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:
- 金额:$ 31.76万
- 依托单位:
- 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:
- 关键词:AccelerationAntifungal AgentsAntifungal TherapyAspergillus nidulansBacteriaBasic ScienceBiological AssayBiomassCell WallCellsCellvibrioChitinChitinaseComplexComplex MixturesContainmentCoupledDataDevelopmentDiseaseDrug resistanceEnsureEnzymatic BiochemistryEnzymesExcisionFungal ComponentsFungi ModelFutureGene DeletionGene ExpressionGenesGlucansGoalsGrowthHealthHumanIn VitroIncidenceIndividualInfectionKineticsKnowledgeMeasurementMedicalMedical DeviceMethodsMicrobial BiofilmsMissionModelingMoldsMutationMycosesNatureNutrientOintmentsOutcomePatternPharmaceutical PreparationsPharmacotherapyPhenotypePolysaccharidesPropertyProteinsProteomeProteomicsPublic HealthRegulationResearchResearch Project GrantsResistanceSaccharomyces cerevisiaeSourceStructureSubstrate SpecificitySystemSystems BiologyTechniquesTestingTherapeuticTimeTreatment CostUnited States National Institutes of HealthYeastscandidate identificationcombatdepolymerizationdesignfitnessfitness testfungusgene complementationgene productgenetic analysisin vivoinnovationinsightmortalitymultiple omicsmutantnovelnovel strategiespathogenic funguspoor health outcomepreventprotein expressionrational designsynergismtranscriptometranscriptomics
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
真菌感染在死亡率和治疗费用方面严重影响人类健康。虽然反-
抗真菌药物一直是真菌感染的主要治疗方法,但耐药的发生率越来越高,
真菌感染是很难治疗的。一种替代方法来破坏真菌细胞壁的合成,
药物是真菌细胞壁多糖的活性降解。然而,有大量的知识
在有效真菌细胞壁降解的要求方面存在差距。这一缺陷阻碍了
开发新的抗真菌疗法,可单独使用或与现有药物联合使用
治疗。本项目的长期目标是发展对多糖的机理理解
解构以产生医学相关的酶。这一特定提案的目标重点是
鉴定和表征真菌细胞壁多糖的降解机制,
细菌纤维弧菌我们的中心假设是,需要一套协调的酶来
有效地降解真菌细胞壁的葡聚糖和几丁质组分。我们将用三个例子来检验这个假设。
具体目的:(1)真菌细胞壁多糖降解过程中的多组学分析,(2)功能性
分析编码真菌细胞壁解构所必需的酶的基因,和(3)定量
真菌细胞壁降解酶的酶学。对于第一个目标,我们将使用已建立的转录组学和
蛋白质组学方法来破译复杂的基因和蛋白表达模式的C。当积极
降解构巢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背景中。第二个目标将决定
用于真菌细胞壁的解构的单个基因产物。我们已经建立了转座子和
高通量靶向突变方法,以鉴定和分析基因是必不可少的,
C. - 是的我们将测试缺乏这些基因的突变株的适应性,
使用不溶性真菌细胞壁多糖和完整真菌生物质的生长测定。第三个目标,我们
将纯化和表征能够降解真菌细胞壁多糖的酶,以确定其
底物特异性、动力学参数,并评估酶协同作用。使用真菌生物质作为
底物将使我们能够确定什么酶组合在解构完整的
真菌细胞壁这些方法是创新的,因为我们使用了一种具有强大多糖的细菌,
降解能力加上新的筛选,使用完整的真菌生物质,包括丝状
真菌和酵母。这个项目是重要的,因为它将表征酶与医学相关的
性质,给出了有效破坏真菌细胞壁的要求的机械见解,和
为发现具有抗真菌潜力的酶提供了一个强大的系统。
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Microbe Profile: Cellvibrio japonicus: living the sweet life via biomass break-down.
微生物概况:日本细胞弧菌:通过生物量分解过上甜蜜的生活。
- DOI:10.1099/mic.0.001450
- 发表时间:2024
- 期刊:
- 影响因子:0
- 作者:Gardner,JeffreyG
- 通讯作者:Gardner,JeffreyG
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