Dear Dr McBride,

Thank you very much for submitting your manuscript "A Conserved Switch Controls Virulence, Sporulation, and Motility in C. difficile" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a revised version that takes into account the reviewers' comments.

The Reviewers were enthusiastic about the finding that Spo0E regulates multiple processes in both C. difficile and B. subtilis. The finding that Spo0E interacts directly or indirectly with Spo0A and RstA in C. difficile is an important contribution, but Reviewers 1 and 3 request additional verification of these mass spectrometry-based co-IP experiments either in the form of validating the AlphaFold-predicted interactions or through another experimental method. They raised additional points regarding alternative interpretations of the data that can be readily addressed through textual changes. If these items are addressed, I hope to make a final decision without needing to send the manuscript out for review again.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Aimee Shen

Guest Editor

PLOS Pathogens

Michael Wessels

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

The Reviewers were enthusiastic about the finding that Spo0E regulates multiple processes in both C. difficile and B. subtilis. The finding that Spo0E interacts directly or indirectly with Spo0A and RstA in C. difficile is an important contribution, but Reviewers 1 and 3 request additional verification of these mass spectrometry-based co-IP experiments either in the form of validating the AlphaFold-predicted interactions or through another experimental method. They raised additional points regarding alternative interpretations of the data that can be readily addressed through textual changes. If these items are addressed, I hope to make a final decision without needing to send the manuscript out for review again.

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 describes new phenotypical observations and mechanistic explanations of how the small protein, Spo0E, impacts sporulation, motility, and toxin production in C. difficile. TargeTron disruption of spo0E was generated, validated by sequencing, and complemented to demonstrate that sporulation frequency and TcdA+TcdB toxin is negatively impacted by Spo0E under lab conditions, and it has a modest negative impact on motility. The mutant displays an enhanced virulence phenotype in an animal model of infection. To identify proteins that interact with Spo0E that could explain phenotypes beyond sporulation, Co-IP was conducted. RstA, a transcription factor, was a top candidate. AlphaFold modeling was used to predict interactions between Spo0E, RstA and Spo0A. Residues known to confer interactions between Spo0E and Spo0A from prior studies in Bacillus subtilis were identified in the model’s protein-protein interface, providing confidence in the Spo0E-Spo0A prediction. The role of Spo0E in B. subtilis motility was investigated, as it had not been questioned before, and indeed found to impact this characteristic. However, cross-complementation studies (placing C.diff spo0E into B.sub, and vice versa) found that they could not substitute for one another. Yet, spo0E-like proteins are identified among a variety of bacteria, based on sequence and structural predictions, and therefore indicate these small proteins are likely to be important regulators.

The primary weakness of this study is seen in its reliance on Alphafold predictions between Spo0E and RstA. No evidence is provided to support the notion that Spo0E and RstA actually interact. Nevertheless, the manuscript is easy to follow, the included experiments are technically sound, and the findings are highly significant.

Reviewer #2: The manuscript by DiCandia et al. examines the phosphatase Spo0E in Clostridium difficile. In B. subtilis, Spo0E represses the function of Spo0A, the master regulator of entry into sporulation, but the function of Spo0E in C. difficile is not known. The authors report that in C. difficile, spo0E also represses sporulation but unexpectedly discovered that Spo0E also increases motility and toxin production. Interestingly, an effect of Spo0E on motility (albeit, the opposite effect, but still Spo0A-independent) was conserved and previously overlooked in B. subtilis. Most strikingly, the authors report that Spo0E-like proteins are broadly conserved in bacteria, suggesting that a similar regulatory mechanism exists in other, non-sporulating, cells.

Reviewer #3: Overall this is a well written manuscript describing the important role of Spo0E in regulating sporulation, virulence, toxin production and motility. This is mostly a descriptive study showing the importance of Spo0E in regulating these phenotypes. The authors use Co-IP mass spec to identify interacting partners of Spo0E and Spo0A. There is not significant follow-up demonstrating the importance of these interactions.

**********

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: Major Comments:

1. Foremost, the AlphaFold prediction needs to be validated experimentally by some method, preferably showing the predicted interface between Spo0E and RstA is true.

2. Line 169; I disagree with the statement, “the number of spo0E mutant bacteria present in cecum was similar to the WT strain.” It seems there are obviously less bacteria in the distribution of mutant-infected hamsters, compared to the wildtype infected animals. This is interesting given the enhanced virulence of the mutant, which is likely due to elevated toxin production.

3. It appears from figure 3 that Spo0E interacts with a monomer of RstA. Is it known whether RstA needs to form a dimer to bind DNA, like most RRNPP transcriptional regulators with a DNA-binding domain? Would the Spo0E-RstA interaction be possible if RstA were in a dimeric complex? If RstA were to require dimer formation to bind DNA (likely) might the mechanism of Spo0E work by preventing RstA dimer formation? Is there a way to easily test whether Spo0E impacts RstA dimerization?

4. As Spo0E was found to interact with both Spo0A and RstA by Co-IP, and because RstA is reported to impact sporulation by an unknown mechanism, a model is put forth (line 206, Fig. S7) that Spo0E serves as an intermediate between the two regulatory proteins. It could be argued that Spo0E doesn’t need to physically bridge RstA to Spo0A for RstA to have an impact on sporulation. Perhaps Spo0E interacts with one partner at a time and the effect on sporulation is merely an outcome of whether Spo0E is free to engage Spo0A. I suggest including this alternative scenario that Spo0E may not need to function as intermediate between RstA and Spo0A.

Reviewer #2: In general, this was a well-written paper with largely well-designed experiments that should be easily accessible for a broad audience. I have only some relatively cosmetic suggestions for the authors.

