This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1. Prepare in Advance
<ol>
	<li>Warm sterile phosphate buffered saline (PBS) to 37 &#176;C.</li>
	<li>Warm Dulbecco&#8217;s Modification of Eagle&#8217;s Medium (DMEM) and Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 I.U./ml penicillin, and 100 &#181;g/ml streptomycin to 37 &#176;C. Note: These represent Normal Growth Media (NGM) for RAW and HeLa cells respectively.</li>
</ol>
2. Preparation of Cells
<ol>
	<li>Grow RAW 264.7 cells to 70-90% confluency in NGM.
	<ol>
		<li>Maintain cells at 37 &#176;C in a humidified 95% air/5% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>Grow HeLa cells to 70-90% confluency in NGM.
	<ol>
		<li>Maintain cells at 37 &#176;C in a humidified 95% air/5% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>Before collection, wash cell monolayer once with sterile PBS.</li>
	<li>Collect pre-confluent cells in a sterile conical tube.
	<ol>
		<li>Gently scrape RAW cells with a rubber policeman in PBS, with repeated pipetting to disperse cell aggregations.</li>
		<li>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.
		<ol>
			<li>Quench dissociation reaction with NGM containing 10% FBS.</li>
			<li>Use at least twice the volume of NGM to trypsin, with repeated pipetting to disperse cell aggregations.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Lightly pellet cells by centrifugation at 900 x g for 4 min.</li>
	<li>Resuspend in same volume as trypsin/quench solution using sterile PBS.</li>
	<li>Count cells using hemocytometer or particle counter.</li>
	<li>Lightly pellet cells by centrifugation at 900 x g for 4 min.</li>
	<li>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.</li>
	<li>Keep cell suspension on ice until electroporation.</li>
</ol>
3. Preparation for Electroporation
<ol>
	<li>Pre-chill electroporation cuvettes (0.4 cm gap) on ice.</li>
	<li>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 &#956;F and 600 &#937; by the manufacturer).</li>
	<li>Fill recovery plates with NGM and equilibrate in humidified 95% air/5% CO2 atmosphere at 37&#176; C.</li>
</ol>
4. Electroporation
<ol>
	<li>Place 400 &#181;l of cell suspension in pre-chilled cuvette and add 20 &#181;g of selected protein to cuvette (50 &#181;g/ml).</li>
	<li>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.</li>
	<li>Dry outside of cuvette with paper towel or other wiper to avoid electrical arcing in the electroporator.</li>
	<li>Electroporate sample at 0.3 kV for 1.5-1.7 msec. Note: This was typical for this study.</li>
	<li>Immediately after electroporation flick cuvette gently ~10 times to mix thoroughly.</li>
</ol>
5. Plating of Cells
<ol>
	<li>Store cuvette with electroporated cells on ice until ready to place in recovery plates.</li>
	<li>For most downstream analyses, wash cells 1x with pre-warmed NGM to remove extraneous effector protein.
	<ol>
		<li>Remove cells for analysis and suspend in 3-5 ml of NGM.</li>
		<li>Pellet cells by centrifugation at 900 x g for 4 min.</li>
		<li>Resuspend in adequate volume of NGM for desired plate size (e.g. 2 ml for 35 mm dish).</li>
	</ol>
	</li>
	<li>Remove appropriate amount of cells for downstream analysis.
	<ol>
		<li>Example 1: plate into glass bottom dishes for microscopy analysis.</li>
		<li>Example 2: plate into cell culture plastic for protein analysis such as affinity purifications.</li>
	</ol>
	</li>
	<li>Allow cells to recover in equilibrated plates in humidified 95% air/5% CO2 atmosphere at 37&#176; C for at least 4 hr.</li>
</ol>
6. Microscopy Analysis
6.1) Fixation/Immunofluorescence Staining
<ol>
	<li>Wash cells 1x with sterile PBS after 4 hr&#160;recovery period.</li>
	<li>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).</li>
	<li>Wash 3x with sterile PBS.</li>
	<li>Permeabilize cells with 0.4% Triton X-100 in PBS for 15 min. For example, use 1 ml for a 35 mm diameter plate.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Block with 5% Bovine Serum Albumin (BSA) in PBS for 1 hr&#160;at RT. For example, use 1 ml for a 35 mm diameter plate.</li>
	<li>Wash 3x with PBS.</li>
	<li>Incubate primary antibody in antibody binding solution (0.1% Triton X-100 and 1% BSA in PBS) overnight at 4&#160;&#176;C with gentle rocking. For example, use 0.5 ml for a 35 mm diameter plate.
	<ol>
		<li>Follow manufacturer&#8217;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.</li>
	</ol>
	</li>
	<li>Wash 4x with PBS.</li>
	<li>Incubate with appropriate fluorescently conjugated secondary antibody in antibody binding solution for 1 hr&#160;at room temperature, protected from light.
	<ol>
		<li>For example, use Alexa 488 or Alexa 647 for 1 hr&#160;at room temperature.
		<ol>
			<li>Follow manufacturer&#8217;s recommendations for antibody dilution. (e.g. about 1:1,000 for this study).</li>
		</ol>
		</li>
		<li>Add other stains as needed, for example, 5 &#956;M Wheat germ agglutinnin (WGA) conjugated to Alexa 647 or DAPI according to manufacturer&#8217;s recommendation, for 1 hr&#160;at room temperature.</li>
	</ol>
	</li>
	<li>Wash 5x with PBS and store at 4 &#176;C, protected from light until ready to image.</li>
</ol>
6.2) Confocal Microscopy and Image Analysis
<ol>
	<li>Image samples on an inverted confocal microscope using a 63x oil immersion objective.
	<ol>
		<li>Image green channel using the 488nm line of an argon laser, with bandwidth emission between 492-542 nm.</li>
		<li>Image red channel using a 633 nm diode laser, with bandwidth emission between 640-718 nm.</li>
		<li>Image blue channel using a 405 nm diode laser, with bandwidth emission between 407-453 nm.</li>
		<li>Ensure the multiple channel z-stacks cover the entirety of the cellular volume.</li>
	</ol>
	</li>
	<li>Process images with appropriate image processing software.</li>
</ol>
7. Affinity Purification
<ol>
	<li>Wash electroporated cells twice with 4 &#176;C PBS after 4 hr&#160;recovery period.</li>
	<li>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.</li>
	<li>Promptly scrape cells with rubber policeman and collect into conical tubes.</li>
	<li>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.
	<ol>
		<li>Sonicate 3 x 30 sec with intermittent vortexing.</li>
	</ol>
	</li>
	<li>Centrifuge at 10,000 x g for 10 min at 4 &#176;C to collect cellular debris and insoluble aggregates; save the supernatant.</li>
	<li>Combine equal volumes (~1.0 ml) of electroporated cell lysate with 50 &#181;l of Streptavidin agarose resin suspension at 4 &#176;C overnight with end-over-end rotation. Note: This resin captures the electroporated protein (and associated complexes) via its streptavidin-binding peptide affinity tag.</li>
	<li>Centrifuge at 2,500 x g for 2 min and discard supernatant.</li>
	<li>Wash twice with 40 bed volumes (~1 ml) PBS.</li>
	<li>Add 30 &#181;l 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 &#181;l diH2O, and 1 &#181;l of 0.5 M tris(2-carboxyethyl)phosphine (TCEP), allowing for some volume to remain in beads.</li>
	<li>Heat at 95 &#176;C for 10 min and cool on ice.</li>
	<li>Spin &#62;10,000 x g at 4 &#176;C for 5 min.</li>
	<li>Collect supernatant.</li>
	<li>Perform a western blot using appropriate antibodies Note: Here, anti-PKN1 primary antibody is used.</li>
</ol>

