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Schistosoma japonicum Histone Acetyltransferase 1 (SjHAT1): A Novel Anti-schistosomal Drug Target

PLOS Pathogens

Dear Dr. Xia,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Mostafa Zamanian

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens-->--> -->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497-->--> -->-->Michael Malim-->-->Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

The reviewers find the experimental work generally sound but request major revisions to support the central conclusions. The authors must more rigorously justify the claim that SjHAT1 is a viable and selective drug target by addressing conservation with host homologues, correcting the phylogenetic and domain analyses, and clarifying the proposed mechanism of action of DW-3-15. In addition, the transcriptomic and integrative components should be strengthened to present a more cohesive, hypothesis-driven study.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: "Schistosoma japonica histone acetyltransferase 1, a novel anti-schistosomal drug target," reports a well-designed project that comprehensively profiles the function of SjHAT1 and explores its potential as a drug target. The experiments are well designed, the statistics are appropriate, and the results are clear. While I have no major concerns with the experimental or methodological approach, I disagree with most of the interpretation and argument that SjHAT1 is a potential drug target. I also have questions about the composite understanding of SjHAT1 function in the context of previous work done by the authors exploring DW-3-15 as a compound that targets this HAT.

My primary critique is that based on the data reported here, I have the opposite conclusion as the authors regarding their interpretation of SJ-HAT1 being sufficiently diverged to be a good drug target. It's clear that the activity of the protein is important to proper development of the worm, and inhibition or knockdown of it seems to have strong deleterious effects on its fitness. However, this is not particularly surprising, given the importance of acetyl transferases on general developmental and physiological processes. While it's clear that the enzyme is important, the authors do not provide evidence that it can actually be targeted by a drug without off-target effects. In line 161, the authors state that there is only 34% identity, when in reality this is actually a fairly high value and indicates strong conservation with a human homologue. In line 164, based on the phylogenetic tree, the authors claim that SJ-hat-1 is in an evolutionarily distant clade, but the phylogenetic tree does not show that, and indeed cannot show that, given that it is unrooted with no outgroup. The authors repeat many of these claims in the discussion, such as lines 369 through 374, and suggest that molecules could be designed to target the unique domains of SJ, but their Figure 1B shows no such unique domains. Absent any other evidence from the authors, such as, for instance, experiments on DW-3-15's effects on mammalian acetyltransferases, or other protein modeling efforts, I interpret the author's own data in the exact opposite direction as they do - I'm convinced that SjHAT1 is not actually druggable.

My second primary comment is on the lack of clarity surrounding the hypothetical model of the mechanism of action of DW-3-15/SjHAT1 knockdown. Based on previous reports, SjHAT1 was proposed to be a target of DW-3-15 because it was identified as down-regulated by tandem mass spectrometry experiments. Here, authors show in an in vitro experiment with recombinant SjHAT1 that DW-3-15 acts on to inhibit its acetyltransferase activity (based on previous reviews). This experimental request is surprising, and the results are even more surprising, as the downregulation of a protein caused by a HAT inhibitor does not at all suggest that the protein is also itsef the target of the molecule. Are the authors suggesting that DW-3-15 inhibits SjHAT1, which then leads to the downregulation of its own gene product? Are the authors aware of other examples in the literature of this proposed feedback loop where an epigenetic modulator acts on its own genetic locus? Based on the results from the multiple papers, it seems like this is the proposed mechanism of action of DW-3-15: SjHAT1 inhibition --> reduced acetylation --> chromatin condensation --> SjHAT1 downregulation --> deleterious effects. Given the amount of work on this molecule and the target(s) over several published papers, I think that this manuscript would be a good place for the authors to summarize the current state of their working hypothesis for how DW-3-15 acts on SjHAT1 to have negative effects on schistosome fitness. The potential for other targets or non-specific events needs to be considered, especially since the molecule was shown to act on other stages while the experiments reported here focus on adults (and, particularly, females).

Reviewer #2: The manuscript focuses on Schistosoma japonicum Histone Acetyltransferase (SjHAT1): A Novel Anti-schistosomal Drug Target. The authors have already demonstrated in prior work that a praziquantel derivative acts though SjHAT1 inhibition. In the current work they cloned SjHAT1, showed that it is active in acetylating histones and that it is inhibited by the drug they have described previously. They conducted a phylogenetic analysis, showed stage and sex-specific patterns with qRT-PCR, and examine localization using microscopy including FISH and EM. Lastly, they undertook an RNA sequencing analysis comparing knockdown to control. Overall their data suggests that downstream effects are on pheromone signaling and reproduction.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: NA

Reviewer #2: 1. The phylogenetic analysis is misinterpreted or misconstrued. The tree shows the evolution that is expected of a vertically inherited schistomsome gene. I think it is fine to report it, the issue is just the result are conveyed as being unusual, when in fact they are quite ordinary.

2. Figure 6 is unnecessary. The results are summarized in the text and nothing new is added. At most they could be summarized in a table. But I think it could be improved by replacing it with a heat map of the differentially expressed genes that shows the regulation and has labels that highlight genes in pathways discussed in manuscript.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Line 60: notbaly --> notably

Line 253: bilogical --> biological

Line 312: comparation --> comparison

Line 313: replicates --> replicate

Line 375: genders --> sexes

Line 414: italics on in vitro

PRJNA1313613 is not available on NIH BioProject

Figure 5C - Given that the images are available, is it possible to get quantitative measurements? The qualitative images are not entirely convincing.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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Dear Prof. Xia,

We are pleased to inform you that your manuscript 'Schistosoma japonicum Histone Acetyltransferase 1 (SjHAT1): A Novel Anti-schistosomal Drug Target' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Mostafa Zamanian

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: I thank the authors for their revisions and congratulate them on a compelling project.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

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PLOS authors have the option to publish the peer review history of their article (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: No