The ability of various intracellular pathogens to spread from cell to cell without having to be exposed to the extra-cellular environment is a fundamental aspect of their pathogenic properties. Intracellular bacterial pathogens, such as Listeria ssp, Rickettsia spp. and Shigella spp., display actin-based motility within the cytosol of infected host cells and spread into neighboring cells through the formation of membrane protrusions. Genetic and biochemical studies revealed that Listeria monocytogenes actin-based motility relies on the expression of ActA, a bacterial factor that recruits the ARP2/3 complex to the bacterial surface and mimics the nucleation-promoting activity of WASP/WAVE family members. To identify host factors involved in pathogen spread, we have implemented a systematic genetic approach based on the combination of the RNA interference methodology, high-throughput fluorescence microscopy and computer-assisted image analysis. We used the approach to screen a siRNA library covering the human kinome and identified CK11 and CK2 as host cell serine/threonine kinases required for L. monocytogenes spread from cell to cell. Our preliminary results indicate that CK2 regulates actin tail formation through phosphorylation of ActA, whereas CK1 regulates membrane protrusion formation through an unknown mechanism. In this application, we propose to further understand the role of ActA phosphorylation in actin tail formation (Aim 1). With the long-term goal of determining the cellular mechanisms supporting actin tail and membrane protrusion formation, we also propose to complete our genome-wide RNAi screen and characterize the role of the identified host factors in cell-to-cell spread (Aim 2). The proposed research program is designed to further our understanding of the mechanisms underlying intracellular pathogen spread from cell to cell. It may also illuminate the native function(s) of host factors involved in complex cellular processes, such as regulation of the actin cytoskeleton and remodeling of the plasma membrane. PUBLIC HEALTH RELEVANCE: Various intracellular pathogens have evolved the ability to manipulate host cell processes in order to spread from infected cells into the neighboring cells. Most of he host factors contributing to the cell-to-cell spread of pathogens are unknown. In this application, we present our plans to develop the first genetic approach of its kind to uncover these host factors. The proposed approach will contribute to our general understanding of the mechanisms underlying microbial pathogenesis and may therefore constitute the foundation for the rational design of preventive and therapeutic interventions.