Name: in vitro and in vivo model to study bacterial adhesion to the vessel wall under flow conditions
Uploaded: 2015-06-11T15:00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions at JoVE.com

This work was supported by the Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen G0466.10, 11I0113N; &#8220;Eddy Merckx Research Grant&#8221; and the &#8220;Sporta research Grant&#8221; for Pediatric Cardiology, UZ Leuven, Belgium (J.C.); the Center for Molecular and Vascular Biology is supported by the Programmafinanciering KU Leuven (PF/10/014), by the &#8220;Geconcentreerde Onderzoeksacties&#8221; (GOA 2009/13) from the University of Leuven and a research grant from Boehringer-Ingelheim.

Animal experiments were approved by the Ethical Committee of the KU Leuven.
1. Preparing Bacteria for&#160;In Vitro Perfusions and In Vivo Experiments
<ol>
	<li>We used S. aureus strain Newman for all experiments described in this manuscript. S. aureus Newman was stored in Brain Heart Infusion (BHI) with 10% glycerol at -80 &#176;C.</li>
	<li>Use a sterile loop to scrape the frozen bacteria off and inoculate in 5 ml Tryptic Soy Broth (TSB) O/N at 37 &#176;C (OD600 &#62;3).</li>
	<li>Wash the bacteria by centrifugation (2,600 g, RT, 5 min) and resuspend the bacterial pellet in 5 ml PBS (phosphate buffered saline).</li>
	<li>Prepare a 1 mg/ml solution of 5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester (carboxy-fluorescein) in ethanol. Dilute the 1 mg/ml carboxy-fluorescein solution to 150 &#181;g/ml in laboratory grade water (e.g., MilliQ water). Protect the tubes from light with aluminum foil and store at -20 &#176;C.</li>
	<li>Centrifuge the bacteria (2,600 x g, RT, 5 min). Resuspend the bacterial pellet in 800 &#181;l PBS and add 200 &#181;l (final concentration of 30 &#181;g/ml for perfusion experiments) or 400 &#181;l (final concentration 50 &#181;g/ml for in vivo experiments) of the 150 &#181;g/ml carboxy-fluorescein solution. Protect the tubes from light with aluminum foil and incubate for 30 min at RT on a shaker.</li>
	<li>After labeling, block with 6% bovine serum albumin (BSA) solution in PBS.</li>
	<li>Dilute bacteria using optical densitometry (OD), an OD600 of 0.65 for in vitro experiments (corresponding to approximately 3 x 108 colony forming units (CFU)/ml for S. aureus) and an OD600 of 1.8 for in vivo experiments (corresponding to approximately 1 x 109 CFU/ml for S. aureus) in PBS. Protect the tubes from light with aluminum foil and leave on ice.</li>
</ol>
2. In Vitro Perfusion Experiments
<ol>
	<li>Coating of Glass Coverslips
	<ol>
		<li>Dilute von Willebrand factor (VWF) (Haemate P, stock concentration 2,400 &#181;g/ml) in laboratory grade water (deionized distilled) to a final concentration of 50 &#181;g/ml.</li>
		<li>Dilute collagen in isotonic glucose solution (SKF solution, pH 2.7-2.9, as supplied by the manufacturer) to a final concentration of 160 &#181;g/ml.</li>
		<li>Coat glass coverslips (24 &#215; 50 mm) with VWF or collagen by dropping 200 &#181;l of the coating on parafilm and place the coverslip on top of the droplet. The droplet will spread along the surface of the coverslip.</li>
		<li>Incubate the coverslip in a humidified container for 4 hr at RT. Carefully lift the coverslips from the parafilm with a blunt needle. Mount the coverslip in the bottom part of the flow chamber.</li>
	</ol>
	</li>
	<li>Coating of Plastic Slips with Endothelial Cells
	<ol>
		<li>Coat plastic slips (1-well PCA cell culture chamber, Sarstedt, Germany) with 1 ml of a 1% gelatin solution in PBS and incubate for 30 min at 37&#176;C. Seed human umbilical vein endothelial cells (HUVECs) on the gelatin coated plastic slips and grow them to 70-80% confluency. Mount the plastic slip in the bottom part of the flow chamber.</li>
	</ol>
	</li>
	<li>Perfusion Experiment 
	NOTE: A schematic overview of the in vitro perfusion model is represented in Figure 1.