Major comments:

1. Fig. 2B. Statistical analysis suggests that the p value is greater than 0.05 (therefore ‘not significant’), and the authors conclude that increase in toxin production was not due to increased colonization or carriage. That said, the difference in the spread of the data between the two groups is striking and it really looks like deleting spo0E results in reduced number of bacteria in the cecum., depending on the particular animal. Is it possible that increased toxin production *causes* a decreased C. difficile burden in a fraction of animals? In other words, what is the virulence/pathogenesis consequence of increased toxin production? Not requesting additional experiments here, just proposing a different interpretation of the results that the authors may or may not wish to address.

2. Fig. 2, Table 1. If modeling predicts that Spo0E, RstA, and Spo0A form a complex, any idea why RstA did not copurify with Spo0A (was RstA present, but below the cutoff value in the LC-MS/MS data for the Spo0A co-IP)?

3. Consider moving the model figure in Fig. S7 to the main text as a new Fig. 5.

Reviewer #3: 1. Some insight into what the important interactions of Spo0E would strengthen the paper Ideally this would be point mutants that disrupt interaction with Spo0A or RtsA specifically but may be outside the scope. A less difficult experiment would be to construct a spo0E rstA double mutant to determine if it resembles a spo0E or rtsA phenotype. This could provide some insight to which interactions are more important.

2. The CO-IP mass spec experiments should be confirmed using another assay.

**********

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: Minor comments.

Could the authors comment on why the motility phenotype is so modest as compared to toxin production if both are thought to be mediated by RstA and SigD?

Line 203. ‘Complement’ probably isn’t the best word choice, maybe state as the converse experiment?

Reviewer #2: Minor comments:

1. Line 106. Please define “RRNPP” upon first use.

2. Fig. 1B, 1D, Fig. 4. Consider eschewing the use of bar graphs and instead use scatter graphs (similar to Fig. 2B-D) so that the reader may appreciate the spread of data.

3. The complementation data in Fig. S2 is very important. Consider moving Fig. S2A-B into Fig. 1.

Reviewer #3: 1. I would like to see the complementation data in the main text.

2. Do Spo0E homologs from other organisms complement either B. subtilis or C. difficile?

3. It would be helpful to know if the flag-tagged proteins are functional (do they complement mutants).

**********

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

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr McBride,

Thank you very much for submitting your manuscript "A Conserved Switch Controls Virulence, Sporulation, and Motility in C. difficile" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The newly added co-immunoprecipitation data greatly strengthened this interesting manuscript. The Reviewers and I are in agreement that the article should be accepted, but there is a request for very slight changes in a revised manuscript that can be easily addressed. It would be nice to include the silver-stained gel of the pull-downs into the supplemental (as Reviewer 3 notes, the methods described silver staining). Reviewer 3 also had questions about the band labeled as "RstA dimer" in the co-IP. For example, there is a band that may run at a similar size in the Spo0A western blot (but it is hard to tell with the MW markers shown).

In addition, to avoid confusion of readers who may be quickly glancing at the figures to indicate that the structures shown are AlphaFold models in the figure legends and figure legend title. Please change the title of Figure 5 so that it reads "C. difficile Spo0E is predicted to interact with functional domains of Spo0A and RstA." Similarly, for supplemental figure 3, please revise the legend title to read "Predicted C. difficile Spo0E interactions with Spo0A and RstA."

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Aimee Shen

Guest Editor

PLOS Pathogens

Michael Wessels

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

The newly added co-immunoprecipitation data greatly strengthened this interesting manuscript. The Reviewers and I are in agreement that the article should be accepted, but there is a request for very slight changes in a revised manuscript that can be easily addressed. It would be nice to include the silver-stained gel of the pull-downs into the supplemental (as Reviewer 3 notes, the methods described silver staining). Reviewer 3 also had questions about the band labeled as "RstA dimer" in the co-IP. For example, there is a band that may run at a similar size in the Spo0A western blot (but it is hard to tell with the MW markers shown).

In addition, to avoid confusion of readers who may be quickly glancing at the figures to indicate that the structures shown are AlphaFold models in the figure legends and figure legend title. Please change the title of Figure 5 so that it reads "C. difficile Spo0E is predicted to interact with functional domains of Spo0A and RstA." Similarly, for supplemental figure 3, please revised the legend title to read "Predicted C. difficile Spo0E interactions with Spo0A and RstA."

Reviewer Comments (if any, and for reference):

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: (No Response)

Reviewer #3: In the initial review I raised two concerns. The first was a confirmation of the interaction between SpoOE and RstA which was provided and convincing. They were unable to confirm with genetics that this interaction is biologically relevant. Otherwise they have addresssed the majority of the concerns well.

**********

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: (No Response)

Reviewer #3: (No Response)

**********

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 #3: I am not convinced based on what the authors have written that what is labeled as RtsA dimer is really a dimer. Is it possible that this has not entered the gel (there are not standards labeled above the "dimer)? If looked at closely there are "dimer" bands present in the Eluate of all 3 samples. It would be beneficial to include the silver stain in the supplemental figures as it is mentioned in the methods but I did not see this in the main or supplemental figures.

**********

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 #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

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.

Dear Dr McBride,

We are pleased to inform you that your manuscript 'A Conserved Switch Controls Virulence, Sporulation, and Motility in C. difficile' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Aimee Shen

Guest Editor

PLOS Pathogens

Michael Wessels

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Thank you for your revised submission. The newly added silver stained gel is a fantastic addition to the paper. We greatly appreciate your attention to the Reviewers' comments and thank you for submitting this excellent study.

Reviewer Comments (if any, and for reference):