Fig. 1

In vitro infection of mouse peritoneal macrophages. Six-week-old female C57BL/6 mouse peritoneal macrophages were seeded on 96-well plates for culturing. Three hours later, non-adherent cells were removed and cell density was adjusted to 20,000 cells per well with RPMI 1640 complete medium without antibiotics and the freshly cultured X4550(pYA3334), X4550(pYA3334-P-SspH2) or X4550(pYA3334-P-SspH2-EscI) were added with MOI 100. 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 and 100 μg/ml streptomycin prior to the start of the subsequent incubation. The uninfected cells were used as control. a Activation of intracellular caspase-1 at different hours post-infection (hpi) using FLICA staining; b Cytotoxicity assay by lactate dehydrogenase (LDH) release at 5 hpi; c Cell morphology observation at 5 hpi. a, uninfected control; b, X4550(pYA3334-P-SspH2) infection; c, X4550(pYA3334-P-SspH2-EscI) infection. d Cell function including reactive oxygen species (ROS), nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and mitochondrial membrane potential (MMP) at 1, 3, 5 hpi stained with DCFH-DA, DAF-FM DA, Fluo-3 AM and rhodamine 123 (Rh123) respectively. e Intracellular pH value at 0, 1, 3, 5 hpi stained with BCECF-AM; f Supernatant cytokines levels at 1, 3, 5 hpi using cytometric bead array system kit. Results are representative of at least three independent experiments (c, d). *P < 0.05; X4550(pYA3334-P-SspH2-EscI) infection vs X4550(pYA3334-P-SspH2) or X4550(pYA3334) infection (e, f)