Structure/function studies of biofilm agents from Aa

Aa 生物膜剂的结构/功能研究

• 批准号: 6964908
• 负责人: NARAYANAN RAMASUBBU
• 金额: $ 23.33万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ DENTAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6964908
• 关键词: Actinobacillus actinomycetemcomitans; N acetylglucosamine; X ray crystallography; bacterial proteins; beta N acetylhexosaminidase; binding sites; biofilm; enzyme activity; enzyme structure; high performance liquid chromatography; hydrolysis; site directed mutagenesis; thin layer chromatography

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.

描述：放线菌和几种致病细菌附着在非生物表面并产生外聚合物，将细菌细胞固定在这些定植表面上。这些细胞的定植导致生物膜具有不同于单个细胞的代谢和生理能力。这些生物膜显著的独特特性是它们对抗菌素的抗性。为了在其他未开发的表面上定居，并克服与人口过剩相关的饥饿，细胞必须从这些表面分离和分散。我们之前已经证明，在Aa中，分散酶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。由于几种致病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，这些新方法侧重于病原菌在表面的初始附着，而不是它们的抗菌素耐药性。在这方面，通过使用细胞外聚合物质来防止细菌附着在表面的酶的研究是一个不发达的领域。我们发现分散蛋白B可以解聚由-1,6-连接n -乙酰氨基葡萄糖（NAG）组成的胞外多糖，是去除许多革兰氏阴性和革兰氏阳性细菌病原体生物膜的潜在候选物质。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：