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

Solid-state nuclear magnetic resonance (ssNMR) measurements of intact cell walls and cellular samples often generate spectra that are difficult to interpret due to the presence of many coexisting glycans and the structural polymorphism observed in native conditions. To overcome this analytical challenge, we present a statistical approach for analyzing carbohydrate signals using high-resolution ssNMR data indexed in a carbohydrate database. We generate simulated spectra to demonstrate the chemical shift dispersion and compare this with experimental data to facilitate the identification of important fungal and plant polysaccharides, such as chitin and glucans in fungi and cellulose, hemicellulose, and pectic polymers in plants. We also demonstrate that chemically distinct carbohydrates from different organisms may produce almost identical signals, highlighting the need for high-resolution spectra and validation of resonance assignments. Our study provides a means to differentiate the characteristic signals of major carbohydrates and allows us to summarize currently undetected polysaccharides in plants and fungi, which may inspire future investigations.

Structural adaptation of fungal cell wall in hypersaline environment.

Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications in harsh industrial processes, such as bioremediation and fermentation under unfavorable conditions of hypersalinity, low water activity, and extreme pH. However, the role of the cell wall in surviving these environmental conditions remains unclear. Here we employ solid-state NMR spectroscopy to compare the cell wall architecture of Aspergillus sydowii across salinity gradients. Analyses of intact cells reveal that A. sydowii cell walls contain a rigid core comprising chitin, ß-glucan, and chitosan, shielded by a surface shell composed of galactomannan and galactosaminogalactan. When exposed to hypersaline conditions, A. sydowii enhances chitin biosynthesis and incorporates α-glucan to create thick, stiff, and hydrophobic cell walls. Such structural rearrangements enable the fungus to adapt to both hypersaline and salt-deprived conditions, providing a robust mechanism for withstanding external stress. These molecular principles can aid in the optimization of halophilic strains for biotechnology applications.

Polysaccharide assemblies in fungal and plant cell walls explored by solid-state NMR.

Structural analysis of macromolecular complexes within their natural cellular environment presents a significant challenge. Recent applications of solid-state NMR (ssNMR) techniques on living fungal cells and intact plant tissues have greatly enhanced our understanding of the structure of extracellular matrices. Here, we selectively highlight the most recent progress in this field. Specifically, we discuss how ssNMR can provide detailed insights into the chemical composition and conformational structure of pectin, and the consequential impact on polysaccharide interactions and cell wall organization. We elaborate on the use of ssNMR data to uncover the arrangement of the lignin-polysaccharide interface and the macrofibrillar structure in native plant stems or during degradation processes. We also comprehend the dynamic structure of fungal cell walls under various morphotypes and stress conditions. Finally, we assess how the combination of NMR with other techniques can enhance our capacity to address unresolved structural questions concerning these complex macromolecular assemblies.

Solid-state NMR analysis of unlabeled fungal cell walls from Aspergillus and Candida species.

Fungal infections cause high mortality in immunocompromised individuals, which has emerged as a significant threat to human health. The efforts devoted to the development of antifungal agents targeting the cell wall polysaccharides have been hindered by our incomplete picture of the assembly and remodeling of fungal cell walls. High-resolution solid-state nuclear magnetic resonance (ss NMR) studies have substantially revised our understanding of the polymorphic structure of polysaccharides and the nanoscale organization of cell walls in Aspergillus fumigatus and multiple other fungi. However, this approach requires 13C/15N-enrichment of the sample being studied, severely restricting its application. Here we employ the dynamic nuclear polarization (DNP) technique to compare the unlabeled cell wall materials of A. fumigatus and C. albicans prepared using both liquid and solid media. For each fungus, we have identified a highly conserved carbohydrate core for the cell walls of conidia and mycelia, and from liquid and solid cultures. Using samples prepared in different media, the recently identified function of α-glucan, which packs with chitin to form the mechanical centers, has been confirmed through conventional ss NMR measurements of polymer dynamics. These timely efforts not only validate the structural principles recently discovered for A. fumigatus cell walls in different morphological stages, but also open up the possibility of extending the current investigation to other fungal materials and cellular systems that are challenging to label.

