Dear Dr. Lebreton,

Thank you very much for submitting your manuscript "Fluorescent secreted bacterial effectors reveal an intravacuolar replication compartment for Listeria monocytogenes" 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 several 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.

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,

Renée M. Tsolis

Section Editor

PLOS Pathogens

Renée Tsolis

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

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

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: In this paper the authors developed a method to track secreted proteins in live microscopy. Authors show application of the new tracking system to two markedly different secretion systems in both Gram positive and Gram negative intracellular pathogens. They then used their tool to measure time of vacuole rupture in Listeria, which is an important step in Listeria infection cycle, freeing bacteria and allowing infection of the neighbouring cell. They observed that during the infection not all of the bacteria succeed in escaping the vacuole. This phenomenon, which occurs despite expression of the listeriolysin O (LLO) pore forming toxin, allows a subpopulation of Listeria to proliferate within these vacuoles reminiscent of macrophage SLAPs, from which they eventually escape. The eSLAP (for epithelial SLAP) therefore represents another replicative niche used by Listeria (and possibly other intracellular bacteria) to a colonize host cell. In addition, this piece of work provides an interesting tool that is relevant and potentially applicable to many bacterial organisms. The work should therefore be of interest for a large community of bacteriologists.

The paper is well written and easy to follow. Overall, this work is of outstanding quality and well supported by the results with appropriate controls and statistical analysis. I reviewed it earlier for another journal and the authors have addressed all the comments I had then. This is now a version that can be published without further delays.

Reviewer #2: This is an interesting work that describes the use of a FAST reporter to study the localization of LLO from Listeria in real time. Using a LLO-FAST reporter, the authors find that, in epithelial cell lines, a subpopulation of Listeria fails to escape from phagosomes and instead replicate in these structures in a manner dependent on LLO. They refer to phagosomes in epithelial cells that contain replicating Listeria as ‘eSLAPS’.

This is a well performed study that provides new insight into the functions of LLO. I provide a few comments and questions below.

**********

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: The paper is acceptable for publication as is, authors have taken on board all comments from a previous round of reviewing in which I happened to take part.

Reviewer #2: 1. I could not find any information on the method of statistical analysis used in this study. In my view, it is critical to mention the statistical test used in each figure so that one can judge if the statistical analysis is appropriate.

2. Does replication in eSLAPs require the pore-forming activity of LLO?

3. Line 251. Just because the PrfA* mutant form of PrfA is constitutively activated in broth culture does not necessarily mean it is more active than wild-type PrfA in host cells, given that PrfA is activated in cells. Are there published data demonstrating that a PrfA* mutant stimulates higher expression of PrfA-dependent genes in host cells compared to wild-type bacteria? Although the images in Fig. S9 give the general impression that LLO-FAST accumulates to a greater extent in eSLAPs from PrfA* bacteria compared to wild-type bacteria, this apparent difference is not quantified. I would suggest quantification from several eSLAPs and the use of statistical analysis to determine if PrfA* does, in fact, cause increased accumulation of LLO-FAST.

**********

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: None

Reviewer #2: 1. Figure 2C. It is difficult for this referee to distinguish between the green points and the blue curve. I would suggest changing blue to another color. Actually, black might work fairly well.

2. Line 191, ‘significantly longer’; line 193 ‘a significant proportion’.l I would avoid the use of ‘significant’ in these contexts, since statistical analysis did not seem to be performed on Fig. 3C or Fig. 3D. In a related matter, I wonder why statistical analysis was not carried out on Fig. 3D. It would seem to be important to determine if the difference between the WT and ∆hlyA strain is significant.

3. Fig. S7A. What do the dashed lines represent?

4. Fig. S7B. How does one account for the fluctuating in LLO-FAST signals in some of the vacuoles (Fig. S7B)? Is LLO-FAST being degraded and then re-synthesized?

5. Line 248. Should be Fig. 4C (not Fig. 3C).

6. Line 251. What is meant by ‘Symmetrically’?

7. Line 279 and Fig. 5A. To this referee, the evidence that eSLAP rupture precedes decoration of bacteria with YFP-CBD does not seem that compelling in this image. The CBD labeling seems quite weak and I’m not convinced that CBD-labeled bacteria appear in the same area of the cytosol that previously was occupied by the vacuolar structure labeled with Lm. Perhaps the authors could select a clearer set of images?

**********

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

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, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Lebreton,

We are pleased to inform you that your manuscript 'Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells' has been provisionally accepted for publication in PLOS Pathogens.

Thank you for your detailed and thoughtful response to Reviewer #2. We look forward to publication of this exciting work!

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,

Renée Tsolis

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

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