FAK in E.coli Pathogenesis

描述（由申请人提供）：细菌感染对公共卫生提出了紧迫的挑战。这些活动包括治疗和预防重大疾病，以及制定预防生物恐怖主义的战略。E.大肠杆菌K1能侵入人脑血管内皮，导致中枢神经系统的严重发病和死亡。粘着斑激酶（pp 125 FAK）被认为是这一过程的促进者。本研究主要探讨FAK在E. coli K1侵袭和破坏血管内皮。第一个具体目标将检查细菌入侵。FAK可以通过增强纤连蛋白原纤维形成和为入侵的原核生物提供附着点，或者通过调节细菌进入细胞所使用的皮层下肌动蛋白网络的动态可塑性来煽动细菌入侵。这些研究将检验FAK表达水平、纤连蛋白和肌动蛋白微结构与E. coliK 1入侵。已经制备了含有显性阴性、野生型和过度活化的FAK的腺病毒载体以改变FAK活性。第二个具体目标将审查机制的E。大肠杆菌诱导的血管内皮功能的变化。FAK激酶活性和RhoA活性的反应和调节将在细菌感染期间被定义。具有不同FAK活性水平的内皮细胞将被E. coli K1.将测量粘附连接中VE-钙粘蛋白和β-连环蛋白的组织变化。将使用电细胞基质阻抗传感（ECIS）定量内皮单层渗透性。将使用微图案化硅柱研究细胞骨架力向细胞外基质的传递，以报告受感染细胞和单层的局部（接近离散侵入事件）和全局变化。这些研究将提供一个更详细的概念框架，为确定治疗靶点，以减少E。大肠杆菌K1病在脑微血管中的作用。对大肠杆菌入侵机制的深入了解可能为研究大肠杆菌提供有用的范例。大肠杆菌病在其他器官系统。

DESCRIPTION (provided by applicant): Bacterial infections present pressing public health challenges. These range from the treatment and prevention of critical illness to the preparation of preventive strategies against bioterrorism. E. coli K1 invades the vascular endothelium of the human brain and causes devastating central nervous system morbidity and mortality. The focal adhesion kinase (pp125FAK) has been implicated as a facilitator of this process. This proposal focuses on the role of FAK in E. coli K1 invasion and disruption of vascular endothelium. The first Specific Aim will examine bacterial invasion. FAK may abet bacterial invasion by either enhancing fibronectin fibrillogenesis and providing points of attachment for invading prokaryotes, or by regulating the dynamic plasticity of subcortical actin networks used by bacteria to enter the cell. These studies will examine the direct relationships between levels of FAK expression, fibronectin and actin microarchitecture, and the success of E. coli K1 invasion. Adenoviral vectors containing dominant negative, wild type, and hyperactive FAK have been prepared to alter FAK activity. The second Specific Aim will examine mechanisms of E. coli-induced changes in vascular endothelial function. The responses and regulation of FAK kinase activity and RhoA activity will be defined during bacterial infection. Endothelial cells with varying levels of FAK activity will be infected with E. coli K1. Changes in the organization of VE-cadherin and beta-catenin in adherens junctions will be measured. Endothelial monolayer permeability will be quantified using an electrical cell-substrate impedance sensing (ECIS). Cytoskeletal force transmission to extracellular matrix will be studied using micropatterned silicon posts to report both local (proximate to discrete invasion events) and global changes in infected cells and monolayers. These studies will provide a more detailed conceptual framework for the identification of therapeutic targets to attenuate E. coli K1 disease in the brain microvasculature. Insights into mechanisms of E .coli invasion that are gained here may provide useful paradigms for approaching E. coli disease in other organ systems.