Membranes were incubated with blocking buffer (5% w/v nonfat milk in PBS) and then with anti-tetra His (Qiagen, Valencia, CA), anti-IpaB, anti-GroEL (Sigma Chemical Co.) antibodies. The activity of a horse peroxidase-labeled secondary antibody was visualized by adding TMB substrate (KPL, Gaithersburg, MD) directly to the membrane. Translocation of ShET-2 was assayed with the β-lactamase reported system as

described (Charpentier & Oswald, 2004) with some modifications. The full-length sen gene was amplified by PCR using wild-type S. flexneri strain 2457T total DNA as a template with primers SenFT-F click here (5′-CCGGAATTCATGCCATCAGTAAATTTAA-3′) and SenFT-R (5′-CGCGGATCCGCTTTTTATATTCTTCATA), and cloned into the BamH1–EcoR1 sites of pTB–TEM-FLAG (Ashida et al., 2007) kindly provided by Drs Chihiro Sasakawa and Hiroshi Ashida. BMN 673 manufacturer The resultant plasmid (pTB-ShET-2–TEM-FLAG) was transferred to S. flexneri wild-type strain 2457T and the T3SS mutant strain BS547. Expression of the fusion protein was assessed by Western blot analysis using an anti-FLAG antibody. To evaluate translocation of ShET-2–TEM-FLAG fusion protein, HEp-2 cells were infected at a multiplicity of infection (MOI) of 100, and plates

were centrifuged at 1000 g for 10 min. After an incubation of 30 min at 37 °C with 5% CO2, the plates were washed three times with PBS and incubated with Dulbecco’s modified Eagle’s medium (DMEM) containing 100 μg mL−1 gentamicin and 0.2 mM isopropyl-β-d-thiogalactopyranoside for 90 min. Cells were washed with PBS and loaded for 90 min with 1 mM CCF2/AM (Invitrogen). Finally, plates were examined on a fluorescence microscope with the appropriate filters. HEp-2 and T84 cells were cultured in minimal essential medium (DMEM and DMEM/F-12, respectively; Invitrogen), supplemented with 10% fetal bovine serum (FBS), at 37 °C under 5% CO2. Gentamicin

protection assays were performed by previously described methods with slight modifications (Noriega et al., 1996). Briefly, semi-confluent HEp-2 cell monolayers on 24-well plates were Tobramycin infected in triplicate wells with S. flexneri wild-type strain 2457T, 2457Tsen and plasmidless strain M4243A at an MOI of ∼100 for 90 min. Extracellular organisms were killed with gentamicin (100 μg mL−1) for 30 min (0-h time point), washed three times with PBS and then incubated with gentamicin (100 μg mL−1) for an additional 4 h. The monolayers were then washed with PBS and lysed by addition of 1% Triton X-100 to liberate the intracellular bacteria. Serial dilutions of the lysates were plated on LB agar plates. The intracellular dissemination of bacteria was evaluated using the plaque-formation assay as described (Oaks et al., 1985). Confluent HEp-2 cells on six-well plates were infected with S. flexneri 2457T, 2457Tsen and M4243A strains at an MOI of ∼100 for 90 min. After washing with PBS, medium containing 10% FBS, gentamicin (100 μg mL−1) and 0.