PPATHOGENS-D-25-02987

Acoziborole resistance associated mutations in trypanosome CPSF3

PLOS Pathogens

Dear Dr. Horn,

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Kind regards,

Christine Clayton

Academic Editor

PLOS Pathogens

Margaret Phillips

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:

Your paper has been reviewed by one expert referee and I have read it myself. Since it is a straightforward paper with clear results, the expert suggests only minor modifications - including fewer claims of "novelty" and clearer acknowledgement that your mutant design was reliant on previous results from other organisms. I agree with all of the reviewer's suggestions.

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Melanie Ridgway, Marketa Novotna, Cesar Mendoza-Martinez, Michele Tinti, Simone Altmann, Graeme Sloan, and David Horn. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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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: This is a very clean study identifying mutations that could impact the activity of acoziborole in T. brucei. The work is technically well-performed and the manuscript well-structured and written. Overall, it is very nice, although the results are not particularly remarkable (but few really are).

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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: none

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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: With the exception of 1 and 2, these are just suggestions with respect to emphasis - things the authors might consider

1. The titles refer to "trypanosome" CPSF3 when only the T. brucei CPSF3 was studied. The results may not apply to other trypanosomes

2. The authors indicate that the approach to making triple mutants was "novel" in several places - which strictly speaking might be true but is perhaps a bit of an oversell. It was an obvious approach. By this criterion, every new experiment/approach that had not been explicitly performed previously is "novel"

3. a little more discussion of why they think the S412 mutant did not confer any detectable resistance might be useful.

4. While the authors don't hide the fact, they also don't emphasize that making the triple mutant was only possible because single mutations in these sites had been discovered in other organisms - but where not discovered using their approach in T. brucei. That is, without these other mutations to test, this paper largely wouldn't exist (the N232 mutant was reported in the Altmann 2022 paper). This represents a potential limitation of this experimental approach (it is rare to have other drug resistance info from so many other organisms in response to the same class of drugs).

5. The difficulty in inducing and selecting for resistance to this class of compounds in trypanosomes is pretty remarkable. The authors note this, but this point feels quite undersold - and I think more comment in this regard would emphasize better the impact of this study (which otherwise is modest).

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Reviewer #1: Yes: Rick L. Tarleton

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Dear Dr. Horn,

We are pleased to inform you that your manuscript 'Acoziborole resistance associated mutations in Trypanosoma brucei CPSF3' 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,

Christine Clayton

Academic Editor

PLOS Pathogens

Margaret Phillips

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):