Structural and functional studies of glycosyl hydrolases governing Vibrio biofilm dispersal

项目总结/摘要 生物膜是由细胞外保护性基质包围的表面附着的细菌群落 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，营养限制和物理损伤，而在 环境水库。与生物膜的形成一样重要的是，分散机制允许细菌 降解生物膜基质并响应于内部或环境线索的变化而逃逸。 了解生物膜是如何分散的，对于开发对抗生物膜相关疾病的新策略非常重要。 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 在分子尺度上理解生物膜形成、粘附和分散机制的方法。 本提案的总体目标是利用该模型了解生物膜扩散的机制 生物膜形成细菌霍乱弧菌。霍乱弧菌生物膜由一种分泌的 一种称为弧菌多糖（VPS）的胞外多糖，沿着分泌的基质蛋白和胞外 DNA.该建议的中心假设是，RbmB，一种分泌的假定糖基水解酶，是一种关键酶， 在Vc生物膜的分散的因素，它破坏VPS，导致细胞外基质的分解。 我们的目标是通过研究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切割的特异性、机制和动力学特性， 来自霍乱弧菌的推定糖基水解酶RbmB; 2）确定三维结构， RbmB在切割VPS中的酶促机制;以及3）使用体外和体内技术，确定RbmB如何切割VPS。 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。我们对拟议工作的理由是，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 获得生物膜如何分散的基本了解。虽然这项建议的重点是霍乱弧菌，我们预计， 这些见解将适用于其他细菌病原体谁生产生物膜使用分泌 胞外多糖和基质蛋白。这项建议的结果将有助于我们了解 糖基水解酶有助于它们在治疗抗肿瘤和恶性肿瘤中的潜在治疗用途 细菌感染这项工作也预示着新的研究可能性，在发展具体的 用于生物工程和生物医学应用的可切割多糖支架。

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.