Project Summary The goal of the proposed research is to identify the structural characteristics of fungal cell walls involved in virulence and drug response. Invasive fungal infection results in fatal diseases in individuals with immunodeficiency. Existing antifungal drugs have been mainly designed to target fungal cell membranes, but they also bind to human cell membranes, thus causing severe side effects. Recent efforts have been devoted to developing agents that bind fungal cell walls because they contain carbohydrates, for example, chitins and β- glucans, that are absent in human. Such biomolecules, however, evade high-resolution structural characterization as they are typically insoluble and polymorphic in structure. Recently, we initiated a project to investigate the native cell wall structure of Aspergillus fumigatus, a major pathogenic fungus causing invasive aspergillosis, using solid-state NMR spectroscopy. This method provides atomic-level insight into the supramolecular assembly of carbohydrates and proteins in native fungal cell walls with minimal perturbation. The central hypothesis is that the large-scale spatial rearrangement of biomolecules directly regulates fungal virulence and drug resistance. This hypothesis will be tested by pursuing two specific aims: 1) identify the structural variation of cell walls from pathogenic and non-pathogenic fungi, and between the young and aged fungi; 2) elucidate the molecular effects of antifungal drugs on the cell wall structure of wildtype and drug-resistant strains. The expected outcomes include the first set of comparative models of Aspergillus cell wall structure with dependence on four variables: fungal type and pathogenicity, age, drug concentration, and genetic mutants. The approaches established in this project will be widely applicable to investigations of many other fungal pathogens. This combined effort of biophysical methods, carbohydrate chemistry, and medical mycology presents a unique and novel contribution as it bridges the gap between molecular structures with phenotype and drug effects. The dynamic and comprehensive view of the functional structure of fungal cell wall obtained here, in the long term, will facilitate the discovery and evaluation of novel therapies that can effectively inhibit a broad spectrum of pathogenic fungi by targeting their cell wall structure or biosynthesis.