Dear Dr %:LAST_NAME%,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: The authors investigated the importance of F. novicida FTN_0096 as an essential factor for intracellular replication of F. novicida within host cells. In this study, the authors showed that an F. novicida FTN_0096 mutant generated by transposon mutation could not escape from phagolysosomes in the initial phases of infection and was subsequently digested within the lysosomes after being captured by autophagosomes. All experimental methods were well designed, scientific results and the logical discussion were well described. This study may help to understand the virulence factors required for intracellular replication of F. novicida within phagocytes after infection, as well as and phagocytic pathways involved in F. novicida entry into host cells. Moreover, it may help to understand the basic aspects of immunological responses and the development of preventive tools in Francisella infection in humans and animals. Considering that Francisella infection is an important zoonosis by an intracellular parasitic pathogen, this study suggests that F. novicida FTN_0096 is an essential factor for intracellular replication and pathogenicity of F. novicida. However, this reviewer believes that the manuscript must be improved in several areas.

1. The abstract is too long and needs to be revise. The authors should delete the general introduction in abstract. In particular, lines 20 to 25 ‘Francisella tularensis is the causative agent of the zoonotic disease tularemia. F. tularensis is a gram-negative, facultative intracellular bacteria. F. tularensis subsp. (F. novicida) is a facultative intracellular pathogen that proliferates within macrophages; it has the minimum incidence of virulence in humans but is pathogenic in mice. Furthermore, F. novicida shares considerable similarities with highly virulent subspecies and is extensively utilized as a surrogate in the investigation of Francisella.’ should be deleted or moved to the Introduction section.

2. The format of whole manuscript must be checked. Especially, the titles of figures and references (Italic font, etc.) must be checked before submission.

3. The authors found that F. novicida FTN_0096 mutant could not escape from the phagosome and FTN_0096 is an essential factor for the intracellular replication of F. novicida within host cells. A more detail description of this finding is needed in the Discussion section to help readers better understand the significance of the study.

Reviewer #2: General Comments

In this paper, the authors investigated the virulence mechanism of F. novicida using a novel infection model, HeLa–FcγRII. They demonstrated that FTN_0096 is involved in the intracellular growth of F. novicida in both HeLa cells and THP-1 cells. While the reviewer agrees that FTN_0096 contributes to the intracellular survival of F. novicida, some parts of the manuscript should be revised for clarity and readability.

Specific Comments

Figs.: "FTN_0096" is probably used as an abbreviation for the FTN_0096 mutant (E12-3), but this is not described anywhere. Please revise this point.

Line 1: The involvement of FTN_0096 in F. novicida pathogenicity has already been reported. Moreover, it is unclear whether FTN_0096 itself is cytotoxic. Therefore, the reviewer suggests considering a revision of the title.

Lines 28, 77: The reviewer does not understand why the authors mentioned the difference in genetic manipulability between HeLa cells and macrophages. Since no gene editing was done in this study, this explanation may be unnecessary.

Lines 35-37, 260-266, 491: Why do the authors consider that the FTN_0096 mutant is captured by autophagosomes at the late stage of infection if this mutant fails to escape from phagolysosomes?

Lines 48-87, 194, 252, 429-555: In this context, what does “Francisella” refer to? It may be used to indicate all F. tularensis subspecies, rather than the genus Francisella. Please clarify the definition.

Lines 100, 196-199: Why were HeLa–FcγRII cells used for screening? In the Introduction, these cells are merely described as a new model. Please explain the advantages of this model and clarify why it was selected for the screening.

Line 118: Please describe the screening procedure.

Line 154: The use of "strains" may not be appropriate. Please correct.

Line 159: Please provide the full name of PMA.

Line 161: Please revise the sentence to indicate that mouse serum contains anti-F. novicida antibodies.

Lines 204, 440: Please revise "FcR HeLa cells" to " HeLa–FcγRII cells".

Lines 211–213: The reviewer infers that the cells were infected individually with each mutant, and that panel B is a representative image of the screening assay. If this is correct, please revise the sentence.

