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Selective sugar transport supports Proteus mirabilis fitness in the urinary tract

PLOS Pathogens

Dear Dr. Pearson,

Thank you for submitting your manuscript to PLOS Pathogens. Your manuscript was evaluated by members of the editorial board and three external reviewers. All agree that it has potential to be an important contribution, but there are concerns that need to be addressed. Therefore, we invite you to submit a substantially revised version of the manuscript that addresses all of the points raised by all three reviewers. Reviewers 1 and 3, in particular, raise a number of points that should be addressed, including the genetic complementation of key mutants in at least some assays.

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Kind regards,

Matthew A Mulvey, PhD

Academic Editor

PLOS Pathogens

D. Scott Samuels

Section Editor

PLOS Pathogens

-->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

-->-->Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript from Shea et al. addresses the role of sugar import systems in the fitness of P. mirabilis, a clinically significant uropathogen. Specifically, they generated 47 mutants lacking various sugar transport systems and tested the role of these genes in fitness under various in vitro conditions and in the mouse model of urinary tract infection. This work unravels the role of several new fitness factors that are involved in the pathogenesis of UTI caused by P. mirabilis. They demonstrate that increased urine glucose, simulating diabetic urinary tract, enhances pathogen colonization in a complex mechanism since there is inverse correlation between urinary glucose levels and bacterial colonization. This Reviewer finds the use of InSeq for in vivo fitness assessment as a robust quantitative approach that overcomes the limitations of bottleneck in the infection model. Their results also raise an important point that genome annotations must be experimentally validated and might not be accurate as presented in databases. The experimental design is rigorous. Methods are described in sufficient detail. The manuscript is well written and organized logically. This work will be of interest to researchers in the fields of host-pathogen interaction and diabetic complications.

Reviewer #2: This manuscript presents a comprehensive investigation into carbohydrate import systems relevant to Proteus mirabilis infection of the urinary tract. Some highlights include the generation of 47 targeted mutants in predicted sugar transporters, screening of these mutants in vitro to evaluate gene function, the use of pooled In seq and co challenge experiments to determine in vivo function, and evaluation of the impact of glucosuria using an SGLT2 inhibitor model. This impressive amount of work led to multiple findings, including the identification of ptsH, ptsI, and xapB as critical fitness genes, the discovery that many transporter genes have been misannotated in P. mirabilis, and the demonstration that induced glucosuria exacerbates P. mirabilis colonization.

Reviewer #3: Here the authors evaluate the contribution of genes predicted to be involved in sugar transport to Proteus mirabilis HI4320 urinary tract infection in a mouse model. Using a pooled infection/InSeq approach, the authors identify genes whose loss results in various colonization defects in the mouse UTI model. The authors attempt to identify substrates for various predicted transport systems but are largely unsuccessful as many of the predicted substrates did not support growth when provided as a sole carbon source leading to mostly inconclusive results. However the authors did identify growth defects for the ptsH mutant on several carbon and nitrogen sources. To try to understand the pathway through which ptsH was acting the authors tested the fitness of triple mutants in PTS systems and identified colonization defects for the ulaA, ptsG and scrA triple mutant. Finally the authors investigated the impact of glucosuria on P. mirabilis colonization in their mouse UTI model and fitness of the ptsH mutant using dapagliflozin. Mice treated with dapagliflozin experienced more severe P. mirabilis UTI and that the ptsH mutant was less competitive in colonization at some experimental endpoints. Overall, this work identifies sugar transport systems contributing to P. mirabilis UTI and examines the effects of SGLT-2 induced glucosuria on P. mirabilis pathogenesis.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Have the Authors investigated the growth of sugar transport mutants in human urine, and urine supplemented with glucose to resemble diabetic urine? This seems like a straightforward experiment that could yield critical insights to strengthen this manuscript.

Do the mutants with severe in vivo fitness defect exhibit changes in known virulence factors such as urease? Adding this information is needed to clarify decreased fitness due to poor in vivo growth as opposed to decreased pathogenicity.

There seems to be bacterial growth in Fig. 3A without a C source? This should be explained.

Fig 8 A&B: Baseline glucose (day -1) is almost double in panel A, compared to B, and also post-treatment differences between these panels. Potential reasons for this change should be included.

Fig. 7A: Error bars should be included to inform the readers of the range of data points

Fig. 1A: Please add gene names at least for the ones that have growth defects.

Reviewer #2: Overall, the data presented are sound, the manuscript is very well written, and the work will be of broad interest to researchers studying urinary tract infections, bacterial metabolism, and host-pathogen interactions.

