The plates were incubated for 1 hr at 37°C under 5% CO2. Cell Titer 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA) was added and incubated for a further 1 hr at 37°C under 5% CO2. Absorbance of each well was measured at 490 nm. The data are presented as percent viability to determine the concentration of toxin causing 50% cell death (EC50) as described previously [23]. Vero cells (3 × 107/mL) were treated with PI-PLC (0.5 U/mL; EMD Biosciences, Darmstadt, Germany) for 2 hr at 37°C in PBS and centrifuged

as described previously [24]. Aliquots of cells and supernatants were used for SDS–PAGE and toxin overlay assay. Vero cells were scraped from 25 cm2 flasks 3-MA in vitro with a rubber policeman and harvested by centrifugation at 1000 g for 5 min. After washing, cells were suspended in 1 mL of cold lysis buffer consisting of 10 mM Tris–HCl buffer (pH 7.0) containing 150 mM NaCl, 1% Triton X-114 (Pierce) and 0.1% protease inhibitor cocktail. After allowing them to stand for 1 hr on ice, the detergent-insoluble fractions were separated from the supernatants (the detergent-soluble fractions) by centrifugation at 15,000 g for 15 min, and finally resuspended in 1 mL of PBS. SDS–PAGE was carried

out in 5–20% gradient gels (ATTO, Tokyo, Japan). After electrophoresis, detergent-soluble and -insoluble fractions from Vero cells were blotted onto PVDF membranes. After blotting, the membranes were blocked with Succinyl-CoA 5% skim milk in PBS for 1 hr at room temperature. After washing three times with PBS-0.01% Tween 20, the membranes were incubated for 1 hr at room temperature in the presence of 10 µg/mL wild-type or mutant alpha-toxin BI 6727 molecular weight in 0.5% skim milk. This was followed by washing and incubation for a further 1 hr at room temperature with 5 µg/mL affinity-purified rabbit anti-alpha-toxin

IgG [25] in 0.5% skim milk. The membranes were treated for 30 min at room temperature with goat anti-rabbit IgG (H + L) conjugated with peroxidase (1:3000 dilution; Cappel, West Chester, PA, USA) in 0.5% skim milk. After washing, the membranes were developed in 20 mL of PBS containing 0.05% 3′3-diaminobenzidine (Dojin Laboratories, Kumamoto, Japan) and 0.02% H2O2. Protein concentrations were determined by the method of Bradford [26] with bovine gamma globulin as a standard. To evaluate the roles of the tryptophan-rich region in the C-terminal domain in the cytotoxic effect of alpha-toxin, we constructed several mutant toxins by individually replacing tryptophan and some residues surrounding tryptophan with other amino acids (Table 1). We individually replaced tryptophan (W307, W309, and W311) with phenylalanine (W307F, W309F, and W311F), which is hydrophobic and also has an aromatic side chain. These tryptophans were also replaced with alanine to create loss of an aromatic side chain and substitution by its minimal side chain (W307A, W309A, and W311A).