Probing Cell‐Surface Interactions in Fungal Cell Walls by High‐Resolution ¹H‐Detected Solid‐State NMR Spectroscopy

Abstract

Solid‐state NMR (ssNMR) spectroscopy facilitates the non‐destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra‐high field 1H‐detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic‐level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom‐forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin‐2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N‐acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make‐up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts.

Structural Adaptation of Fungal Cell Wall in Hypersaline Environment

“…These methods can efficiently provide information on the polymorphic structure, composition, and physical packing of biomolecules in native cell walls, but are also limited by the long experimental time required for finishing a complete analysis. Recent development of proton-detection methods and sensitivity-enhancing dynamic nuclear polarization (DNP) techniques could expedite future analysis of fungal cell walls 23,37,82 .…”

“…Recently, the use of solid-state NMR (ssNMR) spectroscopy has led to a better understanding of the molecular architecture and dynamics of fungal cell walls [19][20][21][22] . Through integrated ssNMR and biochemical analyses of Aspergillus fumigatus, it has been discovered that a poorly hydrated, mechanically stiff core is formed by physically associated chitin and α-1,3-glucan [23][24][25] , which is conserved in both mycelia and conidia 26 , but with altered molecular composition during morphotype transition 27 .…”

“…Recently, the use of solid-state NMR (ssNMR) spectroscopy has led to a better understanding of the molecular architecture and dynamics of fungal cell walls [21][22][23] . Because intact cells are being analyzed, the structural information of the cell wall and its polysaccharides can be directly obtained at atomic resolution, without the need for solubilization or extraction 24,25 .…”

The complex structure of Fomes fomentarius represents an architectural design for high-performance ultralightweight materials

“…We further estimated the relative abundance of each polysaccharide by peak integration analysis (Fig. 5B), as previously described for a 13 C-labeled fungal cell wall model (30). We estimated that the crust layer consists of 55% of both α-glucan and β-glucan, 1% of chitin, and 44% of other polysaccharides.…”

Charting the solid‐state NMR signals of polysaccharides: A database‐driven roadmap

“…Early pioneers in this field explored the use of different ssNMR methods, such as one-dimensional (1D) experiments for identifying the structure and quantifying the content of carbohydrate polymers and allomorphs, and Rotational Echo Double Resonance (REDOR) and multidimensional correlation techniques for revealing polymer cross-linking and spatial proximities. [1][2][3][4][5][6] There has been a substantial increase in high-resolution ssNMR data that rely on 13 C and 15 N 2D/3D correlation experiments, [7][8][9] as well as 1 H detection, [10][11][12] to provide detailed insights into the structure and function of carbohydrates in a variety of living organisms.…”