The authors would like to thank Emilie Mairey and Emmanuel Donnadieu for the initial setting up of the procedure.

1. Human host cell and bacterial culture
<ol>
	<li>Culture HUVECs between passage 1 and 9 at 37&deg; in a humidified incubator with 5% (v/v) CO2. Passage the cells when they approach 80% confluence with trypsin/EDTA to provide maintenance cultures on 75 cm2 culture flasks and experimental cultures in disposable flow chambers (&mu;-Slides VI).</li>
	<li>Withdraw medium from the flasks to be passaged, wash the cells with 10 ml of PBS, withdraw and replace it with 1.5 ml of trypsin/EDTA. Allow the cells to detach in a cell culture incubator for 5 minutes at 37&deg;C.</li>
	<li>Collect the cells with 10 ml of cell culture medium into a 15 ml collection tube. Pellet the cells with a 5 minute centrifugation (at 200 g), at room temperature. Suspend the cells in 4 ml of Endo-SFM supplemented with 10% (v/v) FBS and count them on a Malassez chamber, according to the manufacturer's instructions.</li>
	<li>Introduce 30 &mu;l of a 1x106 HUVEC cells per ml suspension into the channel (3x104 total) and allow the cells to adhere for 3 hours at 37&deg; in a humidified incubator with an atmosphere of 5% (v/v) CO2. Add an additional volume of 120 &mu;l of Endo-SFM to fill the wells. Cells should form a subconfluent monolayer (Figure 2).</li>
	<li>Grow N. meningitidis expressing GFP (in this case strain 8013 expressing GFP under the control of an IPTG-inducible promoter), on GCB agar plates containing Kellogg's supplements and 5 &mu;g/ml of chloramphenicol at 37&deg;C in a moist atmosphere containing 5% (v/v) CO2 for 16 hours.</li>
</ol>
2. Initial adhesion of individual bacteria to host cells
<ol>
	<li>Adjust the concentration of bacteria grown on GCB agar plates to an OD600 of 0.05 with pre-warmed Endo-SFM containing 10% (v/v) FBS and incubate for 120 minutes at 37&deg;C in a moist atmosphere containing 5% (v/v) CO2, under gentle shaking (130 rpm). Induce GFP expression by adding 1 mM IPTG into the culture medium for the whole incubation period.</li>
	<li>Place the disposable &mu;-Slide on the stage of an inverted microscope equipped with a heated platform to maintain the sample temperature at 37&deg;C.</li>
	<li>Pour pre-warmed Endo-SFM supplemented with 2% (v/v) FBS into a sterile glass beaker and fill a sterile 50 ml syringe with this medium. Attach the "entry" tubing to the syringe and fill by introducing medium.</li>
	<li>Connect the "entry" tubing to the &mu;-Slide, with care, in order to avoid introducing air into the chamber. Then, affix the "exit" tube to the other end and carefully fill the channel with medium to approximately 1 cm distance from the chamber "exit".</li>
	<li>Measure the bacterial OD600 and adjust to 0.15 in Endo-SFM containing 2% (v/v) FBS. In order to look at individual bacteria, any aggregates must be disrupted by vigorous vortexing of the bacterial sample.</li>
	<li>Place 100 &mu;l of Endo-SFM supplemented with 2% (v/v) FBS medium into the reservoir and add 100 &mu;l of the bacterial solution taken from the top of the vortexed solution to avoid sampling any remaining bacterial aggregates.</li>
	<li>Carefully introduce the 200 &mu;l volume into the &mu;-Slide by turning the stopcock to inject the bacteria into the chamber.</li>
	<li>Introduce Endo-SFM containing 2% (v/v) FBS, maintained at 37&deg;C with the heated platform, into the chamber using a syringe pump with a shear stress compatible with adhesion of 0.044 dynes/cm2 for 15 minutes.</li>
</ol>
See for example video 1 or figure 3. After this step, either move to sections 3, 4 or 6.
3. Quantification of initial adhesion of individual bacteria to host cells
<ol>
	<li>After initial adhesion for 15 minutes, acquire images of ten fields randomly while liquid is still circulating.</li>
	<li>Analyze the acquired images using the ImageJ software 8, in order to access the mean number of adherant bacteria per field. This is done as follows: first of all, each image is thresholded using the "Threshold" window found in the "Image" menu to highlight individual adhering fluorescent bacteria, while minimizing the background from the cells. Then, every single bacterium is counted, by using the Plug-In "Cell Counter" (<a href="http://rsb.info.nih.gov/ij/plugins/cell-counter.html">http://rsb.info.nih.gov/ij/plugins/cell-counter.html</a>). The data for each image are reported in an Excel spreadsheet and are averaged to quantify the mean number of adherent bacteria per field.</li>
</ol>
4. Measuring the resistance to flow of individual bacteria adherent to host cells
<ol>
	<li>Program the syringe pump set-up to generate shear stress ranging from 3 to 100 dynes/cm2 for 5 minutes.</li>
	<li>At the end of the 5 minutes, acquire ten fields randomly chosen as described in step 3.1, but with the flow stopped prior to the acquisition.</li>
	<li>Analyze the acquired images as described in step 3.2 and quantify the mean number of remaining bacteria per field.</li>
</ol>
