Structure of fungal cell walls and remodeling by antifungal drugs elucidated using solid-state NMR

Understanding the cell wall structure, dynamics, and mechanisms of adaptations are important for the development of cell wall-targeted drugs for the treatment of fatal infections caused by fungi. We first combined ssNMR, and functional genomics analysis to identify the functionality of cell wall carbohydrates in Aspergillus fumigatus and four mutants depleted of major structural polysaccharides. We revealed a rigid inner core of the cell wall formed by tightly associated chitin and α-1,3-glucan, which are embedded in a soft matrix of β-glucans and capped by a mobile outer shell rich in galactosaminogalactan and galactomannan. The distribution of α-1,3-glucan in chemically and dynamically distinct domains supports its dual functionality in structure and pathogenicity. Also, ssNMR revealed the modification of polymer dynamics which suggests that fungi produce a denser and hydrophobic cell wall as a response to structural defects. Second, we characterized the structural responses of A. fumigatus cell walls to the treatment of antifungal drugs. The drug-treated cell walls are highly hydrophobic and stiff to resist external stress. The fungi also increased the content of chitin and produced new forms of α-glucans to compensate for the loss of β-glucan due to drug inhibition. Third, we found that the crystalline structure of chitin bears intrinsic heterogeneity, which is resistant to caspofungin treatment. We documented the unique structural fingerprints of chitin across different fungal species. Finally, we used an innovative approach combing the ssNMR with the dynamic nuclear polarization technique to characterize the cell wall organization in different morphotypes varied along with the life cycle, revealing a highly conserved carbohydrate core in both conidia and mycelia. These studies and the biophysical techniques yield essential information that can serve as potential targets for discovering novel antifungal compounds with broad spectrums.