Lines 222–223: Using "mutation" instead of "mutant" in this title might be more appropriate.

Line 224: Maybe, a modification like "To analyze the function of FTN_0096" is appropriate.

Lines 252–253: The meaning of this sentence is unclear. You might revise the phrase "particularly the mechanism of FTN_0096 escapes" to "particularly in the escape" for clarity.

Lines 335-345: The authors describe the cytotoxicity of F. novicida against THP-1 cells; however, the results only demonstrate intracellular growth. Is F. novicida cytotoxic to THP-1 cells, and is FTN_0096 involved in this process?

Lines 358-359: This sentence is grammatically incomplete. Please revise it.

Which results are the values "50%-75%" and "59%-84%" derived from?

Lines 491-494: The reviewer could not follow the intention of these sentences. What do the phrases "These findings," "The same results," and "These results" refer to?

Lines 501-502: The reviewer could not understand the necessity of this sentence. Could you consider removing it?

Lines 518, 545: Please clarify what "These results" refer to.

Lines 528-529: Are there any contributions of cytophagy or cytotoxicity to the transport function of the Golgi complex? If so, please provide a citation.

Lines 549, 550: This sentence might not match the purpose and results of this manuscript. You might revise this sentence like "In conclusion, we screened a transposon mutant library using HeLa–FcγRII cells and identified FTN_0096 as a pathogenic factor of F. novicida ".

Lines 550-552: Are the authors suggesting that FTN_0096 mainly facilitates the escape of F. novicida from the capture by mitochondria and the Golgi complex? Is there any hypothesis about how FTN_0096 contributes to this escape, and how it is involved in the intracellular growth and cytotoxicity of this bacterium? A discussion of these points might help clarify the manuscript.

Reviewer #3: General Comments

In this paper, the authors identified a virulence factor of F. novicida using a novel infection model, HeLa–FcγRII. They demonstrated that FTN_0096 plays a critical role in the intracellular replication of F. novicida. While the reviewer concurs with this finding, certain sections of the manuscript require revision.

Specific Comments

Methods: The method for calculating the colocalization rate of bacteria and each cell organelle is not provided. Please revise this point.

Line 159: Please provide the full name of PMA.

Line 161: Please revise "with mouse serum" to "with mouse serum contains anti-F. novicida antibodies".

Lines 204, 440: Please replace "FcR HeLa cells" with "HeLa–FcγRII cells".

Lines 200-202, 211–213: In Fig. 1B, the bacterial strain that infected the HeLa-FcγRII cells is not clearly described. The reviewer assumes that HeLa-FcγRII cells were infected with the FTN_0096

mutant strain; is this correct? Please revise the legend of Fig. 1B to include more detailed information about the bacterial strain employed.

Lines 211, 213: "Scale bar = 20 μm" is missing a period, please correct.

Lines 335-345, 347-348: The authors describe the 'cytotoxicity of the FTN_0096 mutant in THP-1 cells'; however, the cytotoxicity assay was only performed using HeLa–FcγRII cells. Furthermore, results of the cytotoxicity assay for THP-1 cells are neither presented nor discussed in the manuscript. Therefore, the term 'cytotoxicity' should be removed from these sentences.

Lines 491-494: The reviewer was unable to clearly understand the intent of these sentences. It is unclear what is meant by the phrases 'These findings,' 'The same results,' and 'These results.'

Lines 549-550: The reviewer finds this sentence confusing. In the context of this study, it would be more appropriate to revise 'and identified FTN_0096 as a pathogenic factor of F. novicida' instead of 'to identify FTN_0096 as a pathogenic factor of F. novicida'.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Identification of the Francisella novicida FTN_0096 as a factor involved in intracellular replication and host response

PONE-D-25-23563R1

Dear Dr. Shimizu,

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.

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Kind regards,

Yung-Fu Chang

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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Authors identified the virulence factor of Francisella novicida and characterize its roles in phagocytes in this study. All comments were addressed and revised in the whole manuscript point by point. It may be accepted in this journal.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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:  Suk Kim

Reviewer #2: No

Reviewer #3: No

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