Electroporation of Functional Bacterial Effectors into Mammalian Cells
| Autor: | Cosmin Mihai, Joshua N. Adkins, Roslyn N. Brown, Tatiana Skarina, Hong Cui, John R. Cort, Ryan L. Sontag, Alexei Savchenko, Galya Orr |
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| Rok vydání: | 2015 |
| Předmět: |
Salmonella typhimurium
Virulence Factors General Chemical Engineering Immunology Context (language use) Biology Transfection General Biochemistry Genetics and Molecular Biology Protein–protein interaction Mice Transduction (genetics) Bacterial Proteins Mutant protein Animals Humans General Immunology and Microbiology Effector General Neuroscience Electroporation Molecular biology Recombinant Proteins Transport protein Cell biology Protein Transport RAW 264.7 Cells Microscopy Fluorescence Host-Pathogen Interactions HeLa Cells |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins. |
| Databáze: | OpenAIRE |
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