FAK in E.coli Pathogenesis

描述(由申请人提供)：细菌感染是公共卫生面临的紧迫挑战。这些措施包括治疗和预防危重疾病，以及制定针对生物恐怖主义的预防性战略。大肠杆菌K1侵入人脑的血管内皮细胞，导致毁灭性的中枢神经系统发病率和死亡率。粘着斑激酶(Pp125FAK)被认为是这个过程的促进器。本研究的重点是FAK在大肠杆菌K1侵袭和血管内皮细胞破坏中的作用。第一个具体目标是检查细菌的入侵情况。FAK可能通过促进纤维连接蛋白纤维生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质下肌动蛋白网络的动态可塑性来辅助细菌入侵。这些研究将考察FAK表达水平、纤维连接蛋白和肌动蛋白微结构与大肠杆菌K1侵袭成功之间的直接关系。含有显性阴性、野生型和高活性FAK的腺病毒载体已经被制备来改变FAK的活性。第二个具体目标将研究大肠杆菌诱导血管内皮功能变化的机制。在细菌感染过程中，FAK激酶活性和RhoA活性的反应和调节将被明确。具有不同水平FAK活性的内皮细胞将被大肠杆菌K1感染。将测量粘连连接中VE-钙粘附素和β-连环素组织的变化。内皮单层通透性将使用电细胞-基质阻抗传感(ECIS)进行量化。细胞骨架力传递到细胞外基质的研究将使用微图案化的硅柱来报告感染细胞和单层中的局部(接近离散的入侵事件)和全局变化。这些研究将为确定治疗靶点以减轻脑微血管中的大肠杆菌K1疾病提供更详细的概念框架。在此获得的对大肠杆菌入侵机制的洞察可能为研究其他器官系统中的大肠杆菌疾病提供有用的范例。

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.