PONE-D-24-50847Transcriptomic analysis of benznidazole-resistant Trypanosoma cruzi clone reveals nitroreductase I-independent resistance mechanismsPLOS ONE

Dear Dr. Ana Maria Mejia-Jaramillo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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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Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

From the perspective of drug resistance, is there a possible correlation between different DTUs or strains of the parasite and their levels of resistance or susceptibility?

What are the genotypes of the strains used in this study? Discussing these aspects could help clarify this issue.

Have the researchers observed any variations in parasite susceptibility based on its biological stage? While epimastigotes are commonly utilized for studies, could trypomastigotes (the infective form in mammals) exhibit different responses? Additionally, have the researchers detected any differences at the transcriptomic level?

Line 88. Please clarify what you mean by "same experimental and environmental conditions."

Line 198. It would enhance the discussion by providing more information about the strains and clones analyzed.

Line 462. Please replace "Trypanosoma cruzi" with "T. cruzi."

Figures: Please enhance the quality of the figures.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The article presents a very novel work. It is approached from a transcriptomic approach to elucidate the possible mechanisms of resistance to BZ in populations of T. cruzi resistant by pressure to induced BZ combined with CRISPR Cas technology for genetic knock out (It was lacking to exploit these results a little more in the discussion). However, the discussion of the results needs to be strengthened in general by comparing with more related literature. Although one of the most relevant conclusions of the article is to reaffirm what has been reported by other authors, NTR I independent and multifactorial resistance mechanisms. It remains to further nurture this discussion by trying to present the possible alternative mechanisms that were rightly mentioned in the introduction (lines 44-50).

>What is the reason why the authors performed the work with BZ and not with NFX or both (not bad, but to know their reasons would be good). Why were IC50 determinations made for both, but only selective pressure against BZ? What is the reason for making this decision?

In the methodology it is mentioned that 10 concentrations of Bz and NFX were used, specify which were those concentrations. It also does not specify the presentation in which NFX and BZ were used, pure, tablet or purified from a tablet?

>What is the justification for performing the determination of the IC50 at 72 h?

>Specify in the methodology what was the concentration of rezarzurin used since only 20 ul/well is mentioned, but this is very ambiguous.

>How do they justify the use of the Epimastigote form and not the trypomastigote form?

>While the focus of the article is not to relate this to intra and inter DTUs genetic variability of T. cruzi (suggested to mention this in the introduction as a possible factor that may influence heterogeneity of response to Bz), how scalable or representable are these results for the other DTU's? Would one expect a big change using another DTU or another TcI isolate?

Reviewer #2: This is the revision of the study entitled 'Transcriptomic analysis of benznidazole-resistant Trypanosoma cruzi clone reveals nitroreductase I-independent resistance mechanisms' by Mejia-Jaramillo et al. The study is robust and well-executed, with the generation of benznidazole-resistant T. cruzi aligning with findings reported in the literature. While the study has some flaws, its findings are valuable to the scientific community as they highlight resistance mechanisms entirely independent of nitroreductase I.

The introduction includes a few ambiguous statements that require clarification. Additionally, the Materials and Methods section lacks crucial details necessary for reproducibility. The Results section also needs refinement to improve the clarity and presentation of the data.

Introduction

Lines 38-43: The end of this paragraph is highly inaccurate. Both Benznidazole and Nifurtimox have satisfactory results in the acute phase of Chagas disease (50-80% efficacy) but debatable efficiency in the chronic phase. The cited percentage of people discontinuing treatment is also unknown to me, as the last published finalized clinical trial BENEFIT (10.1056/NEJMoa1507574) showed a number of 13.4% of treatment discontinuation due to adverse events. If a study showed 20% of treatment discontinuation, please cite it in the text. I would cite clinical trials aiming to test both drugs in a prolonged treatment scheme too (10.1136/bmjopen-2021-052897).

I suggest updating the references and citing more relevant bibliography.

Here are some suggestions:

10.1111/bcp.14700

10.1056/NEJMct1014204

10.3390/vaccines12080870

Lines 45-47: “In resistance to Bz and Nfx, the enzyme nitroreductase I (NTRI) has been implicated as the primary gene responsible for resistance in vitro [4,5] and in patients with treatment failure”. The sentence is confusing; I would rephrase it to make it understandable that it is the gene encoding the enzyme nitroreductase that is associated with drug resistance.

Materials and Methods

Please revise the text to provide catalog numbers, the manufacturer of the kits/reagents used, and complete names and models of the equipment used.

The methods section is quite detailed and dense, which may make it challenging for readers to follow. To enhance clarity and accessibility, I recommend including a visual graphic that summarizes the key steps and analytical processes described in the paper. This could help readers better understand the workflow and overall approach at a glance.

Lines 74-81: In this section, it is not clear to me if you added Bz, G418, and Puromycin in the same media or if they were added into different media comprising different groups (one was treated only with Bz, one only with G418 and one only with Puromycin). Please clarify and explain why the use G418 and Puromycin (positive/negative controls? Or selection of a specific genotype?) If they’re for different experiments, please include the methodology for each experiment separately.

