In vitro adhesion and invasion assay

The cell lines and their culture medium used in this study are listed in Table S1. One day before the assays, cells were seeded into 24-well plates at a density of ~10⁵ cells per well. One hour before the infection, cell culture medium was changed into serum-free medium and ~10⁶ CFU Sf301 from mid-exponential phase was added to the cells together with a titration of HD5 (0–8 μM). Bacteria were centrifuged (2000 rpm, 10 min, RT) onto HeLa cells (MOI 10:1, or indicated MOI) to synchronize the infection. For the adhesion assay, after washing, the cells were lysed with 0.1 % Triton/H₂O and the CFU were enumerated after plating. For invasion and proliferation assays, bacteria/HeLa mixtures were incubated for 40 min after centrifugation and then washed, treated with gentamicin-containing (25 μg/ml) medium for another 1 hour (invasion) or 4 hours (proliferation) before being lysed for plating. Adhesion was defined as the total number of HeLa cell-associated bacteria and is shown as the percentage of input. Invasion and proliferation were defined as the total number of intracellular bacteria in cells (extracellular bacteria were killed by gentamicin, a cell-impermeable antibiotic). Average results of three independent experiments are reported as mean ± SD. For pre-treatment experiments, cells or bacteria were pre-incubated with HD5 at the indicated concentrations for 30 min, washed once with DMEM and then mixed to allow infection. Adhesion assays, invasion assays and proliferation assays were performed as stated above. Schematic illustration of the assays is shown in Fig. S2.