Figure 3

Translocation of SrfN and Pag proteins into the macrophage cytosol. A. RAW264.7 cells were infected with Salmonella expressing CyaA'-tagged proteins and intracellular cAMP was measured at 6 and 18 h post-infection. Wild-type Salmonella (14028s, not expressing CyaA'), 14028s transformed with pMJW1791 [41] expressing β-gal-CyaA' (LacZ, an intracellular protein), and 14028s expressing chromosomal SseJ-CyaA' (SseJ, a well-known SPI-2 effector) were used in infection as controls. B. SrfN and PagK (PagK1)/STM2585A (PagK2)/PagJ were labeled with β-lactamase and RAW264.7 cells were infected with wild-type Salmonella and Bla fusion strains for 18 hours. Cells were loaded with CCF4-AM for 2 hours. CCF4-AM cleaved by translocated Bla-tagged proteins changed emission wavelength from 528 nm (green) to 457 nm (blue). Salmonella strains were transformed with pWKS30-Tomato and shown in red. pWKS30-Tomato encodes an episomal Bla lacking a secretion signal and wild-type Salmonella transformed with pWKS30-Tomato was used as a negative control. As a positive control, SseJ, an effector translocated via SPI-2 T3SS, was tagged with Bla and its translocation is shown in Additional file 12, Figure S8.