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.
