The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence

Teri N. Hreha; Sara Foreman; Ana Duran-Pinedo; Andrew R. Morris; Patricia Diaz-Rodriguez; J. Andrew Jones; Kristina Ferrara; Anais Bourges; Lauren Rodriguez; Mattheos A. G. Koffas; Mariah Hahn; Alan R. Hauser; Blanca Barquera

doi:10.1371/journal.pone.0244142

Peer Review History

Original SubmissionAugust 30, 2020
12 Nov 2020 Decision Letter - Alessandro Giuffrè, Editor PONE-D-20-25026 The three NADH dehydrogenases of Pseudomonas aeruginosa: their roles in energy metabolism and links to virulence PLOS ONE Dear Dr. Barquera, 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. The work is clear, solid and of interest. A concerned was raised by Reviewer #1 about your mice infection data as the results of just one experiment are shown in Fig.8A, although the experiment was repeated twice. Please address this concern, provide the requested details about the mutant strains and revise the manuscript as constructively advised by both reviewers. Please submit your revised manuscript by Dec 27 2020 11:59PM. 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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. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript describes the phenotypes of deletion strains for NDH-2, NQR and NUO in P. aeruginosa. The manuscript is well written and clearly describes differences between the reported results and existing literature. The use of LB and SCFM for experimentation, as well as stationary and log phase is helpful. The authors have used PCR to confirm the identities of the transposon strains used. The finding that MexEF-OprN is differentially regulated in the �Nqr strain is interesting. The finding that Major Comments 1. Please show in the manuscript (or provide to the reviewers) the data for the other replicate of the mouse experiment. Demonstration of only one replicate is not adequate for evaluation. Minor Comments • Line 63, would briefly mention the ability of P. aeruginosa to anaerobically respire. This adds even more branches to the respiratory chain (and is an acceptable limitation to this data set). • Format organism names in the bibliography uniformly. • Table 1: Refer to insertion deletion mutants using ::Tn or something similar to differentiate the from the chromosomal deletions (see https://www.gs.washington.edu/labs/manoil/libraryindex.htm). Are the double mutants done in the ::Tn backgrounds? If so, was the abx cassette from a Tn left in place? For the purposes of the body of the manuscript, the chosen nomenclature is adequate because it improves clarity of the writing. • For growth curves, please present data as log-OD (Figures 1, 2, S1, etc). • Table 3: Doublecheck the > and < signs in the ��ndh column • Line 541 and 542: comment still present in document from Word. Reviewer #2: Hreha et al. characterize the function of the three NADH dehydrogenases in P. aeruginosa. They determine that NQR has the greatest dehydrogenase activity and that its absence increases the cytotoxin pyocyanin, macrophage death, and mouse mortality. Their results suggest a model in which NADH dehydrogenase activity through NQR negatively regulates pyocyanin and virulence. Overall, the topic is very interesting and the results provide further evidence for a causal link between NADH metabolism and pathogenesis. My only comments are requests for further clarity on the issues below. Minor issues: 1. Line 308: ref 29 does not appear to be an appropriate reference for SCFM medium. 2. Fig. 7. The y-axis appears to be the fold-change in macrophage death compared to the bacteria-free control. However, the extent of death in bacteria-free control should be the minimal and the death in the other groups is therefore difficult to interpret. I suggest the authors plot something more intuitive such as % cell death or % viability instead. 3. Section starting at line 656: Title mentions biofilm formation but this is not discussed in the paragraph that follows. 4. Line 229: “4V%” looks like a typo. 5. Lines 446 & 447: “propodium” should be “propidium” 6. Line 473: There are no stars in Fig. 5 ****** 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. 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Revision 1
2 Dec 2020 Author Response Reviewer #1: This manuscript describes the phenotypes of deletion strains for NDH-2, NQR and NUO in P. aeruginosa. The manuscript is well written and clearly describes differences between the reported results and existing literature. The use of LB and SCFM for experimentation, as well as stationary and log phase is helpful. The authors have used PCR to confirm the identities of the transposon strains used. The finding that MexEF-OprN is differentially regulated in the DNqr strain is interesting. The finding that Major Comments 1. Please show in the manuscript (or provide to the reviewers) the data for the other replicate of the mouse experiment. Demonstration of only one replicate is not adequate for evaluation. We have included data for the replica of the mouse experiments (Figure 8). These data are included in S7 Figure. Minor Comments • Line 63, would briefly mention the ability of P. aeruginosa to anaerobically respire. This adds even more branches to the respiratory chain (and is an acceptable limitation to this data set). We have modified the text to include the referee’s comment. • Format organism names in the bibliography uniformly. This was corrected. • Table 1: Refer to insertion deletion mutants using ::Tn or something similar to differentiate the from the chromosomal deletions (see https://www.gs.washington.edu/labs/manoil/libraryindex.htm). Are the double mutants done in the ::Tn backgrounds? If so, was the abx cassette from a Tn left in place? For the purposes of the body of the manuscript, the chosen nomenclature is adequate because it impr9ves clarify of the writing. We have now included a clarification of the nomenclature used for the mutants (Line 11.) The transposon mutants were also described in Table 1 using the “ ::Tn nomenclature”. • For growth curves, please present data as log-OD (Figures 1, 2, S1, etc). We have included new Figures 1 and 2 presenting the growth curves in log scale. The original figures are included as S2 and S3 Figures. • Table 3: Doublecheck the < and > signs in the Dndh column This was corrected. • Line 541 and 542: comment still present in document from Word. This was corrected. Reviewer #2: Hreha et al. characterize the function of the three NADH dehydrogenases in P. aeruginosa. They determine that NQR has the greatest dehydrogenase activity and that its absence increases the cytotoxin pyocyanin, macrophage death, and mouse mortality. Their results suggest a model in which NADH dehydrogenase activity through NQR negatively regulates pyocyanin and virulence. Overall, the topic is very interesting and the results provide further evidence for a causal link between NADH metabolism and pathogenesis. My only comments are requests for further clarity on the issues below. Minor issues: 1. Line 308: ref 29 does not appear to be an appropriate reference for SCFM medium This was corrected. 2. Fig. 7. The y-axis appears to be the fold-change in macrophage death compared to the bacteria-free control. However, the extent of death in bacteria-free control should be the minimal and the death in the other groups is therefore difficult to interpret. I suggest the authors plot something more intuitive such as % cell death or % viability instead. We have included a new Figure 7. This figure shows the % of cell death. 3. Section starting at line 656: Title mentions biofilm formation but this is not discussed in the paragraph that follows. The title has been modified. 4. Line 229: “4V%” looks like a typo. This was corrected. 5. Lines 446 & 447: “propodium” should be “propidium” This was corrected. 6. Line 473: There are no stars in Fig. 5. This was corrected. Attachments Attachment Submitted filename: Reviewers commments and response 12.2.20.docx https://doi.org/10.1371/journal.pone.0244142.r002
4 Dec 2020 Decision Letter - Alessandro Giuffrè, Editor The three NADH dehydrogenases of Pseudomonas aeruginosa: their roles in energy metabolism and links to virulence PONE-D-20-25026R1 Dear Dr. Barquera, 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, Alessandro Giuffrè Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0244142.r003
Formally Accepted
9 Dec 2020 Acceptance Letter - Alessandro Giuffrè, Editor PONE-D-20-25026R1 The three NADH dehydrogenases of Pseudomonas aeruginosa: their roles in energy metabolism and links to virulence Dear Dr. Barquera: 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 Alessandro Giuffrè Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244142.r004

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