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 interaction was shown in the nucleus for the first time, offers additional support that the protein was trafficked correctly by the host.
Secreted effectors from pathogenic bacteria have evolved to function within the host cell environment and thus it is helpful to study them in situ within the host. Introduction of specific effectors of interest into host cells allows the relevant pathogen-host interactions to be studied in isolation without interference from other bacterial proteins. The goal was to explore electroporation as a means to introduce bacterial effector proteins into eukaryotic host cells, thereby avoiding some of the challenges associated with transfection or transduction. Green fluorescent protein was used as a control and Salmonella effector proteins were tested. Because of interest in bacterial pathogens that target host epithelial cells as well as immune cells, a human epithelial-like cell line (HeLa) and mouse macrophage-like cells (RAW 264.7) were used. Electroporation can deliver exogenous proteins into host cells with no appreciable decrease in cell viability, and the delivered proteins were detectable in cells for up to four days.
To isolate the effects of electroporated protein, pure protein is required. In this study, proteins were expressed in E. coli strain BL21 with N-terminal polyhistidine and streptavidin binding-peptide tags. Proteins were purified with Ni-NTA resin, assayed for concentration (generally between 5-25 mg/ml) and purity, and stored at -80 °C until electroporation. Commercially produced proteins would also be acceptable for electroporation, as they tend to be of high purity and well characterized.
Key steps in this protocol included completely removing the cell culture medium by washing and suspending cells in protein-free buffer. This ensures that any downstream phenotypes observed are due to the exogenous protein of interest and not to proteins present in the culture medium. Better efficiency of electroporation was observed when cell density was higher (e.g. 6 x 106 vs