roscopy without excessive membrane associated aggregates) were obtained at 50 μg/ml for GtgE and thus no greater concentrations were tested.
Figure 4: Protein internalization and lack of co-localization with endosome markers. Consecutive optical slices (z-stacks) obtained by confocal microscopy to visualize if the electroporated protein was internal. RAW cells were electroporated with 50 µg/ml GtgE. (A) shows optical slice 6 of 36, the z-stack (2.1 micrometers) above the bottom of the dish. (B) shows the same field of view but at slice 26 of 36. The intracellular foci extend throughout the cell indicating that the protein is truly intracellular and not aggregated on the surface of the cell. Light blue is the combination of red WGA, green secondary antibody, and blue DAPI. Co-localization assay with markers of endosomes/lysosomes, Rab5, (C) or a lysosome marker, LAMP1 (D). The effector protein has been stained green, the markers (Rab5 or LAMP1) stained red, and the nuclei delineated with DAPI, blue. There is no evidence of co-localization with either LAMP1 or Rab5 indicating that the protein was not phagocytized.
Figure 5: Protein persistence after electroporation. Confocal micrograph showing the persistence of electroporated GtgE over time. 50 µg/ml GtgE was electroporated into HeLa cells. The cells were then fixed and stained with an anti-SBP-tag antibody (green) and the nuclei mask, DAPI (blue). 4 hr (A) was determined to be the minimum amount of time to allow cells to recover and as expected shows maximum intracellular protein. After 24 hr (B) and 96 hr (C), green foci, both intracellular and on the cellular surface, show effector persistence indicating that the protein is not degraded days after the initial treatment. The light blue color in this image is the overlay of blue DAPI and the green antibody staining.
Figure 6: Cell surface protein aggregation and an electroporation phenotype. RAW macrophage-like cells were either incubated (A) or electroporated (B) with 50 µg/ml GtgE and stained with anti-SBP-tag antibody (Green), WGA (Red), and with DAPI (Blue). Both RAW 264.7 and to a lesser degree HeLa cells, tended to aggregate effector on the surface of the cell; all electroporated cells were shown to have increased internal protein (HeLa not shown). Yellow is the overlay of red from WGA and green from target protein. RAW (C) and HeLa (D) cells showing an altered phenotype after incubation with GtgE (shown). Several experiments showed a phenotype consisting of smaller cells with differential DAPI staining and a loss of cytoplasm. Light blue is the overlay from the red WGA, green secondary antibody, and blue DAPI. Confocal microscopy was used to show the target protein accumulating in the nucleus. Since this phenotype of exogenous protein-laden cells appeared after both incubation and electroporation, one could hypothesize this phenotype to be suggestive of apoptosis.
Figure 7: Host protein interactions with electroporated Salmonella SspH1 - HeLa cells. Cells were electroporated at the listed concentrations with SspH1, allowed to recover for 4 hr before performing a modified affinity purification followed by western blot (A) to enrich for and detect the known eukaryotic interacting partner: serine/threonine protein kinase N1 (PKN1). Note that the far right lane (2x EP) includes 2 pooled cuvettes, as the signal from one cuvette blended into background, yet PKN1 was still enriched above the lack of binding seen in the no-bait control. The western blot was run with native HeLa lysate, purified SspH1, and the affinity purification samples before being probed with a monoclonal antibody to PKN1. Target detection was obtained via a horseradish peroxidase conjugated secondary antibody with reactivity against the isotype of the primary antibody. HeLa cells (B) showing co-localization (yellow) between PKN1 (red) and SspH1 (green) via by confocal microscopy after electroporation with 50 µg/ml SspH1. This optical overlap indicates that the effector and the host protein are close enough to physically interact. The fact that the in