Structural Interactions of Bacterial Adhesin with Glycosaminoglycans

描述（由申请人提供）：抗生素耐药性正在成为迫在眉睫的健康威胁。对抗这种情况的一个策略是抑制微生物对人体细胞的粘附。然而，微生物与靶细胞之间的特定结构相互作用并没有很好地表征，从而阻碍了新的抗微生物药物的开发。本研究旨在通过对细菌粘附素与糖胺聚糖（GAGs）之间相互作用的深入结构研究来克服这一缺陷。糖胺聚糖是一种普遍存在于细胞表面的多糖，是细菌粘附素的主要靶点。该提案的重点将放在decorin binding protein （dbp）上，这是一种来自莱姆病病原伯氏疏螺旋体（Borrelia burgdorferi）的粘附素。为了研究GAGs和dbp之间的相互作用，dbp在细胞质及其天然脂蛋白形式中的结构将使用溶液核磁共振进行解析。利用这些结构作为碱基，dbp与异相和均相GAG低聚物的相互作用将被确定。本研究的主要目标是获得dbp与GAG复合物的高分辨率结构，并从这些结构中推断出GAG与dbp之间的特异性相互作用，从而可以靶向阻止细菌对人体细胞的粘附。最后，由于gag通常以蛋白聚糖的形式存在，我们将尝试表征dbp与完整的decorin （dbp主要靶向的蛋白聚糖）之间的相互作用。这部分建议的主要目标是仅利用核磁共振衍生的取向约束和完整装饰素的二聚体性质来构建具有完整装饰素的dbp的复杂结构。拟议的工作将在复杂碳水化合物研究中心进行，这是一个多学科研究机构，也是该国首屈一指的糖生物学研究中心之一。候选人的长期职业目标是了解控制gag与其蛋白质靶点相互作用的规则，并制定策略，将这些相互作用引导到期望的方向。在指导阶段，候选人的直接目标将是熟练掌握用于纯化GAG低聚物的化学和生化方法，并学习使用哺乳动物表达系统，这对于获得完整的decorin至关重要。这些技术的知识将使候选人能够在建议的领域进行独立的研究。

DESCRIPTION (provided by applicant): Antibiotic resistance is becoming an imminent health threat. One strategy for combating this is to inhibit the adhesion of microbes to human cells. However, specific structural interactions between microbes and target cells are not well characterized, thus hampering the development of new anti-microbial agents. This proposal strives to overcome this deficiency by conducting an in-depth structural study of the interactions between bacterial adhesins and glycosaminoglycans (GAGs), a ubiquitous polysaccharide found on the surface of cells and a primary target for bacterial adhesins. The focus of the proposal will be on the decorin binding protein (dbp), an adhesin from Borrelia burgdorferi, the etiological agent of Lyme disease. To study the interactions between GAGs and dbp, the structures of dbp in both cytoplasmic and its native lipoprotein form will be solved using solution NMR. Using these structures as bases, dbp's interactions with heterogeneous and homogeneous GAG oligomers will be determined. The main goal of this research is to obtain high resolution structures of dbp complexed to GAG and deduce from these structures the specific interactions between GAG and dbp that can be targeted to prevent the adhesion of bacteria to human cells. Finally, as GAGs often exist in the form of proteoglycans, an attempt will be made to characterize the interactions between dbp and intact decorin, the proteoglycan that dbp primarily targets. The main goal for this portion of the proposal will be to construct the complex structure of dbp with intact decorin utilizing only NMR derived orientational restraints and the dimeric nature of intact decorin. The proposed work will be carried out at the Complex Carbohydrate Research Center, a multidisciplinary research institute and one of the country's premier glycobiology research centers. The candidate's long term career goal is to understand the rules governing the interactions of GAGs with its protein targets and devise strategies for steering these interactions in a desired direction. The candidate's immediate goal during the mentored phase will be to acquire proficiency with the chemical and biochemical methods used to purify GAG oligomers and learn the use of mammalian expression systems that will be crucial for obtaining intact decorin. The knowledge of these techniques will enable the candidate to conduct independent research in the proposed area. Relevance: The results of this proposal will be invaluable for the design of a new class of anti-microbial agents that specifically inhibits the adhesion of microbials to human cells. It also adds to our understanding of how sulfated polysaccharides interact with proteins to initiate important biological events.