Are M-RATTUS, CO, 91, GAL-80 and 61.SUC different T. cruzi genotypes or clones? Please specify in the text, it is not clear. In that case, was the IC50 for Bz exactly the same value for all strains?

Lines 90-91: Cite a reference for the alamarBlue method for the determination of IC50.

Lines 101-102: From my understanding, only the 61.SUC clone was selected for Benznidazole pressure experiments, but that is not clear in the text. Please clarify how that decision was made and what led you to that decision.

Line 152: The numbers do not need to be written; they can be used as numerical numbers.

Lines 161: Include the name of the kit used for library preparation.

Results

Overall, if it is possible, I would change the colors to red for up-regulated and blue for down-regulated genes. This is what we most see in omics papers, and I believe it would make it easier for understanding.

Line 187: It’s ‘passages’ instead of ‘passes’.

Lines 187-198: Please mention the IC50 for each of the groups, corroborating fold-change resistance to Bz. If you mentioned it for Bz, mention the values for Nfx as well.

The 61S_Cas9, SKO_NTR1, and DKO_NTRI groups were not mentioned in the results, only in the figure legend. You either exclude this data from the graph or present the results in your text.

Lines 223-229: I see that the Figure 1 61S_Cas9, SKO_NTR1, and DKO_NTRI groups are mentioned here. However, I suggest separating the graphs and making another figure (or maybe Figure 1C) to fit this part of the results.

Line 240: Why was the Log2FC chosen of 0.58, when usually the Log2FC chosen for genes is 1?

Line 252: What is the importance of showing that the DRGs are annotated as conserved

hypothetical protein (CHP) and unspecified product (UnP)?

Figure 2C: Please increase the font size for the gene names, as they will be unreadable in the paper.

Figure 4. I recommend separating each functional group in (A) Transmembrane Transport, (B) Ribosome and (C) Nucleobase metabolic process, and mentioning it separately in the text.

S4 Fig. Legend description and presentation of results need to be improved. What does the blue, pink and yellow colors in the subunits mean? What question you plan to answer with this analysis?

Figure 6. Please increase the font size for readers.

Reviewer #3: The manuscript by Mejia-Jaramillo et al.describes the results of an exhaustive study centered in the characterization by RNA-seq analysis to identify genes other than nitroreductase-I (NTRI) involved in benznidazole and nifurtimox activity against Trypanosoma cruzi, the causative agent of American Trypanosomiasis (Chagas disease). The authors conclude that besides intrinsic NTRI toxicity, upregulation of heat shock proteins (HSPs) and of CDC45 in resistant clones could favor more efficient detoxification, DNA replication and repair under Bz/Nfx genotoxic stress. These are novel and very relevant findings in the characterization of molecular mechanisms involved in the resistance to the only two drugs currently available for the etiological treatment of Chagas disease, in terms of the potential prevention of such resistance via combination treatments.

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Reviewer #1: No

Reviewer #2: Yes:  Priscila Silva Grijo Farani

Reviewer #3: Yes:  Julio A. Urbina

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PONE-D-24-50847R1

Transcriptomic analysis of benznidazole-resistant Trypanosoma cruzi clone reveals nitroreductase I-independent resistance mechanismsPLOS ONE

Dear Dr. Ana Maria Mejia-Jaramillo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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.

Please submit your revised manuscript by Mar 05 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Claudia Patricia Herrera, Ph.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Thank you for submitting the revision and addressing the reviewers' comments. I believe the paper is ready for publication; however, I would appreciate it if you could take the last comment from Reviewer 2 into account before final acceptance.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I have read the revised version, and it is much better and more scientifically robust, I would just like to point out two things to the authors.

1. in relation to resarsurin, the authors state the use of 20 uL of the above mentioned reagent but they still omit the concentration they have of the compound in those 20 uL, please specify.

2. Has recently been published the work; Ochoa-Martínez P, López-Monteon A, López-Domínguez J, Manning-Cela RG, Ramos-Ligonio A. Expression Analysis of Thirteen Genes in Response to Nifurtimox and Benznidazole in Mexican Isolates of Trypanosoma cruzi by Digital PCR. Acta Parasitol. 2025 Jan 7;70(1):15. doi: 10.1007/s11686-024-00986-w. Where a review of the activation of various genes in response to stress exerted by BZN and NFX is made, what can the authors discuss their results in relation to this work?

Reviewer #2: The author has thoroughly addressed all the suggestions and recommendations provided. Upon further review, I am confident that this article is now suitable for publication in PLOS ONE.

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7. 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.

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Reviewer #1: No

Reviewer #2: Yes:  Priscila Silva Grijo Farani

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Transcriptomic analysis of benznidazole-resistant Trypanosoma cruzi clone reveals nitroreductase I-independent resistance mechanisms

PONE-D-24-50847R2

Dear Dr. Ana Maria Mejia-Jaramillo

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Kind regards,

Claudia Patricia Herrera, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: