Shigella flexneri, the causative agent of bacillary dysentery, uses its type III secretion system (TTSS) as a conduit through which effector proteins are shuttled from bacterial to host cell cytoplasm where they subvert normal host functions. While much about TTSS function has been worked out, details on initial host interaction are not well understood. It has been shown that integral components of the type III secretion apparatus (TTSA) interact with cholesterol (Chol) and recent evidence indicates that liposomes containing Chol and sphingomyelin (Sm) induce TTS. These and other data suggest a role for lipid raft microdomains in Shigella contact, stable interaction, and effector injection into host cells, however, there has been no sustained attempts to directly explore the link between the pathogen and lipid raft-like domains. It is my hypothesis that lipid raft- like domains in human cells provide a site at which the Shigella TTSS can anchor to the surface of the cell while simultaneously supplying the signal that induces TTS and allows the injection of effector proteins into the host cell's cytoplasm. To better understand the role of lipid rafts in the virulece of Shigella, the specific aims of this investigation are to: 1) determine the role of host cell lipd raft components and membrane phases on S. flexneri initial attachment to and entry into host cells; and 2) determine the effects of lipid membrane dynamics on the interaction between live Shigella and model membranes. These studies will use a diverse array of techniques and approaches including: biochemical study of purified proteins using model membrane systems, real-time imaging of Shigella interaction with cultured cells, and developing methods for improving imaging of macromolecular assemblies. In completing these aims, I will provide the first comprehensive examination of this pathogen's microenvironment as it first encounters a host cell and the influence that lipids have on this process. This will provide insight into the roe of the TTSS as both an anchor on the host cell surface and as a signaling platform that is needed for host-pathogen crosstalk. The information obtained from these studies will provide basic information on the pathogenic mechanisms of Shigella TTS with likely implications with regard to other TTSS. This could eventually lead to the development of practical compounds or methods for preventing infections by TTSS-expressing human pathogen.