β-1,6-glucan plays a central role in the structure and remodeling of the bilaminate fungal cell wall

Reviewer #1 (Public Review):

Summary:

The fungal cell wall is a very important structure for the physiology of a fungus but also for the interaction of pathogenic fungi with the host. Although a lot of knowledge on the fungal cell wall has been gained, there is a lack of understanding of the meaning of ß-1,6-glucan in the cell wall. In the current manuscript, the authors studied in particular this carbohydrate in the important human-pathogenic fungus Candida albicans. The authors provide a comprehensive characterization of cell wall constituents under different environmental and physiological conditions, in particular of ß-1,6-glucan. Also, β-1,6-glucan biosynthesis was found to be likely a compensatory reaction when mannan elongation was defective. The absence of β-1,6-glucan resulted in a significantly sick growth phenotype and complete cell wall reorganization. The manuscript contains a detailed analysis of the genetic and biochemical basis of ß-1,6-glucan biosynthesis which is apparently in many aspects similar to yeast. Finally, the authors provide some initial studies on the immune modulatory effects of ß-1,6-glucan.

Strengths:

The findings are very well documented, and the data are clear and obtained by sophisticated biochemical methods. It is impressive that the authors successfully optimized methods for the analyses and quantification of ß-1-6-glucan under different environmental conditions and in different mutant strains.

Weaknesses:

However, although already very interesting, at this stage there are some loose ends that need to be combined to strengthen the manuscript. For example, the immunological studies are rather preliminary and need at least some substantiation. Also, at this stage, the manuscript in some places remains a bit too descriptive and needs the elucidation of potential causalities.

Reviewer #2 (Public Review):

Summary:

The authors provide the first (to my knowledge) detailed characterization of cell wall b-1,6 glucan in the pathogen Candida albicans. The approaches range from biochemistry to genetics to immunology. The study provides fundamental information and will be a resource of exceptional value to the field going forward. Highlights include the construction of a mutant that lacks all b-1,6 glucan and the characterization of its cell wall composition and structure. Figure 5a is a feast for the eyes, showing that b-1,6 glucan is vital for the outer fibrillar layer of the cell wall. Also much appreciated was the summary figure, Figure 7, which presents the main findings in digestible form.

Strengths:

The work is highly significant for the fungal pathogen field especially, and more broadly for anyone studying fungi, antifungal drugs, or antifungal immune responses.

The manuscript is very readable, which is important because most readers will be cell wall nonspecialists.

The authors construct a key quadruple mutant, which is not trivial even with CRISPR methods, and validate it with a complemented strain. This aspect of the study sets the bar high.

The authors develop new and transferable methods for b-1,6 glucan analysis.

Weaknesses:

The one "famous" cell type that would have been interesting to include is the opaque cell. This could be included in a future paper.

Reviewer #3 (Public Review):

Summary:

The cell wall of human fungal pathogens, such as Candida albicans, is crucial for structural support and modulating the host immune response. Although extensively studied in yeasts and molds, the structural composition has largely focused on the structural glucan b,1,3-glucan and the surface exposed mannans, while the fibrillar component β-1,6-glucan, a significant component of the well wall, has been largely overlooked. This comprehensive biochemical and immunological study by a highly experienced cell wall group provides a strong case for the importance of β-1,6-glucan contributing critically to cell wall integrity, filamentous growth, and cell wall stability resulting from defects in mannan elongation. Additionally, β-1,6-glucan responds to environmental stimuli and stresses, playing a key role in wall remodeling and immune response modulation, making it a potential critical factor for host-pathogen interactions.

Strengths:

Overall, this study is well-designed and executed. It provides the first comprehensive assessment of β-1,6-glucan as a dynamic, albeit underappreciated, molecule. The role of β-1,6-glucan genetics and biochemistry has been explored in molds like Aspergillus fumigatus, but this work shines an important light on its role in Candida albicans. This is important work that is of value to Medical Mycology, since β-1,6-glucan plays more than just a structural role in the wall. It may serve as a PAMP and a potential modulator of host-pathogen interactions. In keeping with this important role, the manuscript rigor would benefit from a more physiological evaluation ex vivo and preferably in vivo, assessment on stimulating the immune system within in the cell wall and not just as a purified component. This is a critical outcome measure for this study and gets squarely at its importance for host-pathogen interactions, especially in response to environmental stimuli and drug exposure.

Author response:

Response to reviewers (Public review):

We thank all the three reviewers for their opinion on our work on Candida albicans β-1,6-glucan, which highlights the importance of this cell wall component in the biology of fungi. Here are our responses to their comments for public reviews:

(1) Indeed, the data presented for immunological studies is preliminary. It has been acknowledged by the reviewers that our analysis providing insights into the biosynthetic pathways involved in comprehensive in dealing with organization and dynamics of the β-1,6-glucan polymer in relation with other cell wall components and environmental conditions (temperature, stress, nutrient availability, etc.). However, we anticipated that there would be immediate curiosity as to what the immunological contribution of β-1,6 glucan and we therefore felt we needed to initiative these studies and include them. We therefore performed immunological studies to assess whether β-1,6-glucans act as a pathogen-associated molecular pattern (PAMP), and if so, what its immunostimulatory potential is. Our data clearly suggest that β-1,6-glucan is a PAMP, and consequently lead to several questions: (a) what are the host immune receptors involved in the recognition of this polysaccharide, and thereby the downstream signaling pathways, (b) how is β-1,6-glucan differentially recognized by the host when C. albicans switches from a commensal to an opportunistic pathogen, and (c) how does the host environment impact the exposure of this polysaccharide on the fungal surface. We believe addressing these questions is beyond the scope of the present manuscript and aim to present new data in future manuscript. Nonetheless, in the revised manuscript, suggest approaches that we can take to identify the receptor that could be involved in the recognition of β-1,6-glucan. Moreover, we have modified the discussion presenting it based on the data rather than being descriptive.

(2) It will be interesting to assess the organization of β-1,6-glucan and other cell wall components in the opaque cells. It is documented that the opaque cells are induced at acidic pH and in the presence of N-acetylglucosamine and CO2. Our data shows that pH has an impact on β-1,6-glucan, which suggests that there will be differential organization of this polysaccharide in the cell wall of opaque cells. As suggested by the reviewer, we will include analysis of opaque cells (and other C. albicans cell types) in future studies.

With the exception of these major new avenues for this research, our revision can address each of the comments provided by the reviewers.