting their final version: L73: control. L127: show. L170: remove the primer sequence since it is already in the supplementary table and no other primer sequences are shown. L265: A citation may be missing here for “as reported previously”. ****** 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: Yes: Wael A. H. Hegazy Reviewer #3: Yes: Francisco Javier Marcos-Torres ******** [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". 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Revision 2
17 Jun 2024 Author Response June 17, 2024 Rajesh P. Shastry, Ph.D Academic Editor PLOS ONE In this document, we respond to the comments and suggestions made by the reviewer for the manuscript PONE-D-24-00964R1 entitled ‘The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1’ by Luis Fernando Montelongo-Martínez, Miguel Díaz-Guerrero, Verónica Roxana Flores-Vega, Martín Paolo Soto-Aceves, Roberto Rosales-Reyes, Sara Elizabeth Quiroz-Morales, Bertha González-Pedrajo, Gloria Soberón-Chávez, Miguel Cocotl-Yañez. Best regards Miguel Cocotl-Yañez Corresponding author. 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 #3: (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 #3: Yes 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: 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 #3: 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 #3: 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 #3: In this MS Cocotl-Yanez et al. delve into the role of QS in regulating T3SS in Pseudomonas aeruginosa. Since QS has branching global effects on virulence in this pathogen, this is a very relevant topic and the results provided by this group shed some light on T3SS regulation and provide highly needed clarifications to some experimental discrepancies found regarding this topic. The authors make a sound analysis on the regulation of the T3SS and provide solid data that support most of their conclusions. Although I consider it a high quality work and I would support this MS for its acceptance, there are a few concerns that I think need to be addressed regarding some of their conclusions: Major concerns: The authors conclude that, as overexpressing exsA restores ExoS production in a rhlR mutant, RhlR controls the T3SS expression via ExsA. This is not necessarily the case, they can both act independently, and overexpressing one of the two independent activators may still correct the lack of the other. Their data proves that RhlR does not act downstream of ExsA, but there is still the question if RhlR acts upstream of ExsA as they claim or if they act in different signaling pathways. This would be clarified if they introduced their rhlR overexpression plasmid in a exsA mutant background. If overproducing RhlR can also restore ExoS production they might be independent activators. On the other hand, if ExoS production is not restored, it may indicate that both regulators act hierarchically as they describe. R: Thank you for your suggestion. We overexpressed rhlR in a strain defective in the T3SS and transcription of exsCEBA and exoS were measured, the results are described in lines 378-387. I have trouble relating ExoS quantifications in figure 5b with their associated western blot data. What I see in the graphs is not an increase in ExoS protein levels in the rhlI mutant background as claimed in the main text (L399) and glimpsed in the western blot (in any case average levels in the graphs seem to be higher in the pqsE mutant although not significant). Also the statistical analysis does not seem to indicate that any of the mutants besides rhlR is significantly different from the wild type to make any of these statements. Please clarify these discrepancies. R: Thank you for your observation. We have corrected it (lines 416-417) and also it was discussed (lines 556-561). Minor concerns: Also regarding ExsA and RhlR relationship, it would be interesting to see if the internal PexsA promoter is also affected by the rhlR mutation. R: Thanks for your comment. We found that exsA internal promoter is barely expressed and is not activated by induction conditions, thus we focused on determining the role of the QS systems on the T3SS genes that were activated in induction conditions. L118: I fail to understand why are there discrepancies on T3SS regulation by the Las system. It is clear why Rhl role in T3SS needs further clarification, but all evidence discussed up to this point about Las system seems to agree that it is not involved. I would suggest changing this to “there are some discrepancies in the virulence and regulation of the T3SS by QS systems” so it is more accurate. R: It was changed (Line 118) L302: The first section of the results jumps from figure 1 to figure 3. If the authors consider that discussing this mutant at this point of the MS is important I would suggest to rename it as figure 2. Otherwise they can try to introduce this result at a later point. R: Figure 3 was renamed as Figure 2. L395: As they also mentioned in the introduction, RhlR is known to be able to act independently of C4. I would rephrase this to “can be dependent” or other formulation that better reflects that this is not an absolute dependence. R: It was changed (Line 414) L401-404: It is a bit hard to understand why this result is discussed here, and it also introduces a bit of difficulties in following the reasoning of the experimental procedure. I would move this phrase to a different point, maybe after discussing the results on Fig 6, so it is easier to follow and shows in a clear way why there are differences between exsC promoter activities in Fig 6 and S5. R: The section was modified according to your suggestion. L438, 442, 551, and 553: The authors probably mean a rhl box or a las-rhl box? R: Thank you. It was changed to las-rhl box. L461: The authors should clarify how their results show dependence on C4 and PqsE. By looking at figure 8 one can see PqsE dependence, but this is not discussed in the text, and C4 dependence is equally not properly described in this section despite being shown in the picture. Please reformulate this whole section so it is clear to the reader why your data supports your claims (which does). R: Thank you for your comment. The section was modified and also the results were discussed (Lines 586-591). Minor errata and things the authors may want to check before submitting their final version: L73: control. R: Corrected L127: show. R: Corrected L170: remove the primer sequence since it is already in the supplementary table and no other primer sequences are shown. R: Corrected L265: A citation may be missing here for “as reported previously”. R: Corrected 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: Yes: Wael A. H. Hegazy Reviewer #3: Yes: Francisco Javier Marcos-Torres Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0307174.r004
2 Jul 2024 Decision Letter - Rajesh P. Shastry, Editor The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1. PONE-D-24-00964R2 Dear Dr. Cocotl-Yanez, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Rajesh P. Shastry, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #3: 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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: 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 #3: 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 #3: 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: From the first revision; all the raised points have been adressed and it can be published in the current form Reviewer #3: With this new version of the MS, Cocotl-Yanez et al have made a remarkable work addressing all previous concerns and strengthening their conclusions. Considering the quality of the resulting work I fully recommend for its publication without further modifications. ****** 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: Yes: Wael A. H. Hegazy Reviewer #3: Yes: Francisco Javier Marcos-Torres ******** https://doi.org/10.1371/journal.pone.0307174.r005
Formally Accepted
6 Aug 2024 Acceptance Letter - Rajesh P. Shastry, Editor PONE-D-24-00964R2 PLOS ONE Dear Dr. Cocotl-Yanez, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Rajesh P. Shastry Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307174.r006

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