Translocation of SteA into RAW264.7 cells.

<p>(A) RAW264.7 cells were infected with <i>Salmonella</i> strains expressing SteA-3xFLAG that were derivatives of the wild-type strain (wt), the <i>prgH</i> mutant (lacking T3SS1), or the <i>ssaV</i> mutant (lacking T3SS2). 2 h, 6 h and 12 h post-infection cells were lysed with 0.1% Triton and submitted to centrifugation. The pellets and the filtered and concentrated supernatants (cytosol) were analyzed by immunoblotting. The membranes were cut in three parts. The top was incubated with anti-GroEL antibodies as a control of contamination of the cytosol fraction with non-secreted bacterial proteins. The middle was incubated with antibodies against the cytosolic protein β-actin to measure the number of eukaryotic cells that was present in each condition. The bottom was incubated with anti-FLAG antibodies to detect the SteA protein that was present in the intracellular bacteria (pellet) and that was translocated into the cytosol of the eukaryotic cells (cytosol). (B) Macrophages RAW264.7 were infected with <i>S. enterica</i> serovar Typhimurium wild-type, <i>prgH</i>, <i>ssaV</i>, or <i>prgH ssaV</i> mutant strains, all of them harboring the plasmid pIZ1886 that expresses full length SteA in fusion with the catalytic domain of CyaA from <i>B. pertussis</i>. To analyze translocation, the level of cAMP was measured 6 h post-infection as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026930#s4" target="_blank">Materials and Methods</a> section. It is expressed as % of the maximum level of cAMP that was measured per intracellular bacterium.