Summary Plasma membrane disruption by pore-forming toxins (PFTs) is a most common and ancient strategy used by bacterial pathogens to infect their host and evade the host’s immune responses. Listeriolysin O (LLO) is a PFT produced by the foodborne pathogen Listeria monocytogenes (Lm). Lm is a Gram-positive bacterium responsible for listeriosis, a severe illness leading to 99% hospitalization and up to 30% fatality despite treatment. Lm is a facultative intracellular pathogen that infects a large array of cells including macrophages. Although Lm produces numerous virulence factors, LLO is uniquely known to be indispensable for pathogenesis. Therefore, LLO and the host pathways targeted by this toxin are promising targets for the development of therapeutic interventions. LLO is secreted at all stages of the Lm intracellular lifecycle and binds cholesterol to assemble a large transmembrane pore complex. This virulence factor has long been known to perforate the membrane of the Lm- containing phagosome to release Lm into its replicative niche, the cytosol. It was recently established that LLO also perforates the host cell plasma membrane, which facilitates cell invasion and phagosomal escape. Monocyte/Macrophages, which specialize in the capture and destruction of microbes, evolved cytoprotective mechanisms (referred to as the macrophage repairome) to maintain cell homeostasis and survival despite LLO attack. How macrophages repair their plasma membrane and prevent toxin attack is not fully understood. The goal of this R03 proposal is to develop tools to discover the “macrophage repairome” using unbiased whole- genome screening. Our lab successfully developed fluorescence-based screening methods to analyze the repair machineries of cells exposed to LLO. To establish the macrophage repairome in an unbiased fashion, we will perform a whole-genome screen using CRISPR/Cas9 genome editing. We will generate a CRISPR/Cas9 library in THP-1 cells (human monocyte-like cell line) and screen the library for cells unable to maintain their integrity upon LLO exposure using florescence-activated cell sorting (FACS)-selectable phenotype (positive screen). Indeed, damaged cells with deficient cell repair will be fluorescent, whereas intact cells will exclude the fluorescent dye. Deep sequencing will generate a list of candidate genes required for maintaining macrophage integrity. We will perform pathway analysis, select the most novel and promising pathways, and validate the selected pathways in THP-1 cells. These pathways will be studied in detail in the context of L. monocytogenes infection via future R01 funding. Understanding the mechanisms used by macrophages to counteract bacterial pore-forming toxins is expected to facilitate the development of novel antimicrobial treatments to alleviate the burden of infectious diseases.