The L. monocytogenes ΔactA strain [10403s background; gift from D. Portnoy (79)] was grown overnight in BHI medium in a shaker at 37°C and washed three times in serum- and additive-free DMEM before use. Infections were performed at multiplicities of 5 to 10 bacteria per host cell and synchronized by centrifugation (750g for 10 min). Gentamicin (100 μg/ml) was added 1 hour after infection for 1 hour, after which cells were washed and incubated in complete DMEM containing gentamicin (20 μg/ml) for a further 8 hours. Wells were washed in DMEM and lysed in PBS containing 0.5% Triton X-100, and serial fivefold dilutions were plated (10 μl) on BHI agar plates to enumerate colony-forming units (CFU). Fold replication of bacteria between 10 and 2 hours after infection was calculated from technical triplicates.

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