Molecular Responses of the Eukaryotic Cell Line INT407 on the Internalized Campylobacter jejuni—The Other Side of the Coin

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

This revised version of the manuscript has a little change over the first version. This reviewer is convinced the topic investigated in this study is interesting. The authors are encouraged to address the questions below.

First of all, the authors provide a descriptive summary of the huge dataset in this work. It would be very helpful if the authors could analyze the data and come up with the key take-home message for this work. For example, at the end of Summary, the authors conclude that this work emphasizes the pathological significance of this organism. This is a generic statement and we all know this is a true pathogen.

Related to the above comments, I could not find out that, in this study, how many repeats of the bacterial infections have been performed. With the huge number of genes analyzed in this work, if a single study was performed, the variation would be huge and then the conclusion would be less solid from this work. Besides, with several replicates, you can perform statistical analysis, between the groups.

The authors can further improve the writing, and here are some examples:

Line 138: 3hours shall be 3 hours;

Line 150: theC shall be the C;

In table 4, "function" is misspelled;

line 367: Up- shall be spelt out as upregulation;

 

 

Comments on the Quality of English Language

A significant language polishing is appreciated.

Author Response

Reviewer 1

Comments and Suggestions for Authors

This revised version of the manuscript has a little change over the first version. This reviewer is convinced the topic investigated in this study is interesting. The authors are encouraged to address the questions below.

• Dear Reviewer, thank You for your still positive opinion about the manuscript.

First of all, the authors provide a descriptive summary of the huge dataset in this work. It would be very helpful if the authors could analyze the data and come up with the key take-home message for this work. For example, at the end of Summary, the authors conclude that this work emphasizes the pathological significance of this organism. This is a generic statement and we all know this is a true pathogen.

• Yes the data with that we worked is fairly huge. For this reason in frame of this experimental setup, we just could flash up those clearcuts, or those molecular changes that occured during the invasion process. With this manusript our intention was to reveal some changes that could be interesting and that could open new fields of research on this area. By this researchers could answer about on such crucial questions why forexample C. jejuni can survive in epithelial cells while not in macrophages. Actually what is (are) the turning point(s) of this process and which factors influence it. This manuscript offers a repertoire of potential genes that are candidates for further analyses.
• The take home message of this work is that the invaded eukaryotic cell oscillates between two major possibilities, like death or survival. This study only could show one timeslot in this probably stormy game, on that we hinted in Conclusion part.
• Yes the pathological significance of C. jejuni to evoke certain posinfectious diseases is partially known. We correted our statement as we now „confirm” the expression of some of those genes that are hypothetised to confer to pathological conditions. same is certainly true about the potential Pathological consequences. We think that this is important because expression of some of these factors could already be detected at the beginning of the invasion process of ths organism. We do not want to make firm conclusion from this finding, but actually supports earlier observations of ypotheses.             

Related to the above comments, I could not find out that, in this study, how many repeats of the bacterial infections have been performed. With the huge number of genes analyzed in this work, if a single study was performed, the variation would be huge and then the conclusion would be less solid from this work. Besides, with several replicates, you can perform statistical analysis, between the groups.

• No, it was not a single experiment. The experiments were performed in three parallels in 24 well TC plates, that we described in Materials and Methods (2.3). But, the basic concept behind this was, that the three RNA samples for sequencing were gained from one timepoint. Each of the three RNA samples was the pool of 6 wells, or with other words 6 parallel invasion experiments. Based on that the gained WTA results are the summaries of 18 (3x6) wells / invasion expriments. The three RNA samples were parallel sequenced and the three gained RNA seq results were averaged and analysed.

The authors can further improve the writing, and here are some examples:

Line 138: 3hours shall be 3 hours; - corrected

Line 150: theC shall be the C; - corrected

In table 4, "function" is misspelled; - corrected

line 367: Up- shall be spelt out as upregulation; - corrected

 

Dr. Gyorgy Schneider / corresponding author

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

 I agree the authors have made changes to the manuscript and it reads and looks better than before. However,  I still beleive this is just an observational study and it would have been more appealing if the authors had validated some of this using C. Jejuni mutants and validating using invasion/interaction assay or /and additional rt-qPCR.

 

 

 

Comments on the Quality of English Language

Authors should go through the text and make corrections.

Author Response

Dear Reviwer 1

 

Thank You for your commences and certainly for the critical review toward our manuscript.

 

 

Comments and Suggestions for Authors

 I agree the authors have made changes to the manuscript and it reads and looks better than before. However,  I still beleive this is just an observational study and it would have been more appealing if the authors had validated some of this using C. Jejuni mutants and validating using invasion/interaction assay or /and additional rt-qPCR.

• Yes, we agree that because of its inherent complexity our results can only flash up a wider spectrum of novel research directions on this field and do not go in detail. However we have to consider that by presenting the results of this study we could attract more attention on this area and we offer some starting points for other researchers intended to focus on the clarification of the roles of certain genes during invasion, survival and cell-to-cell communication. I think our results could show the major clearcuts and help to choose out a repertoire candidate genes for further studies.  

Authors should go through the text and make corrections.

