Fig. 2

In vitro infection of mouse peritoneal macrophages. C57BL/6 mouse peritoneal macrophages seeded on 96-well plates were pre-stimulated with 1 μg/ml E. coli lipopolysaccharide to induce the expression of pro-IL-1β for 3 h. The freshly cultured X4550(pYA3334-SspH2-EscI), X4550(pYA3334-SspH2) and X4550(pYA3334) were then added with the desired multiplicity of infection (MOI). The cell plate was centrifuged to enhance the contact of bacteria with the cells and the infected cells were then incubated for 30 min. The supernatants containing uninfected bacteria were replaced with RPMI 1640 complete medium (100 μl/well) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml LPS prior to the start of the subsequent incubation. The uninfected cells were used as control. a Supernatant IL-1β and IL-18 levels at 4 h post-infection (hpi) with MOI 10, 50 and 100; b LDH release at 1, 3, 5 hpi with MOI 100 and 24 hpi with MOI 10, 50 and 100; c Intracellular caspase-1 activation at 1 h hpi with MOI 100; d Cell morphology at 24 hpi with MOI 100 (a, uninfection; b, infected with X4550(pYA3334); c, infected with X4550(pYA3334-SspH2); d, infected with X4550(pYA3334-SspH2-EscI), arrows show pyroptotic cell death); e The count of cells and intracellular bacteria at 24 hpi with MOI 100. The data shown are representative of three replicate experiments