Figure 1.

Analysis of the virulence of the Salmonella mlc mutant strain. All of the cell infection experiments were carried out with wild-type (SL1344) and mlc⁻ mutant (SR1304) strains. HEp-2 (A) and HeLa cells (B) were infected with Salmonella grown to exponential phase without shaking. Infected cells were lysed 2 h after infection, and dilutions of the suspension were plated onto LB agar medium, to enumerate colony-forming units (CFUs). The data are presented as percentages of the CFU of the wild-type strain. (C) For the cytotoxicity assay, RAW 264.7 macrophage cells were infected with Salmonella grown to exponential phase without shaking, and then assayed for LDH release. (D) The intracellular replication assay was carried out with opsonized bacteria grown to stationary phase under aerobic conditions. After 2 and 18 h of infection, the eukaryotic cells were lysed and the viable intracellular (gentamicin-protected) bacteria were counted. The values shown represent fold-increases, which were calculated as the ratios of intracellular bacteria 2 and 18 h after bacterial entry. These assays were performed at least twice in triplicate, to allow calculation of means and standard deviations.