Electroporation of Functional Bacterial Effectors into Mammalian Cells

Summary

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

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.

Introduction

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 macrophage-like cells and human epithelial cells. The method is quick, efficient, and inexpensive, with no appreciable decrease in cellular viability. The introduced proteins can be visualized via immunofluorescence microscopy or used for functional assays. This has been demonstrated using green fluorescent protein (GFP) as a non-toxic standard, as well as two Salmonella effector proteins, SspH1 and GtgE. We propose protein electroporation as an additional tool in the repertoire for the study of bacterial virulence proteins and their functions in eukaryotic host cells.

Protocol

1. Prepare in Advance

1. Warm sterile phosphate buffered saline (PBS) to 37 °C.
2. Warm Dulbecco’s Modification of Eagle’s Medium (DMEM) and Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 I.U./ml penicillin, and 100 µg/ml streptomycin to 37 °C. Note: These represent Normal Growth Media (NGM) for RAW and HeLa cells respectively.

2. Preparation of Cells

1. Grow RAW 264.7 cells to 70-90% confluency in NGM.
   1. Maintain cells at 37 °C in a humidified 95% air/5% CO₂ atmosphere.
2. Grow HeLa cells to 70-90% confluency in NGM.
   1. Maintain cells at 37 °C in a humidified 95% air/5% CO₂ atmosphere.
3. Before collection, wash cell monolayer once with sterile PBS.
4. Collect pre-confluent cells in a sterile conical tube.
   1. Gently scrape RAW cells with a rubber policeman in PBS, with repeated pipetting to disperse cell aggregations.
   2. Detach HeLa cells with 0.25% trypsin solution until visual examination shows dissociation from culture surface. For example, use 2 to 3 ml for a T-75 flask. Adjust volume accordingly to ensure trypsin solution covers entire growth surface.
      1. Quench dissociation reaction with NGM containing 10% FBS.
      2. Use at least twice the volume of NGM to trypsin, with repeated pipetting to disperse cell aggregations.
5. Lightly pellet cells by centrifugation at 900 x g for 4 min.
6. Resuspend in same volume as trypsin/quench solution using sterile PBS.
7. Count cells using hemocytometer or particle counter.
8. Lightly pellet cells by centrifugation at 900 x g for 4 min.
9. Resuspend in adequate volume of PBS for 5.5 x 10⁶ to 6.0 x 10⁶ cells/ml. Note: For example, one T-75 flask will yield approximately 7.5 x 10⁶ cells ²⁹.
10. Keep cell suspension on ice until electroporation.

3. Preparation for Electroporation

1. Pre-chill electroporation cuvettes (0.4 cm gap) on ice.
2. Turn on electroporation apparatus and set the voltage to 0.3 kV.
   NOTE: Capacitance and resistance were not adjustable settings on our electroporator (set at 10 μF and 600 Ω by the manufacturer).
3. Fill recovery plates with NGM and equilibrate in humidified 95% air/5% CO₂ atmosphere at 37° C.

4. Electroporation

1. Place 400 µl of cell suspension in pre-chilled cuvette and add 20 µg of selected protein to cuvette (50 µg/ml).
2. Flick cuvette gently ~10 times to mix without damaging cells. Note: The cuvette may also be inverted several times to mix thoroughly, but do not pipet up and down or vortex to avoid damaging cells.
3. Dry outside of cuvette with paper towel or other wiper to avoid electrical arcing in the electroporator.
4. Electroporate sample at 0.3 kV for 1.5-1.7 msec. Note: This was typical for this study.
5. Immediately after electroporation flick cuvette gently ~10 times to mix thoroughly.

5. Plating of Cells

1. Store cuvette with electroporated cells on ice until ready to place in recovery plates.
2. For most downstream analyses, wash cells 1x with pre-warmed NGM to remove extraneous effector protein.
   1. Remove cells for analysis and suspend in 3-5 ml of NGM.
   2. Pellet cells by centrifugation at 900 x g for 4 min.
   3. Resuspend in adequate volume of NGM for desired plate size (e.g. 2 ml for 35 mm dish).
3. Remove appropriate amount of cells for downstream analysis.
   1. Example 1: plate into glass bottom dishes for microscopy analysis.
   2. Example 2: plate into cell culture plastic for protein analysis such as affinity purifications.
4. Allow cells to recover in equilibrated plates in humidified 95% air/5% CO₂ atmosphere at 37° C for at least 4 hr.

6. Microscopy Analysis

6.1) Fixation/Immunofluorescence Staining

1. Wash cells 1x with sterile PBS after 4 hr recovery period.
2. Fix cells in 100% methanol for 2 min at room temperature. Use enough methanol to completely cover cells (e.g. 2 ml for 35 mm dish).
3. Wash 3x with sterile PBS.
4. Permeabilize cells with 0.4% Triton X-100 in PBS for 15 min. For example, use 1 ml for a 35 mm diameter plate.
   1. Adjust length of permeabilization and strength of Triton X-100 according to epitope and location of target protein. Note: Best results will need to be empirically determined for each target, however the above conditions should be adequate for most cytosolic targets.
5. Block with 5% Bovine Serum Albumin (BSA) in PBS for 1 hr at RT. For example, use 1 ml for a 35 mm diameter plate.
6. Wash 3x with PBS.
7. Incubate primary antibody in antibody binding solution (0.1% Triton X-100 and 1% BSA in PBS) overnight at 4 °C with gentle rocking. For example, use 0.5 ml for a 35 mm diameter plate.
   1. Follow manufacturer’s recommendations for antibody dilution. Note: For example, the streptavidin-binding peptide tag (SBP-tag) antibody was used at 1:1,000 dilution, while the PKN1 antibody was used at 1:200 dilution.
8. Wash 4x with PBS.
9. Incubate with appropriate fluorescently conjugated secondary antibody in antibody binding solution for 1 hr at room temperature, protected from light.
   1. For example, use Alexa 488 or Alexa 647 for 1 hr at room temperature.
      1. Follow manufacturer’s recommendations for antibody dilution. (e.g. about 1:1,000 for this study).
   2. Add other stains as needed, for example, 5 μM Wheat germ agglutinnin (WGA) conjugated to Alexa 647 or DAPI according to manufacturer’s recommendation, for 1 hr at room temperature.
10. Wash 5x with PBS and store at 4 °C, protected from light until ready to image.

