Revealing the Cell Wall Organization of Fungal Pathogens and Structural Responses to Antifungal Drugs Using Cellular Solid-State NMR

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Project Summary Invasive fungal infection has high occurrence and mortality among immunocompromised patients. Most of the currently available antifungal agents targeting the cell membrane have limited efficacy, relatively high toxicity, and an observed increase in drug resistance. The cell wall of fungal pathogens is a promising target for antifungal drugs due to its absence in humans. Still, such efforts have been hindered by our inadequate knowledge of the cell wall organization. The long-term goal of our research is to understand the structures of cell walls of prevalent fungal pathogens at the molecular level to promote the development of wall- targeting therapeutics against infections. This project aims to develop solid-state nuclear magnetic resonance (ssNMR) technology that allows understanding the nanoscale remodeling of fungal cell wall structures involved in fungal virulence and drug resistance. The central hypothesis is that the fungal cell walls’ structural dynamics and mechanical properties are major factors contributing to virulence and drug resistance. This central hypothesis will be tested by three specific aims. We will interrogate the polymorphic structure of major structural polysaccharides and their supramolecular packing in the cell walls of three major fungal pathogens, including Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. Second, we will determine the structural dynamics of the cell wall and the remodeling process of the biopolymer composite induced by antifungal echinocandins and nikkomycin that target, respectively, β- glucan and chitin, two of the major cell wall components. Third, we will examine cell wall structures of naturally occurring mutant strains of A. fumigatus and C. albicans showing modulated susceptibility to antifungal agents and the superbug Candida auris to identify the structural mechanisms underlying altered drug responses. We will pursue these aims by establishing an innovative approach that combines ssNMR and dynamic nuclear polarization (DNP) techniques to enable high-resolution and non-destructive characterization of intact and living fungal cells. The project is significant because it will identify the essential carbohydrate components and their structures or packing interfaces that serve as the potential targets for discovering novel antifungal compounds with broad spectrums and improved efficacy. Our efforts will yield a collection of structural diagrams of cell walls across three major fungal pathogens and their responses to antifungal agents and mutations. The research will define a new biophysical direction bridging the long- standing gap between biomedical observations of fungal phenotypes and the fundamental understanding of structures and interactions of biomolecules at atomic levels. The methodology established here will also permit the high-resolution assessment of structural effects of antifungal agents that are available or being developed, which will revolutionize antifungal drug design.