Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence

Bruno Maras; Anna Maggiore; Giuseppina Mignogna; Maria D’Erme; Letizia Angiolella

doi:10.1371/journal.pone.0252555

Peer Review History

Original SubmissionFebruary 19, 2021
22 Mar 2021 Decision Letter - Aijaz Ahmad, Editor PONE-D-21-05206 Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence PLOS ONE Dear Dr. Angiolella, 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 May 03 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 We look forward to receiving your revised manuscript. Kind regards, Aijaz Ahmad, 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 thank “Cooperativa Comunale Logistica Industriale Integrata” for the donation to support research.' 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. a. 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: 'LA, GM, BM. Grant number: RM 1181641E9150B3 Sapienza University of Rome https://wwwuniroma1.it The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.' b. Additionally, because some of your funding information pertains to commercial funding, we ask you to provide an updated Competing Interests statement, declaring all sources of commercial funding. In your Competing Interests statement, please confirm that your commercial funding does not alter your adherence to PLOS ONE Editorial policies and criteria by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests. If this statement is not true and your adherence to PLOS policies on sharing data and materials is altered, please explain how. c. Please include the updated Competing Interests Statement and Funding Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: 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: 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: No ****** 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: 1) Line no. 20,21,176,234, 240: There are either typing error or some spelling mistakes in words like microbiome, population, micafungin etc. Please modify. 2- Why the author used different temperature conditions for the solid (Line-98) and liquid media (Line-108) for adapting and calculating the MICs for the strains. Please explain the rationale behind this. 3-What is status of other echinocandin drugs like Caspofungin, Anidulafungin on these clinical isolates which showing resistant to micafungin. 4- Cdr1 is known to be promiscuous transporters involve in transport of various substrates drug specially azoles however there is no report for Cdr1 as transporter for Micafungin. What are the possible reasons for the upregulation of CDR1 in CO23RFK which are resistant to Micafungin and sensitive to Fluconazole? 5- What is the status of expression profile of other CDRs like Cdr4 and Cdr3 in these strains taken under study? 6- Has the author tried gene other than ACT1 as internal control since the CT value may vary from strains to strains also there is no mentioning of primer validation was done before final experiments? 7- Author did not mention the which method (for example Livak method etc.) used for the calculation of relative expression profiling of different gene. 8- Author mentioned the role of MFS transporters specifically MDR1 however the MFS is family containing large numbers of proteins so it’s not possible to check the expression profile for all. Some of the known transporters like QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins need to be mentioned or investigate since the QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. So, it can provide better insight of MFS superfamily in these strains. 9- In discussion section author mentioned the role of gain of function mutation in different Tfs have you observed any change after the adaptation in LIR strains also the change in Erg11 sequence or any mutation analysis done? Reviewer #2: The authors have tried to correlate the virulence of Candida albicans with overexpression of genes related to drug efflux and cell surface adhesion. -The data does not support the conclusion - Discussion is majorly speculative - More studies can be done using other virulence related genes - Should increase the number of clinical strains and resistant strains - The choice of abbreviations for the strains (resistant and susceptible) makes the understanding this work difficult -Tables and figures should have information regarding each strain as footnote - There are too many typo/spelling mistakes - Sentence formation is not appropriate at several places throughout the manuscript ****** 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: Yes: Dr. Hammad Alam Reviewer #2: Yes:** Dr Nikhat Manzoor [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. https://doi.org/10.1371/journal.pone.0252555.r001