	<ol>
		<li>Perform in vitro bacterial adhesion studies in a micro-parallel plate flow chamber at a laminar shear stress between 2.5 dyne/cm2 and 20 dyne/cm2 to simulate different physiological flow conditions.</li>
		<li>The flow chamber (in-house design) consists of a metal frame and a perfusion chamber made out of plexiglas (poly(methyl) methacrylate (PMMA)). By connecting it to a high-accuracy infusion pump (PHD 2000 Infusion, Harvard Apparatus, USA), we can generate flow rates between 0.0001 &#181;l/min and 220.82 ml/min.</li>
		<li>Connect the tubing to the upper part of the flow chamber and inject medium in the tubing. Gently place the upper part of the flow chamber on top of the bottom part and assemble the flow chamber. Be careful to avoid air bubbles. Inject 1 ml of medium through the chamber to make sure that the chamber is not leaking and to remove excess coating solution. Avoid air bubbles.</li>
		<li>Place the mouse on a thermo-controlled heating pad at 37 &#176;C on a microscope tray. Since this is a terminal procedure, there is no need for strict asceptic procedures. Make an incision near the jugular vein, gently remove the right side of the cervical muscle and isolate the jugular vein from the surrounding tissue.
		</li>
		<li>Set up the infusion pump and fluorescence microscope. Infusion pump settings depend on the syringe diameter and the desired flow rate (See section 2.4). From now on, work in a dark room.</li>
		<li>VWF Coating:
		<ol>
			<li>Fill a syringe with fluorescently labeled bacteria and connect it to the inlet tube. Avoid air bubbles. Start the infusion pump for 10 min. The infusion time depends on the shear rate and the coating, bacteria and medium used and should represent the steady state of adhesion.</li>
			<li>After 10 min, wash away unbound bacteria by connecting a syringe with PBS to the inlet tube and starting the infusion pump.</li>
			<li>Take at least 15 images or movies at different locations after the wash process. Bacteria are small and potentially difficult to focus on. Prior to the in vitro flow experiment, the appropriate focal plane can be retrieved by placing a drop of fluorescently labeled bacteria on a coverslip and placing the coverslip in the flow chamber. Then, search for the appropriate focal plane and save the settings. 
			NOTE: During the in vitro flow experiment, capturing the images during the washing step (&#177; 5 min after start) ensures that only the signal for adherent bacteria is captured.</li>
		</ol>
		</li>
		<li>Collagen Coating:
		<ol>
			<li>Add 60 &#181;g/ml VWF to the fluorescently labeled bacteria just before starting the perfusion. Fill a syringe with fluorescently labeled bacteria or fluorescently labeled bacteria supplemented with 60 &#181;g/ml VWF and connect it to the inlet tube. Avoid air bubbles. Repeat steps 2.3.5.2 to 2.3.5.3</li>
		</ol>
		</li>
		<li>Endothelial Cells:
		<ol>
			<li>Activate the endothelial cells by perfusion with a 0.1 mM solution of the Ca2+-ionophore A23187 (stock solution 10 mM dissolved in dimethyl sulfoxide (DMSO)) in DMEM at the same shear rate as the bacterial perfusion for 10 min by perfusion with a 0.1 mM. Repeat steps 2.3.5.2 to 2.3.5.3.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calculate shear rate and shear stress as follows. 
