Gentamicin protection assay

The assay was performed as described before [20]. Briefly, RAW264.7 cells were seeded 24 h or 48 h prior infection in a surface-treated 24-well plate (TPP) to reach confluency (~4 x 10⁵ cells per well) on the day of infection. U937 cells were seeded 72 h prior infection in surface-treated 24-well plates (TPP) to reach confluency (~4 x 10⁵ cells per well) on the day of infection and were treated with 50 ng x μl^-1 phorbol 12-myristate 13-acetate (PMA) for differentiation and cell attachment. HeLa cells were seeded 48 h prior infection in surface-treated 24-well plates (TPP) to reach confluency (~2 x 10⁵ cells per well) on the day of infection. For infection of RAW264.7 and U937 cells, bacteria were grown overnight (~ 20 h) aerobically in LB medium. For infection of HeLa cells, fresh LB medium was inoculated 1:31 with overnight cultures of STM and incubated for 3.5 h with agitation. For subculture of STY and SPA, 10 ml fresh LB medium was inoculated 1:100 with aerobic over day cultures and grown static under microaerophilic conditions for 16 h [51]. Then the bacteria were adjusted to an OD₆₀₀ of 0.2 in PBS and further diluted in DMEM (RAW and HeLa cells) or RPMI medium (U937 cells) for infection of cells at MOI of 1. Bacteria were centrifuged onto the cells for 5 min at 500 x g, and the infection was allowed to proceed for 25 min. After three washing steps with PBS, medium containing 100 μg x ml^-1 gentamicin was added for 1 h to kill extracellular bacteria. Afterwards the cells were incubated with medium containing 10 μg x ml^-1 gentamicin for the ongoing experiment. Cells were washed three times with PBS and lysed using 0.1% Triton X-100 at 2 h and 24 h post infection (p.i.). Colony forming units (CFU) were determined by plating serial dilutions of lysates and inoculum on Mueller-Hinton II agar and incubated overnight at 37°C. The percentage of phagocytosed bacteria as well as the replication rate was calculated.