The Role of Rv1476 in Regulating Stress Response and Intracellular Survival of Mycobacterium tuberculosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research article “The role of Rv1476 in regulating stress response and intracellular survival of Mycobacterium tuberculosis” by Reheman and co-authors explored the function of Rv1476, a putative transmembrane protein, in the pathogenesis of Mtb. The gene rv1476 was discovered from a CRISPR campaign and the knock-down strain of rv1476 showed an attenuated survival rate in macrophages. In this current work, the authors utilized an over-expression strain of Rv1476 and characterized its growth rate under different stress conditions. The authors discovered that by over expressing Rv1476, Mtb is more susceptible to ROS, SDS and lysozymes, however the bacteria showed a slightly increased survival rate in macrophages. By profiling the transcriptome of THP-1 cells infected with the Rv1476 over-expression strain, the authors reasoned that Rv1476 affects the cell proliferation, carbon metabolism, cytokine related response and immune response pathways of THP-1 cells. However, the precise function of Rv1476 has yet to be elucidated. The authors linked to the over expression of Rv1476 to the downregulation of a series of chemokine and antibacterial genes in macrophages but I think there need to be more elucidations on the functions of Rv1476 by linking the overexpression to some chemokines on a molecular level, ie quantify the cytokines secreted by THP-1 under different conditions. The paper overall is well written and is easy to follow. Another point is that it seems a bit puzzling to me that the overexpression of Rv1476 sensitize the bacteria to different stress conditions in vitro but the survival rate of the recombinant strain was increased in macrophages which is considered to provide different stress conditions to eliminate the infection. Can you authors elaborate more on that? Does it seem contradictory?

 

Comments on the Quality of English Language

n/a

Author Response

Dear reviewer, thank you for reviewing our manuscript and providing constructive comments, which greatly helped us improve it. The manuscript has been carefully revised and the point-by-point responses are as follows. We want your comments to be processed accurately. The revised manuscripts appear in red font and responses in blue font.

Once again, thank you very much for your critical comments and inspiring

suggestions.

 

Best regards,

Aikebaier Reheman

 

The research article “The role of Rv1476 in regulating stress response and intracellular survival of Mycobacterium tuberculosis” by Reheman and co-authors explored the function of Rv1476, a putative transmembrane protein, in the pathogenesis of Mtb. The gene rv1476 was discovered from a CRISPR campaign and the knock-down strain of rv1476 showed an attenuated survival rate in macrophages. In this current work, the authors utilized an over-expression strain of Rv1476 and characterized its growth rate under different stress conditions. The authors discovered that by over expressing Rv1476, Mtb is more susceptible to ROS, SDS and lysozymes, however the bacteria showed a slightly increased survival rate in macrophages. By profiling the transcriptome of THP-1 cells infected with the Rv1476 over-expression strain, the authors reasoned that Rv1476 affects the cell proliferation, carbon metabolism, cytokine related response and immune response pathways of THP-1 cells.

 

However, the precise function of Rv1476 has yet to be elucidated. The authors linked to the over expression of Rv1476 to the downregulation of a series of chemokine and antibacterial genes in macrophages but I think there need to be more elucidations on the functions of Rv1476 by linking the overexpression to some chemokines on a molecular level, ie quantify the cytokines secreted by THP-1 under different conditions.

Response: Thank you for your comments. Your suggestions are valuable for enhancing the quality of this study. However, due to certain constraints, we have not conducted an extensive investigation in this study. In this manuscript, we have solely examined the mRNA expression of certain chemokine and antibacterial genes, without delving into the molecular level. In future research, we intend to construct a gene knockout strain and conduct cell and animal infection experiments to further validate the influence of Rv1476 on chemokine secretion.

 

The paper overall is well written and is easy to follow. Another point is that it seems a bit puzzling to me that the overexpression of Rv1476 sensitize the bacteria to different stress conditions in vitro but the survival rate of the recombinant strain was increased in macrophages which is considered to provide different stress conditions to eliminate the infection. Can you authors elaborate more on that? Does it seem contradictory?

Response: Thank you for your comments. We are very grateful to the reviewers for their affirmation and recognition of our work. Like you, we were confused when we got the test results; However, through literature review, it was found that Ruan et al., also reported similar experimental results. Their observed that Rv0426c recombinants became more susceptible to various stresses by increasing cell wall permeability, however with elevated early survival rate within macrophages, and they suggest that the enhanced intracellular survival was not related to the resistance of the stresses tested (DOI:10.1016/j.meegid.2019.104070).