	Shear rate = 6Q/wh2 
	Where: Q: flow rate in ml/min, w: width in cm, h: height in cm 
	Shear stress (&#964;) = shear rate x viscosity (&#181;) 
	Where &#181;: medium: 0.01 dynes x sec/cm2 , whole blood: 0.04 dynes x sec/cm2 
	&#160;</li>
	<li>Image analysis
	<ol>
		<li>Obtain live images using an inverted fluorescence microscope with a black and white camera and develop using imaging software. Use exposure time of 1.5 sec. Take multiple snapshots (at least 15) randomly spread over the coated surface of the flow chamber and save them in the appropriate file format.</li>
		<li>Perform image analysis with ImageJ. Subtract the background to remove smooth continuous backgrounds from the image (Process &#8211; Substract Background) and define the threshold to set lower and upper threshold values, segmenting gray scale images into features of interest. Measure the area limited to the threshold.</li>
		<li>Compare bacterial adhesion, expressed as fluorescent area, e.g., using statistical analysis software. Compare the groups using one-way ANOVA or two-tailed Student&#8217;s t- test. Report all values as mean &#177; standard error of the mean (SEM). Consider a p-value of &#60;0.05 significant (* p &#60;0.05; ** p &#60;0.01; *** p &#60;0.001).</li>
	</ol>
	</li>
</ol>
3. In Vivo Mesenteric Perfusion Model
<ol>
	<li>Preparation/surgery of the mouse
	<ol>
		<li>Fast the mouse the night before the experiment in order to limit bowel movement.</li>
		<li>Give a 6-8 week old mouse (C57Bl/6) pre-operative analgesia by a subcutaneous injection of buprenorphine (0.1 mg/kg body weight (BW)) 20-30 min&#160;prior to the surgery.</li>
		<li>Anesthetize the mouse by intra-peritoneal injection of ketamine (125 mg/kg BW) and xylazine (12.5 mg/kg BW). Check by pedal reflex. Apply vet ointment to prevent dryness.</li>
		<li>Place the mouse on a thermo-controlled heating pad at 37 &#176;C on a microscope tray. Since this is a terminal procedure, there is no need for strict asceptic procedures. Make an incision near the jugular vein, gently remove the right side of the cervical muscle and isolate the jugular vein from the surrounding tissue. &#160;</li>
		<li>Insert a 2 French intravenous catheter into the right jugular vein for infusion of fluorescently labeled bacteria or other solutions. Open the peritoneal cavity via a midline abdominal incision and use cotton swabs to spread the mesenterium and to visualize the mesenteric arteriolar and venular circulation.</li>
		<li>Place the mouse on the right side on a transparent plate and secure the cannula with tape. Use a hot pack to prevent hypothermia. To prevent dehydration of the tissue, drop 500 &#181;l 0.9 % NaCl on the intestines.</li>
	</ol>
	</li>
	<li>Fluorescence microscopy of bacterial adhesion to the mesenteric circulation
	<ol>
		<li>Work in a dark room. Use cotton swabs to immobilize the vessels and visualize them under an inverted microscope.</li>
		<li>Topically apply 5 &#181;l of a 10 mM solution of the Ca2+-ionophore A23187 dissolved in DMSO. After 10 sec, inject 100 &#181;l labeled bacteria (see step 1) through the jugular catheter. Take time-lapse images.&#160;After the experiment is finished, euthanize the mouse according to institutional approved guidelines.</li>
	</ol>
	</li>
	<li>Image Analysis
	<ol>
		<li>Obtain live images using an inverted fluorescence microscope, captured using a black and white camera and developed using any imaging software. Apply automated exposure time and contrast optimization specific to the equipment used.</li>
		<li>Acquire time-lapse images using the &#8216;Acquisition&#8217; tool in the toolbar (Multidimensional Acquisitions &#8211; Time) using 40 cycles of 1,000 images/sec. Save the images in an appropriate image file format.</li>
		<li>Process images using ImageJ analysis software to measure the area of fluorescent signal per image. Define the threshold to set lower and upper threshold values, segmenting gray scale images into features of interest. Identify the region of interest (blood vessel) and measure the area limited to the threshold and the region of interest. Compare bacterial adhesion, expressed as fluorescent area using any statistical or graphing software.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Shear stress is a crucial factor for the early bacterial adhesion to the vessel wall and for the subsequent generation of endovascular or endocardial vegetations and metastatic infections4,5. We described complementary in vitro and in vivo models to study the pathogenesis of endovascular infections under physiological shear stress. These models have allowed us to identify von Willebrand factor-binding protein (vWbp) as the major S. aureus protein to interact under flow with an injured vascular wall exposing VWF4.
