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.

项目概要/摘要 生物膜是表面附着的细菌群落，周围有细胞外保护基质 由多糖、蛋白质、核酸组成。生物膜保护细菌病原体免受抗生素侵害 和宿主免疫系统以及捕食、营养限制和身体伤害 环境水库。与生物膜的形成一样重要的是，扩散机制允许细菌 降解生物膜基质并响应内部或环境线索的变化而逃逸。 了解生物膜如何分散对于制定对抗生物膜相关的新策略非常重要 感染和抗生素耐药细菌。这项研究的长期目标是使用结构/功能 方法在分子尺度上了解生物膜形成、粘附和分散的机制。 该提案的总体目标是使用该模型了解生物膜分散的机制 生物膜形成细菌霍乱弧菌。霍乱弧菌生物膜由分泌的 称为弧菌多糖 (VPS) 的胞外多糖，以及分泌的基质蛋白和细胞外 脱氧核糖核酸。该提案的中心假设是 RbmB（一种假定的分泌型糖基水解酶）是关键 Vc 生物膜分散的因素，并且它消化 VPS 导致细胞外基质分解。 我们的目标是通过探究 RbmB 相关 VPS 消化的结构和机制 以下三个具体目标： 1) 了解 VPS 裂解特异性、机制和动力学特性 假定的来自霍乱弧菌的糖基水解酶 RbmB； 2）确定三维结构并 RbmB裂解VPS的酶促机制； 3) 使用体外和体内技术，确定如何 RbmB 活性导致生物膜降解和霍乱弧菌扩散。我们将使用以下组合 活霍乱弧菌的酶测定、定点诱变、X 射线晶体学和成像 生物膜来完成这些目标。我们提出的工作的基本原理是，通过了解结构和 RbmB 的机制、其对 VPS 的特异性以及基质蛋白在生物膜分散中的作用，我们将 对生物膜如何分散有基本的了解。虽然该提案重点关注霍乱弧菌，但我们预计 这些见解将适用于使用分泌物产生生物膜的其他细菌病原体 胞外多糖和基质蛋白。该提案的结果将有助于我们理解 糖基水解酶有助于其在治疗抗生素耐药性和恶性疾病中的潜在治疗用途 细菌感染。这项工作还为开发专门的研究提供了新的可能性 用于生物工程和生物医学应用的可裂解多糖支架。