6.2) Confocal Microscopy and Image Analysis

1. Image samples on an inverted confocal microscope using a 63x oil immersion objective.
   1. Image green channel using the 488nm line of an argon laser, with bandwidth emission between 492-542 nm.
   2. Image red channel using a 633 nm diode laser, with bandwidth emission between 640-718 nm.
   3. Image blue channel using a 405 nm diode laser, with bandwidth emission between 407-453 nm.
   4. Ensure the multiple channel z-stacks cover the entirety of the cellular volume.
2. Process images with appropriate image processing software.

7. Affinity Purification

1. Wash electroporated cells twice with 4 °C PBS after 4 hr recovery period.
2. Lyse with ~1.0 ml of lysis buffer (1% Triton X-100 with protease inhibitor cocktail and phosphatase inhibitor in PBS) on ice. Use inhibitors according to manufacturer instructions. Note: For example, both the phosphatase and protease inhibitors used in this study were supplied at 100x, but other formulations should work equally well.
3. Promptly scrape cells with rubber policeman and collect into conical tubes.
4. Lyse by vigorous vortexing and sonication. Note: Other lysis methods may work equally as well, but efficiency will need to be empirically determined as to the suitability of assay requirements.
   1. Sonicate 3 x 30 sec with intermittent vortexing.
5. Centrifuge at 10,000 x g for 10 min at 4 °C to collect cellular debris and insoluble aggregates; save the supernatant.
6. Combine equal volumes (~1.0 ml) of electroporated cell lysate with 50 µl of Streptavidin agarose resin suspension at 4 °C overnight with end-over-end rotation. Note: This resin captures the electroporated protein (and associated complexes) via its streptavidin-binding peptide affinity tag.
7. Centrifuge at 2,500 x g for 2 min and discard supernatant.
8. Wash twice with 40 bed volumes (~1 ml) PBS.
9. Add 30 µl 4x LDS loading buffer (141 mM Tris base, 2% LDS, 10% glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G, 0.175 mM phenol red, pH 8.5), 20 µl diH₂O, and 1 µl of 0.5 M tris(2-carboxyethyl)phosphine (TCEP), allowing for some volume to remain in beads.
10. Heat at 95 °C for 10 min and cool on ice.
11. Spin >10,000 x g at 4 °C for 5 min.
12. Collect supernatant.
13. Perform a western blot using appropriate antibodies Note: Here, anti-PKN1 primary antibody is used.

Results

As an initial proof of concept, purified green fluorescent protein was successfully introduced into mammalian cells using electroporation. GFP, an approximate 27 kD a molecular weight protein is commonly introduced into mammalian cells (normally expressed from plasmid DNA) as a molecular biology tool without significant cellular toxicity. HeLa cells were incubated (Figure 1A) or electroporated (Figure 1B) with 25 µg/ml GFP, followed by immunofluorescence confocal microscopy to check...

Discussion

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 asso...

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA Solution	Cellgro	25-050-Cl
0.4 cm Gap-disposable electroporation cuvettes	Bio-Rad	165-2088
100% Methanol	Any	N/A	Flammable, Toxic
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4919
Cell Counting Apparatus - Hemocytometer or Coulter Counter	Beckman Coulter	Model Z1
Cell Culture Incubator	Any	N/A	Humidified 95% air/5% CO2 atmosphere at 37 °C
Cell Culture Plastic	Any	N/A	Cell culture flasks/plates, pipets, tubes, rubber policeman
Dulbecco's Modification of Eagle’s Medium (DMEM)	Cellgro	10-013	Warm to 37 °C
Electroporator	Bio-Rad	E. coli Pulser
Fetal Bovine Serum (FBS)	Cellgro	35-016-CV
Fluorescent confocal microscope	Ziess	Model  LSM 710
Glass Bottom Dishes for Microscopy	Wilco Wells	HBSt-3522
HALT Protease Inhibitor Cocktail	Pierce	78430	Corrosive, Toxic
HeLa Cell Line	ATCC	ATCC CCL-2
LDS 4X Loading Buffer	Invitrogen	NP0007
Minimal Essential Medium (MEM)	Cellgro	10-010	Warm to 37 °C
Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent	Any	N/A
Penicillin/Streptomycin	Cellgro	30-002-Cl
RAW 264.7 Cell line	ATCC	TIB-71
Primary Antibody Against Target of Interest	Any	N/A
Secondary Antibody Conjugated to Fluorophore	Any	N/A
Phosphate Buffered Saline	Any	N/A	Chill to 4 °C
Sterile Phosphate Buffered Saline	Any	N/A	Warm to 37 °C
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Streptavidin Agarose Resin Suspension	Pierce	20353
Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)	Any	N/A
TCEP	Sigma-Aldrich	646547	Corrosive, Toxic
Triton X-100	Sigma-Aldrich	T8585	Irritant, Toxic