Endovascular infections, and infective endocarditis in particular, are of concern not only because of sepsis-induced organ failure and death, but also because of local and distant (&#8216;metastatic&#8217;) complications. To cause infective endocarditis and metastatic infections, bacteria have to adhere to the vessel wall and thus resist the shear stress of flowing blood. Most studies on bacteria virulence factors have been performed in static conditions. However, these established interactions might not withstand shear forces and studies under flow conditions can reveal new, previously unrecognized factors in bacteria-host interplay.
Using the micro-parallel flow chamber, we and others have shown the importance of VWF for vascular adhesion. Under shear stress, VWF progressively unfolds from its resting globular structure, and exposes the A1 domain that interacts with platelets via its GPIb receptor6. Flow chambers have been extensively used to study platelet function7.
Remarkably, also S. aureus adhesion under flow requires VWF, and in particular the A1 domain that is exposed upon shear. We identified vWbp to mediate VWF binding. vWbp is a coagulase that contributes to S. aureus pathophysiology by activating the host&#8217;s prothrombin. Staphylothrombin, the resulting complex of a bacterial coagulase and prothrombin, converts fibrinogen into insoluble fibrin8,9. Our studies have shown that vWbp does not only activate prothrombin, but triggers the formation of bacteria-fibrin-platelet aggregates, which enhance the adhesion to blood vessels under flow4,10,11.
The in vitro flow chamber model allows to study the different players in bacterial adhesion to cellular or matrix components. Bacterial virulence factors can be studied by using mutants or innocuous bacteria expressing specific surface proteins. Alternatively, pharmacologic inhibitors or blocking antibodies can be added to the medium in the flow chamber. The role of host factors such as different constituents of extracellular matrix can be studied by using coverslips with different coatings. The coverslips can also be covered with endothelial cells, of which the activation status can be modulated by adding specific stimulators. Apart from the vascular wall, the contribution of host blood cells and plasma proteins can be studied by adding these factors to the flowing medium. Thus, different conditions of increasing complexity can be studied under standardized conditions of laminar flow to unravel the interactions that allow bacteria to adhere to the vessel wall in vivo.
Interactions identified in the in vitro model are subsequently studied in an animal model to test their relevance in a complex organism. Other in vivo models to study dynamic interactions under flow have been described, such as the hamster dorsal skinfold chamber12 and the cremaster model13. In comparison, the mesenteric perfusion model described here offers several advantages because of its ease of use, the possibility to vary host genetic background of the mice and to evaluate pharmacological interventions.
In conclusion, the described models offer the possibility to study surface proteins not only of S. aureus, but of many other microorganisms in different host backgrounds, to better understand the pathogenesis of vascular infections.

To establish endovascular infections, pathogens require a mechanism to adhere to the endothelium, which lines the vessel wall and the inner surface of the heart, and to persist and establish an infection despite being exposed to the shear stress of rapidly flowing blood. The most frequent pathogen causing life-threatening endovascular infections and infective endocarditis is Staphylococcus aureus (S. aureus)1. 
Various bacterial surface-bound adhesive molecules mediate adhesion to host tissue by interacting with extracellular matrix components. These MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) recognize molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF). MSCRAMMs are important virulence factors of S. aureus and are implicated in the colonization and invasion of the host2. Most studies on these virulence factors have been performed in static conditions, and thus may not be representative for human infections where initial adhesion of the bacteria occurs in flowing blood. 
In the case of bloodstream infections, bacteria need to overcome the shearing forces of flowing blood in order to attach to the vessel wall. Models that investigate the interaction between bacteria and endothelium or subendothelium under flow conditions are therefore of particular interest. 
A recent study showed that the adhesion of S. aureus to blood vessels under shear stress is mediated by VWF3. VWF, a shear stress-operational protein, is released from endothelial cells upon activation. Circulating VWF binds to collagen fibers of the exposed subendothelial matrix. Our group reported that the von Willebrand factor-binding protein (vWbp) of S. aureus is crucial for shear-mediated adhesion to VWF4. 
