Identification of Attenuated Yersinia pseudotuberculosis Strains and Characterization of an Orogastric Infection in BALB/c Mice on Day 5 Postinfection by Signature-Tagged Mutagenesis

Phenotypes of ST strains with cells in culture. (A) Percentage of ST strains recovered after bacteria were grown at 26°C in 2xYT containing 5 mM CaCl₂ and exposed to HeLa cells at a multiplicity of infection of 10:1. After a 30-min infection, gentamicin was added to the medium (first box in each pair) for an additional 90 min, the cells were washed and lysed with 1% Triton X-100, and the bacteria were plated. In a side-by-side control, gentamicin was not added to the medium cells were lysed with a final concentration of 1% Triton X-100 and bacteria were plated (second box in each pair). All experiments were done in triplicate, and each strain was tested at least two times in both assays. (B) Percentage of gentamicin-resistant bacteria of various ST strains after infection of HeLa cells with bacteria pregrown at 37°C for 2 h. HeLa cells were infected as described above and in Materials and Methods. (C) Percentage of RAW264.7 macrophage cell death due to infection with Y. pseudotuberculosis relative to complete lysis of the macrophages. The bacteria were pregrown at 37°C for 2 h, the cells were infected for 2 h, gentamicin was added to the medium, and culture supernatants were assayed 7 h after the addition of gentamicin. All experiments were performed in triplicate and repeated twice. The strain labeled lcrR is YPIIIpIB71, which does not secrete any Yops.