Name: a bacterial infection assay for studying anaerobic bacteria-host cell interactions
Uploaded: 2023-11-30T12:47:41.000+00:00
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Description: The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...

a bacterial infection assay for studying anaerobic bacteria-host cell interactions

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

For culture and experimentation with Porphyromonas gingivalis, a vinyl anaerobic chamber is maintained with an artificial atmosphere containing 80% nitrogen, 10% hydrogen, and 10% carbon dioxide.
Place 30 blood agar plates, 500 milliliters of PBS, and milliliters of vascular endothelial growth factor, or VEGF medium, inside the anaerobic chamber 24 hours in advance to equilibrate the reagents.
Seed 4 times 10 to the 5 human umbilical vein endothelial cells, or HUVECs, per well in six-well plates in VEGF medium and leave them in the cell culture incubator overnight for attachment. Prepare Porphyromonas gingivalis cultures in brain-heart infusion, or BHI medium, as described in the accompanying text, and allow the cultures to reach log phase while the optical density of the culture at 660 nanometers ranges between 0.5 to 0.7.
Transfer the bacteria to a 15-milliliter tube and wrap a piece of parafilm around the cap to prevent aerobic exposure. Then, centrifuge the bacteria at 5,000 times g for 10 minutes. Inside the anaerobic chamber, wash the pelleted bacteria with anaerobic PBS, and spin down again at 5,000 times g for 10 minutes. Resuspend the bacteria in VEGF medium.
To infect the HUVECs with Porphyromonas gingivalis, transfer the six-well plates from the incubator to the anaerobic chamber. Aspirate the medium from the test wells, and wash three times with anaerobic PBS.
Determine the cell number from one representative well by trypan blue exclusion assay. Then, add 2 milliliters per well of anaerobic VEGF medium and place the plates at 37 degrees Celsius in the anaerobic incubator for 20 minutes to equilibrate.
Determine the optical density of the bacteria and resuspend them in an adequate volume of VEGF medium to infect cells at a multiplicity of infection of 100 to 1 bacteria-to-cells ratio. Add the bacterial suspension to the cells and incubate for 30 minutes at 37 degrees Celsius for infection. For cell lysis, inside the anaerobic chamber, prepare a 1% weight per volume saponin solution in BHI medium, and filter through a 0.2-micron filter.
To study bacterial interactions with host cells, remove the six-well plate from the incubator and aspirate the infection medium. Wash three times with anaerobic PBS. Then, add 2 milliliters of saponin solution to each well and incubate for 15 minutes to allow cell lysis.
Next, scrape the cells from the bottom of the well, and dilute the cell lysate 1 to 1 with BHI medium. Then, make serial dilutions starting at 1 to 100. Plate 200 microliters of a suitable dilution on blood agar plates. Wrap the plates with parafilm, and incubate them in a 37 degrees Celsius anaerobic incubator for seven days to allow colony formation. Then, using a light box, count the colony-forming units, or CFUs on each plate.

