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

项目摘要 侵袭性真菌感染在免疫功能低下的患者中发生率和死亡率较高。大部分 目前可用的针对细胞膜的抗真菌药物疗效有限，相对较高 毒性，以及观察到的抗药性增加。真菌病原体的细胞壁是一个很有希望的目标。 抗真菌药物，因为它在人类身上不存在。尽管如此，这些努力由于我们的不足而受到阻碍。 细胞壁组织的知识。我们研究的长期目标是了解这些结构 在分子水平上对流行的真菌病原菌的细胞壁进行研究，以促进细胞壁的发育 针对感染的治疗药物。该项目旨在开发固态核磁 共振(SsNMR)技术，可以了解真菌细胞壁的纳米级重构 参与真菌毒力和耐药性的结构。中心假设是真菌细胞 墙体的结构动力学和力学性能是致病和致毒的主要因素。 抵抗。这一核心假设将通过三个具体目标进行检验。我们将审问多态的 三种主要结构多糖的结构及其在细胞壁中的超分子堆积 真菌病原体，包括烟曲霉、白色念珠菌和新生隐球菌。 第二，我们将确定细胞壁的结构动力学和细胞的重塑过程 抗真菌棘球菌素和尼可霉素诱导的生物聚合物复合材料分别靶向β- 葡聚糖和甲壳素，两种主要的细胞壁成分。第三，我们将研究细菌的细胞壁结构 自然产生的烟曲霉菌和白色念珠菌突变株对 抗真菌药物和超级细菌金黄色假丝酵母菌鉴定改变的结构机制 药物反应。我们将通过建立一种结合单核磁共振的创新方法来实现这些目标 和动态核极化(DNP)技术，以实现高分辨率和无损 完整的和活的真菌细胞的特征。该项目意义重大，因为它将确定必要的 作为潜在目标的碳水化合物成分及其结构或包装界面 发现具有广谱和提高药效的新型抗真菌化合物。我们的努力将会取得成果 三种主要真菌病原体的细胞壁结构图及其对 抗真菌药物和突变。这项研究将定义一个新的生物物理方向，将长期和 真菌表型的生物医学观察与对真菌表型的基本认识之间的差距 生物分子在原子水平上的结构和相互作用。这里建立的方法论也将 允许对现有或正在使用的抗真菌药物的结构效应进行高分辨率评估 开发的，这将彻底改变抗真菌药物的设计。

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.