Deletion of the Candida albicans TLO gene family results in alterations in membrane sterol composition and fluconazole tolerance

James O’Connor-Moneley; Jessica Fletcher; Cody Bean; Josie Parker; Steven L. Kelly; Gary P. Moran; Derek J. Sullivan

doi:10.1371/journal.pone.0308665

Peer Review History

Original SubmissionJuly 3, 2024
23 Jul 2024 Decision Letter - Rajendra Upadhya, Editor PONE-D-24-27239Deletion of the Candida albicans TLO gene family results in alterations in membrane sterol composition and fluconazole tolerancePLOS ONE Dear Dr. Sullivan, 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 Sep 06 2024 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. 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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 would like to acknowledge support from Science Foundation Ireland (SFI, Grant No. 19/FFP/6422 [DJS & GPM]) and the Dublin Dental University Hospital. The funders did not play any role in study design, data collection, analysis or in the decision to publish." Please note that 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. 3. Please note that your Data Availability Statement is currently missing the direct link to access each database. If your manuscript is accepted for publication, you will be asked to provide these details on a very short timeline. We therefore suggest that you provide this information now, though we will not hold up the peer review process if you are unable. 4. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical. 5. Please include all 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. 6. 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: Dear Drs. Sullivan and Moran, We appreciate your submission of the work to PLOS ONE. This manuscript has undergone a thorough examination by two reviewers, both of whom appreciated the work and acknowledged that the results of the manuscript offer a valuable dataset that could potentially contribute to our understanding of the role of the TLO gene family in azole tolerance. I believe that addressing a few minor concerns identified by one of the reviewers will enhance the clarity of the work for the readers. [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: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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 ****** 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: The intention of the authors of the manuscript was to investigate the influence of the TLO gene family in Candida albicans on its resistance to fluconazole. The publication showed that the deltatlo mutant is more resistant to fluconazole than the initial strain (WT). The next stage of the research was to obtain transcriptomes of the tested strains growing without and with fluconazole and to analyse gene expression. Based on the transcriptome analysis, the authors selected possible mechanisms of C. albicans resistance to fluconazole and used the qPCR method to examine the expression of the selected genes and the activities of the proteins encoded by them. It can be concluded that the research methods adopted by the authors is absolutely correct. As is often the case, the results obtained did not provide a clear answer to the question of what mechanism is responsible for the resistance of deltatlo mutants to fluconazole. The effect appears to occur through pleiotropic changes in the cell, including perhaps as yet unknown mechanisms. The work submitted for publication indicates a number of issues whose explanation should be continued and deepened. The ms is therefore rather an introduction to further research. However, this type of work undoubtedly contributes to the development of science in the field of fungal resistance to azoles and that's why I recommend ms to be published in Plos One. Line 608-612 – the authors should write what they want to present in a different way, because now it seems that these two sentences contradict each other. Reviewer #2: This article describes the characterization of a Candida albicans mutant deleted for all members of the TLO gene family, encoding a subunit of the mediator, regarding fluconazole tolerance. The authors use transcript profiling comparison to uncover the molecular basis for drug tolerance in the mutant and show a link with respiration; they also analyze cell membrane composition in parallel with expression studies on individual ERG genes. Overall, the data presented here are rather convincing even if they do not uncover a major pathway linking TTLO genes and Fluconazole tolerance. However, I have several concerns that need to be addressed: In the result section, the authors should mention that the tlo mutant displays some resistance toward fluconazole, as experiments clearly show an increase of MIC (from 0.25 to 0.75 µg/ml). The KO mutant is complemented by either one of 3 genes, alpha1, beta2 or gamma11, representative of the three gene clades. A discussion about their similarities is missing. Moreover, to my knowledge, no NLS could be predicted for TLO11. Maybe another gene from that clade could have been used, or the grounds for the choice discussed. In the discussion, the authors suggest that the observed phenotype could be linked with a reduced expression/activity of Erg251. Yet, overexpressing ERG251 in the tlo KO had no impact, suggesting the involvement of other proteins. There is a discrepancy between the results section and legend of figure 6, which states that cells were grown in YPD. Which is correct? Why have the authors