The Rv1476 studied in this article is also a cell wall-associated transmembrane protein, so we speculate that overexpression of Rv1476 may increase the permeability of the bacterial cell wall, making the bacteria more sensitive to stress. In addition, this study found in the transcriptome data that Rv1476 affects certain chemokine and antibacterial gene expression level, so overexpression of Rv1476 may be beneficial to the early survival of bacteria in macrophages.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Language:

The text is scientifically sound and understandable. As a non-native English speaker, I do not feel qualified to judge the text, however, at several instances I felt that some small language polishing is needed.

General considerations:

The manuscript deals with a problem of a great importance – clarifying the mechanisms of pathogenicity of M. tuberculosis. The introduction is concise and very well focused on the research problem. The Materials and methods section is well-written in a reproducible manner with some exceptions. Elements of discussion are found within the Results section, otherwise, the results are adequately presented. The Discussion section is adequate.

 

Major issues:

1. Please, provide more details for subsection 2.6.

 

Minor issues:

1. I do not advise the use of the abbreviation “M.tb.” – please, use instead “M. tuberculosis”.

2. Provide in subsection 2.7. dates when the online tools were accessed as well as the versions of the software.

3. “Statistical analysis" should be n umbered as subsection 2.8.

4. Lines 141-147, 175-177,185-186, 204-206, 227-229 – discussion (even including references!). Please, move to the appropriate section.

Author Response

Dear reviewer, thank you for reviewing our manuscript and providing constructive comments, which greatly helped us improve it. The manuscript has been carefully revised and the point-by-point responses are as follows. We want your comments to be processed accurately. The revised manuscripts appear in red font and responses in blue font.

Once again, thank you very much for your critical comments and inspiring

suggestions.

 

Best regards,

Aikebaier Reheman

 

Language:

The text is scientifically sound and understandable. As a non-native English speaker, I do not feel qualified to judge the text, however, at several instances I felt that some small language polishing is needed.

Response: Thank you for your comments. According to your suggestions, we have re-edited and checked the language organization of the full text.

 

General considerations:

The manuscript deals with a problem of a great importance – clarifying the mechanisms of pathogenicity of M. tuberculosis. The introduction is concise and very well focused on the research problem. The Materials and methods section is well-written in a reproducible manner with some exceptions. Elements of discussion are found within the Results section, otherwise, the results are adequately presented. The Discussion section is adequate.

Response: Thank you very much for your recognition and affirmation of our work. According to your valuable suggestions, we have revised the article and fully discussed the results in the discussion section.

 

Major issues:

1. Please, provide more details for subsection 2.6.

Response: Thank you for your comments. We have written subsection 2.6 of the manuscript in detail, and appear in red font, as shown on line 118-127 of the manuscript.

 

Minor issues:

1. I do not advise the use of the abbreviation “M.tb.” – please, use instead “M. tuberculosis”.

Response: Thank you for your opinion. With the exception of strain names, we have modified all abbreviations in the main text, and appear in red font.

 

2. Provide in subsection 2.7. dates when the online tools were accessed as well as the versions of the software.

Response: Thank you for your comments. Following the reviewer’s suggestion, we have added the version number of the R package used for data visualization, and appear in red font, as shown on line 129-138 of the manuscript.

 

3. “Statistical analysis" should be n umbered as subsection 2.8.

Response: Thank you for your comments. We have made revised in the manuscript and appear in red font.

 

4. Lines 141-147, 175-177,185-186, 204-206, 227-229 – discussion (even including references!). Please, move to the appropriate section.

Response: Thank you for your comments. Following your suggestion we have moved these sections to the appropriate sections, and appear in red font, as shown on line 244-248, 254-256, 260-262, 268-269, 281-282 of the manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled "The role of Rv1476 in regulating stress response and intracellular survival of Mycobacterium tuberculosis" is devoted to the study of the biological function of the membrane protein Rv1476 in Mycobacterium tuberculosis. For that purpose a strain with overexpression of Rv1476 was constructed and studied mainly through transcriptome analysis. Although the exact role of the studied gene product remains unclear, the work provides valuable insights into the function of the protein, making the manuscript suitable for publication in CIMB.

Major comments:

1. The Introduction mentions the construction of a strain with Rv1476 knockdown, but there is no further information about this strain in the cited reference (#15, DOI: 10.1016/j.gpb.2021.01.008). Providing more details about previous studies on this gene product would enhance the context of the current study.

