PPATHOGENS-D-26-01006

An emergent biofilm program from inactivation of Candida albicans master regulators Efg1 and Ndt80

PLOS Pathogens

Dear Dr. Mitchell,

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Biao Ren

Academic 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

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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: The submission from Do et al considers the complex genetic determinants of biofilm formation in the medically important fungal pathogen Candida albicans. Filamentation and biofilm formation are important phenotypes of many fungal pathogens that enable them to avoid certain aspects of the host immune response and to resist clearance by antibiotics. These properties can represent significant problems with catheterization or implant surgeries. Therefore, understanding the molecular genetic underpinnings of this process could provide significant insights into the causes and prevention of serious fungal infections. Previous work had identified two key regulators of the gene expression cascade the leads to filamentation and biofilm formation, Efg1 and Ndt80. Disruption of the genes encoding these transcription factors prevents the formation of biofilms. Remarkably, in this manuscript the authors surprisingly report that creation of the double knockout rescues both filamentation and biofilm formation, and even enhances these phenotypes under certain conditions, including biofilm formation in a mouse catheterization model. The authors go on to characterize many aspects of the knockout lines, including gene expression profiles and interactions with other loci previously shown to be important for these phenotypes. The properties described here are somewhat strain specific and the paper does not solve what is clearly an exceptionally complex regulatory hierarchy, nonetheless the manuscript provides new insights into the gene expression cascades that determine biofilm formation and adds to our understanding of the regulatory network of transcription factors controlling it.

Reviewer #2: This manuscript reports on new mechanistic insight into C. albicans biofilm formation, which is significant because biofilms commonly underlie the development of lethal systemic infections. The key result of this manuscript is the surprising discovery that combining two mutations (efg1 and ndt80) that individually cause defects in biofilm formation results in the opposite phenotype – biofilm formation that is better than either single mutant alone. This surprising genetic effect was most obvious in the SC5314 strain background, but the efg1 ndt80 combination resulted in improved hyphal growth in 5 other clinical isolates, strengthening the significance of this genetic interaction. Further genetic analysis showed that biofilm formation in the efg1 ngt1 strain was regulated by only a subset of the genes that have been identified previously to contribute to biofilm formation (BCR1, UME6, and HGC1), which correlated with the increased expression of these genes in the efg1 ndt80 strain. Interestingly, comparison of RNAseq data revealed that one of the commonly regulated genes was FLO9, a putative adhesin. Deletion of FLO9 essentially blocked biofilm in the efg1 ndt80 strain, highlighting a key role in this strain background. Altogether, these data show that a “stripped down” version of the complex biofilm circuitry is sufficient to drive biofilm formation, which now provides a new avenue for identifying regulators of this complex process.

Reviewer #3: The study by Do et al. reports the unexpected finding that the efg1Δ/Δ ndt80Δ/Δ double mutant of Candida albicans, despite the severe biofilm defects of each single mutant, is capable of forming robust biofilms in both in vitro and in vivo models. The authors define this as an “emergent biofilm” phenotype and provide evidence that it is mechanistically distinct from the conventional biofilm regulatory program. Through transcriptomic and genetic analyses, the study identifies key roles for Hgc1, Bcr1, Ume6, and the adhesin Flo9 in supporting this alternative biofilm state, while demonstrating independence from several canonical biofilm regulators, including Brg1, Rob1, Tec1, and Wor3. Overall, the work reveals an unexpected plasticity in the C. albicans biofilm regulatory network and suggests the existence of a simplified or primordial surface colonization program. The findings are interesting and provide new insight into how fungal virulence regulatory networks can adapt and reorganize.

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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: None

Reviewer #2: 1. It is interesting that the authors refer to the efg1 ndt80 genetic interaction as “emergent biofilm” formation. However, for clarity the authors should also state that it is a variation on the “synthetic” genetic interactions that have been widely used to study positive and negative interactions between genes.

2. The SC5314 strain differs from other clinical isolates in that it the two different alleles of ROB1 that differ in how they promote filament formation. I think it would be interesting for the authors to comment on the fact that deletion of ROB1 did not impact the efg1 ndt80 phenotype, indicating that the ROB1 alleles were not the critical difference for the SC5314 strain phenotype caused by efg1 ndt80.

3. FLO9 was described as a “putative adhesin”. The authors should supply more information about the basis for this description. Perhaps a supplemental figure showing the structural features on which this assessment is based? Is FLO9 conserved in other biofilm-forming species?

4. The NCBI SRA accession number PRJNA1453028 did not work. Please check it is correct.

Reviewer #3: Major comments:

1. Is Flo9 overexpression sufficient to drive biofilm formation in the efg1 or ndt80 single mutants?

2. Does the deletion of the Ume6 interacting protein, Upc2, affect the emergent biofilm formation? That would tell whether new binding partners are required.

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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: Overall, the manuscript is thorough, and the data presented in a clear and easily understood manner. I have only minor comments for the authors to consider.

1. In the paragraph beginning on line 87, the authors mention that both efg1 and ndt80 knockout lines display defects in biofilm formation and filamentation, leading the reader to assume that they phenocopy each other. However, from the last sentence of this paragraph, it is clear that the two mutants share some characteristics and differ in others. For unfamiliar readers, it would be helpful to know how similar these mutant lines are. If there are additional data from previous characterizations of these single mutant lines, it would be great if the authors could provide a brief summary.

2. In the section beginning on line 163, the authors compare RNA-seq data sets from a wild type line and the efg1/ndt80 double mutant. This is, of course, both interesting and key to the analysis that the authors are presenting. However, it would also be interesting to know how the double mutant compares to each single mutant (and perhaps how the single mutants compare to each other). Ideally, the RNA-seq analyses of all these lines would be performed in parallel, however this might be prohibitive at this point. Are there preexisting RNA-seq datasets for each of the single mutant lines that could be used for comparison, even if the comparison is qualitative rather than quantitative (e.g. are similar/different pathways up/down regulated)?

Reviewer #2: • Lines 63-65. Sentence unclear – missing a word?

• Line 355 to around Line 390 – define YFG1 and YFG

• Lines 411-412. Unclear sentence. What is OD0.5?

• define ddH2O

Reviewer #3: Minor comments:

1. It would be good to also include the phenotype of the single mutants in Fig. 4A, 4B, and 4C.

2. In Fig. 5, please add what RNA-seq conditions from other studies were used to generate 5B.

3. It would be good to add to the discussion why the emergent biofilm of the double mutant is only in SC5314, but not in other strain background. In addition, it would be good to discuss what makes Flo9 so special to be required for emergent biofilm formation, but not other adhesins. Is the regulation of Flo9 much different compared to other adhesins?

4. Line 64: add “condensates” after “phase-separated”

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Reviewer #2: No

Reviewer #3: No

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Dear Professor Mitchell,

We are pleased to inform you that your manuscript 'An emergent biofilm program from inactivation of Candida albicans master regulators Efg1 and Ndt80' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

Biao Ren

Academic 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

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Reviewer Comments (if any, and for reference):

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 manuscript provides intriguing and important information regarding the mechanisms regulating biofilm formation in the fungal pathogen Candida albicans. The authors employed a genetic approach to identify key determinants of this virulence associated property. The information included in the manuscript solidly supports the conclusions of the authors.

Reviewer #2: The authors have addressed my comments in a satisfactory manner.

Reviewer #3: The authors had a lovely first submission and have fully addressed any concerns.

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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: The authors have suitably addressed my previous comments. I have no further suggestions.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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: The authors have suitably addressed my previous comments. I have no further suggestions.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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

Reviewer #3: No