Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

Review- manuscript ijms-3936144

Toxoplasma gondii is a common intracellular parasite, and research on its genes is essential for understanding the mechanisms of infection and host–pathogen interactions. CRISPR-Cas9 technology enables the rapid generation of knockout mutants; however, genetic manipulation can lead to pleiotropic effects, including changes in both parasite and host gene expression. The aim of the reviewed study was to determine the extent to which the knockout of selected genes affects the parasite and host cell proteomes more broadly, which is crucial for the reliable interpretation of functional studies and highlights the significance of the research undertaken.

The study demonstrated that knockout of single genes (ORF 297720 and ORF 319730) in Toxoplasma gondii not only results in the absence of the targeted proteins but also causes widespread changes in the expression of other parasite and host cell genes. Dozens of common differentially expressed proteins were identified in both knockout lines, including proteins related to the host antiviral response. These changes were pleiotropic and unrelated to the direct function of the deleted genes. The findings challenge the straightforward interpretation of knockout phenotypes as solely the result of gene loss and underline the importance of using appropriate controls and comprehensive proteomic analyses to avoid misinterpretation.

The use of robust bioinformatics and statistical analyses was both appropriate and effective. These methods enabled a reliable comparison of wild-type and knockout proteomes and the precise identification of differentially expressed proteins. Network and functional enrichment analyses linked the observed proteomic changes to relevant biological processes, such as the host antiviral response. This integrated approach allowed the authors to distinguish between specific and pleiotropic effects, ensuring a sound interpretation of the data and confirming the validity of the analytical methods applied.

Overall, the study is methodologically solid, addresses an important research question, and provides valuable insights. 

 

 

Author Response

Thanks you for your positive assessment

We have mad some changes to abstract, introduction and the methods sections (see report reviewer 2).

Reviewer 2 Report

Comments and Suggestions for Authors

Peer Review Report

Manuscript Title: Pleiotropic effects on tachyzoite and host cell proteomes in knock-out clones of the open reading frames 297720 and 319730 constitutively expressed in T. gondii ShSp1 tachyzoites

1. Overall Assessment

Firstly, I would like to express my gratitude to the authors for their efforts in this manuscript.

 I appreciate their work and this manuscript presents a rigorous proteomic investigation of Toxoplasma gondii knockout clones targeting two open reading frames (ORF 297720 and ORF 319730) to evaluate pleiotropic effects in both parasite and host cell proteomes. The study is conceptually strong, technically detailed, and provides valuable insights into the unintended systemic consequences of genetic manipulation in apicomplexan parasites. However, I have a few constructive comments to enhance the clarity and quality of the paper

2. Title and Abstract

Strengths: The title is precise and reflects the core study. The abstract clearly summarizes the rationale, methodology, and key findings.

Suggestions:
a- Add a concluding sentence summarizing the main finding that KO manipulations can affect both parasite and host proteomes.( as-----

 Overall, this study highlights that genetic manipulations in *T. gondii* can lead to system-wide proteomic shifts in both parasite and host, emphasizing the need for cautious interpretation of knockout-based functional analyses

3. Introduction

The introduction provides good context on T. gondii genetics and CRISPR/Cas9 use. However, it is dense and should be streamlined. Clearly state the hypothesis: to test whether knockout effects extend beyond targeted genes to global proteomic changes. Consider reorganizing paragraphs for smoother flow and eliminate historical redundancy.

(Toxoplasma gondii* is an obligate intracellular apicomplexan parasite and a valuable model for studying host–parasite interactions. Advances in genome sequencing and the adoption of CRISPR/Cas9 have greatly facilitated gene function analysis in this organism. Despite the precision of these tools, evidence suggests that gene knockouts (KOs) may trigger secondary, system-wide effects beyond the intended target. Such pleiotropic outcomes can complicate data interpretation and limit the reliability of genotype–phenotype correlations.

