2.6 | In vitro type 3 secretion assay
EPEC strains were grown overnight in LB supplemented with the appropriate antibiotics in a shaker at 37°C. The cultures were diluted 1:40 into pre-heated DMEM (Biological Industries) and grown statically for 6 hr in a tissue culture incubator (with 5% CO2), to an OD of 0.7 at 600 nm (OD600). The cultures were then centrifuged at 20000 × g  for 5 min to separate the bacterial pellets from the supernatants; the pellets were dissolved in SDS-PAGE sample buffer, and the supernatants were collected and filtered through a 0.22-μm filter (Millipore). The supernatants were then precipitated with 10% (v/v) trichloroacetic acid (TCA) overnight at 4°C to concentrate proteins secreted into the culture medium. The volume of the supernatants was normalized to the bacterial cultures at OD600 to ensure equal loading of the samples. The samples were then centrifuged at 18000 × g for 30 min at 4°C, the precipitates of the secreted proteins were dissolved in SDS-PAGE sample buffer, and the residual TCA was neutralized with saturated Tris. The T3SS activity of C. rodentium was determined similarly to that described for EPEC. For EHEC, we cultured double the amount of EPEC (8 mL cultures instead of 4 mL) due to lower amounts of secreted proteins of EHEC relative to EPEC. T3SS activity of Salmonella was determined as previously described (Kujat Choy et al., 2004).