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The authors have nothing to disclose.

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...

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 &#8220;transfection&#8221; reagents 15, calcium phosphate precipitations 16, and electroporation 17-20. The introduced molecules range from nucleic acids including DNA, RNA, &#38; 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.

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			<td height="21" style="height:21px;width:532px;">Material or Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:315px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA Solution</td>
			<td>Cellgro</td>
			<td>25-050-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4cm Gap-disposable electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Methanol</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Flammable, Toxic</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Cell Counting Apparatus - Hemocytometer or Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>Model Z1</td>
			<td></td>
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			<td height="25" style="height:25px;">Cell Culture Incubator</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Humidified 95% air/5% CO2 atmosphere at 37 &#176;C&#160;</td>
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			<td height="20" style="height:20px;">Cell Culture Plastic</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Cell culture flasks/plates, pipets, tubes, rubber policeman</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modification of Eagle&#8217;s Medium (DMEM)</td>
			<td>Cellgro</td>
			<td>10-013</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator</td>
			<td>Bio-Rad</td>
			<td>E. coli Pulser</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-016-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent confocal microscope&#160;</td>
			<td>Ziess</td>
			<td>Model&#160; LSM 710&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Dishes for Microscopy</td>
			<td>Wilco Wells</td>
			<td>HBSt-3522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HALT Protease Inhibitor Cocktail</td>
			<td>Pierce</td>
			<td align="right">78430</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HeLa Cell Line</td>
			<td>ATCC</td>
			<td>ATCC CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDS 4X Loading Buffer</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimal Essential Medium (MEM)&#160;</td>
			<td>Cellgro</td>
			<td>10-010</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 Cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primary Antibody Against Target of Interest</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Chill to 4 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
			<td align="right">20353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
		</tr>
	</tbody>
</table>

electroporation of functional bacterial effectors into mammalian cells

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.

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 &#181;g/ml GFP, followed by immunofluorescence confocal microscopy to check...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

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.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K Views.  Pacific Northwest National Laboratory. 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. 

Video: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...