Request for 5-year extension of Al - 36929 under MERIT award Listeria monocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns, and immunocompromised or older adults. From the initial site of Infection in the intestine, the bacteria are able to spread systemically while avoiding the antibody-mediated arm of the hose immune response. These bacteria grow directly in the cytoplasm of infected host cells and move rapidly throughout and between infected cells using a form of actin-based motility. This remarkable ability allows the bacteria to spread from the intestinal epithelial cells into circulating macrophages, which then carry the bacteria throughout the body and are thought to mediate their spread into distal tissues including the liver, the brain, and the placenta (in pregnant women). The intracellular actin-based motility of L. monocytogenes has served as an important model system for understanding the molecular and biophysical mechanisms of eukaryotic processes driven by actin polymerization, including whole-cell crawling in the immune system and in cancer metastasis. Through our interdisciplinary work involving biochemical reconstitution of motility, biophysical measurement of force-generating processes operating at the bacterial surface, molecular genetic dissection of the bacterial and host contributions to motility, and mathematical modeling of this complex process, we have developed a very detailed understanding of the intracellular motility phase of the infection cycle. In our current work, we are expanding our interdisciplinary analysis to other steps in the infection, including host cell invasion, bacterial growth and surface polarization, and cell-to-cell spread. 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 interactions between ActA and host cell cytoplasmic factors have been well-studied, but the behavior of ActA itself is less explored. This is a very large intrinsically disordered protein, whose size is such that it should not be able to diffuse through the nanometer-scale pores in the thick, cross-linked Gram-positive bacterial cell wall. Nevertheless, it does extend through the wall while remaining anchored in the membrane. Very recently, we have developed a conceptual breakthrough that can explain quantitative features of ActA translocation as an entropy-driven process. Our new model is highly relevant for surface presentation of other virulence factors in Gram-positive organisms. Over the next five years of this ongoing project, we propose to use L monocytogenes as a genetic system to identify bacterial genes involved in determination of the physical properties of the cell wall (such as thickness and pore size) that govern entropy-driven protein secretion, and expand our analysis to other Gram-positive virulence factors that are structurally related to ActA, particularly in Staphylococcus aureus. For our studies of invasion and cell-to-cell spread, we are focusing on interactions between L. monocytogenes and the endothelial cells that line blood vessels, as these cells should represent a critical barrier to systemic dissemination of the bacteria. The most likely mechanisms of bypassing the barrier properties of the endothelium include: direct infection of endothelial cells, infection of endothelial cells via cell-to-cell spread from infected circulating immune system cells, and transmigration of infected immune cells across an uninfected endothelium. In tissue culture, we have been able to replicate each of these processes, and have used systematic siRNA screening to identify host cell factors uniquely involved in each step. These initial results have yielded many surprises, and have demonstrated that invasion of endothelial cells is mechanistically distinct from invasion of intestinal epithelial cells. Over the next five years, we propose to continue and expand our molecular dissection of these processes and test the role of the molecules we identify in mouse models of L monocytogenes infection. RELEVANCE AND SIGNIFICANCE Listeria monocytogenes bacteria cause a rare but very serious form of food poisoning, which can lead to meningitis (infection of the brain and central nervous system) in newborns, immunocompromised people, and older adults, and can also cause late-term miscarriage in pregnant women. These bacteria grow directly inside of human host cells and use their niche inside of human cells to "hide out" from antibodies that the immune system normally generates to fight infections. The short-term goal of this research is to understand how Listeria monocytogenes are able to invade human cells and move from the inside of one human cell to another, with a long-term goal of finding better methods to prevent or treat serious bacterial infections.