Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study is the first to simultaneously compare acute and chronic Toxoplasma gondii infection at the liver tissue level, revealing a biphasic regulatory pattern of the Akt/mTOR pathway characterized by widespread suppression during acute infection and selective activation during chronic infection. These findings provide a foundational basis for understanding liver pathology caused by T. gondii and for the development of targeted therapies. Some major or minor issues should be clarified before its acceptable for publication.

1.In general, the English language of the MS is good enough but needs improvement in grammar, sentence structure and other language issues.

2.Regarding the establishment of the chronic infection model, type II T. gondii tachyzoites or tissue cysts should be used for infection.

3.The T. gondii GT1 strain is a type I genotype. It is questionable whether its tachyzoites can form tissue cysts in mice. In addition, using MAG1 antibody levels to represent cyst burden is not rigorous and lacks sufficient validity.

 

Author Response

I am grateful for the reviewers’ constructive and insightful suggestions, which have significantly enhanced my manuscript. My responses to these comments are as follows.  Revisions made in the manuscript are underlined for their easy identification.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Differential Regulation of Hepatic Akt/mTOR Signaling During Acute and Chronic Toxoplasma gondii Infection in a Murine Model” written by Jianchun Xiao, demonstrating meaningful insights into the regulation of Akt/mTOR signaling during Toxoplasma gondii infection and highlights important stage-dependent host-pathogen interactions. The study is scientifically sound, and is suitable for publication after minor revisions.

In abstract, 6 out of 8 markers were downregulated, the others two are not mentioned.

Graphical abstract shows 7 markers were suppressed as highlighted by color in acute case. Whereas, results showed 6 out of 8 markers were suppressed. Could the author elaborated it?

Material and methods 2.1 and 2.2: The statement suggests reuse of previously collected specimens, yet detailed experimental procedures (infection with Toxoplasma gondii, chemotherapy, and sacrifice at 5 months post-infection) imply that animals were actively used and euthanized. This contradicts the claim that no animals were sacrificed for the present study. The authors should explicitly state whether: the data and samples were entirely derived from a previously completed and ethically approved study, OR new experimental procedures were conducted as part of the current work.

The infection of T. gondii was checked by ELISA, how the author set the standard of acute and chronic stages of infection based on antibodies production?

In 2.4 and 2.5: Is this protocol belonging to current research or it was done previously?

Line 154: phosphorylation levels of eight pathway components in liver tissues was compared Or were compared?

In result section 3.1: p70S6KThr389 is not mentioned in abstract which is downregulated.

The authors are encouraged to quote result of each section in Results by figures or tables as reference.

The authors are encouraged to change all the scientific names of organisms to italic form. Check throughout manuscript.

Line 237, 265, 294, 300, 332: remove hyphen (-).

Minor grammatical and typographical corrections are needed throughout the manuscript.

Comments on the Quality of English Language

Minor grammatical and typographical corrections are needed 

Author Response

I am grateful for the reviewers’ constructive and insightful suggestions, which have significantly enhanced my manuscript. My responses to these comments are as follows.  Revisions made in the manuscript are underlined for their easy identification.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript examines whether Toxoplasma gondii differentially alters hepatic Akt/mTOR signaling during acute versus chronic infection in mice. The central observation—broad phospho-suppression during acute infection and selective activation during chronic infection—is interesting, plausible, and potentially important for understanding host–parasite interactions.

The paper is well organized and the stage-specific framing is a strength. However, the study is descriptive and based on phosphoprotein measurements in bulk liver homogenate. Several mechanistic claims in the Discussion and Abstract go beyond what the data directly show. The manuscript would be substantially stronger with more cautious interpretation and fuller discussion of methodological limitations.

Major Comments

1. The manuscript over-interprets correlative phosphoprotein data as direct mechanism. Several claims regarding apoptosis, autophagy, immune evasion, insulin signaling, and oncogenic risk are not directly tested and should be softened or explicitly framed as hypotheses consistent with prior literature rather than demonstrated outcomes.
2. Use of whole-liver homogenate is a major limitation. The observed phosphoprotein changes cannot be assigned specifically to infected hepatocytes because infiltrating immune cells, stromal cells, endothelial cells, and shifts in tissue composition may contribute substantially, especially in acute infection.
3. The chronic model requires clearer discussion of confounding factors. Sulfadiazine treatment, survivor effects, and the long interval to five months post-infection may all influence hepatic signaling independently of bradyzoite persistence or cyst burden.
4. Statistical reporting should be tightened. The distinction between significant results and trends should remain explicit; the framework for subgroup testing in the chronic experiment should be clearer; and the correlation analysis should address multiple-testing concerns.
5. The MAG1-based subgroup analysis is useful but still indirect. MAG1 antibody levels are a proxy for chronic burden rather than a direct liver cyst count, and the manuscript should avoid implying stronger burden dependence than the data warrant, particularly because none of the eight markers significantly correlate with MAG1 continuously.
6. Interpretation of individual phosphosites needs more nuance. For example, pPTEN Ser380 and pmTOR Ser2448 are not simple one-to-one readouts of pathway activity, and the paper itself notes that pAkt and pBAD are not significantly correlated across groups.

Minor Comments

The title and graphical abstract currently imply a stronger mechanistic conclusion than the data support; consider slightly softer wording.

The Abstract contains some awkward phrasing, especially the sentence about “strong correlations in signaling changes between inter-components”; revise for clarity.

The limitations section should be expanded beyond missing clinical chemistry markers to include bulk tissue analysis, lack of total protein normalization, lack of orthogonal validation, and absence of functional assays.

Please check first-person phrasing for consistency with journal style (for example, “I stratified” and “I explored”).

Author Response

I am grateful for the reviewers’ constructive and insightful suggestions, which have significantly enhanced my manuscript. My responses to these comments are as follows.  Revisions made in the manuscript are underlined for their easy identification.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been well revised and is recommended for acceptance and publication.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Author, thank you for your efforts to correct the manuscript. I have no other comments.