A 13C three-dimensional DQ-SQ-SQ correlation experiment for high-resolution analysis of complex carbohydrates using solid-state NMR.

Complex carbohydrates are the key components of the protective cell walls of microbial pathogens and the bioenergy reservoir in plants and algae. Structural characterization of these polymorphic molecules requires assistance from multidimensional 13C correlation approaches. To facilitate the analysis of carbohydrate structure using solid-state NMR, we present a three-dimensional (3D) 13C-13C-13C experiment that includes a double-quantum (DQ) dimension and is thus free of the cube's body diagonal. The enhanced resolution supports the unambiguous resonance assignment of many polysaccharides in plant and fungal cell walls using uniformly 13C-labeled cells of spruce and Aspergillus fumigatus. Long-range structural restraints were effectively obtained to revisit our understanding of the spatial organization of plant cellulose microfibrils. The method is widely applicable to the investigations of cellular carbohydrates and carbon-based biomaterials.

A molecular vision of fungal cell wall organization by functional genomics and solid-state NMR.

Vast efforts have been devoted to the development of antifungal drugs targeting the cell wall, but the supramolecular architecture of this carbohydrate-rich composite remains insufficiently understood. Here we compare the cell wall structure of a fungal pathogen Aspergillus fumigatus and four mutants depleted of major structural polysaccharides. High-resolution solid-state NMR spectroscopy of intact cells reveals a rigid core formed by chitin, ß-1,3-glucan, and α-1,3-glucan, with galactosaminogalactan and galactomannan present in the mobile phase. Gene deletion reshuffles the composition and spatial organization of polysaccharides, with significant changes in their dynamics and water accessibility. The distribution of α-1,3-glucan in chemically isolated and dynamically distinct domains supports its functional diversity. Identification of valines in the alkali-insoluble carbohydrate core suggests a putative function in stabilizing macromolecular complexes. We propose a revised model of cell wall architecture which will improve our understanding of the structural response of fungal pathogens to stresses.

Structural Polymorphism of Chitin and Chitosan in Fungal Cell Walls From Solid-State NMR and Principal Component Analysis.

Chitin is a major carbohydrate component of the fungal cell wall and a promising target for novel antifungal agents. However, it is technically challenging to characterize the structure of this polymer in native cell walls. Here, we recorded and compared 13C chemical shifts of chitin using isotopically enriched cells of six Aspergillus, Rhizopus, and Candida strains, with data interpretation assisted by principal component analysis (PCA) and linear discriminant analysis (LDA) methods. The structure of chitin is found to be intrinsically heterogeneous, with peak multiplicity detected in each sample and distinct fingerprints observed across fungal species. Fungal chitin exhibits partial similarity to the model structures of α- and γ-allomorphs; therefore, chitin structure is not significantly affected by interactions with other cell wall components. Addition of antifungal drugs and salts did not significantly perturb the chemical shifts, revealing the structural resistance of chitin to external stress. In addition, the structure of the deacetylated form, chitosan, was found to resemble a relaxed two-fold helix conformation. This study provides high-resolution information on the structure of chitin and chitosan in their cellular contexts. The method is applicable to the analysis of other complex carbohydrates and polymer composites.

Solid-state NMR of plant and fungal cell walls: A critical review.

The cell walls of plants and microbes are a central source for bio-renewable energy and the major targets of antibiotics and antifungal agents. It is highly challenging to determine the molecular structure of complex carbohydrates, protein and lignin, and their supramolecular assembly in intact cell walls. This article selectively highlights the recent breakthroughs that employ 13C/15N solid-state NMR techniques to elucidate the architecture of fungal cell walls in Aspergillus fumigatus and the primary and secondary cell walls in a large variety of plant species such as Arabidopsis, Brachypodium, maize, and spruce. Built upon these pioneering studies, we further summarize the underexplored aspects of fungal and plant cell walls. The new research opportunities introduced by innovative methods, such as the detection of proton and quadrupolar nuclei on ultrahigh-field magnets and under fast magic-angle spinning, paramagnetic probes, natural-abundance DNP, and software development, are also critically discussed.