Cellular and Extracellular Vesicle RNA Analysis in the Global Threat Fungus Candida auris

ABSTRACT Emerging and reemerging pathogens are a worldwide concern, and it is predicted that these microbes will cause severe outbreaks. Candida auris affects people with weakened immune systems, particularly those who are hospitalized or are in health care facilities. Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all domains of life. EVs can deliver functional molecules to target cells, including proteins and nucleic acids, especially RNA molecules. EVs from several pathogenic fungi species play diverse biological roles related to cell-cell communication and pathogen-host interaction. In this study, we describe a data set which we produced by sequencing the RNA content of EVs from C. auris under normal growth conditions and in the presence of the antifungal caspofungin, a first-line drug to treat this fungus. To generate a more complete data set for future comparative studies, we also sequenced the RNA cellular content of EVs under the same conditions. This data set addresses a previously unexplored area of fungal biology regarding cellular small RNA and EV RNA. Our data will provide a molecular basis for the study of the aspects associated with antifungal treatment, gene expression response, and EV composition in C. auris. These data will also allow the exploration of small RNA content in the fungal kingdom and might serve as an informative basis for studies on the mechanisms by which molecules are directed to fungal EVs. IMPORTANCE Candida auris, a relevant emerging human-pathogenic yeast, is the first fungus to be called a global public health threat by the WHO. This is because of its rapid spread on all inhabited continents, together with its extremely high frequency of drug and multidrug resistance. In our study, we generated a large data set for 3 distinct strains of C. auris and obtained cellular small RNA fraction as well as extracellular vesicle RNA (EV-RNA) during normal growth conditions and after treatment with caspofungin, the first-line drug used to treat C. auris infection.

Munhoz da Rocha and colleagues described their attempts in characterizing the secreted vesicular and cellular small RNAs from the three strains of Candida auris with-and without the presence of the lipopeptide antifungal drug Caspofungin. They described mainly the experimental procedures and parameters of their finding. Their effort in attempting to provide a dataset benefiting the research field is worthy of applauding; however, as the report's current strands, the effort would not be able to benefit anyone.

Main comments:
This is a transcriptomic study of secreted and cellular small RNAs from C auris, but no data annotation, such as analysis of functional gene ontology (GO) terms or biological pathways (KEGG pathways), was performed. Without it, the study is not publishable.
The report listed figure and table numbers in the text but no data interpretation was provided. Most of the Results and Discussions is just the extended description of Materials and Methods.

Specific comments:
Lines 24-26: The preceding lines are statements on extracellular vesicles, but the sentences here do not follow through and suddenly turn into small cellular RNAs.
Lines 278-30: Need to be specific, such as "sRNA-mediated regulation of the gene expression in response to antifungals." Line 75: why "viral infection" is mentioned here?
Line 79: "The proteins of RNA silencing pathway were lost in a significant number of fungal species." The statement makes no sense to be here and is not supported by reference (no reference was provided).
Lines 87-97: These statements are somewhat erroneous. Authors may wish to initiate a literature search to be up to date with the current study progress.
Reviewer #2 (Comments for the Author): Dear Editor, The manuscript by Rocha et al describes the composition and identity of the small RNA fraction isolated from cellular and extracellular vesicles (EVs) compartments from Candida auris. The authors describes a slight alteration in the EVs profile of three distinct C. auris strains exposed to the antifungal drug caspofungin. In addition, employing small RNA-seq analysis, the authors found that yeast cells treated with caspofungin displayed a substantial difference in the small RNA composition in EVs compared to cellular compartment, as revealed by PCA analysis. They also found that caspofungin itself caused an alteration in the small RNA profile in such strains. While the manuscript is well written, it lacks proper analysis of the results and consequent data-dependent generation of hypothesis. There are some major points that are illustrative of my previous comment: I'm sorry for the previous confusion. As the editor, I had missed the important notation that your manuscript was submitted as a "Resource Report" and not a "Research Article". For this I sincerely apologize and I am glad that you appealed my initial decision. In your response to the reviewer comments, please note that your manuscript is a resource that is being provided to the research community. I would also recommend that you include additional language in the abstract, introduction, and discussion to highlight to the research community that these data are being provided as a resource since this is a new format for our community and will prevent confusion such as occurred here in the future when your paper is published. Again, I'm very sorry for the mistake on my part.
Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed information on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Munhoz da Rocha and colleagues described their attempts in characterizing the secreted vesicular and cellular small RNAs from the three strains of Candida auris with-and without the presence of the lipopeptide antifungal drug Caspofungin. They described mainly the experimental procedures and parameters of their finding. Their effort in attempting to provide a dataset benefiting the research field is worthy of applauding; however, as the report's current strands, the effort would not be able to benefit anyone.

