FAK in E.coli Pathogenesis

大肠杆菌发病机制中的 FAK

• 批准号: 6926935
• 负责人: LEWIS H ROMER
• 金额: $ 24.36万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6926935
• 关键词: Escherichia coli; actins; active sites; bacteria infection mechanism; cadherins; cell adhesion; cytoskeleton; digital imaging; electron microscopy; enzyme activity; enzyme mechanism; extracellular matrix; fibronectins; focal adhesion kinase; gene expression; guanine nucleotide binding protein; host organism interaction; immunofluorescence technique; intercellular connection; molecular assembly /self assembly; protein structure function; single cell analysis; transfection /expression vector; vascular endothelium; vascular endothelium permeability

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.

描述（由申请人提供）：细菌感染是迫在眉睫的公共卫生挑战。这些工作范围从治疗和预防危重疾病到制定针对生物恐怖主义的预防战略。大肠杆菌K1侵入人脑的血管内皮，导致毁灭性的中枢神经系统发病率和死亡率。焦点粘附激酶（pp125FAK）被认为是这一过程的促进者。本研究的重点是FAK在大肠杆菌K1侵袭和破坏血管内皮中的作用。第一个特异性目标是检查细菌入侵。FAK可能通过增强纤维连接蛋白纤维形成并为入侵的原核生物提供附着点，或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促进细菌的入侵。这些研究将检验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。腺病毒载体含有显性阴性、野生型和过度活跃的FAK，可以改变FAK的活性。第二个特定目标将研究大肠杆菌诱导血管内皮功能改变的机制。FAK激酶活性和RhoA活性的响应和调控将在细菌感染期间确定。具有不同水平FAK活性的内皮细胞将被大肠杆菌K1感染。将测量粘附连接中VE-cadherin和β -catenin组织的变化。内皮单层渗透性将使用电细胞-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硅柱来报告局部（接近离散入侵事件）和感染细胞和单层的全局变化。这些研究将为确定减轻脑微血管中大肠杆菌K1疾病的治疗靶点提供更详细的概念框架。对E机制的洞察。这里获得的大肠杆菌入侵可能为研究其他器官系统的大肠杆菌疾病提供有用的范例。