Bhi medium

L. monocytogenes was cultivated in the BHI medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and grown at 37 °C with agitation at 200 rpm. Plasmid-bearing strains were grown in the presence of 10 µg mL⁻¹ erythromycin (Sigma-Aldrich, USA) to maintain the plasmid. To prepare a culture for infection, bacteria were grown to the mid-exponential phase, washed with PBS (Amresco, Cochran Rd, Solon, OH, USA) three times, aliquoted, and frozen in the presence of 10% glycerol (Sigma-Aldrich, USA). The growth rate was estimated using its optical density at 600 nm. The strains were grown overnight in BHI medium (Becton, Dickinson and Company, USA) at 37 °C and subsequently diluted 1:100 (the volume of night culture: the volume of the fresh media) in the same media. Optical density was measured every hour on an Ultrospec™ 10 Cell Density Meter spectrophotometer (Biochrom, Cambridge, UK). E. coli BL21 strains encoding recombinant idInlBs were grown in the LB medium complemented with 100 µg mL⁻¹ kanamycin and 1 mM IPTG.

L. monocytogenes strains were stored at –80°C in brain heart infusion (BHI) medium containing 50% glycerol and cultured in BHI medium (BD Biosciences). Selected colonies were grown overnight at 30°C with shaking. Bacteria were pelleted, washed, and resuspended in phosphate-buffered saline (PBS). The bacterial inocula were estimated based on the optical density at 600 nm (OD₆₀₀) and verified by plating serial dilutions on plates to determine the CFU.
L. monocytogenes WT (10403S), Δhly (DP-L2161, ΔLLO), and ΔactA (DP-L1942, ΔActin) strains, as well as the L. monocytogenes RFP (DP-L5538) strain in which the RFP tag is expressed under the actA promoter, were saved in our laboratory (17 (link)). DCs of 8 × 10⁴ cells/well were plated into 96-well plates (eight or four independent wells according to experiment needs) and cultured for 8 h. DCs were infected with L. monocytogenes strains, which were cultivated in BHI medium (BD Biosciences) to an OD₆₀₀ of 0.4 at a multiplicity of infection (MOI) of 20. At 1 h postinfection, the culture medium was replaced with 100 μg/ml gentamicin for 30 min to kill extracellular bacteria, followed by treatment with 10 μg/ml gentamicin to prevent reinfection. At each time point, DCs were lysed with 0.1% Triton (Sigma) for 10 min and then serially diluted and plated on BHI plates (the samples were tested in triplicate) to verify the colony count.

HT-29 cells (ATCC HTN-38) were used to determine the virulence of the Listeria strains [37 (link)]. L. monocytogenes strains (L. monocytogenes Scott A, isogenic deletion mutants ΔLMOf2365_1875 and ΔLMOf2365_1877, and the parental LMOf2365) and L. innocua were used for the invasion assays performed as described previously [36 (link)]. In brief, HT-29 cells were grown in 24-well tissue culture plates for 5 days to obtain almost confluent monolayers. Strains of L. monocytogenes (L. monocytogenes Scott A, isogenic deletion mutants ΔLMOf2365_1875 and ΔLMOf2365_1877, and the parental LMOf2365) and L. innocua were grown to log-phase (OD_600nm ~0.3) at 37°C. HT-29 cell monolayers incubated in medium without antibiotics for 24 h were infected for 1 h at 37°C with 10⁷ CFU bacterial cells in 300 μl BHI medium (Becton Dickinson and Co., Sparks, MD). The cell monolayers were washed with DMEM and incubated in DMEM containing gentamicin (100 μg/ml) for 1.5 h at 37°C. HT-29 cell monolayers were gently washed three times with phosphate buffered saline (pH 7.4) and then disrupted with 1 ml cold sterile water (4°C). Viable bacteria were counted after plating serial dilutions onto TSA. The results were expressed as the percentage of CFU recovered after 2 h relative to the number of bacterial cells deposited per well. Three independent experiments were performed for each strain.