Characterization of bacterial enzymes that depolymerize fungal cell wall polysaccharides

项目总结 真菌感染在死亡率和治疗成本方面都对人类健康造成了重大影响。同时反- 真菌药物一直是真菌感染的主要治疗方法，耐药的发生率也在不断上升 难以治疗的真菌感染。另一种扰乱真菌细胞壁合成的方法 药物是真菌细胞壁多糖的活性降解产物。然而，有一项重要的知识 在有效降解真菌细胞壁的要求方面存在差距。这一不足之处使 可单独或与现有药物联合使用的新的抗真菌疗法的开发 治疗。这个项目的长期目标是发展对多糖的机理理解。 解构以产生医学上相关的酶。这项特别提案的目标集中在 真菌细胞壁多糖降解机理的鉴定与表征 日本细胞弧菌。我们的中心假设是，需要一套协调的酶来 有效降解真菌细胞壁中的葡聚糖和甲壳素成分。我们将用三个例子来检验这一假设 具体目标：(1)真菌细胞壁多糖降解过程中的多组学分析；(2)功能 分析编码真菌细胞壁解构所必需的酶的基因，以及(3)定量 真菌细胞壁降解酶的酶学研究。对于第一个目标，我们将使用已建立的转录和 蛋白质组学方法破译日本血吸虫活跃时的复杂基因和蛋白质表达模式 降解尼杜拉曲霉和酿酒酵母的真菌细胞壁。新的目标将是 通过随后的遗传分析将其置于功能范围内。第二个目标将决定 用于解构真菌细胞壁的单个基因产物。我们已经建立了转座子和 高通量靶向突变方法，用于识别和分析基因 日本对虾多糖的降解研究。我们将测试缺乏这些基因的突变菌株的适合性 使用不溶性真菌细胞壁多糖和完整的真菌生物量进行生长分析。对于第三个目标，我们 将纯化和鉴定能够降解真菌细胞壁多糖的酶以确定它们的 底物专一性，动力学参数，并评估酶的协同作用。利用真菌生物质作为一种 底物将允许我们确定什么酶组合在解构完整结构方面最有效 真菌细胞壁。这些方法是创新的，因为我们使用了一种含有强健多糖的细菌 降解能力与使用包括丝状菌在内的完整真菌生物量的新型筛子相结合 真菌和酵母菌。这个项目意义重大，因为它将表征与医学相关的酶 属性，提供了对有效破坏真菌细胞壁的要求的机械洞察，以及 产生一个强大的系统来发现具有抗真菌潜力的酶。

PROJECT SUMMARY Fungal infections significantly impact human health, both in terms of mortality and treatment cost. While anti- fungal drugs have been the leading therapy for fungal infections, there is an increasing incidence of resistant fungal infections that are difficult to treat. An alternative approach to disrupting fungal cell wall synthesis with drugs is the active degradation of fungal cell wall polysaccharides. However, there is a substantial knowledge gap in regards to the requirements for effective fungal cell wall degradation. This shortfall prevents the development of new anti-fungal therapies that could be used alone or in combination with current drug treatments. The long-term goal of this project is to develop mechanistic understanding of polysaccharide deconstruction to produce medically relevant enzymes. The objective of this particular proposal is focused on identifying and characterizing the mechanisms for the degradation of fungal cell wall polysaccharides by the bacterium Cellvibrio japonicus. Our central hypothesis is that a coordinated suite of enzymes is required to effectively degrade the glucan and chitin components of fungal cell walls. We will test this hypothesis with three Specific Aims: (1) Multiomic analyses during degradation of fungal cell wall polysaccharides, (2) Functional analysis of genes that encode enzymes essential for fungal cell wall deconstruction, and (3) Quantitative enzymology of fungal cell wall degrading enzymes. For the first Aim, we will use established transcriptomic and proteomic methods to decipher the complex gene and protein expression patterns of C. japonicus when actively degrading the fungal cell walls of Aspergillus nidulans and Saccharomyces cerevisiae. Novel targets will be placed in a functional context by subsequent genetic analysis. The second Aim will determine the contribution of individual gene products for the deconstruction of fungal cell walls. We have established both transposon and high-throughput targeted mutational approaches to identify and analyze genes that are essential for polysaccharide degradation in C. japonicus. We will test the fitness of mutant strains lacking these genes with growth assays using insoluble fungal cell wall polysaccharides and intact fungal biomass. For the third Aim, we will purify and characterize enzymes capable of degrading fungal cell wall polysaccharides to determine their substrate specificity, kinetic parameters, and to assess enzyme synergy. The use of fungal biomass as a substrate will allow us to determine what enzyme combinations are maximally effective at deconstructing intact fungal cell walls. These approaches are innovative because we use a bacterium that has a robust polysaccharide degrading capability coupled with a novel screen that uses intact fungal biomass, which includes filamentous fungi and yeasts. This project is significant because it will characterize enzymes with medically-relevant properties, give mechanistic insight into the requirements for the effective disruption of fungal cell walls, and generate a powerful system for the discovery of enzymes that have anti-fungal potential.