5. Measuring the growth of an isolated adherent bacterium to a microcolony
<ol>
	<li>Introduce Endo-SFM containing 10% (v/v) FBS, maintained at 37&deg;C with the heated platform, into the chamber using a syringe pump with the chosen shear stress for several hours. Record images for real-time video microscopy at one frame every 5 minutes.</li>
	<li>Following video microscopy, stop the shear stress by turning off the syringe pump, acquire 2-3 additional images and then stop the image acquisition sequence on the software. An example of bacterial proliferation can be seen on video 2.</li>
</ol>
6. Measuring the resistance to flow of adherent bacterial microcolonies
<ol>
	<li>After the formation of large microcolonies on the cellular surface (step5.2) acquire 2-3 images of both cells (by phase contrast) and bacteria (by GFP-fluorescence) before flow application. For the rest of the experiment, the acquisition protocol will monitor only the fluorescence.</li>
	<li>Applying high shear stress for 5 minutes (3-100 dynes/cm2) and record images at one frame every 5 seconds.</li>
	<li>Stop the shear stress by turning off the syringe pump, acquire 2-3 additional images and then stop the image acquisition sequence on the software.</li>
	<li>Next, remove the "exit" tubing first and carefully, to avoid emptying the channel and then add pre-warmed fresh medium to fill the wells before removing the "entry" tubing.</li>
	<li>To quantitatively determine the effect of shear stress on bacterial resistance, a plating assay can be performed as follows: collect the remaining medium and wash the &mu;-Slide twice with 120 &mu;l of PBS, which removes the remaining medium from the channel (both washes are also collected).</li>
	<li>Detach the infected cells with the addition of 50 &mu;l of trypsin/EDTA for 5 minutes at 37&deg; and mix this detached sample with the suspension collected in the previous step.</li>
	<li>Perform serial dilutions in PBS and plate a 10 &mu;l fraction onto GCB agar plates, in triplicate, in order to determine the number of colony forming units (CFU) on the next day, after incubation of the plates at 37&deg;C in an incubator with 5% (v/v) CO2.</li>
</ol>
Videos 3 and 4 compare the wild type strain with the pilV mutant.
7. Bacterial detachment from microcolonies
<ol>
	<li>Infect cells in the flow chamber by repeating steps 2.1 to 2.8 and allow the infection to proceed for 30 minutes.</li>
	<li>Remove unbound bacteria by increasing flow to 10 dynes/cm2 for a period of 2 minutes and allow infection to continue for 2h.</li>
	<li>Reduce flow to 0.15 dynes/cm2.</li>
	<li>Every hour, collect a drop of medium coming out of the flow chamber.</li>
	<li>Perform serial dilutions of the samples and plate them on GCB agar plates.</li>
	<li>Determine the number of colony forming units (CFU) on the next day, after incubation of the plates at 37&deg;C in an incubator with 5% (v/v) CO2.</li>
</ol>
8. Representative results
<img alt="Figure 1" src="/files/ftp_upload/3241/3241fig1.jpg" /> 
Figure 1: Different steps of a cellular monolayer that can be observed and measured in the presence of flow in the procedure.
<img alt="Figure 2" src="/files/ftp_upload/3241/3241fig2.jpg" /> 
Figure 2: Endothelial cell monolayer in the flow chamber at the beginning of an experiment (absence of shear stress). About 50 endothelial cells are organized in a sub-confluent monolayer.
<img alt="Figure 3" src="/files/ftp_upload/3241/3241fig3.jpg" /> 
Figure 3: Graphic representation of initial adhesion of bacteria on the cellular surface. Values for three fields are indicated to give an idea of the field-to-field variation that is expected (0,044 dynes/cm2).
Video 1. Visualization of initial adhesion of bacteria on the cellular monolayer as a function of time (0,044 dynes/cm2). GFP-expressing bacteria are following the direction of the flowing medium. The film is accelerated 60-fold, the real duration of the video is 10 minutes. 
<a href="https://www.jove.com/files/ftp_upload/3241/3241video1.mov">Click here to watch the video.</a>
Video 2. After initial adhesion bacteria were allowed to proliferate on the cellular surface for a period of 7 hours (accelerated 1000-fold). 
<a href="https://www.jove.com/files/ftp_upload/3241/3241video2.mov">Click here to watch the video.</a>
Video 3. After Proliferation on the cellular surface the mechanical resistance of microcolonies was tested by increasing the shear stress level to 10 dynes/cm2 for 5 minutes but wild type microcolonies are resistant accelerated 60-fold. 
<a href="https://www.jove.com/files/ftp_upload/3241/3241video3.mov">Click here to watch the video.</a>
Video 4. Microcolonies formed by the pilV mutant are disrupted by flow increase (10 dynes/cm2). This mutant fails to induce the remodeling of the plasma membrane under microcolonies and is thus more highly sensitive to increased shear stress (video is accelerated 60-fold)5. 
<a href="https://www.jove.com/files/ftp_upload/3241/3241video4.mov">Click here to watch the video.</a>

