NDLI: Electroporation of functional bacterial effectors into mammalian cells.

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Electroporation of functional bacterial effectors into mammalian cells.

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Electroporation of functional bacterial effectors into mammalian cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Sontag, Ryan L. Mihai, Cosmin Orr, Galya Savchenko, Alexei Skarina, Tatiana Cui, Hong Cort, John R. Adkins, Joshua N. Brown, Roslyn N.
Description	Author Affiliation: Sontag RL ( Biological Sciences Division, Pacific Northwest National Laboratory.); Mihai C ( Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory.); Orr G ( Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory.); Savchenko A ( Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto.); Skarina T ( Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto.); Cui H ( Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto.); Cort JR ( Biological Sciences Division, Pacific Northwest National Laboratory.); Adkins JN ( Biological Sciences Division, Pacific Northwest National Laboratory); Brown RN ( Center for Bioproducts and Bioenergy, Washington State University)
Abstract	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.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/52296
Journal	Journal of Visualized Experiments
Issue Number	95
Language	English
Publisher	MyJove Corp.
Publisher Date	2015-01-19
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Bacterial Proteins Genetics Metabolism Electroporation Virulence Factors Animals Biosynthesis Hela Cells Host-pathogen Interactions Physiology Mice Microscopy, Fluorescence Protein Transport Raw 264.7 Cells Recombinant Proteins Salmonella Typhimurium Transfection Research Support, N.i.h., Extramural Research Support, U.s. Gov't, Non-p.h.s. Video-audio Media
Content Type	Text
Resource Type	Article

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