Summary of significant Tn-seq hits.

<div> <i>Listeria monocytogenes</i> is a Gram-positive pathogen that causes the severe foodborne disease listeriosis. Following oral infection of the host, <i>L. monocytogenes</i> disseminates from the gastrointestinal tract to peripheral organs, including the gallbladder, where it replicates to high densities, establishing the gallbladder as the primary bacterial reservoir. Despite its importance in pathogenesis, little is known about how <i>L. monocytogenes</i> survives and replicates in the gallbladder. In this study, we assessed the <i>L. monocytogenes</i> genes required for growth and survival in <i>ex vivo</i> non-human primate gallbladders using a transposon sequencing approach. The screen identified 43 genes required for replication in the gallbladder, some of which were known to be important for virulence, and others had not been previously studied in the context of infection. We evaluated the roles of 19 genes identified in our screen both <i>in vitro</i> and <i>in vivo</i>, and demonstrate that most were required for replication in bile <i>in vitro</i>, for intracellular infection of murine cells in tissue culture, and for virulence in an oral murine model of listeriosis. Interestingly, strains lacking the mannose and glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS) permeases Mpt and Mpo exhibited no defects in intracellular growth or intercellular spread, but were significantly attenuated during murine infection. While the roles of PTS systems <i>in vivo</i> were not previously appreciated, these results suggest that PTS permeases are necessary for extracellular replication during infection. Overall, this study demonstrates that <i>L. monocytogenes</i> genes required for replication in the gallbladder also play broader roles in disease. </div>