A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocy-togenesmutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified genemenD or pepPwere defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus vali-dating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of themenD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, themenD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both themenD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multipl