Revision 1
7 May 2021 Author Response General points. i) English has been revised as well as mis-typings all over the text. Referee 1 Point 1 Mis-typing: See general point. Point 2 Temperature condition: we apologize for the mistake. Temperature conditions of the solid media for adapting and the liquid media for MIC were both set at 28° C. Corresponding sentence in Material and Methods, at line 109, has been amended, accordingly. Point 3 Echinocandin status: the CI1 and CI2 isolates were assayed for caspofungin and MIC value was 8 �g/mL for both isolates, overlapping the results obtained for micafungin. Point 4 CDR1 upregulation in RFK: we agree that the increase of CDR1 and CDR2 in micafungin resistant strain is not explained with the efflux mechanism usually involved in fluconazole resistant. However, an increase in CDR1 and 2 has been reported as associated with a caspofungin resistance in Candida albicans clinical isolates (Schuetzer-Muehlbauer M, Willinger B, Krapf G, Enzinger S, Presterl E, Kuchler K. The Candida albicans Cdr2p ATP-binding cassette (ABC) transporter confers resistance to caspofungin. Mol Microbiol. 2003 Apr;48(1):225-35. doi: 10.1046/j.1365-2958.2003.03430.x). To confirm CDRs role, authors expressed these genes in Saccharomyces cerevisiae and observed an increased MIC for caspofungin. It is worth notice that the authors stated that resistance is probably not due to the direct effect of these proteins as efflux pumps but more reasonably: “as a consequence of distinct mechanisms that may operate simultaneously”. A similar scenario may be tentatively proposed for our micafungin resistant strain. Point 5 CDR3 and CDR4: we missed the analyses for these two drug transporters because, to our knowledge, they seem not to have a critical role in resistance to fluconazole. CDR3 was found highly expressed in WO-1 opaque cells and its overexpression was not linked to an increase in fluconazole resistance. This data suggested a role for this protein in the white-opaque switch rather than in drug efflux. (Balan I, Alarco AM, Raymond M. The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. J Bacteriol. 1997 Dec;179(23):7210-8. doi: 10.1128/jb.179.23.7210-7218.1997.). A similar consideration may be proposed for CDR4. CDR4 mutant revealed not hypersusceptible to fluconazole and furthermore Candida isolates from AIDS patients treated with fluconazole did not reveal any involvement of CDR4 in resistance (Franz R, Michel S, Morschhäuser J. A fourth gene from the Candida albicans CDR family of ABC transporters. Gene. 1998 Oct 5;220(1-2):91-8. doi: 10.1016/s0378-1119(98)00412-0.) Point 6 RT-PCR internal control: We tried to assess the expression rate using 18S rRNA as housekeeping gene for CDR1 and CDR2 (unfortunately, we had not a suitable amount left for screening the other genes). Analyses showed a similar pattern obtained with the 18S rRNA to that reported in the paper obtained with actin gene. Furthermore, these results agree with those reported in a previous paper where TEF3 gene was used as housekeeping (Angiolella L, Stringaro AR, De Bernardis F, Posteraro B, Bonito M, Toccacieli L, Torosantucci A, Colone M, Sanguinetti M, Cassone A, Palamara AT. Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans. Antimicrob Agents Chemother. 2008 Mar;52(3):927-36. doi: 10.1128/AAC.01223-07). Data are reported in the figures below. (Please note that only two replicates for CDR2 were performed due to the small availability of this sample and thus statistical analysis was missed). Expression levels of C. albicans CDR1 and CDR2 genes determined by quantitative Real-Time PCR. The expression levels of the CDR1 and CDR2 with Actin as housekeeping (A and C) and CDR1 and CDR2 with 18S rRNA as houskeeping (B and D) in the wild-type (CO23), resistant (CO23RFK, CO23RFLC and CO23RR) and clinical isolated (CI1 and CI2) strains, represented as n-fold increase or decrease relative to the level of the control strain (CO23). Data for CDR1s and CDR2 with Actin housekeeping and for CDR1 with 18S rRNA are shown as mean±SEM from three independent experiments performed in triplicate. p< 0.05, p< 0.01, p< 0.001, *p<0.0001 vs CO23S and #p< 0.05, ##p< 0.01, ###p< 0.001, ####p< 0.0001 vs CO23RR. Point 7 