• We performed this.

 

Dr. György Schneider

Corresponding author

Author Response File: Author Response.docx

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors took into account the comments - suggestions of the reviewers.

Please add "space" after some words in the added text

 

Author Response

Dear Reviwer 2

 

Thank You for your commences and certainly for the critical review toward our manuscript during the review process!

 

 

Comments and Suggestions for Authors

The authors took into account the comments - suggestions of the reviewers.

• thank You for accepting our answers!

Please add "space" after some words in the added text

• we went through the text and corrected these mistakes.

 

Dr. György Schneider

Corresponding author

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Anital et al. investigated the molecular responses of an eukaryotic cell line to the internalized Campylobacter jejuni. The research topic is interesting, and the manuscript is overall well written. The authors are encouraged to address the comments below.

This reviewer failed to find out how many repeats were performed in this study. Or a single experiment was run? It is important that the investigators did at least triple repeats for this study; then, the statistical analysis was performed to compare the level of these target molecules in different groups. Otherwise, the conclusion from this work is not solid. This point needs to well address before this manuscript can be considered to be published.

The conclusion of this study solely relies on the WTA. It is expected that some confirmation assay will be used to verify the data and conclusion drawn from WTA.

Minor comments:

The word Campylobacter needs to be spelt out in the manuscript title.

Line 15: it is 41,769 not 41.769.

Line 24: change X to folds. Again, without the statistical analysis and comparison, we can conclude which marker is significant higher than others even we see a change of 10 folds.

Each of the figures need to have a detailed figure legend together with the figure title.

 

Comments on the Quality of English Language

It is Ok overall, and I have some comments for the authors regarding the English writing.

Reviewer 2 Report

Comments and Suggestions for Authors

The study investigates transcriptomic changes in the INT407 human cell line following invasion by Campylobacter jejuni. Transcriptome analysis reveals significant upregulation and downregulation of genes associated with immune response, metabolic functions, stress responses, apoptosis, and potential pathologies. Key findings include the activation of immune functions, induction of apoptosis, metabolic reprogramming, and stress responses. The study sheds light on host-pathogen interactions and identifies potential targets for future research and therapeutic interventions.

 

Introduction:

·      Providing more specific details about the gaps in current knowledge or unanswered questions in the field would enhance the justification for the study.

 

Methods:

·   Providing more specific details about certain experimental procedures, such as the conditions for C. jejuni infection or the bioinformatics tools used for transcriptome analysis, would enhance reproducibility.

 

·  Including information on quality control measures or validation steps for the transcriptomic data would add credibility to the results.

 

Discussion:

·      While the conclusion provides a good overview, it could benefit from a more in-depth discussion of the potential clinical or therapeutic implications of the findings. Exploring how the insights gained from this study could inform the development of new treatment strategies or diagnostic approaches would enhance the impact of the conclusion.

·      It would be helpful for the conclusion to acknowledge any limitations or challenges encountered during the study and discuss how these limitations could be addressed in future research.

·      While the conclusion mentions future research directions, it could be strengthened by providing more specific recommendations or hypotheses for follow-up studies. Clearly articulating the research questions that arise from the current findings would guide future investigations in a more focused manner.

 

 Major concerns:

·      Were any validation experiments conducted to confirm the findings from the transcriptomic analysis? Without this how are the authors sure about these changes?

 

·      How was the infection protocol standardized to ensure consistency across experiments? The authors mention cfu, but for this study the authors report on epithelail cells and not bacterial cells, I struggle to understand the rationale here

 

·      Can you discuss any potential limitations or biases associated with the transcriptomic analysis, such as variations in gene expression due to cell culture conditions or sequencing artifacts?

 

·      Can you provide additional information about the reproducibility of the experimental results, such as the number of independent replicates performed for each experimental condition?

 

·      Statistical analysis methods? Can you provide details about the experimental design, including the number of replicates for each experimental condition?

·      Were the experiments performed in triplicate, and if so, were the replicates independent biological replicates or technical replicates?

·      What statistical methods were used to analyze the data and determine differential gene expression?

·      Were any adjustments made for multiple testing to control the false discovery rate?

·      Can you describe how the variability between replicates was assessed, and were any outliers identified and removed from the analysis?

 

 

Comments on the Quality of English Language

Quality of English was ok, needs some editing.

Reviewer 3 Report

Comments and Suggestions for Authors

In the paper titled "Molecular Responses of The Eukaryotic Cell Line INT407 on the Internalized C. jejuni – the Other Side of the Coin," the authors elucidate the molecular alterations within eukaryotic cells during the initial stages of C. jejuni internalization through comprehensive transcriptomic analyses.

 

The findings showcased in this study, both in the main body and supplementary materials, provide intriguing insights into the genetic expression dynamics following C. jejuni invasion into cells.

 

Please consider these suggestions for improving this manuscript:

 

Minor:

 

Introduction:

 

Line74: immune response

 

Material and Methods:

 

Line 117:  “cells, wells” instead of “cells wells”

 

Discussion:

 

Line 313: good example 

 

Line 349: mystery

 

Line 349: “route” instead of “issue”