Life-threatening invasive fungal infection occurs to more than two million patients annually with high mortality (20-95%). The inefficiency of antifungal drugs necessitates the development of new agents with novel targets or mechanisms. The carbohydrate-rich fungal cell wall is a promising target of antifungal drugs, and new compounds have recently been developed to disrupt glucan-synthesis and perturb cell wall integrity with reduced toxicity. Due to the technical difficulty in characterizing the highly polymorphic and disordered structure of complex carbohydrates, our understanding of the fungal cell wall structure remains elusive on the molecular level. Here we present three solid-state NMR and Dynamic Nuclear Polarization (DNP) studies of the cell wall of Aspergillus fumigatus, a major fungal pathogen, in intact cells and with atomic resolution. First, we have measured a series of 2D 13C-13C/15N experiments and successfully determined the sub-nanometer packing, ns-μs motion and site-specific hydration of biomolecules in intact cell walls. The fungal cell wall is found to contain a hydrophobic scaffold of chitin and alpha-1,3-glucan, which is surrounded by a hydrated matrix of diversely linked beta-glucans and capped by a dynamic, outer layer rich in glycoproteins. Second, based on this structural frame, we further tested how drug inhibition affects the composition and supramolecular assembly of carbohydrates in cell walls. The presence of Echinocandins drug substantially reduces the amount of beta-glucans but enhances the synthesis of chitin instead. The remaining carbohydrates exhibit higher rigidity and hydrophobicity, thus efficiently prevents the water-soluble antifungal drugs from penetrating this layer of carbohydrate armor and increase the chance of survival. Third, we will also present the high-resolution spectra collected on the world-record 1.5 GHz magnet. The substantially improved 13C resolution allows us to better resolve the heavily overlapped signals of cell wall biomolecules in wild-type A. fumigatus, its genetic mutants, and drug-treated cultures. These studies provide invaluable insight into the functional structure of carbohydrates and the drug response and resistance of A. fumigatus, which has the potential to promote the development of better wall-targeted antifungals.

Reveal the functional structure of complex carbohydrates in intact fungal cell walls using solid-state NMR

ACS Spring Meeting 2021

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