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The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols may be used for a number of anaerobic pathogens. Although HUVECs are used, this protocol may be used for other eukaryotic cells both immune and non-immune.
1. Anaerobic Chamber Use and Maintenance
Note: P. gingivalis is an anaerobe sensitive to normal levels of oxygen encountered in ambient air. A controlled anaerobic environment is vital for the cultivation of P. gingivalis.
<ol>
	<li>Here, maintain an artificial atmosphere designated as mixed anaerobic gas (80% N2, 10% H2, 10% CO2) in a vinyl anaerobic chamber (Figure 1A). Use an airlock (Figure 1B) for transferring items from the laboratory environment to the anaerobic chamber. The airlock operates manually, twice purging with N2 gas before introducing the mixed anaerobic gas.</li>
	<li>Use a hydrogen sulfide removal column (Figure 1C) for maintenance-free removal of the undesirable hydrogen sulfide. Place a dehumidifier within the chamber to remove H2O created by the catalyst and to avoid aerosols that facilitate the spread of contamination. 
	Note: Hydrogen sulfide is a natural metabolic byproduct of many anaerobic bacteria and its accumulation is toxic to bacteria and can result in damage to electronics and decrease the lifetime of a catalyst.</li>
	<li>Use a fan box to circulate the chamber&#39;s atmosphere through a palladium catalyst, which removes oxygen in the presence of hydrogen (Figure 1D). 
	Note: A recirculating atmospheric (high-efficiency particulate air or HEPA) filter removes airborne contaminants with a size of 0.22 &#181;m or larger.</li>
	<li>Culture anaerobic bacteria in a 37 &#176;C incubator that is located inside the anaerobic chamber. Use standard aseptic techniques when working inside the anaerobic chamber.</li>
</ol>
2. Preparation of Anaerobic Bacteria
Note: P. gingivalis is aerotolerant and can be stored in aerobic conditions but it will not grow in the presence of oxygen at levels higher than 6%. An anaerobic chamber is necessary for the proper cultivation of P. gingivalis and other anaerobic species (Figure 1). Proper training and education on anaerobic chamber use are required before working with micro anaerobes.
<ol>
	<li>Equilibrate all liquid media and plates to anaerobic conditions for at least 12 hr prior to experimentation to remove residual oxygen.</li>
	<li>Transfer P. gingivalis from a -80 &#176;C freezer to an anaerobic chamber, and let thaw.</li>
	<li>Streak P. gingivalis on trypticase soy blood agar plates (TSA II with 5% sheep blood). Wrap plates in parafilm and store at 37 &#176;C in an anaerobic incubator for 4-7 days.</li>
	<li>Inoculate P. gingivalis into 3 ml brain heart infusion (BHI) broth supplemented with hemin and menadione, an enriched non-selective liquid media for the isolation and culture of anaerobic and fastidious microorganisms, using sterile loops. 
	Note: For long-term storage, mix bacterial cultures prepared in BHI with glycerol or DMSO (10-20% final concentration) and place in a -80 &#176;C freezer.</li>
	<li>Prepare a starter culture of P. gingivalis by making a 1:10 dilution and allowing bacteria to grow until the mid-log phase.</li>
</ol>
Note: The optical density of the bacterial suspension is determined and the bacterial concentration for each strain to be examined is adjusted. For P. gingivalis a suspension at OD660 of 0.7 corresponds to mid-log phase and ~7 x 108 cells/ml. Growth conditions described in the protocol above are specific for P. gingivalis and may need to be adapted for other bacterial strains.
3. Endothelial Cell Culture
Note: Purchase pooled primary HUVECs and culture in basal medium containing vascular endothelial growth factors (VEGF) at 37 &#176;C in 5% CO2 according to manufacturer&#39;s instructions.
<ol>
	<li>Seed HUVECs in T-75 flasks at 2.5 x 105 cells/flask in 15 ml VEGF media. 
	Note: Check viability via a 1:1 dilution with 4.0% trypan blue. Cells with a compromised membrane will retain trypan blue, and healthy cells with intact membranes will appear white when viewed under a binocular light microscope. Count 100 cells, and ensure that over 80% of the cells are viable.</li>
	<li>Replace media every 2 days with pre-warmed fresh VEGF media until cells reach ~80% confluency.</li>
	<li>Wash cells once with pre-warmed phosphate-buffered saline (PBS). Liberate cells from the T75 flask by incubating with 2 ml trypsin-EDTA (0.25%) for 5 min followed by 2 ml trypsin neutralizing solution.</li>
	<li>Collect suspended HUVECs in a 50 ml conical tube. Wash out any extra cells from T-75 flasks with PBS and transfer to 50 ml conical tubes.</li>
	<li>Centrifuge cells at 200 x g for 10 min.</li>
	<li>Remove supernatant, and suspend cell pellet in 10 ml pre-warmed VEGF media.</li>
	<li>Determine cell concentration using a hemocytometer or similar cell counting device.</li>
	<li>Calculate the amount of cell suspension to add to either a 6-well plate (400,000/well) or a 12-well plate with coverslips (50,000/well). HUVECs will be ready for experimentation the next day.</li>
</ol>
4. Survival Assay Invasion/Interaction (Plating)
Note: When performing this assay, prepare two 6-well plates of endothelial cells seeded at 400,000 cells/well. One plate will be used to assess bacteria attached to and internalized by host cells. The other plate will account for intracellular bacteria. The 6-well plate allows for triplicates of two samples to be performed in one experiment. For an outline of this protocol please refer to the survival assay flowchart (Figure 2).
<ol>
	<li>Prepare anaerobic bacteria as described above (see section 1) until they reach mid-log growth (OD660 0.5-0.7).</li>
	<li>Centrifuge bacteria at 5,000 x g for 10 min. 
	Note: If the centrifuge is outside an anaerobic chamber, carry bacterial samples in a tightly sealed 15 ml tube, and wrap the cap with parafilm to prevent oxygen leakage.</li>
	<li>Place pelleted P. gingivalis back in the chamber, and discard supernatant. Wash with PBS, and pellet bacteria again before resuspending in VEGF media. Prepare suspensions for all bacterial strains to be tested at OD660 of 0.7 which corresponds to the mid-log phase (~7 x 108 cells/ml). The bacteria are now ready for infection.</li>
	<li>Transfer 6-well plates containing HUVECs from the tissue culture incubator into the anaerobic chamber. Remove media and wash three times with anaerobic PBS. Add 2 ml of anaerobic VEGF media to each well and place the plates at 37 &#176;C in the anaerobic incubator for 20 min to equilibrate the temperature for infection. 
	Note: Plate bacteria on blood agar plates to ensure the ones used for infection are homogenous and not contaminated upon infection.</li>
	<li>Infect host cells with bacteria at a multiplicity of infection (MOI bacteria: host) of 100:1. 
	Note: HUVEC cell number is determined by performing a trypan exclusion test on a single well before infection. Bacterial cell number is determined via optical density (e.g., OD of 0.5 = 5 x 108 cells/ml). Bacterial concentration is adjusted to proper MOI based on HUVEC&#39;s concentration.</li>
	<li>Place 6-well plates with infected HUVECs into the anaerobic incubator and allow bacteria to interact with host cells for 30 min.</li>
	<li>Prepare saponin in BHI (1.0% w/v) inside the anaerobic chamber and filter through a 0.2 &#181;m filter.</li>
	<li>Survival of both attached and internalized bacteria.
	<ol>
		<li>Remove plates from the incubator, aspirate media, wash three times with anaerobic PBS, and add 2 ml filtered 1.0% saponin (prepared as described in step 4.8). Incubate for 15 min to allow host cell lysis.</li>
		<li>Scrape the bottom of each well with a cell scraper. Collect the cell mixture from each well and make a 1:1 dilution in BHI.</li>
		<li>Proceed to make serial dilutions of the sample. Depending on bacterial species and concentration, adjust serial dilutions. Start with 1:100 or 1:1,000 dilutions.</li>
		<li>Plate 200 &#181;l of desired dilution on blood agar plates. Wrap the plates in parafilm and place them in the anaerobic incubator at 37 &#176;C.</li>
		<li>Following seven days of incubation at 37 &#176;C, remove the plates and count colony forming units (CFUs) using a lightbox to manually count colonies. 
		Note: CFUs are enumerated. For larger quantities of CFUs, images may be taken and computer software can be used to facilitate the enumeration of CFUs.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vinyl Anaerobic Chamber-Type B</td>
			<td>Coy Laboratory Products</td>
			<td></td>
			<td>Model 2000 incubator</td>
		</tr>
		<tr>
			<td>TSA II Trypticase Soy Agar w/5% Sheep Blood</td>
			<td>BBL</td>
			<td>221261</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells 10-donor Pool</td>
			<td>LifeLine Technology</td>
			<td>FC-0044</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF Medium Complete Kit</td>
			<td>LifeLine Technology</td>
			<td>LL-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypKit</td>
			<td>LifeLine</td>
			<td>LL-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Riedel-de Haen</td>
			<td>16109</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