<ol>
	<li>Mairey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microcirculation+shear+stress+levels+determine+Neisseria+meningitidis+attachment+sites+along+the+blood-brain+barrier.">Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.</a> J Exp Med. 203, 1939-1950 (2006).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+Principles+of+Vascular+Beds.">Design Principles of Vascular Beds.</a> Circ Res. 77, 1017-1023 (1995).</li><li>Zarbock, A., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesion+and+activation+under+flow.">Neutrophil adhesion and activation under flow.</a> Microcirculation. 16, 31-42 (2009).</li><li>Tissot, O., Pierres, A., Foa, C., Delaage, M., Bongrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motion+of+cells+sedimenting+on+a+solid+surface+in+a+laminar+shear+flow.">Motion of cells sedimenting on a solid surface in a laminar shear flow.</a> Biophys J. 61, 204-215 (1992).</li><li>Mikaty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+bacterial+pathogen+induces+host+cell+surface+reorganization+to+resist+shear+stress.">Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress.</a> PLoS Pathog. 5, e1000314-e1000314 (2009).</li><li>Chamot-Rooke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranslational+modification+of+pili+upon+cell+contact+triggers+N.+meningitidis+dissemination.">Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.</a> Science. 331, 778-782 (2011).</li><li>Soyer, M., Dum&#237;nil, G., Christodoulides, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesion+under+shear+stress.">Bacterial adhesion under shear stress.</a> Neisseria meningitidis: Methods and Protocols (Methods in Molecular Biology). , (2011).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Thomas, W. E., Trintchina, E., Forero, M., Vogel, V., Sokurenko, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesion+to+target+cells+enhanced+by+shear+force.">Bacterial adhesion to target cells enhanced by shear force.</a> Cell. 109, 913-923 (2002).</li><li>Tchesnokova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear-enhanced+binding+of+intestinal+colonization+factor+antigen+I+of+enterotoxigenic+Escherichia+coli.">Shear-enhanced binding of intestinal colonization factor antigen I of enterotoxigenic Escherichia coli.</a> Mol Microbiol. 76, 489-502 (2010).</li><li>Konto-Ghiorghi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+role+for+pilus+in+adherence+to+epithelial+cells+and+biofilm+formation+in+Streptococcus+agalactiae.">Dual role for pilus in adherence to epithelial cells and biofilm formation in Streptococcus agalactiae.</a> PLoS Pathog. 5, e1000422-e1000422 (2009).</li></ol>

No conflicts of interest declared.

The importance of shear stress and generally of mechanical aspects in biology is increasingly being recognized. For instance the highly adapted adhesive properties of the Selectin family of proteins in the process of lymphocytes adhesion and rolling on the vascular wall has been recognized by introducing shear stress in the process. The procedure described above was applied to the Gram-negative bacteria Neisseria meningitidis but should be applicable to a wide range of pathogens. The importance of shear stress has been shown also for other pathogens and other infection sites. Bacterial adhesion conditioned by shear stress has been described by the study of the FimH adhesin found on uropathogenic Escherichia coli (UPEC) 9. Similar to Selectins, the interaction between FimH and its host cell receptor was shown to be strengthened by shear-induced mechanical forces 9 and the CfaE adhesin of enterotoxigenic E.coli has been reported also to mediate adhesion to intestinal epithelial cells via a shear-dependant mechanism 10. We reported, using the laminar-flow chamber assay protocol described in this chapter that Streptococcus agalactiae pili were essential for adherence of this pathogen to epithelial cells under flow conditions 11. Such studies confirm that our flow chamber assay is a useful tool for investigating host cell-pathogen interactions under conditions of shear stress.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>&mu;-Slide VI0.4 flow kit </td>
			<td>IBIDI</td>
			<td>80606</td>
		</tr>
		<tr>
			<td>Syringe Plastipak 50 ml Luer-Lock</td>
			<td>Becton Dickinson</td>
			<td>300865</td>
		</tr>
		<tr>
			<td>Tygon Tubing R3603 3.2 x 4.8mm</td>
			<td>Fisher-Scientific</td>
			<td>R3603</td>
		</tr>
		<tr>
			<td>3-way stopcock, 2 female luer to male luer</td>
			<td>Bio-Rad</td>
			<td>7328103</td>
		</tr>
		<tr>
			<td>Syringe pump </td>
			<td>Harvard Apparatus</td>
			<td>PHD 2000</td>
		</tr>
		<tr>
			<td>Inverted microscope, Nikon </td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Hamamatsu</td>
			<td>ORCA 285 CCD or ORCA 3-CCD</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td>Freeware 
			(<a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a>)</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Introducing Shear Stress in the Study of Bacterial Adhesion at JoVE.com

Introducing Shear Stress in the Study of Bacterial Adhesion

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 ...

introducing-shear-stress-in-the-study-of-bacterial-adhesion

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Immunology and Infection

15.6K Views.  INSERM U970. 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.

Video: Introducing Shear Stress in the Study of Bacterial Adhesion

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 ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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 ...

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.

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

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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 ...

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 ...