PONE-D-21-21871

Candida albicans PPG1, a serine/threonine phosphatase, plays a vital role in central carbon metabolisms under filament-inducing conditions: a multi-omics approach.

PLOS ONE

Dear Dr. Al Bataineh,

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.

Two experts in this field thoroughly review this manuscript. Both of them appreciated the quantity and quality of this work, but raised a series of editorial and experimental concerns that the authors should pay attention to. In particular, the authors may need to further analyze their transcriptome and metabolomics data to obtain a better picture of PPG1-dependent signaling networks. 

Please submit your revised manuscript by Sep 11 2021 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: http://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,

Yong-Sun Bahn, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"The authors wish to acknowledge the generous support of the Research Institute for Medical and Health Sciences, University of Sharjah UAE. We are grateful to David Kadosh (U.T. Health, San Antonio), USA, to provide C. albicans strains. This work was supported by research grants 1701090226-P/MTA, 1901050144/M.H., University of Sharjah, Sharjah, UAE."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"MTA/1701090226-P, MH/1901050144, University of Sharjah, Sharjah, UAE.

the Research Institute for Medical and Health Sciences, University of Sharjah UAE. This work was supported by research grants. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript"

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

[Note: HTML markup is below. Please do not edit.]

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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: No

**********

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

**********

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: This study performed combined RNA sequencing and metabolomics assay to check the detailed function of PPG1 in filamentation and virulence. Generally, this study showed plenty of data with appropriate analysis, which gives useful information. The manuscript is well written. And the author discussed the potential mechanism by which PPG1 regulates filamentation and virulence logically. But I have some suggestions and comments which may be useful for improving the manuscript.

1. The manuscript supplied enough data but didn't end with a clear story or conclusion. Actually, I still don't know how PPG1 regulate filamentation and virulence. Just saw some genes upregulated and some others downregulated. Therefore, more extended analysis is necessary to tell a logical story.

2. The author directly compares the gene expression between WT and PPG1 KO strains under the FBS inducing conditions. But to find filamentation related genes that are regulated by PPG1, it is better to know the FBS induced transcription in WT group first, and see what kind of genes are not induced or suppressed in KO group. So, I suggest the author should set the groups as WT vs WT+FBS and PPG1KO vs PPG1KO+FBS, if they have the original data.

3. The whole paper supplied enough omics information but with no confirmation. qRT PCR is necessary to confirm the transcription even they used 3 biological replicates, in view of that they neglect the P value or FDR when chosing the changed genes. In particular, the potential targets for PPG1 mentioned in discussion part need to be confirmed.

4. I believe PPG1 regulates the transcription of HWP1, ECE1, ALS3, but the high expression of HSGs, is a common effect by deleting many filamention related genes. And the author try to link the transcription data to metabolomics data, such as the author suggested ALA may function through a Ppg1- dependent mechanism. But I think it is not enough. Some bench work is needed to support the prediction, such as overexpression the targets in PPG1 KO strain and detect the metabolisms.

5. A working model of PPG1 may help to understand its role in filamentation and virulence. PPG1 itself is a serine/threonine phosphatase, a graph contains the substrate and their filamentation targets as well as metabolisms will be helpful for understanding.

Reviewer #2: Summary: The authors have previously characterized Ppg1, a PP2A-type protein phosphatase that controls filament extension and virulence in C. albicans. This study is a follow up analysis of the ppg1Δ/Δ strain in regulating transcriptome relevant to morphogenesis using RNA sequencing analysis. The authors identified that downregulation of well-characterized genes linked to filamentation and virulence as well as the genes involved in the central carbon metabolisms were down regulated in the mutant strain. Their subsequent metabolomics analysis of C. albicans ppg1Δ/Δ strain revealed a negative enrichment of metabolites with carboxylic acid substituents and a positive enrichment of metabolites with pyranose substituents. The authors concluded that Ppg1 is a link between metabolites substituents and filament formation controlled by a phosphatase to regulate morphogenesis and virulence. Overall, this manuscript is descriptive, and no functional validation was undertaken to validate the RNA-seq/metabolomics finding. The authors make a case that Ppg1 controls carbon metabolism and filamentation, but the connection to hyphae based on metabolism is not clear.

