Structural and functional studies of glycosyl hydrolases governing Vibrio biofilm dispersal

控制弧菌生物膜分散的糖基水解酶的结构和功能研究

• 批准号: 10795423
• 负责人: RICHARD A OLSON
• 金额: $ 47.12万
• 依托单位: WESLEYAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10795423
• 关键词: Active Sites; Adhesions; Affect; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Biochemical; Biochemistry; Biological Assay; Biological Models; Biomedical Engineering; Biophysical Process; Cells; Communicable Diseases; Communities; Complement; Creativeness; Crystallization; Cues; DNA; Development; Digestion; Environment; Extracellular Matrix; Extracellular Matrix Proteins; Fostering; Goals; Growth; Homologous Gene; Human; Hydrolase; Image; Immune system; In Vitro; Infection; Kinetics; Knowledge; Learning; Microbe; Microbial Biofilms; Mission; Modeling; Molecular; Mutagenesis; Mutation; Nucleic Acids; Nutrient; Outcome; Pathogenicity; Phenotype; Polysaccharides; Predatory Behavior; Process; Property; Protein Secretion; Proteins; Proteolysis; Public Health; Research; Resolution; Role; Site; Site-Directed Mutagenesis; Specificity; Structure; Surface; Techniques; Therapeutic; Therapeutic Uses; United States National Institutes of Health; Vibrio; Vibrio cholerae; Work; X-Ray Crystallography; crosslink; drug resistant bacteria; experimental study; extracellular; fighting; human disease; human pathogen; in vivo; insight; molecular scale; mutant; pathogen; pathogenic bacteria; physical insult; reconstitution; response; scaffold; therapy design; three dimensional structure; tool

PROJECT SUMMARY/ABSTRACT Biofilms are surface-attached communities of bacteria surrounded by an extracellular protective matrix composed of polysaccharides, proteins, and nucleic acids. Biofilms protect bacterial pathogens from antibiotics and the host immune system as well as from predation, nutrient limitation, and physical insults while in environmental reservoirs. As important as the formation of biofilms, dispersal mechanisms allow bacteria to degrade the biofilm matrix and escape in response to changes in internal or environmental cues. Understanding how biofilms disperse is important in developing new strategies for combatting biofilm-related infections and antibiotic-resistant bacteria. The long-term goal of this research is to use a structure/function approach to understand the mechanisms of biofilm formation, adhesion, and dispersal at the molecular scale. The overall objective of this proposal is to understand the mechanism of biofilm dispersal using the model biofilm-forming bacterium Vibrio cholerae. Vibrio cholerae biofilms are composed of a secreted exopolysaccharide called Vibrio polysaccharide (VPS), along with secreted matrix proteins and extracellular DNA. The central hypothesis of this proposal is that RbmB, a secreted putative glycosyl hydrolase, is a key factor in the dispersal of Vc biofilms and that it digests VPS leading to breakdown of the extracellular matrix. We aim to understand the structure and mechanism of RbmB-associated VPS digestion by pursuing the following three specific aims: 1) Understand the VPS cleavage specificity, mechanism and kinetic properties of the putative glycosyl hydrolase RbmB from Vibrio cholerae; 2) Determine the three-dimensional structure and enzymatic mechanism of RbmB in cleaving VPS; and 3) Using in vitro and in vivo techniques, determine how RbmB activity leads to degradation of the biofilm and Vibrio cholerae dispersal. We will use a combination of enzymatic assays, site-directed mutagenesis, X-ray crystallography, and imaging of living Vibrio cholerae biofilms to complete these aims. Our rationale for the proposed work is that by understanding the structure and mechanism of RbmB, its specificity towards VPS, and the role of matrix proteins in biofilm dispersal, we will gain a basic understanding of how biofilms disperse. While this proposal focuses on Vibrio cholerae, we expect that these insights will be applicable to other bacterial pathogens who produce biofilms using secreted exopolysaccharides and matrix proteins. Results from this proposal will contribute to our understanding of glycosyl hydrolases aiding their potential therapeutic use in the treatment of antibiotic-resistant and pernicious bacterial infections. This work also promises new research possibilities in the development of specifically cleavable polysaccharide scaffolds for bioengineering and biomedical applications.

项目摘要/摘要 生物膜是被细胞外保护基质包围的细菌表面附着的社区 由多糖，蛋白质和核酸组成。生物膜保护细菌病原体免受抗生素 以及宿主免疫系统以及捕食，营养限制和身体侮辱 环境水库。与生物膜的形成一样重要，分散机制使细菌得以实现 降解生物膜基质并响应内部或环境线索的变化而逃脱。 了解生物膜分散在制定与生物膜相关的新策略中如何重要 感染和抗生素耐药菌。这项研究的长期目标是使用结构/功能 理解生物膜形成，粘附和分散在分子尺度上的机制的方法。 该提案的总体目的是使用模型了解生物膜扩散的机制 生物膜形成细菌弧菌霍乱。弧菌霍乱生物膜由分泌的 外多糖称为弧菌多糖（VPS），以及分泌的基质蛋白和细胞外 脱氧核糖核酸。该提议的中心假设是RBMB是一种分泌的推定糖基水解酶，是关键 VC生物膜分散的因素，并消化VPS导致细胞外基质的崩溃。 我们旨在通过追求RBMB相关VP的消化的结构和机制 以下三个具体目的：1）了解VPS裂解特异性，机制和动力学特性 来自Vibrio Cholerae的假定糖基水解酶RBMB； 2）确定三维结构和 RBMB在切割VPS中的酶促机制； 3）使用体外和体内技术，确定如何 RBMB活性导致生物膜和弧形霍乱分散的降解。我们将结合 酶试验，定点诱变，X射线晶体学和活弧霍乱的成像 生物膜完成这些目标。我们对拟议工作的理由是，通过了解结构和 RBMB机制，其对VPS的特异性以及基质蛋白在生物膜扩散中的作用，我们将 对生物膜分散的方式有基本的了解。虽然该提议重点是霍乱弧菌，但我们希望 这些见解将适用于其他细菌病原体，这些病原体使用分泌的生物膜产生生物膜 外多糖和基质蛋白。该提案的结果将有助于我们对 糖基水解酶有助于其在治疗抗生素耐药和恶性治疗方面的潜在治疗用途 细菌感染。这项工作还有望在专门开发方面进行新的研究可能性 可切合的多糖支架，用于生物工程和生物医学应用。