PPATHOGENS-D-24-02306Vacuolar sterol β-glucosidase EGCrP2/Sgl1 deficiency in Cryptococcus neoformans : dysfunctional autophagy and Mincle-dependent immune activation as targets of novel antifungal strategiesPLOS Pathogen Dear Dr. Ito, 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 Please submit your revised manuscript within 60 days Jan 23 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers '. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below. * 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. We look forward to receiving your revised manuscript. Kind regards, Kirsten Nielsen, Ph.DGuest EditorPLOS Pathogens Michal OlszewskiSection EditorPLOS Pathogens Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064 Additional Editor Comments : The concerns regarding controls raised by Reviewer 2 and 3 need to be addressed. Both reviewers 1 and 2 were concerned about the biological relevance of the studies and these concerns should be addressed with proposed experiments or similar ones that provide the necessary clarification. Journal Requirements:

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: Takashi Watanabe, Masayoshi Nagai, Yohei Ishibashi, Mio Iwasaki, Masaki Mizoguchi, Masahiro Nagata, Takashi Imai, Koichi Takato, Akihiro Imamura, Yoshimitsu Kakuta, Takamasa Teramoto, Motohiro Tani, Junko Matsuda, Hideharu Ishida, Sho Yamasaki, Nozomu Okino, and Makoto Ito. 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: In this study from Watanabe et al, the authors aim to understand the mechanisms underlying decreased virulence that has been previously observed in Cryptococcus neoformans strains deficient for sterol-beta-glucosidase. The authors suggest a two-fold mechanism: 1) Sgl1-deficient Cn results in increased rates of yeast cell death when cultured at 37C due to defective autophagy, and 2) Deletion of Sgl1 results in accumulation of ergosterol-beta-glucoside as wells as acetylated ergosterol-beta-glucoside, the latter of which can function as a ligand for the C-type lectin receptor Mincle when secreted by EVs. Overall, the results demonstrating defective autophagy in vitro and accumulation of EGs and AEGs are clear. The identification of AEGs as Mincle ligands is also an important insight.

However, the significance of the autophagy defect vs Mincle activation in vivo, and the actual activation of Mincle by the KO Cn strain are less convincing. The authors generally rely on ultracentrifugation of supernatants to isolate EVs and then test their ability to activate DCs, however, this approach may result in non-physiological enrichment of trace amounts of lipid activity.

Major Comments:

1. The authors show clearly that 37C culture of KO Cn results in increased yeast cell death and that this correlates with decreased expression of autophagy genes and localization of ATG8 with vacuoles. What is less clear to me is whether the observed effects on autophagy are a cause or a consequence of the cell death at 37C? Much of the analyses done for testing autophagy function are done in nitrogen starvation media – how does this relate to the late increase in cell death when culturing in YPD? Are there ways to rescue/increase autophagy in the KO cells to see if this rescues?

2. I’m convinced that KO Cn produces more EG and AEGs, and that AEGs will activate Mincle when you add them to BMDCs or into reporter lines. My concern with the EV experiments is that these may be added in supraphysiological concentrations in the functional assays. In Fig 12, the KO EVs give only a 2-fold increase in production of MIP-2 from BMDCs compared with WT, which is inconsistent with almost non-existent concentrations of EG and AEGs in WT Cn compared to KO. As a very simple experiment, if you just challenge BMDCs or the Mincle reporter line with WT vs KO Cn, do the KO cells actually secrete enough AEG to activate? If so, can you observe the same effect with conditioned supernatants? These sorts of experiments are crucial to perform before doing ultracentrifugation on supernatants.

3. Can the authors perform some experiments to assess the relative contributions of autophagy defects vs increased Mincle production to the virulence defect in vivo? The authors show an increase in CFU of KO Cn at Day 3 post-infection in Mincle-deficient mice compared to WT mice. This is a crucial line of experiments in that it asks how much of the in vivo defect is due to increased Mincle ligand production vs autophagy defects – I think the manuscript would be well-served to do this experiment as a survival curve or CFU time course to see how much the infection outcome is altered in Mincle KO mice. Alternatively, since Card9 deletion has a much stronger phenotype in vitro for loss of activation in response to AEGs, it might be better to see how much of the CFU defect of Sgl1KO Cn is rescued in Card9-/- mice.

