The Himar1 mariner transposon system was used to construct a random transposon library in wild-type L. monocytogenes carrying bacteriolysis reporter pBHE573 (8) as outlined in references 65 and 66. To perform the genetic screen, random mutant colonies from the Himar1 bacteriolysis library were isolated and grown in wells from a 96-well plate overnight in BHI media at 30°C. In parallel, iIFNAR−/− macrophages were seeded in opaque 96-well plates at 1 × 105 cells per well and then allowed to incubate at 37°C in a 5% CO2 atmosphere for approximately 16 h. iIFNAR−/− macrophages were infected at an estimated multiplicity of infection (MOI) of 10. At 1 h postinfection, the culture medium was exchanged for media containing 50 µg/ml gentamycin. At 6 h postinfection, the culture medium was replaced with TNT buffer to lyse cells. Luciferase reagent was added and measured for luciferase activity using a luminometer (BioTek Synergy HT).