FIG. 4.

SseL is secreted by the SPI2 type III secretion system and translocated into the host cytoplasm during infection. (A) Western blot analysis of bacterial cell lysates (pellet, right panel) and secreted proteins (left panel) from wild type (wt) and ssaR and ssaL mutants (7) grown in LPM medium. Protein fractions were probed for DnaK, SseL-HA, and SseB. The positions of molecular mass standards (in kilodaltons) are indicated to the left of the panel. (B) Fractionation of host cells following infection reveals translocation and retention of SseL in the host cell cytoplasm, dependent on SPI2 T3SS activity. HeLa cells were infected with either wild-type Salmonella or a strain defective in SPI2-mediated type III secretion (ΔssaR), each expressing SseL-HA. Following infection, host cells were biochemically fractionated into pellet, membrane, and cytosol fractions and probed using antibodies to HA, DnaK (bacterial cytosolic protein), tubulin (host cell cytosolic protein), and calnexin (a host cell membrane protein).