Despite major advances in understanding how cells detect pathogens in the cytosol (1), little is known about the factors that protect the cytosol from invasion by bacterial pathogens. Likewise, despite a number of studies over the past two decades demonstrating that cells protect their cytosol from bacterial invasion (2–4), how cytosolic pathogens avoid these host defenses and utilize the cytosol as a replication niche remains largely unknown. Listeria monocytogenes is a deadly food-borne pathogen (5) that invades the cytosol of a wide variety of cell types and maintains its intracellular niche through coordinated expression of well-characterized virulence factors (6). Maintenance of an intracellular niche is essential for L. monocytogenes pathogenesis, and induction of host cell death highly attenuates bacterial virulence (7). We had previously identified a highly conserved protein of unknown function (YvcK) required for survival of L. monocytogenes in the macrophage cytosol (8). Bacterial killing in the cytosol resulted in the release of DNA from lysed bacteria, activation of the AIM2 inflammasome, and induction of a programmed cell death process known as pyroptosis (8–11). Pyroptosis attenuates infection by eliminating the replication niche of L. monocytogenes (12, 13); as such, cytosolic survival and avoidance of detection by the AIM2 inflammasome are critical for L. monocytogenes pathogenesis.