LDH cytotoxicity assay.

HeLa cells were seeded at 7.5 × 10⁴ cells/ml in 24-well plates. For RAW 264.7 macrophages, cells were seeded at 5 × 10⁵ cells/ml. Overnight cultures of V. proteolyticus were normalized to an OD₆₀₀ of 0.1 in 5 ml MLB and grown for 2 h at 30°C with agitation. When necessary, 200 µg/ml kanamycin and 0.1% (wt/vol) l-arabinose were added to maintain plasmids and induce protein expression. Infection solutions with an MOI of 25 were prepared in DMEM free of supplements and phenol red (clear DMEM). Cells were washed twice with 1 ml of clear DMEM, and then 1 ml infection solution was added in triplicate for each strain. Duplicates of high and low control wells were also included for each time point. Further methodological details on LDH release measurements can be found in Text S1 in the supplemental material. Experiments were performed at least twice with similar results. Results of a representative experiment are shown.