In this article, we present an in vitro flow chamber model where bacterial adhesion to different components of the extracellular matrix or to endothelial cells can be evaluated. To validate the findings from in vitro data, we have developed an in vivo model that visualizes and quantifies the direct interaction of bacteria with the vessel wall and the formation of bacteria-platelet thrombi in the mesenteric circulation of mice, using real-time intravital vascular microscopy.

<table border="0" cellpadding="0" cellspacing="0" style="border-collapse:
 collapse;width:192pt">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr>
			<td>Brain Heart Infusion (BHI)</td>
			<td>BD Plastipak&#160;</td>
			<td align="right">237500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth (TSB)</td>
			<td>Oxoid</td>
			<td>CM0129</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190-169</td>
			<td>D-PBS</td>
		</tr>
		<tr>
			<td>5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21878-25MG-F</td>
			<td>fluorescent labeling&#160;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V (BSA)</td>
			<td>Roch</td>
			<td colspan="2">10 735 086 001</td>
		</tr>
		<tr>
			<td>Haemate-P</td>
			<td>CSL Behring</td>
			<td>PL 15036/0010</td>
			<td>VWF</td>
		</tr>
		<tr>
			<td>Horm collagen</td>
			<td>Takeda</td>
			<td align="right">10500</td>
			<td>collagen</td>
		</tr>
		<tr>
			<td>1-well PCA cell culture chambers</td>
			<td>Sarstedt</td>
			<td align="center" class="xl63">########</td>
			<td>plastic slips</td>
		</tr>
		<tr>
			<td>Temgesic</td>
			<td>Reckitt Benckiser</td>
			<td align="right">283716</td>
			<td>bruprenorphine</td>
		</tr>
		<tr>
			<td>Anesketin (Ketamin hydrochloride 115 mg/ml (100 mg/ml ketaminum))</td>
			<td>Eurovet</td>
			<td>BE-V136516</td>
			<td>ketamin</td>
		</tr>
		<tr>
			<td>XYL-M 2% (xylazine hydrochloride 23.32 mg/ml (20 mg/ml xylazine))</td>
			<td>VMD Arendonk</td>
			<td>BE-V170581</td>
			<td>xylazine</td>
		</tr>
		<tr>
			<td>2 french intravenous catheter green</td>
			<td>Portex</td>
			<td colspan="2">200/300/010</td>
		</tr>
		<tr>
			<td>0,9% Sodium chloride (NaCl)</td>
			<td>Baxter Healthcare</td>
			<td>W7124</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton swabs</td>
			<td>International Medical Product</td>
			<td align="right">300230</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+-ionophore solution A23187</td>
			<td>Sigma-Aldrich</td>
			<td colspan="2">C7522-10 MG</td>
		</tr>
		<tr>
			<td>26 gauge 1 ml syringe&#160;&#160;</td>
			<td>BD Plastipak&#160;</td>
			<td align="right">300013</td>
			<td></td>
		</tr>
		<tr>
			<td>26 gauge 1 ml syringe&#160; with needle</td>
			<td>BD Plastipak&#160;</td>
			<td align="right">300015</td>
			<td>intra-peritoneal injection</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810-R</td>
			<td>Eppendorf</td>
			<td colspan="2">5811 000.320</td>
		</tr>
		<tr>
			<td>Glass cover slips (24x50)</td>
			<td>VWR</td>
			<td>BB02405A11</td>
			<td>Thickness No, 1</td>
		</tr>
		<tr>
			<td>PHD 2000 Infusion</td>
			<td>Harvard Apparatus</td>
			<td align="right">702100</td>
			<td>High-accuracy Harvard infusion pump</td>
		</tr>
		<tr>
			<td>Axio-observer DI</td>
			<td colspan="2">Carl-Zeiss</td>
			<td>Inverted fluorescence microscope</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td colspan="2">National Institute of Health</td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>Graphpad Prism 5,0</td>
			<td colspan="2">Graphpad Software</td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>AxioCam MRm</td>
			<td colspan="2">Carl-Zeiss&#160;</td>
			<td>Black and white camera</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo model to study bacterial adhesion to the vessel wall under flow conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

S. aureus adhesion to VWF, subendothelial matrix and endothelial cells is a shear stress dependent phenomenon
To emphasize the role of shear stress in the interaction between S. aureus and VWF, we performed perfusions over VWF coated coverslips at different shear rates (a schematic overview of the in vitro perfusion model is given in Figure 1. Adhesion of S. aureus to VWF increased with increasing shear rates from 250 sec-1 to 2,000 sec-1 (Figure 2), indicating that high shear forces do not inhibit but reinforce the adhesion of bacteria to VWF.
