Zygomycetous fungal infections pose an emerging medical threat among individuals with compromised immunity and metabolic abnormalities. Our pathophysiological understanding of these infections, particularly the role of fungal cell walls in growth and immune response, remains limited. Here we conducted multidimensional solid-state NMR analysis to examine cell walls in five Mucorales species, including key mucormycosis causative agents like Rhizopus and Mucor species. We show that the rigid core of the cell wall primarily comprises highly polymorphic chitin and chitosan, with minimal quantities of β-glucans linked to a specific chitin subtype. Chitosan emerges as a pivotal molecule preserving hydration and dynamics. Some proteins are entrapped within this semi-crystalline chitin/chitosan layer, stabilized by the sidechains of hydrophobic amino acid residues, and situated distantly from β-glucans. The mobile domain contains galactan- and mannan-based polysaccharides, along with polymeric α-fucoses. Treatment with the chitin synthase inhibitor nikkomycin removes the β-glucan-chitin/chitosan complex, leaving the other chitin and chitosan allomorphs untouched while simultaneously thickening and rigidifying the cell wall. These findings shed light on the organization of Mucorales cell walls and emphasize the necessity for a deeper understanding of the diverse families of chitin synthases and deacetylases as potential targets for novel antifungal therapies.

Invasive candidiasis affects 1.6 million people annually, with high mortality among immunocompromised and hospitalized patients. Echinocandins are frontline antifungals, but rising resistance limits their efficacy. Here, we show that Candida albicans and multidrug-resistant Candida auris share a conserved cell wall architecture yet differ markedly in their adaptive responses to echinocandins. Solid-state NMR reveals that both species possess a rigid inner layer of tightly associated chitin microfibrils and β−1,3-glucans, supported by a flexible matrix of β−1,6-glucans and additional β−1,3-glucans. Outer mannan fibrils rely on α−1,2-linked sidechains to maintain contact with the inner wall. In both species, caspofungin rigidifies β−1,6-glucans and mannan sidechains and reduces water permeability during β−1,3-glucan depletion; however, C. albicans undergoes wall thickening and alterations in chitin and glucan dynamics, whereas C. auris maintains integrity through β−1,6-glucan upregulation. Deletion of KRE6a, which encodes β−1,6-glucan synthase, reduces echinocandin susceptibility in C. auris, further highlighting β−1,6-glucan’s critical role in adaptive remodeling.

Carbohydrate polymers in their cellular context display highly polymorphic structures and dynamics essential to their diverse functions, yet they are challenging to analyze biochemically. Proton-detection solid-state NMR spectroscopy offers high isotopic abundance and sensitivity, enabling the rapid and high-resolution structural characterization of biomolecules. Here, an array of 2D/3D ¹H-detection solid-state NMR techniques are tailored to investigate polysaccharides in fully protonated or partially deuterated cells of three prevalent pathogenic fungi: Rhizopus delemar, Aspergillus fumigatus, and Candida albicans, representing filamentous species and yeast forms. Selective detection of acetylated carbohydrates reveals 15 forms of N-acetylglucosamine units in R. delemar chitin, which coexists with chitosan, and associates with proteins only at limited sites. This is supported by distinct order parameters and effective correlation times of their motions, analyzed through relaxation measurements and model-free analysis. Five forms of α-1,3-glucan with distinct structural origins and dynamics were identified in A. fumigatus, important for this buffering polysaccharide to perform diverse roles of supporting wall mechanics and regenerating a soft matrix under antifungal stress. Eight α-1,2-mannan side chain variants in C. albicans were resolved, highlighting the crucial role of mannan side chains in maintaining interactions with other cell wall polymers to preserve structural integrity. These methodologies provide novel insights into the functional structures of key fungal polysaccharides and create new opportunities for exploring carbohydrate biosynthesis and modifications across diverse organisms.

The fungal cell wall plays a critical role in regulating cellular integrity and communication, and serves as a frontline defense against stress. It is also a prime target for the development of antifungal agents. The cell wall is comprised of diverse polysaccharides and proteins and poses a challenging target for high-resolution structural characterization. Recently, the solid-state nuclear magnetic resonance (ssNMR) analysis of intact Aspergillus fumigatus cells has provided atomic-level insights into the structural polymorphism and functional assembly principles of carbohydrate components within the cell wall. This physical perspective, alongside structural information from biochemical assays, offers a renewed understanding of the cell wall as a highly complex and dynamic organelle. Here, we summarize key conceptual advancements in the structural elucidation of A. fumigatus mycelial and conidial cell walls and their responses to stressors. We also highlight underexplored areas and discuss the opportunities facilitated by technical advancements in ssNMR spectroscopy.

The plant secondary cell wall, a complex matrix composed of cellulose, hemicellulose, and lignin, is crucial for the mechanical strength and water-proofing properties of plant tissues, and serves as a primary source of biomass for biorenewable energy and biomaterials. Structural analysis of these polymers and their interactions within the secondary cell wall has been heavily relying on ¹³C-based solid-state NMR techniques. In this study, we explore the application of ¹H-detected solid-state NMR techniques for rapid, high-resolution structural characterization of polysaccharides and lignin, demonstrated on the stems of hardwood eucalyptus. We explored the use of synthesized 2D spectra to resolve central ¹H resonances and the combined application of 3D hCCH and hCHH experiments for complete resonance assignment and unambiguous identification of lignin-carbohydrate interactions. Our findings emphasize the central role of acetylated 3-fold xylan conformers, rather than 2-fold, in stabilizing the carbohydrate-lignin interface, with glucuronic acid side chains in eucalyptus glucuronoxylan colocalizing with lignin. We also observed cellulose-lignin interactions involving uncoated microfibril surfaces and detected pectin-lignin interactions indicative of early stage lignification. These results present a novel approach for rapid structural analysis of lignocellulosic biomaterials without the need for solubilization or extraction.