YopE Translocation Assay in Yersinia

The YopE translocation assay was performed to examine the YopE secretion ability of Y. pseudotuberculosis strains carrying complementation constructs (pGM930) and pMK-bla (YopE-TEM, [89 (link)] using LiveBLAzer-FRET B/G Loading Kit (Life Technologies, Carlsbad, USA). Strains were pregrown in LB containing 0.1% (w/v) L-arabinose, 1 mM CaCl₂, ampicillin and kanamycin for 2 h at 25°C. The bacteria were then shifted to 37°C, incubated for additional 2 h, washed and adjusted in PBS buffer to an OD₆₀₀ of 1. HEp-2 cells (1.7x10⁴) seeded in μ-Slides (8-well, Ibidi, Gräfelfing, Germany) were infected with Y. pseudotuberculosis strains at MOI of 50 and centrifuged for 5 min (400 g, RT). This was followed by incubation for 60 min at 37°C and three washing steps of the cells with PBS buffer. Then, 200 μL of infection buffer (RPMI, 20 mM HEPES, 0.4% BSA) containing gentamicin (25 μg/mL) were added and HEp-2 cells were stained with CCF4-AM loading dye according to the manufacturer’s protocol. YopE-TEM translocation was visualized using a fluorescence microscope (BZ-9000 Fluorescence Microscope, Keyence, Osaka, Japan).