4. Along the same lines as above, the ASG treatment along with WT Cn infection is a nice experiment in that it tests how much of an effect Mincle activation alone has on CFU in the absence of an autophagy defect. However, the actual result is not very convincing. Was this just done once? This experiment warrants a repeat and looking at later time points (if you give repeated ASG doses).

Minor comments:

1. The OD600 defects actually seem more pronounced at 25C and 30C (at least at day 7) compared to 37C. The authors suggest that this may be due to leakage of cellular contents at 37C. However, they also observe very small increased in dead Cn at 25 and 30, despite the strong OD600 defect. Can they comment on this?

2. For the model in Fig 14, I understand some of this is linking in previously published data. However, for this specific manuscript, I think the authors should remove the images from the model that are not specifically based on experiments shown in this body of work. For example, the link to gamma delta T cells is entirely speculative.

Reviewer #2: In general, this paper is a succession of explorations related to the EGCrP2/Sgl1 KO mutant. I fell that there is not much link between the different parts. For example, the autophagy is not really related to the other explorations such as immunology and then EVs. We are missing here a united line for the message of this paper.

Reviewer #3: In this very nice study, the authors investigated the mechanisms responsible for the loss of virulence of a slg1delta mutant in Cryptococcus neoformans. This gene has been identified by this group and that of M. del Poeta, and this deletion has been found to be associated with the accumulation of ergosterol beta-glucoside (EG), but the reason for the loss of virulence remained unknown. In this paper, they showed that Slg1 accumulates in the vacuole and controls autophagy gene expression and protein localization. The authors used a battery of assays to show that a concomitant accumulation of EG acetylated derivatives (AEGs) is probably more important than that of EG. They presented convincing experiments showing that AEGs bind to the C-type lectin receptor Mincle in a very affine manner, activating the production of cytokines that ultimately trigger the elimination of the yeast pathogen. They confirmed the importance of both this lipid and the receptor using animal models coupled with AEGs treatments. It is a very nice story and a very convincing set of data.

**********

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: 1. Challenge WT vs Mincle-/- vs Card9-/- BMDCs with WT vs KO Cn live yeasts to see if the KO strain produces enough AEGs to activate the Mincle pathway.

2. Infect Mincle-/- and/or Card9-/- mice with WT vs KO Cn and perform either survival curves or time course (day 3, 7, 10, 14) CFUs

3. Infect WT vs Mincle-/- mice with WT Cn along with ASGs and perform time course CFUs.

Reviewer #2: All the experiments are performed after 3 days in YPD with agitation. The cells are at this step starved and so not in a normal metabolism. Is it really physiological and do the findings could be generalized to LOG or stationary phase?

How far Phloxin B have been validated in Cryptococcus neoformans we need controls together with the experiment (Dead control 65°C 1h, H2O2 2 to 24hrs).

RNASeq have been perform as simplicates per condition with prevent a accurate analysis of the results in particular normalization and adequate calculation of fold changes. DeSeq 2 in the best method/pipeline to analyze RNASeq and qhould be done like this.

Relative expression by qPCR should not use 2-∆∆Cq as individual qPCR have a different efficiency and should use at least 3 reference genes (see 1. Vandesompele J, Preter K de, Pattyn F, Poppe B, Roy N van, Paepe A de, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome biology. 2002 Jun 18;3(7):RESEARCH0034. )

Considering that more than 60% of the cell population is dead (Fig 2C), it is difficult to make sens of the transcriptome analysis of the cells cultivated in the same condition. Are the ATG8 transcript decreased because of death? I would assume so.Probably the half-life of those transcript are much lower than that of the reference gene explaining this decrease Iin the expression.

Enrichment score is between 1 and 1.5 which is not high. Then, is it really meaningful?

I am not sure that the finding on fumigatus are really bringing something to the message on cryptococcus (Aspergillus ascomycetes and Cryptococcus basitiomycetes are indeed very different organisms in terms of biology.)

Reviewer #3: The following comments are intended to improve the quality of this manuscript.

