Listeria monocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns and immunocompromised adults. The bacterium grows directly in the cytoplasm of infected host cells and moves rapidly throughout and between infected cells using a form of actin-based motility. The L. monocytogenes surface protein, ActA, is expressed in a polarized fashion and interacts with host cell cytoskeletal factors to induce the polymerization of an actin "comet tail" structure that pushes the bacterium through the host cell cytoplasm. The overall goal of this project is to understand the mechanism and biological significance of the actin-based motility of L. monocytogenes. We use a combination of three complementary approaches to studying this form of motility-biophysical, biochemical, and cell biological. The full set of basic protein components required for actin-based motility by L. monocytogenes have now been identified and the field is beginning to agree on a general physical framework for understanding force generation during steady-state movement. The next set of conceptual and experimental challenges lie in rebuilding and coming to grips with the mechanisms underlying the full complexity of the biological behavior. We will focus our mechanistic studies on bacterial actin-based motility in two areas that are not adequately addressed by the existing steady-state models: understanding the biochemical and biophysical mechanism of bacterial movement initiation and exploring the determinants responsible for regulating path persistence and curvature. In examining the cell biology of infection in the context of bacterial cell-to-cell spread, we will develop new techniques for direct observation of bacterial spread in fully polarized epithelial monolayers in tissue culture and heterotypic spread between epithelial cells and macrophages. In addition, we will address the cell biology of the bacterial cell in determining the mechanism of ActA protein polarization. Successful completion of our research goals would give significant insight into the mechanisms by which pathogenic bacteria such as L. monocytogenes communicate specifically with the cells of their human hosts. In addition, since L. monocytogenes actin-based motility is a simple model system for force generation by actin polymerization, the results of our research would contribute to our understanding of a wide variety of basic biological processes involving actin-based cell movement, including wound healing, inflammation, embryonic development, and cancer metastasis.