Structure/function studies of biofilm agents from Aa

产品说明：伴放线放线杆菌和几种病原菌附着在非生物表面，并产生外聚合物，使这些定殖表面上的细菌细胞粘附。这些细胞的这种定殖导致具有不同于单个细胞的代谢和生理能力的生物膜。这些生物膜的显著独特性质是它们对抗菌剂的抗性。为了在其他原始表面上定居并克服与过度繁殖相关的饥饿，细胞必须从这些表面分离和分散。我们以前已经表明，在Aa中，酶分散素B能够协助分散过程。我们小组的其他研究表明，分散素B能够阻止几种相关和非相关细菌物种的表面附着。由于几种病原性感染是由生物膜引起的，因此控制生物膜形成的新方法是必要的，该方法集中于病原性细菌在表面上的初始附着，而不是它们的抗微生物剂抗性。在这方面，通过使用细胞外聚合物物质来防止细菌附着到表面的酶的研究是一个欠发达的领域。我们发现，分散素B解聚胞外多糖，是由β-1，6-连接的N-乙酰葡糖胺（NAG），是一个潜在的候选人，以消除许多革兰氏阴性和革兰氏阳性细菌病原体的生物膜。因此，我们的长期目标是为分散素B作为广谱抗生物膜剂的开发奠定基础。我们目前的重点是对这种酶的结构功能研究，以阐明其水解活性所必需的结构决定因素。我们建议通过使用X射线晶体学和突变分析的综合方法来实现这一目标。具体目标是： 1.确定决定分散素B作用机制的结构决定因素。 2.使用β-1，6-连接NAG的寡聚底物通过动力学分析绘制底物结合位点。 3.明确特定氨基酸残基在分散素B酶活性中的作用。

DESCRIPTION: Actinobacillus actinomycetemcomitans and several pathogenic bacteria attach to abiotic surfaces and produce exopolymers that immobilize the bacterial cells on these colonized surfaces. Such colonization of these cells leads to biofilms with metabolic and physiological capabilities distinct from individual cells. Notable unique property of these biofilms is their resistance to antimicrobials. To colonize other virgin surfaces and to overcome starvation associated with overpopulation, cells must detach and disperse from these surfaces. We have previously shown that in Aa, the enzyme dispersin B is capable of assisting in the dispersal process. Additional studies by our group have shown that dispersin B is capable of preventing surface attachment of several related and non-related bacterial species. Since several pathogenic infections are caused by biofilms, new approaches in controlling the biofilm formation that focus on initial attachment of pathogenic bacteria on to the surfaces, in lieu of their antimicrobial resistance, are necessary. In this regard, study of enzymes that prevent the attachment of bacteria to surfaces through the use of extracellular polymeric substances is an underdeveloped area. We show that dispersin B depolymerizes exopolysaccharides that are made up of beta-1,6-linked N-acetylglucosamine (NAG), and is a potential candidate to remove biofilms of many Gram negative and Gram positive bacterial pathogens. Therefore, our long-range goals are to lay the foundation in the development of dispersin B as a broad spectrum anti-biofilm agent. Our immediate focus is on the structure-function studies on this enzyme to elucidate structural determinants that are necessary for its hydrolytic activity. We propose to achieve this through an integrated approach using x-ray crystallographic and mutational analysis. The Specific Aims are: 1. Identify structural determinants governing the mechanism of action of dispersin B. 2. Map the substrate binding site through kinetic analysis using oligomeric substrate of beta-1,6-linked NAG. 3. Define the roles of specific amino acid residues in the enzymatic activity of dispersin B.