1) The use of Phloxin B staining to determine cell viability needs to be validated. In the image shown (Figure 2B), both mother and budding cells are stained, which is strange for a dying cell.

2) Figure 5 shows not only degradation but also production of Atg8, which according to the qPCR experiments also decreases over time. This needs to be better explained.

3) Sgl1 localization (Figure 6): showing one cell at high magnification is good, but it should be accompanied by an image showing multiple cells to convince the reviewer that this co-localization is representative.

4) Line 280 "significantly higher should be confirmed by a statistical test and a p-value should be given.

5) The paragraph starting on 309 describing the mouse mincle docking models with AEG or AG is interesting, but the conclusion would need more experiments to be confirmed. The calcium implication comes out of nowhere for the reader. Its implication and role could be confirmed experimentally. This paragraph could be deleted as well.

6) Same for the next paragraph. The point mutation mutant should be tested if one wants to reach a conclusion.

7) Figure 11C, E, F: EG and AEG are detectable in WT and RE strain. This is surprising as it was stated earlier that it is not detectable in the WT cell. This should be discussed.

8) Line 368: There is probably a word missing in this sentence and in the next one, since EVs do not produce MIP-2 or IL-8.

**********

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: N/A

Reviewer #2: (No Response)

Reviewer #3: none

**********

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Reviewer #3: No

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PPATHOGENS-D-24-02306R1

Vacuolar sterol β-glucosidase EGCrP2/Sgl1 deficiency in Cryptococcus neoformans : dysfunctional autophagy and Mincle-dependent immune activation as targets of novel antifungal strategies

PLOS Pathogens

Dear Dr. Ito,

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. Specifically: 1) Move the RNAseq figure(s) to supplemental data and clearly state in the manuscript that the studies were preliminary and had only a single replicate. 2) Address Reviewer 3's concerns regarding the validity of the results from the PhloxinB results based on the current studies. An additional experiment may be warranted.

Please submit your revised manuscript within 60 days May 04 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Kirsten Nielsen, Ph.D

Guest Editor

PLOS Pathogens

Michal Olszewski

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

Journal Requirements:

1) We ask that a manuscript source file is provided at Revision. Please upload your manuscript file as a .doc, .docx, .rtf or .tex. If you are providing a .tex file, please upload it under the item type u2018LaTeX Source Fileu2019 and leave your .pdf version as the item type u2018Manuscriptu2019.

2) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines: 

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If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

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: The authors have satisfactorily addressed my previous comments.

Reviewer #3: The paper has been improved, but there are still a few issues that need to be addressed.

**********

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: N/A

Reviewer #3: 1) The experiment to validate the PhloxinB assay as a means of assessing cell viability is not the one expected. The author should sort out the PhloxinB positive cells and plate them to check that they are dead. Again, the fact that both the mother cell and the bud can be positive suggests that the assay is not valid.

2) I missed this in my first review, but having a single replicate for the RNA-Seq experiment is a real problem. I understand that the authors see this experiment as a way to identify potentially regulatory genes that would be validated by qRT-PCR. However, the description of the RNA-Seq result should be different. For example, it is possible to write and RNA-seq data analysis 'revealed' something. This paragraph should be changed and the fact that a single replicate was generated should be clearly stated in the results paragraph. The figure 3A and B should be deleted.

**********

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: N/A

Reviewer #3: 1) The name of the gene should be changed. The official name is SLG1, as should be a gene name in Cryptococcus (3 letters and a number). It cannot be EGCrP2/Sgl1. This should be changed in the manuscript.

2) The strains cannot be named KO Cn or RE Cn. slg1delta should identify the mutant strain. The reconstituted strain should be named slg1delta::SLG1.

**********

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Reviewer #1: No

Reviewer #3: No

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Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Ito,

We are pleased to inform you that your manuscript 'Vacuolar sterol β-glucosidase EGCrP2/Sgl1 deficiency in Cryptococcus neoformans : dysfunctional autophagy and Mincle-dependent immune activation as targets of novel antifungal strategies' 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,

Kirsten Nielsen, Ph.D

Guest Editor

PLOS Pathogens

Michal Olszewski

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

***********************************************************

The authors adequately responded to the reviewer and editor comments.

Reviewer Comments (if any, and for reference):