Comments:

1. The authors conducted large scale transcriptome and metabolome analysis and generated significant amount of data set. They would be able to articulate more defined biological implications of Ppg1 function with a thorough data analysis on the valuable resources they already possess. For example, it would be interesting to see how the transcriptome changes in the wild type or ppg1 mutant strains responding to 30 C control vs. 37 C serum conditions. Just for an overview, they would be able to conduct a PCA analysis (or a hierarchical clustering) of 8 samples presented in Table 1 to compare the overall transcriptome of the wild-type and the ppg1 mutant strain under different conditions.

2. The authors analyzed the transcriptome of the wild type and the ppg1 mutant strains at 3 and 5 hrs post hyphal induction. Based on their previous study published in 2014, the ppg1 mutant demonstrated a similar expression pattern of ALS3, HWP1, and ECE1 at 3 and 5 hrs. However, the expression of these genes (and NRG1) was quite distinctive at 1 or 2 hrs post-induction compared to the wild-type strain. Thus, it is expected that ppg1 mutation would have impacted the transcription of genes at the earlier stage of hyphal induction. I am wondering why the authors did not choose earlier time points for transcriptome analysis. Is there any clear rationale why the authors chose 3 and 5 hrs only? Of those two time points, the authors only used the transcriptome at 5 hrs for their data analysis and the reason was not clearly stated either.

3. Even though the authors demonstrated that Ppg1 is involved in hyphal induction, it is also possible that Ppg1 would play roles in carbon metabolism under yeast condition. Since the authors already have transcriptome data at 30 C, I would recommend comparing the transcriptome of the wild type and the mutant strains under yeast condition as well.

4. What is the control condition for the metabolomic analysis? Is it the same as the control condition for the transcriptome analysis (30C no serum)? The comparison of the control and inducing conditions were not clearly stated in the method or result sections. Please change “PPG1 null” to “ppg1 delta/delta” to be consistent with the transcriptome data.

5. Metabolite normalization: if the ppg1 strain is a slower grower, then the final cell number at the harvest time (after 3 and 5 hrs) would have been different. The presentation of the metabolites/total protein would be more appropriate for normalization.

1. In #360-374, the authors claimed that Ppg1 is involved in central carbon metabolism and cell wall architecture. However, the discussion is descriptive and has no supporting evidence. In S. cerevisiae, Ppg1 homolog is involved in glycogen accumulation. Have the authors tested glycogen accumulation in the mutant strain? If Ppg1 plays a role as a master regulator, as the authors speculated, we would expect to see altered cell wall architecture or cell wall integrity. Have the authors tested for cell wall stressor susceptibility or chitin staining to see any compensatory chitin upregulation in the mutant strain?

6. #214 at the 37C non-inducing condition – 30C?

7. #216 can you further elaborate what “comparable results” means?

8. In consistent wording for time point description: #218, 5 hours; 3219, 5-hour; #223, 5hr.

9. #237 and # 238; PPG1 (Gene), Ppg1 for protein

10. #238, “essential genes” are often used for their functional relevance to cell viability. Did you refer the genes that are directly impacted by Ppg1? Then please use a different term.

11. Table 1, could you include functional categories at the front row of this table? Carbon metabolism, hyphal specific genes, etc.

12. #266 wording in the section title: grown under filament-inducing “condition”

13. #307 “ppg1 mutants” to “ppg1 Δ/Δ”

14. #319 C. albicans to strain, please revise the sentence in #318-320.

15. #325 “increased metabolism” can you specify?

16. #336 Italicize “Candida”

17. #362 add space between “table” and “1”

18. GEO submission numbers are not found.

**********

6. 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

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Candida albicans PPG1, a serine/threonine phosphatase, plays a vital role in central carbon metabolisms under filament-inducing conditions: a multi-omics approach.

PONE-D-21-21871R1

Dear Dr. Al Bataineh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Yong-Sun Bahn, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Two original reviewers re-evaluated the revised manuscript, and both agreed that it was properly revised. Although the reviewer 2 made a very minor comment in Figure 3 labeling, I believe that it could be easily fixed during proofreading.

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: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

**********

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

**********

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: The author addressed my corcerns well, unless some tables inculding Table1 and Table s3 seem incomplete.

Reviewer #2: Just a really minor comment on the Figure 3 data label; please switch the order to be WT control, WT serum, ppg1 null control and ppg1 null serum. Otherwise, the authors addressed the reviewers comments well with clarity.

**********

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.

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

Reviewer #2: No