In order to investigate the contribution of VWF to bacterial adhesion to collagen, the main component of the subendothelial matrix, we perfused fluorescently labeled S. aureus over collagen in the presence or absence of VWF. In the absence of VWF, adhesion of S. aureus to collagen decreased with increasing shear rates. However, when VWF was present in the medium, the adhesion of S. aureus increased with increasing shear rates (Figure 3).
The in vitro flow model also allows us to examine the adhesion of bacteria to endothelial cells under flow. We perfused HUVECs with fluorescently labeled S. aureus at shear rates from 500 to 2,000 sec-1. Where indicated, HUVECs were activated with a Ca2+-ionophore, to cause release of VWF. Endothelial cell activation and the subsequent VWF release, increased adhesion of S. aureus (Figure 4A), which formed typical &#8220;string-like&#8221; patterns of fluorescently labeled bacterial clusters aligned in the direction of the shear force (Figure 4B), suggesting the binding of bacteria along a linear-stretched VWF molecule.
Initial in vivo bacterial adhesion in splanchnic veins is mediated by VWF
Since S. aureus is able to adhere to VWF, we used wildtype mice (Vwf+/+) and VWF-deficient mice (Vwf&#8722;/&#8722;) to investigate bacterial adhesion to the activated vessel wall in vivo. Real-time videomicroscopy of splanchnic veins allowed the in vivo visualization of circulating fluorescently labeled S. aureus (Schematic overview of the in vivo perfusion model is represented in Figure 5).
After pharmacological activation of the endothelium by the Ca2+-ionophore, we observed rapid local accumulation of individual bacteria and aggregates of bacteria to the vessel wall of WT mice (supplemental Videos 1 and 2). Almost no adhesion of bacteria was observed on the activated vessel wall of Vwf-deficient mice (supplemental Video 3) compared with adhesion in WT mice (Figure 6). The absence of VWF decreases the ability of S. aureus to adhere to the activated vessel wall.
<img alt="Figure 1" src="/files/ftp_upload/52862/52862fig1.jpg" /> 
Figure 1.&#160;A schematic representation of the in vitro flow model. The in vitro flow model is a multifunctional model, which allows the study of different shear dependent mechanisms such as bacterial adhesion to the subendothelial matrix but also thrombus formation. The micro-parallel flow chamber is placed on a coverslip (plastic or glass) with different coatings of proteins and endothelial cells. The adhesion of different bacteria (orange and grey dots) can be analyzed, and the impact of the presence of plasma proteins, platelets and whole blood can be evaluated. Fluorescent markers for platelets (blue ovals) or fibrinogen (blue strings) can be used in combination with different inhibitors (black ovals) to distinguish bacterial and host factors. Representative images of bacterial adhesion of S. aureus to collagen coating in the presence (bottom) or absence (top) of VWF are shown (scale bar is 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52862/52862fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52862/52862fig2.jpg" /> 
Figure 2.&#160;Adhesion of S. aureus to VWF increases with increasing shear rates. Micro-parallel flow chamber perfusion over coated VWF (50 &#181;g/ml) with fluorescently labeled S. aureus Newman at shear rates of 250 to 2,000 sec-1 (sec-1) in medium (n &#62;5). All results are expressed as mean &#177; SEM. *p &#60;0.05, **p &#60;0.01.
