Summary Listeria monocytogenes is a facultative intracellular pathogen responsible for the life-threatening disease listeriosis. Although Lm produces numerous virulence factors, the secreted pore-forming toxin LLO is indispensable for pathogenesis. LLO is secreted at all stages of the Listeria intracellular life cycle and binds to cholesterol to form transmembrane pores. This virulence factor perforates the membrane of the Listeria- containing endocytic vacuoles to release the bacterium into its replicative niche, the cytosol. It was recently established that LLO also perforates the host cell plasma membrane, which promotes host cell invasion. It remains to elucidate how infected cells maintain viability despite perforation of their plasma membranes and how this low-grade perforation impacts the course of Listeria infection. The work performed in Aim 1 will establish novel mechanisms that maintain viability of infected cells despite perforation of their plasma membranes by LLO. Preliminary studies, via screening of a large siRNA library, led to the identification of novel families of host proteins that were not previously known to repair the plasma membrane of toxin-perforated cells. The Aim 1 studies will establish the mechanisms of action of these novel proteins. Specifically, they will determine the role plasma membrane depolarization plays in organizing calcium-dependent lysosome exocytosis, leading to the release of cytoprotective cathepsins on the cell surface. Aim 1 will also establish a new role for the septins, a family of cytoskeletal proteins, in plasma membrane repair. These studies will employ high-speed and super- resolution microscopy to analyze the molecular assemblies that orchestrate plasma membrane repair. The Aim 2 studies will establish the impact of plasma membrane perforation by LLO on Listeria intracellular survival and the innate immune response of antigen-presenting cells. We showed that transient plasma membrane perforation by LLO triggers Ca2+ influx-dependent activation of conventional PKCs on the endosomal network, a signaling event that is critical for Listeria phagosome escape. Aim 2 will identify the PKCs effectors by SILAC- based quantitative proteomic approach and how they contribute to the release of Listeria into the cytosol. Plasma membrane perforation also causes K+ efflux, which is known to activate the NLRP3 inflammasome. Aim 2 will dissect the role of low-grade plasma membrane perforation in the maturation of antigen presenting cells to enhance T cell immunity, in vitro and in vivo. This work is expected to broadly impact the development of vaccines and novel therapeutic strategies for a wide range of diseases in which pore-forming toxins are employed by pathogens.