Translocation of SrfN and PagK1/PagK2/PagJ independent of SPI-1/2 T3SSs and flagella system. Genes of invA, ssaK, and flgB were deleted individually or in combination to block SPI-1 T3SS (A), SPI-2 T3SS (B), flagella system (C), or all three secretion machineries (D), respectively in Salmonella strains expressing CyaA'-tagged SrfN, or PagK1/PagK2/PagJ. Macrophage cells infected with Salmonella were lysed at 18 h post-infection to examine translocation and expression levels of SrfN and PagJ/K1/K2. Translocation of CyaA' fusions into the macrophage cytosol was measured using cAMP assay (top graphic figures) and the intracellular expression levels of CyaA'-tagged proteins were compared in parallel using Western blot analysis (bottom gel figures). DnaK levels were measured to normalize protein amounts loaded between lanes in Western blotting. Salmonella without SPI-2 T3SS was attenuated in intracellular growth and exhibited low DnaK levels compared with wild-type Salmonella.