<img alt="Figure 3" src="/files/ftp_upload/52862/52862fig3.jpg" /> 
Figure 3.&#160;Adhesion of S. aureus to subendothelium is shear and VWF dependent. Micro-parallel flow chamber perfusion over coated collagen (160 &#181;g/ml) with fluorescently labeled S. aureus Newman at shear rates of 250 to 2,000 sec-1 in medium (n &#62;5). VWF (60 &#181;g/ml) was present in the medium where indicated. All results are expressed as mean &#177; SEM. **p &#60;0.01.
<img alt="Figure 4" src="/files/ftp_upload/52862/52862fig4.jpg" /> 
Figure 4.&#160;Adhesion of S. aureus to activated endothelial cells is shear dependent. Micro-parallel flow chamber perfusion over endothelial cells. (A) Human umbilical vein endothelial cells were activated with the Ca2+-ionophore A23187 (0.1 mM) followed by a 10 min perfusion of fluorescently labeled S. aureus Newman at shear rates of 500 to 2,000 sec-1 in medium (n &#62;5). All results are expressed as mean &#177; SEM. *p &#60;0.05. (B) Image of micro-parallel flow chamber perfusion over activated HUVECs with S. aureus at a shear rate of 1,000 sec-2. S. aureus forms strings of &#177; 200 microns length, suggesting adhesion to VWF multimers (scale bar is 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52862/52862fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52862/52862fig5.jpg" /> 
Figure 5.&#160;A schematic overview of the in vivo mesenteric perfusion model. A right jugular vein catheter (yellow line) is inserted for the administration of fluorescently labeled bacteria (orange dots), additional anesthetics or other components such as pharmaceutical inhibitors and antibodies. The peritoneal cavity is opened and the mesenterium is spread to visualize the blood vessels (venous and arterial) under a fluorescence microscope. After pharmacological activation of the endothelium by a Ca2+-ionophore, which induces the release of VWF, bacteria can be injected through the jugular vein catheter. Real-time intravascular video microscopy allows the in vivo visualization of circulating fluorescently labeled bacteria and the resulting formation of bacteria-platelet thrombi. <a href="https://www.jove.com/files/ftp_upload/52862/52862fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52862/52862fig6.jpg" /> 
Figure 6.&#160;The initial adhesion of S. aureus to activated endothelium in vivo is mediated by VWF. In vivo venous mesenteric perfusion model with C57Bl/6-Vwf+/+ and C57Bl/6-Vwf-/- mice. Adhesion of fluorescently labeled S. aureus to the locally activated vessel wall is significantly lower in Vwf-/- mice. All results are expressed as mean &#177; SEM. ***p &#60;0.001, n &#62;7.
<img alt="Video Figure 1" src="/files/ftp_upload/52862/52862figV1.jpg" /> Video 1: Real-time adhesion of S. aureus to activated vessel wall in Vwf+/+ mice. <a href="https://www.jove.com/files/ftp_upload/52862/Video1JorienClaes.mov" target="_blank">Please click here to view this video.</a>
<img alt="Video Figure 2" src="/files/ftp_upload/52862/52862figV2.jpg" /> Video 2: Real-time aggregate formation and embolization of S. aureus in Vwf+/+ mice. <a href="https://www.jove.com/files/ftp_upload/52862/Video2JorienClaes.mov" target="_blank">Please click here to view this video.</a>
<img alt="Video Figure 3" src="/files/ftp_upload/52862/52862figV3.jpg" /> Video 3: Real-time adhesion of S. aureus to activated vessel wall in Vwf-/- mice. In vivo mesenteric perfusion model with Vwf+/+ and Vwf-/- mice. Five &#181;l of a Ca2+-ionophore (10 mM) was applied to the region of the visualized vascular bed. A suspension of carboxy-fluorescein-labeled S. aureus was injected through the jugular catheter. The mesenteric circulation was visualized under an inverted microscope. <a href="https://www.jove.com/files/ftp_upload/52862/Video3JorienClaes.mov" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions at JoVE.com

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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Research

JoVE Journal

Immunology and Infection

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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...