I have no substantive concerns regarding the data or major conclusions. I cannot fault the work and congratulate the authors on an excellent study.

Reviewer #3: 1. There is no complementation of any of the mutants evaluated in this study. For mutants for which major study conclusions are based, complementation should be demonstrated at least for in vitro (growth) phenotypes if not for in vivo UTI phenotypes. This is imperative to ensure the most important phenotypes are not due off target or polar effects.

2. There are several instances where data to support a conclusion is not provided. For example, the data supporting that there was no variation in input CFUs (line 180) is not provided/referenced in the manuscript. The authors should carefully revise the manuscript making sure to include data for all conclusion and that it is referenced appropriately in the manuscript (e.g. Biolog data is mentioned to in line 207 but there is no dataset referenced here.)

3. Without comparing to double deletion strains, I am not sure if the authors can conclude that both ulaC and scrA along with ptsG are responsible for the ptsI/ptsH in vivo mutants. At the minimum comparisons to double ulaC/ptsG and scrA/ptsG double deletions would be needed to state this and ideally some sort of epistasis analysis would be performed to confirm that these genes are indeed acting in the same pathway as ptsH or ptsI. The phenotypes may be similar, but that does not necessarily mean they are acting through the same pathway.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: Fig 8, S3, S8. I assume the horizontal lines indicatesthe median, but please check and add to the figure legend as appropriate.

Reviewer #3: 1. Please use exact p-values instead of asterisks throughout the manuscript. It is more transparent. Also please avoid using the language "trending towards significance". It is more useful to discuss why the current hypothesis test may have failed (low n-value due to dying mice at that experimental for example) than to suggest possible future signficance.

2. Why was there a change in the duration of the UTI models between the pooled infection/InSeq experiment and the mutant versus wild-type competition experiments. There is no rationale provided for switching from a 24 hour to a 7 day long infection timescale.

3. Line 72 needs a reference

4. Line 97 - please clarify that the findings suggest elevated glucose may impact UTI outcomes in mice as there is not strong data supporting this yet in human cohorts

5. For Figure 1A, either the functional gene names used in the text needed to be added to the figure or the locus tags used in the figure need to be referenced in the text. It is impossible to determine which ptsG or crr or ptsI, etc from how it is currently presented.

6. The current figure 5 may be more well suited as a supplemental figure that can also be referenced earlier in the manuscript when PtsH, PtsI and Crr are first mentioned in line 161.

7. For current figures S1 and S2 please indicate which statistical test was used in the figure legends.

8. Figure S3 data seems very important for interpreting CI data in figure 2 and should be included in the main text as part of figure 2. There should be plenty of room in the current figure 2 to add these data.

9. Throughout the manuscript it is unclear if Biolog results represent growth or metabolic activity. The primary output of Biolog phenotype arrays is usually metabolic activity via reduction of a tetrazolium-based redox dye. Is this what the authors are reporting or is it the OD600 (growth) in the absence of the dye?

10. Please include the recipe for Minimal A in the methods section instead of just a reference.

11. Figure S4 A and B look like two individual runs of the same experiment. These should be combined into a single graph that should be included in the main text in figure 4. Also there is no data presented to support the statement that no CFUs were recovered at the endpoint in line 223.

12. The red/yellow arrows in the current figure 5 are confusing and it would be helpful if they could be explained in the legend or in the text.

13. The references in line 266 need to be corrected. Reference 48 is in Proteus vulgaris and not Proteus mirabilis so it should be removed. Instead the authors should reference https://doi.org/10.3389/fcimb.2021.803409 which demonstrated degradation and utilization of chondroitin sulfate for growth by multiple strains of Proteus mirabilis.

14. The data in Figure S5 seem like they should be included in the main text. It would also be important to give a more detailed explanation for the conclusion on line 280 - for example, what is the nitrogen source in Minimal a and why exactly does this explain the glucose defects in that medium?

15. Overall, there is a difference between a given sugar not being a PTS substrate and it not being able to serve as a sole carbon source. Just because it cannot act as a sole carbon source does not necessarily mean it is not a substrate for a given PTS system. Perhaps in a different condition, it would stimulate growth and this growth would be reduced by deletion of that PTS component. I think maybe some of the conclusions could be updated with this point in mind.

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Reviewer #1: No

Reviewer #2: Yes:  Mark Schembri

Reviewer #3: Yes:  Nicole Janell De Nisco

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Dear Dr. Pearson,

We are pleased to inform you that your manuscript 'Selective sugar transport supports Proteus mirabilis fitness in the urinary tract' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

Matthew A Mulvey, PhD

Academic Editor

PLOS Pathogens

D. Scott Samuels

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):