2. The manuscript mentions subjecting the strain with Rv1476 overexpression to various stresses (surface stress, lysozyme, and peroxide stress). However, for a more comprehensive conclusion on the potential of Rv1476 as a target for tuberculosis treatment, it would be beneficial to include a comparison of antibiotic susceptibility among the wild type, knockdown, and overexpression strains.

Author Response

Dear reviewer, thank you for reviewing our manuscript and providing constructive comments, which greatly helped us improve it. The manuscript has been carefully revised and the point-by-point responses are as follows. We want your comments to be processed accurately. The revised manuscripts appear in red font and responses in blue font.

Once again, thank you very much for your critical comments and inspiring

suggestions.

 

Best regards,

Aikebaier Reheman

 

 

The manuscript titled "The role of Rv1476 in regulating stress response and intracellular survival of Mycobacterium tuberculosis" is devoted to the study of the biological function of the membrane protein Rv1476 in Mycobacterium tuberculosis. For that purpose a strain with overexpression of Rv1476 was constructed and studied mainly through transcriptome analysis. Although the exact role of the studied gene product remains unclear, the work provides valuable insights into the function of the protein, making the manuscript suitable for publication in CIMB.

Major comments:

 

1. The Introduction mentions the construction of a strain with Rv1476 knockdown, but there is no further information about this strain in the cited reference (#15, DOI: 10.1016/j.gpb.2021.01.008). Providing more details about previous studies on this gene product would enhance the context of the current study.

Response: Thank you for your comments. We are very sorry， we may not have written it clearly in the manuscript, which caused your confusion. The previous research group utilized the endogenous type III-A CRISPR/Cas10 system of M. tuberculosis to efficiently perform gene editing and RNA interference (RNAi), they successfully constructed a whole-genome CRISPRi library of M. tuberculosis and identified regulatory genes both in vitro and intracellularly. The article primarily focuses on discussing the potential applications and feasibility of this technology in editing genes of M. tuberculosis, without providing detailed information on each gene. Furthermore, this article conducted further verification and study on the function of RV1476 using the previously screened information of M. tuberculosis genes.

 

2. The manuscript mentions subjecting the strain with Rv1476 overexpression to various stresses (surface stress, lysozyme, and peroxide stress). However, for a more comprehensive conclusion on the potential of Rv1476 as a target for tuberculosis treatment, it would be beneficial to include a comparison of antibiotic susceptibility among the wild type, knockdown, and overexpression strains.

Response: Thank you for your valuable opinion. We appreciate your suggestions for improving the quality of this study. Unfortunately, we have encountered some challenges in constructing a gene knockout strain, which has hindered our progress. However, once we successfully construct the gene knockout strain, we plan to conduct cell and animal infection experiments to comprehensively explore the biological functions of Rv1476. In addition, your suggestion of testing the sensitivity of different mutant strains to antibiotics is a very valuable test and will be of great help to our research, and it aligns with our future research plans. We sincerely appreciate your suggestion.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the response from the authors explaining the potential link between the stress resistance and the bacterial survival rate in macrophages. 

Author Response

Dear reviewer, thank you for reviewing our manuscript and providing constructive comments, which greatly helped us improve it. The manuscript has been carefully revised and the point-by-point responses are as follows. We want your comments to be processed accurately. The revised manuscripts appear in red font and responses in blue font.

Once again, thank you very much for your critical comments and inspiring

suggestions.

 

Best regards,

Aikebaier Reheman

 

I appreciate the response from the authors explaining the potential link between the stress resistance and the bacterial survival rate in macrophages.

Response: Thank you for your comments. We are very grateful to the reviewers for their encouragement and we will further improve the quality of the manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The Introduction section has to be expanded, previous studies on the biological function of RV1476 should be discussed in detail.

Author Response

Dear reviewer, thank you for reviewing our manuscript and providing constructive comments, which greatly helped us improve it. The manuscript has been carefully revised and the point-by-point responses are as follows. We want your comments to be processed accurately. The revised manuscripts appear in red font and responses in blue font.

Once again, thank you very much for your critical comments and inspiring

suggestions.

 

Best regards,

Aikebaier Reheman

 

The Introduction section has to be expanded, previous studies on the biological function of RV1476 should be discussed in detail.

Response: Thank you for your comments. According to your suggestions, we have revised the introduction of the manuscript, and appear in red font, as shown on line 49-57 of the manuscript.

 

Author Response File: Author Response.pdf