Effects of Shear on Specific Adhesion Receptor Expression and Binding in the Bact

描述（由申请人提供）：通过受体：配体与吸附分子（如血浆蛋白、碳水化合物和糖缀合物）的相互作用，细菌对生物（宿主组织）和合成基质（如生物医学设备）的特异性粘附是导致生物膜形成的一系列过程中的关键第一步；宿主组织侵袭、毒力和感染；甚至可能导致死亡。医院感染是美国第四大死亡原因，每年有200万例（约占美国医院患者的10%）。大约60-70%的此类感染与植入医疗设备有关，2002年造成45亿美元的医疗费用，每年约有99,000人死亡。生物膜形成的第一步是细菌细胞通过“粘附素”（或受体）与目标表面的配体分子的特异性粘附。预防这种最初的粘附结合可能潜在地消除生物膜的形成和任何随后的感染。开发小分子疗法或疫苗以防止生物医学设备的特异性粘附，或者相反，制造旨在促进粘附和识别特定病原体的“芯片实验室”阵列，将需要在相关的流体动力学条件下提供细菌特异性结合事件的详细信息。在体外鉴定细菌受体及其固定化配体上的结合表位的尝试，由于以下原因而变得复杂：(1)无法以确定和一致的方向“呈现”配体；(2)缺乏粘附受体表达的详细动力学，作为细胞生长和环境水动力条件的功能，基于先前的工作。我们假设，要精确量化细菌特异性粘附将需要(1)控制结合配体的方向和表面密度；(2)测定细菌粘附受体的数量、亲和力和亲和力；(3)受体结合相互作用的表征：配体对-所有这些都是在生长和流体剪切的相关条件下进行的。我们的最终目标是开发一种通用方案，以定义细菌粘附受体：配体相互作用，在其天然状态下作为流体剪切的功能-用于纯和混合培养生物膜。公共卫生相关性：我们的最终目标是开发一种通用方案，以定义细菌粘附受体：配体相互作用，在其天然状态下作为流体剪切的功能-用于纯和混合培养生物膜。在NIH的支持下，我们将使用表皮葡萄球菌（SE）结合固定化纤维连接蛋白（FN）的模型纯培养系统开发该方案。在这个为期两年的项目中，我们的具体目标是：1。量化SE菌株通过纤维连接蛋白结合受体（FNBR）与固定在受控方向和已知表面密度下的FN的特异性结合，作为主流流体剪切的函数。2. 量化细菌生长条件和主流流体剪切作用下FNBR粘附素受体在SE表面的表达动力学；浮游细胞和贴壁细胞。

DESCRIPTION (provided by applicant): Bacterial specific adhesion to biological (host tissue) and synthetic substrata (e.g., biomedical devices) through receptor:ligand interactions with adsorbed molecules (e.g., blood plasma proteins, carbohydrates, and glycoconjugates) is a critical first step in a cascade of processes leading to biofilm formation; host tissue invasion, virulence and infection; and potentially death. Nosocomial infections are the fourth leading cause of death in the U.S. with >2 million cases annually (or ~10% of American hospital patients). About 60-70% of all such infections are associated with an implanted medical device causing >$4.5 billion medical costs in 2002 and ~99,000 deaths annually. One of the first steps in biofilm formation is the specific adhesion of bacterial cells via `adhesins' (or receptors) to ligand molecules present on the target surface. Prevention of this initial adherence binding could potentially abrogate biofilm formation and any subsequent infection. Development of small molecule therapies or vaccines to prevent specific adhesion to biomedical devices or, conversely, the fabrication of "lab-on-a-chip" arrays designed to promote adhesion and identify specific pathogens will require detailed information on bacterial specific binding events, under pertinent hydrodynamic conditions. Attempts to identify binding epitopes on both bacterial receptors and their immobilized ligands, in vitro, are complicated by (1) the inability to "present" the ligand in a defined and consistent orientation and (2) lack of detailed kinetics for adhesion receptor expression, as a function of cell growth and ambient hydrodynamic conditions, Based on prior work, we hypothesize that to precisely quantify bacterial specific adhesion will require (1) controlling the orientation and surface density of the binding ligand; (2) determination of the number, affinity and avidity of bacterial adhesion receptors; and (3) characterization of the binding interactions of the receptor:ligand pair - all under pertinent conditions of growth and fluid shear. Our ultimate goal is to develop a general protocol that will define bacterial adhesion receptor:ligand interactions, in their native states as a function of fluid shear - for both pure and mixed culture biofilms. PUBLIC HEALTH RELEVANCE: Our ultimate goal is to develop a general protocol that will define bacterial adhesion receptor:ligand interactions, in their native states as a function of fluid shear - for both pure and mixed culture biofilms. With NIH support, we will develop this protocol using the model pure culture system of Staphylococcus epidermidis (SE) binding to immobilized fibronectin (FN). Our specific aims in this two-year project will be: 1. Quantify, as a function of prevailing fluid shear, the specific binding of SE strains via fibronectin binding receptors (FNBR) to FN immobilized in a controlled orientation and known surface density. 2. Quantify the kinetics of FNBR adhesin receptor expression on SE surfaces, as function of bacterial growth condition and prevailing fluid shear; both as planktonic and adherent cells.