Structural and functional studies of glycosyl hydrolases governing Vibrio biofilm dispersal

项目摘要/摘要 生物膜是附着在表面的细菌群落，被细胞外保护基质包围 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的侵害 和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的是，扩散机制允许细菌 降解生物膜基质，并对内部或环境线索的变化做出反应而逃逸。 了解生物膜是如何分散的，对于制定对抗生物膜相关的新策略非常重要 感染和抗药性细菌。这项研究的长期目标是使用一种结构/功能 从分子水平上了解生物膜的形成、黏附和扩散的机制。 这项建议的总体目标是利用该模型来理解生物膜扩散的机理。 形成生物被膜的霍乱弧菌。霍乱弧菌生物膜由一种分泌的 胞外多糖，称为弧菌多糖(VPS)，以及分泌的基质蛋白和胞外 DNA这一提议的中心假设是RbmB，一种分泌的假定糖基水解酶，是一个关键 Vc生物膜扩散的因素和它消化VPS导致细胞外基质分解的因素。 我们的目的是通过研究RbmB相关的VPS消化的结构和机制 以下三个具体目标：1)了解VPS裂解的特异性、机制和动力学特性 霍乱弧菌可能的糖基水解酶RbmB；2)确定其三维结构和 RbmB在切割VPS中的酶机制；以及3)利用体外和体内技术，确定如何 RbmB活性导致生物膜的降解和霍乱弧菌的扩散。我们将结合使用 活霍乱弧菌的酶分析、定点突变、X射线结晶学和成像 生物膜来完成这些目标。我们对拟议工作的基本原理是，通过理解结构和 RbmB的机制，其对VPS的特异性，以及基质蛋白在生物被膜扩散中的作用，我们将 对生物膜如何分散有一个基本的了解。虽然这项提案的重点是霍乱弧菌，但我们预计 这些见解将适用于其他使用分泌物产生生物膜的细菌病原体 胞外多糖和基质蛋白。这项提案的结果将有助于我们理解 糖基水解酶在抗生素耐药和恶性疾病治疗中的潜在应用 细菌感染。这项工作还有望在特定领域的发展中提供新的研究可能性 生物工程和生物医学应用的可裂解多糖支架。

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.