Dear Dr Albuquerque:

Thank you very much for submitting your manuscript "A hidden battle in the dirt: soil amoebae interactions with Paracoccidioides spp" (PNTD-D-19-00966) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

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Sincerely,

Todd B. Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases

Todd Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The methods are clear, concise and well designed to address the study objective. The sample size was enough to ensure the results were statistically valid. Ethical requirements were met.

It would be good if the authors give references for all their methods i.e. electron microscopy (EM) work, etc.

For EM, it would have been good to include a control of cryptococcal cells alone to exclude the harsh preparatory steps of EM as the reason for collapsed cells.

Reviewer #2: Yes.

Reviewer #3: The authors should not include results in the Introduction section "These studies showed that amoebae efficiently ingest and kill Paracoccidioides spp. yeast cells, and that this interaction selects for fungi with increased virulence. Our results support the hypothesis that interaction with neighbor soil predators selects for traits that allow survival of Paracoccidioides spp. in mammalian hosts and add to the existing evidence for the amoeboid predator-animal virulence hypothesis [13].".

The objectives of the study are clearly articulated

The methods were described with sufficient detail and there is information to replicate.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis match the analysis plan. However, lack of a control for EM work questions the findings.

To harvest the cryptococcal cells, amoeba had to be lysed. Could the latter see amoeba also contributing to the observed levels of glucans?

How did the authors normalise the mRNA levels of genes of interest?

Reviewer #2: Yes.

Reviewer #3: The results are clearly and completely presented, and the figures are sufficient quality.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The public health relevance is adequately addressed. The conclusions that reached confirm what is already known in literature.

Reviewer #2: Could be modified.

Reviewer #3: The limitations of study were not described. I suggest Include and discuss the limitations of your study.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Minor revision

Reviewer #2: Lines 186-187: the original strain is part of the comparison, so not a control. Could be “with strains Pb18-Ac or the non-passaged form Pb18.”

Line 203: space “glucan, and”.

Line 206: “analyses” plural.

Line 234: “hits at GenBank”.

Line 274: “were” [bacteria plural].

Line 354: “through mechanisms other than mRNA accumulation”.

Line 386: “1970s”.

References: check the formatting is correct, as the journal will be unlikely to do so, e.g. italics on species or gene names.

Lines 728-729: change to be “Figure 7- Changes in P. brasiliensis Pb18 gene expression after cycles of interaction with amoeba. Transcript accumulation…” the first sentence in bold.

Reviewer #3: “Minor Revision”

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The presented speak to the idea that amoebal predatory pressure may select Paracoccidioides to become pathogenic. This (selection) has also been observed elsewhere for other fungal species. Therefore, there is no novelty in the work. Nonetheless, the work still highlights the medical importance of the fungus and acquisition of its virulence.

The authors should consider sufficiently addressing the issues that are raised.

Reviewer #2: The manuscript reports on the interaction of Paracoccidioides strains with three amoeba species isolated from armadillo burrows, which are associated with Paracoccidioides. Passage of fungal isolates through amoeba lead to ‘evolved’ strains with increased virulence in both mice and an insect model, Galleria mellonella, leading to support of the hypothesis that amoeba (or other predators) are important in maintaining the virulence of Paracoccidioides when it encounters humans.

This is a substantial body of work. One reflection of this is that mouse studies went for 12 months. The electron microscopy adds the visual appeal of the work, and the level of replication to infer statistical significance is noteworthy. The work will appeal to the medical mycology community, especially those working on pathogens that have environmental reservoirs. Probably the main point of concern is how the results are interpreted overall.

1) ‘in controlled laboratory conditions using mostly pure and axenic cultures of

laboratory-adapted predators; this very informative system is nonetheless an extreme

simplification of the complex ecosystem soil saprophytes find in nature’ is a very valid point, and it is relevant that here new amoeba strains were isolated and characterized for their interaction with Paracoccidioides strains. However, counter to this the authors then rely on the standard A. castellanii strain ATCC 30234 [which was identified as a natural predator of another pathogenic fungus, Cryptococcus neoformans, so not as artificial as other amoeba strains] for subsequent characterization of the impact of amoeba on Paracoccidioides by passaging.

2) ‘Sequential co-cultivation of Paracoccidioides with A. castellanii selected for

phenotypical traits related to the survival of the fungus within a natural predator as well as in

murine macrophages and in vivo (Galleria mellonella and mice). This increase in virulence…’ However; Pb18 is already an attenuated strain, so while certainly the passaging has changed the virulence properties, it is unclear that this is not just back to the baseline level.

3) A point that needs addressing more explicitly, counter to statements like “the higher susceptibility of Paracoccidioides to amoeba”, is that some survive predation to become more fit in causing disease. The data (such as figure 2) show that a considerable proportion of the fungal isolates survive.

Reviewer #3: The manuscript "A hidden battle in the dirt: soil amoebae interactions with Paracoccidioides spp." is well written in an engaging and the level is appropriate to readership of PLoSNTD. I have no hesitation in recommending that it be accepted for publication after a few minor details have been attended to, without the need for further experimental work.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[EXSCINDED]

Dear Dr Albuquerque,

We are pleased to inform you that your manuscript, "A hidden battle in the dirt: soil amoebae interactions with Paracoccidioides spp", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please note: your manuscript will not be scheduled for publication until you have made the required changes.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Todd B. Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases

Todd Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases

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