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.
Many Gram-negative bacteria employ specialized secretion systems to inject virulence-related proteins (referred to as effectors) directly into host cells 1-5. These effectors have a wide range of biological functions including: suppression of host immunity, cytoskeletal changes, modification of intracellular trafficking and signaling, transcriptional changes, and host proteasome alterations 6-9. The functions of some effectors are known, however the host targets and biochemical action(s) of many others remains to be determined. While comparing wild-type and recombinant bacterial infections is a valid approach to study intracellular effector virulence mechanisms, it is often advantageous to introduce an individual effector into the host cell. Thus, simple methods for introducing and characterizing bacterial effector proteins in the context of host cells is highly desirable.
Simplifying the experimental analysis with a single effector is critical as other effectors may have opposing or redundant functions. To accomplish this simplification, researchers have previously introduced macromolecules into cells by many different methods, including viral transduction 10, microinjection 11, scrape loading 12,13, cell fusion with chemically induced microinjection 14, proprietary protein “transfection” reagents 15, calcium phosphate precipitations 16, and electroporation 17-20. The introduced molecules range from nucleic acids including DNA, RNA, & RNAi species to proteins, cell-impermeable dyes, and antibodies for intracellular targets 21,22. Some methods have limitations including the type of macromolecule that can be introduced, and particular downstream analyses may be limited due to high cellular toxicity, damaging mechanisms of action, low efficacy, or introduction efficiency. Transfection, an often-used method for expressing bacterial genes in mammalian cells, also suffers the limitation that some relevant host cell types, such as macrophages and primary cells, are particularly resistant towards transfection. Beyond this, it is difficult to control the levels of bacterial protein produced upon introduction of foreign DNA.
Much work has established electroporation of nucleic acids into both bacterial and mammalian cells as a common laboratory technique; however, there is ongoing research into the best methods for delivering proteins into cells under physiological conditions. Reports on protein transfection are promising, but require expensive reagents and optimization. The desire to introduce potentially toxic bacterial effectors into a wide variety of cell targets with minimal cost led us to consider electroporation as a method for studying these proteins in vivo.
Protein electroporation 23-25 is a method to introduce proteins into living cells via electropermeabilization, also known as electro-transfection or electro-injection 26. This technique uses high-intensity electrical pulses to create pores in cell membranes. These reversible pores allow macromolecules that are normally excluded from intracellular space to enter the cell. Upon removal of the external electric field, the membrane can reseal, allowing the cell to retain molecules that passed through the pores 27,28.
A standard electroporator was used in this study to consistently introduce bacterial effectors into both mouse mac