Salmonella-Based β-Lactamase Translocation Assay

The β-lactamase translocation assay was performed as previously described (18 (link)). Briefly, HeLa cells, seeded in black-walled 96-well plates (BD Biosciences), were infected with Salmonella containing pWSK29-Spec (7 (link)) encoding TEM1-tagged effectors (Table S2) at a multiplicity of infection (MOI) of 100. Infected cells were centrifuged at 500 g for 5 min and incubated for 60 min at 37°C and 5% CO₂. The culture medium was replaced with 100 μl of 3 mM probenecid (Sigma-Aldrich), 20 mM Hepes in HBSS (Gibco), and 20 μl CCF2-AM LiveBLAzer-FRET B/G Loading Kit (Invitrogen) and incubated at room temperature, in the dark until 3 h postinfection. The cells were washed before the fluorescence was measured using a FLUOstar Optima plate reader (BMG Labtech) with an excitation wavelength of 410 nm and emission wavelengths of 450 and 520 nm. Response ratios were calculated by first subtracting the average background fluorescence for both 450 and 520 nm wavelengths from the fluorescence reading for each sample. The ratio of fluorescence at 450 nm to fluorescence at 520 nm for each sample was then divided by the uninfected ratio of fluorescence at these wavelengths.