> **Therefore, this study aimed to investigate whether knockout of two *T. gondii* open reading frames (ORF 297720 and ORF 319730) leads to broader proteomic alterations in both parasite and host cells.** By comparing proteomic profiles of knockout and wild-type tachyzoites, we sought to determine the extent of off-target or compensatory effects and to highlight the complex biological responses induced by genetic manipulation).

4. Materials and Methods

a-The methods are comprehensive and transparent but overly detailed in some parts. Move long protocol descriptions (e.g., primer design, buffer compositions) to supplementary material.

b-Define abbreviations such as DDA, DIA, IQ, and Top3 at first mention.

5. Results

Results are well-structured but text-heavy.

a-Figures and tables should be improved for readability.

b-Statistical significance should be shown for major comparisons.

c-Clarify how "common DE proteins" were identified across datasets and summarize findings in concise bullet points within the text.

6. Discussion

The discussion interprets data thoughtfully but includes redundancy and speculative analogies. Condense paragraphs and focus on mechanistic implications. Avoid non-scientific metaphors such as ecosystem analogies. Highlight study limitations, including the number of KO clones and lack of direct functional assays.

7. Figures and Tables

Ensure high-resolution figures with clearly labeled axes, legends, and statistical annotations. Tables should be self-explanatory and numerical units clearly defined. Summarize key patterns instead of listing full datasets in the main text.

a-Use **high-resolution images (≥300 dpi)** when exporting from R, Perseus, or GraphPad.

✅b-Ensure **axes and legends** include:

* Full variable names (e.g., *log₂ Fold Change*, *–log₁₀ p-value .

* Units (e.g., *Intensity (LFQ, log₂) c-.

  ✅ Include **statistical markers**: e.g., “*p* < 0.05”, “FDR < 0.05”.

d- For group comparisons, add **error bars** and indicate the number of replicates (e.g., *n = 3

8. Language and Style

The manuscript requires careful English language revision. Common issues include long sentences, article misuse, and tense inconsistency. Use concise, direct sentences. Example corrections:
a- “The genes could thus not be regarded as essential.” → “These genes are therefore non-essential.”
b-- “It cannot be ruled out that in the plant-related apicomplexans similar processes may occur.” → “Similar processes may occur in plant-related apicomplexans.”

9. Ethical and Data Statements

Ethical and data availability statements are appropriate. Ensure that datasets are accessible at the time of submission.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English could be improved to more clearly express the research

Author Response

Comment 1: Firstly, I would like to express my gratitude to the authors for their efforts in this manuscript.

 I appreciate their work and this manuscript presents a rigorous proteomic investigation of Toxoplasma gondii knockout clones targeting two open reading frames (ORF 297720 and ORF 319730) to evaluate pleiotropic effects in both parasite and host cell proteomes. The study is conceptually strong, technically detailed, and provides valuable insights into the unintended systemic consequences of genetic manipulation in apicomplexan parasites. However, I have a few constructive comments to enhance the clarity and quality of the paper

Response 1: Thank you for the positive assessments. Responses are shown below. Textual changes are marked with yellow background for being easily recognised. 

Comment 2: Abstract and Title.  Strengths: The title is precise and reflects the core study. The abstract clearly summarizes the rationale, methodology, and key findings. Suggestions:
a- Add a concluding sentence summarizing the main finding that KO manipulations can affect both parasite and host proteomes.( as----- Overall, this study highlights that genetic manipulations in *T. gondii* can lead to system-wide proteomic shifts in both parasite and host, emphasizing the need for cautious interpretation of knockout-based functional analyses

Response 2: The sentence suggested by the reviewer was added at the end of the abstract (lanes 35-38, marked in yellow).

Comment 3: Introduction. The introduction provides good context on T. gondii genetics and CRISPR/Cas9 use. However, it is dense and should be streamlined. Clearly state the hypothesis: to test whether knockout effects extend beyond targeted genes to global proteomic changes. Consider reorganizing paragraphs for smoother flow and eliminate historical redundancy.  

