Proton-Detected Solid-State NMR for Deciphering Structural Polymorphism and Dynamic Heterogeneity of Cellular Carbohydrates in Pathogenic Fungi

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: <i>Rhizopus delemar</i>, <i>Aspergillus fumigatus</i>, and <i>Candida albicans</i>, representing filamentous species and yeast forms. Selective detection of acetylated carbohydrates reveals 15 forms of <i>N</i>-acetylglucosamine units in <i>R. delemar</i> 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 <i>A. fumigatus</i>, 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 <i>C. albicans</i> 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.