Main comments:
This is a transcriptomic study of secreted and cellular small RNAs from C auris, but no data annotation, such as analysis of functional gene ontology (GO) terms or biological pathways (KEGG pathways), was performed. Without it, the study is not publishable.
The report listed figure and table numbers in the text but no data interpretation was provided. Most of the Results and Discussions is just the extended description of Materials and Methods.

Specific comments:
Lines 24-26: The preceding lines are statements on extracellular vesicles, but the sentences here do not follow through and suddenly turn into small cellular RNAs.
Lines 278-30: Need to be specific, such as "sRNA-mediated regulation of the gene expression in response to antifungals." Line 75: why "viral infection" is mentioned here?
Line 79: "The proteins of RNA silencing pathway were lost in a significant number of fungal species." The statement makes no sense to be here and is not supported by reference (no reference was provided).
Lines 87-97: These statements are somewhat erroneous. Authors may wish to initiate a literature search to be up to date with the current study progress.
Reviewer #2: The manuscript by Rocha et al describes the composition and identity of the small RNA fraction isolated from cellular and extracellular vesicles (EVs) compartments from Candida auris. The authors describes a slight alteration in the EVs profile of three distinct C. auris strains exposed to the antifungal drug caspofungin. In addition, employing small RNA-seq analysis, the authors found that yeast cells treated with caspofungin displayed a substantial difference in the small RNA composition in EVs compared to cellular compartment, as revealed by PCA analysis. They also found that caspofungin itself caused an alteration in the small RNA profile in such strains. While the manuscript is well written, it lacks proper analysis of the results and consequent data-dependent generation of hypothesis. There are some major points that are illustrative of my previous comment: 1 -Based on Candida albicans previous studies (https://pubmed.ncbi.nlm.nih.gov/23114781/), caspofungin leads to apoptosis in yeast cells. The increased EV content in B8441 and MMC1 strains could be potentially associated with apoptosis. This is not discussed in the manuscript and could be easily evaluated by the TUNEL assay. Moreover, in Figure 2, there is no statistical analysis to compare the groups. Despite described in the text, there is no images of the isolated EVs. 2 -The authors generated a large dataset of small RNA sequences from C. Auris that certainly will be useful to the community. However, the analysis is merely descriptive about the general aspects of size distribution and content of such libraries. The authors could use such data to infer what are the underlying mechanisms about the (i) distinct sensitivity of the strains to caspofungin and (ii) distinct profile of EVs produced by the strains. Moreover, the author could produce a description of distinct classes of sRNAs (miRNA like, tRNA derived fragments, etc), as well as their differential abundance in the treatments and the strains. In addition, the authors could compare their data to their previous publication (doi.org/10.1016/j.csbj.2021.09.007) in order to correlate the transcription profiling of mRNAs and sRNAs of C. auris in response to caspofungin.
Minor points 3 -Why the fraction of unmapped reads is so high? 4 -Based on which criteria the RIN was determined? There is some variation in the parameters according to the organism used. 5 -Why did the authors mapped the reads against two distinct libraries? The sentence that describes such information is not clear (lines 132-135). If this was made for annotation purposes, Why did not the authors merge such annotations in a single annotation file (gff or gtf) and process the alignments files? This would also filter reads spanning multiple aligned reads that were considered in the proposed approach. 6 -Lines 103 -105. The authors stated that they conducted RNA-seq to evaluate the drug effect on living cells. Despite a clear signal could be observed in MIC assays, it is expected that some cells are dead. Does the authors evaluated the viability of the cells exposed to such drugs concentrations used for RNA seq analysis?
Staff Comments:

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Thank you for submitting your paper to Microbiology Spectrum. The authors appropriately responded to the reviewers comments. I requested modifications because the manuscript includes several areas where the authors still include their "(ref)" annotation without the appropriate reference included. Assuming the authors replace these annotations with appropriate citations, the manuscript should be acceptable for publication.
Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues I raised in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed information on submitting your revised paper are below.

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Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,

Kirsten Nielsen
Editor, Microbiology Spectrum Reviewer comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • point-by-point responses to the issues I raised in your cover letter • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.