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

ABSTRACT: 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.

SUBMITTER: Fernando LD

PROVIDER: S-EPMC9310124 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The native cellulose of bacterial, algal, and animal origins has been well studied structurally using X-ray and neutron diffraction and solid-state NMR spectroscopy, and is known to consist of varying proportions of two allomorphs, I? and I?, which differ in hydrogen bonding, chain packing, and local conformation. In comparison, cellulose structure in plant primary cell walls is much less understood because plant cellulose has lower crystallinity and extensive interactions with matrix polysaccharides. Here we have combined two-dimensional magic-angle-spinning (MAS) solid-state nuclear magnetic resonance (solid-state NMR) spectroscopy at high magnetic fields with density functional theory (DFT) calculations to obtain detailed information about the structural polymorphism and spatial distributions of plant primary-wall cellulose. 2D (13)C-(13)C correlation spectra of uniformly (13)C-labeled cell walls of several model plants resolved seven sets of cellulose chemical shifts. Among these, five sets (denoted a-e) belong to cellulose in the interior of the microfibril while two sets (f and g) can be assigned to surface cellulose. Importantly, most of the interior cellulose (13)C chemical shifts differ significantly from the (13)C chemical shifts of the I? and I? allomorphs, indicating that plant primary-wall cellulose has different conformations, packing, and hydrogen bonding from celluloses of other organisms. 2D (13)C-(13)C correlation experiments with long mixing times and with water polarization transfer revealed the spatial distributions and matrix-polysaccharide interactions of these cellulose structures. Celluloses f and g are well mixed chains on the microfibril surface, celluloses a and b are interior chains that are in molecular contact with the surface chains, while cellulose c resides in the core of the microfibril, outside spin diffusion contact with the surface. Interestingly, cellulose d, whose chemical shifts differ most significantly from those of bacterial, algal, and animal cellulose, interacts with hemicellulose, is poorly hydrated, and is targeted by the protein expansin during wall loosening. To obtain information about the C6 hydroxymethyl conformation of these plant celluloses, we carried out DFT calculations of (13)C chemical shifts, using the I? and I? crystal structures as templates and varying the C5-C6 torsion angle. Comparison with the experimental chemical shifts suggests that all interior cellulose favor the tg conformation, but cellulose d also has a similar propensity to adopt the gt conformation. These results indicate that cellulose in plant primary cell walls, due to their interactions with matrix polysaccharides, and has polymorphic structures that are not a simple superposition of the I? and I? allomorphs, thus distinguishing them from bacterial and animal celluloses.

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

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