Characterization of bacterial enzymes that depolymerize fungal cell wall polysaccharides

解聚真菌细胞壁多糖的细菌酶的表征

基本信息

批准号：
10675071
负责人：
Jeffrey Gardner
金额：
$ 31.76万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE COUNTY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10675071
关键词：
Acceleration Antifungal Agents Antifungal Therapy Aspergillus nidulans Bacteria Basic Science Biological Assay Biomass Cell Wall Cells Cellvibrio Chitin Chitinase Complex Complex Mixtures Containment Coupled Data Development Disease Drug resistance Ensure Enzymatic Biochemistry Enzymes Excision Fungal Components Fungi Model Future Gene Deletion Gene Expression Genes Glucans Goals Growth Health Human In Vitro Incidence Individual Infection Kinetics Knowledge Measurement Medical Medical Device Methods Microbial Biofilms Mission Modeling Molds Mutation Mycoses Nature Nutrient Ointments Outcome Pattern Pharmaceutical Preparations Pharmacotherapy Phenotype Polysaccharides Property Proteins Proteome Proteomics Public Health Regulation Research Research Project Grants Resistance Saccharomyces cerevisiae Source Structure Substrate Specificity System Systems Biology Techniques Testing Therapeutic Time Treatment Cost United States National Institutes of Health Yeasts candidate identification combat depolymerization design fitness fitness test fungus gene complementation gene product genetic analysis in vivo innovation insight mortality multiple omics mutant novel novel strategies pathogenic fungus poor health outcome prevent protein expression rational design synergism transcriptome transcriptomics

项目摘要

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.

项目摘要在死亡率和治疗成本方面，真菌感染都会显着影响人类健康。而反 - 真菌药物一直是真菌感染的领先疗法，耐药性越来越多很难治疗的真菌感染。与使用真菌细胞壁合成合成的另一种方法药物是真菌细胞壁多糖的主动降解。但是，有很大的知识关于有效真菌细胞壁降解的要求的差距。此不足阻止了开发可单独使用或与当前药物结合使用的新抗真菌疗法治疗。该项目的长期目标是发展对多糖的机械理解解构以产生医学相关的酶。该特定提议的目的集中在识别和表征通过真菌细胞壁多糖降解的机制细菌细菌japonicus。我们的中心假设是需要协调的酶套件有效地降解真菌细胞壁的葡聚糖和几丁质成分。我们将用三个具体目的：（1）真菌细胞壁多糖降解期间的多素分析，（2）功能分析编码对真菌细胞壁解构必不可少的酶的基因，以及（3）定量真菌细胞壁降解酶的酶学。为了第一个目标，我们将使用已建立的转录组和蛋白质组学方法是在主动地破译Japonicus的复杂基因和蛋白质表达模式降解曲霉和酿酒酵母的真菌细胞壁。新颖的目标将是通过随后的遗传分析放置在功能环境中。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的靶向突变方法，以识别和分析对于对 Japonicus C. japonicus中的多糖降解。我们将测试缺乏这些基因的突变菌株的适应性使用不溶性真菌细胞壁多糖和完整的真菌生物量的生长测定。对于第三个目标，我们将纯化并表征能够降解真菌细胞壁多糖的酶底物特异性，动力学参数并评估酶协同作用。使用真菌生物量作为底物将使我们能够确定哪些酶组合在完整的解构上具有最大有效真菌细胞壁。这些方法具有创新性，因为我们使用具有强大多糖的细菌降解功能以及使用完整真菌生物量的新颖屏幕，其中包括丝状真菌和酵母。该项目很重要，因为它将表征与医学相关的酶属性，对有效破坏真菌细胞壁有效破坏的要求进行机械洞察力，以及生成一个有力的系统，以发现具有抗真实潜力的酶。