<img alt="Figure 1" src="/files/ftp_upload/21795/21795_Figure1.jpg" /> 
Figure 1. Anaerobic vinyl chamber and its components. (A) A vinyl anaerobic chamber sealed completely from atmospheric oxygen provides workspace for two individuals at a time (32 in x 78 in). It contains an incubator set at 37 &#176;C (back middle). (B) An airlock is used for the transfer of items from the lab environment to the anaerobic chamber. Pictured is an ...

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...

Begin with a culture of endothelial cells.
Replace the media with anaerobic media. Incubate in an anaerobic chamber for temperature equilibration.&#160;
Add Porphyromonas gingivalis, a pathogenic anaerobic bacterium, and incubate briefly.
The bacteria possess proteinaceous projections, known as fimbriae, which bind to specific adhesion molecules on the endothelial cells, facilitating bacterial adherence.
This binding triggers intracellular signaling cascades, resulting in actin cytoskeleton rearrangement and bacterial internalization into phagosomes.
Post-incubation, remove media. Wash with an anaerobic buffer to remove non-adhered extracellular bacteria.
Add a mild detergent to permeabilize the host cell membranes and induce cell lysis, releasing internalized and surface-attached bacteria without causing damage.
Dilute the lysate-containing bacteria with media and prepare serial dilutions.
Plate these dilutions on blood agar plates. Incubate under anaerobic conditions to allow Porphyromonas gingivalis to proliferate and form colonies.
Post-incubation, count the colony-forming units (CFUs) on each plate to quantify surface-attached and internalized bacteria, indicating bacteria-host cell interactions.

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A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

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A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions