Listeria Invasion Assay in HT-29 Cells

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.