used the CDG C. albicans Assembly 21 to analyze the RNA-sequencing data, and yet use the systematic names of Assembly 22? The genotypes should be properly written at least in the parental strain, and each new mutant also described, including selection markers. The med3 KO mutant is not in the table. And please use either gene names (eg TLO1) or phylogenetic names (TLOalpha1), and be consistent throughout the article, including figures and legends. Same for strain names DSY, and not Dsy. Fig1A and 1C: replace “RPMI only” by “Fluconazole”, and “RPMI containing …” by “Fluconazole + …”, as in Figure 5. S Figure 2: ergosta-5,7-dienol should be in bold. Typos: line 140 and Fig 4A (Fluconaozle); line 178 (50 mls); lines 245, 246, 249 (an MIC); lines 296, 332 (preformed); line 452 (cholestra). Check syntax line 140-141, line 164-165. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
26 Jul 2024 Author Response Academic Editor/Journal Requirements: 1. PLOS ONE style requirements We have reviewed the document and made some minor adjustments in line with the recommended templates. All submitted files have been named as recommended by PLOS ONE. 2. Acknowledgements We have removed any reference to funding in the text. Therefore, there is no longer a need for a separate acknowledgements section, and this has now been deleted (see lines 640-643). 3. Data Availability Statement The URL which links directly to the RNA-sequencing data submitted to the National Centre for Biotechnology Information (NCBI) sequence read archive has been added to the Data Availability Statement as requested. 4. Abstract The discrepancy between the two abstracts was the result of the online submission form being unable to recognise Greek lettering. We have now removed these from both abstracts, and they should now be identical. 5. Supporting Information file captions. We had omitted the captions for the Tables included as Supporting Information and these have now been added (see lines 802-810). 6. Reference list The reference list is complete and correct. No changes have been made from the original submission. Reviewer # 1 The first sentence refers specifically to the ∆tlo mutant, while the second sentence was originally describing the ERG-overexpressing strains. However, to remove any ambiguity we have deleted the second sentence (see lines 621-625), as this is not essential. Reviewer # 2 1. Resistance phenotype Deletion of the TLO gene family results in an increase in tolerance and resistance of C. albicans to fluconazole. We felt that we had highlighted this in the original text, but we have reiterated the increase in resistance (see lines 252-253) to ensure this is now clearer. 2. Comparison of the TLO clades We have included additional text (see lines 102-105) in the Introduction highlighting differences that we have previously observed between the gamma-clade genes and the apha- and beta-genes. In a previous study (reference [22]) we compared TLOy11 and another gamma gene, TLOy5, and both were found to have identical effects, suggesting that the choice of TLOy11 as a gamma-clade representative is sound. We have also added a sentence at the end of the Discussion (see lines 634-638) stating that future experiments will include additional members of each of the alpha- and gamma-TLO clades to see if there is intra-clade variability in Tlo protein functionality. As there is only one member of the beta clade there is no need for any further analysis of this clade. 3. ERG251 We agree with the reviewer (and with Reviewer #1), that the tolerance phenotype is almost certainly pleiotropic and that additional proteins are likely to be implicated. We had originally included this in the text, but have specifically referenced ERG251 in this context (see line 597-598) to emphasise this point. 4. Figure 6 The data described in Fig 6C are based on the analysis of a dataset from a previously published study (i.e. reference [22]), in which similar RNA-seq experiments were conducted on strains grown in liquid YPD medium. This has now been explicitly highlighted in the text (see lines 432-434) 5. Genome assemblies. We have corrected the text (see line 165) to refer to the CGD C. albicans Genome Assembly 22, not Assembly 21, as originally stated. 6. Genotypes S1 Table, the supplementary table detailing the genotypes of strains used in the study, has been amended, to include the addition of the med3 deletion mutant and selection markers. Gene and strain names are now used consistently throughout the text and figures as suggested. 7. Figures Fig 1A and 1C have been amended, however, S2 Fig: ergosta-5,7-dienol is already in bold 8. Typos and syntax All suggested corrections have been made, although we believe that “an MIC” is correct. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0308665.r002
29 Jul 2024 Decision Letter - Rajendra Upadhya, Editor Deletion of the Candida albicansTLO gene family results in alterations in membrane sterol composition and fluconazole tolerance PONE-D-24-27239R1 Dear Drs. Sullivan and Moran, We’re pleased to inform you that your revised 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Rajendra Upadhya Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0308665.r003
Formally Accepted
1 Aug 2024 Acceptance Letter - Rajendra Upadhya, Editor PONE-D-24-27239R1 PLOS ONE Dear Dr. Sullivan, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Rajendra Upadhya Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0308665.r004

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