(Toxoplasma gondii* is an obligate intracellular apicomplexan parasite and a valuable model for studying host–parasite interactions. Advances in genome sequencing and the adoption of CRISPR/Cas9 have greatly facilitated gene function analysis in this organism. Despite the precision of these tools, evidence suggests that gene knockouts (KOs) may trigger secondary, system-wide effects beyond the intended target. Such pleiotropic outcomes can complicate data interpretation and limit the reliability of genotype–phenotype correlations.

> **Therefore, this study aimed to investigate whether knockout of two *T. gondii* open reading frames (ORF 297720 and ORF 319730) leads to broader proteomic alterations in both parasite and host cells.** By comparing proteomic profiles of knockout and wild-type tachyzoites, we sought to determine the extent of off-target or compensatory effects and to highlight the complex biological responses induced by genetic manipulation).

Response 3: We appreciate the comments on streamlining the introduction, as well as the reviewer’s suggestions. We have incorporated some of these suggestions, as indicated in lanes 71-75 and 82-83

Comment 4: Materials and methods. a-The methods are comprehensive and transparent but overly detailed in some parts. Move long protocol descriptions (e.g., primer design, buffer compositions) to supplementary material.  b-Define abbreviations such as DDA, DIA, IQ, and Top3 at first mention. 

Response 4: As indicated by the reviewer, Table 10 was deleted and is now moved into supplementary files as TableS8. For other methods, we have actually mostly referred to already published references, but ensuring that the findings can be reproduced by others requires some detailed explanation on what was done. Several abbreviations used in the text are now defined at first mentioning as required. Some in M&M, other already earlier in the results section.

Comment 5: Results. Results are well-structured but text-heavy. a-Figures and tables should be improved for readability.  b-Statistical significance should be shown for major comparisons.  c-Clarify how "common DE proteins" were identified across datasets and summarize findings in concise bullet points within the text. 

Response 5: Thank you for these suggestions.

• We checked the figures, and they are actually well-readable, and x and y axes are clearly defined and explained in the figure legends. Where necessary, we have adjusted the figure legends. We cannot see any ways for improvement without more concrete details on what should be changed.
• Where applicable, statistically significant differences are indicated by an asterisk.
• The statistical approach leading to the identification of DE proteins is described in section 4.9. A rigorous approach was used in order to identify true differences. Common DE proteins were identified through comparison of the datasets. The findings are indicated in Table 3. We don’t see that it makes sense to reiterate this in the text using bullet points

Comment 6: Discussion. The discussion interprets data thoughtfully but includes redundancy and speculative analogies. Condense paragraphs and focus on mechanistic implications. Avoid non-scientific metaphors such as ecosystem analogies. Highlight study limitations, including the number of KO clones and lack of direct functional assays. 

Response 6: The nature of a discussion is to embark on some limited speculation based on the data available. We have tried to avoid redundancy and not reiterate statements already found in the results, although in some instances this is actually necessary to explain the facts for a better understanding. Some sentences were slightly modified for clarity. The analogy to changes in ecosystems is not a non-scientific metaphor but refers to a phenomenon that occurs in another scientific field. We here kept it in but can also remove it if deemed necessary by the editor.

Comment 7: Figures and Tables. Ensure high-resolution figures with clearly labeled axes, legends, and statistical annotations. Tables should be self-explanatory and numerical units clearly defined. Summarize key patterns instead of listing full datasets in the main text. 

Response 7: Thank you for this comment. We re-checked all figures and figure legends according to the reviewer’s recommendations and made some changes where necessary.

Comment 8: Language and style.  The manuscript requires careful English language revision. Common issues include long sentences, article misuse, and tense inconsistency. Use concise, direct sentences. Example corrections: a- “The genes could thus not be regarded as essential.” → “These genes are therefore non-essential.” b-- “It cannot be ruled out that in the plant-related apicomplexans similar processes may occur.” → “Similar processes may occur in plant-related apicomplexans.” 

Response 8: The manuscript was read over again and some changes in English style, including those suggested by the reviewer, were made.