Calculation of relative expression profiles: A comparative threshold cycle (CT) method was used to analyze the RT-PCR data. Sensitive Candida albicans sample (CO23s) was used as a control and target gene Ct values were normalized against actin. Data were analyzed by using the 2-ΔΔCT method and expressed as fold change with respect to control (Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8. doi: 10.1006/meth.2001.1262.). This sentence has been added in Material and Method in section: Extraction of RNA and RT-PCR on lines 131-134 Point 8 MSF-QDR: a sentence suggesting an involvement of QDR family in the remarkable hypha development observed in CI1 has been added in the Discussion, lines 311-314 along with the reference (Shah AH, Singh A, Dhamgaye S, Chauhan N, Vandeputte P, Suneetha KJ, Kaur R, Mukherjee PK, Chandra J, Ghannoum MA, Sanglard D, Goswami SK, Prasad R. Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans. Biochem J. 2014 Jun 1;460(2):223-35. doi: 10.1042/BJ20140010.) ). Point 9 Mutations: genetic analysis of fluconazole resistant strains was missing. We analysed RT-PCR expression of ERG11 in sensitive, flucanozole and micafungin resistant strains and we found no difference in gene expression between these strains (Angiolella L, Stringaro AR, De Bernardis F, Posteraro B, Bonito M, Toccacieli L, Torosantucci A, Colone M, Sanguinetti M, Cassone A, Palamara AT. Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans. Antimicrob Agents Chemother. 2008 Mar;52(3):927-36. doi: 10.1128/AAC.01223-07.). In the same paper, we reported a S645Y mutation on FSK1 gene in micafungin resistant strain. Referee 2 Point 1 Data do not support conclusion Overexpression of CDRs and MDR is a usual event occurring in fluconazole resistant strains. The increased expression of this efflux pumps is usually correlated with an increased virulence of the fungus. In this paper we found an evidence, the hyperexpression of these genes and HWP1, that opposes to this common view. For this reason, we believe that this report may be a worthwhile information in this research field. Point 2 Discussion is speculative Unfortunately, data available in literature on regulatory pathways responsible for altered gene expression and reported in the Discussion, do not allow to find a common link in the regulation of the hyperexpression of the three analysed genes. Therefore we refrained from fully correlate reduction of Candida lethality to the altered gene expression rather suggesting that it may be just an epiphenomenon of a vaster rearrangement occurring in these strains. Point 3 Virulence genes We agree that the panel of virulence genes could be increased. However, focusing on gene expression, CDRs and MDR are frequently overexpressed in fluconazole resistant strains along with ALS1 and HWP1 as far as filamentation and adhesiveness is concerned. Point 4 Number of clinical strains We believe that the clinical isolate with the hyperexpression of CDR1, CDR2 and HWP1, found by serendipity, represents a very rare event and then it is unlikely to fish again a resistant clinical strains with a similar phenotype even increasing the number of clinical strains analysed. Point 5-6: Abbreviations -Tables and figures We agree that it is difficult to memorize all strain abbreviations and therefore we decided to insert a new Table (Table 2) in the Result section, reporting information and abbreviation for each strain. Point 7-8 English: see general points. Funding information: we delete in the Acknowledgements section thanks to “Cooperativa Comunale Logistica Industriale Integrata” without sending the founding statement and therefore competing interest statement remain the same as previously. Founders’ roles have been updated. Attachments Attachment Submitted filename: Response to reveiwers.docx* https://doi.org/10.1371/journal.pone.0252555.r002
18 May 2021 Decision Letter - Aijaz Ahmad, Editor Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence PONE-D-21-05206R1 Dear Letizia Angiolella 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, Aijaz Ahmad, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252555.r003
Formally Accepted
21 May 2021 Acceptance Letter - Aijaz Ahmad, Editor PONE-D-21-05206R1 Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence Dear Dr. Angiolella: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Aijaz Ahmad Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252555.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .