Figure 4. Identification of a SopB domain required for its membrane localization.

A. Membrane association of SopB after type III secretion mediated translocation. Henle-407 cells were infected with a S. Typhimurium strain expressing FLAG epitope-tagged wild-type SopB (upper panel) or its mutant SopB^Δub (lower panel) and their presence in the membrane insoluble (P) or soluble (S) fractions of infected cells before and after extraction with NaCl (1 M), Na₂C0₃ (pH11), or Triton X100 was examined by western immunoblot. The star denotes the predicted mobility of unmodified SopB. B. TTSS-mediated secretion, and translocation into host cells of the SopB^Δ288-309 mutant. Whole cell bacterial lysates, bacterial culture supernatants, or TTS-protein translocated fraction of cultured epithelial cells infected with the indicated strains of S. Typhimurium expressing FLAG epitope-tagged SopB^Δ288-309 mutant or wild type SopB (as control) were analyzed by western immunoblot. The star denotes the predicted mobility of unmodified SopB. C. Subcellular localization of the SopB^Δ288-309 mutant. Henle-407 cells, were infected with a S. Typhimurium strain expressing an epitope-tagged SopB^Δ288-309 mutant and examined by immunofluorescence confocal microscopy with an antibody directed to the epitope tag (green) or actin (red). Bar = 10 μM. D. Ability of wild type SopB and the SopB^288-309 mutant to mediate bacterial entry into cultured epithelial cells. Henle-407 cells were infected with a S. Typhimurium ΔsopE ΔsopE2 ΔsopB mutant strain (as control indicated “ΔsopE” in the panel) or a S. Typhimurium ΔsopE ΔsopE2 mutant expressing wild type SopB, or SopB^Δ288-309 (as indicated), and the levels of internalized bacteria were determined as indicated in the Material and Methods. Values are the mean ± standard deviation of three independent experiments and represent the percentage of the original bacterial inoculum that survived the antibiotic treatment due to internalization. E. Ability of wild type SopB and the SopB^288-309 mutant to activate Akt. Henle-407 cells were infected with a S. Typhimurium expressing FLAG epitope tagged wild-type SopB, or the SopB^Δ288-309 mutant, and the levels of phosphorylated (activated) or total Akt 1 hr after infection were examined by western immunoblot analysis. The levels of translocated epitope-tagged SopB, or SopB^Δ288-309 were determined by western immunoblot analysis of the fraction of infected cells containing the translocated protein (upper panel). F. Comparison of the in-vitro phosphoinositide phosphatase activity of purified wild type SopB, the SopB^Δ288-309 mutant, and the phosphatase-inactive SopB^C460S mutant. The phosphatase activity was calculated based on the standard curve (shown in inset) for inorganic phosphate standards using a malachite green chromogenic assay.