This work was supported by grants from the Foundation of Anna and Edwin Berger, Lars Hierta, the O.E. and Edla Johansson Foundation, the Swedish Medical Research Council (grant number K2015-57X-03163-43-4, www.vr.se), the Cancer Foundation at the University Hospital in Malm&#246;, the Physiographical Society (Forssman&#39;s Foundation), and the Sk&#229;ne County Council&#39;s Research and Development Foundation.

1. Analysis of Vn as a Bacterial Surface Protein Ligand
<ol>
	<li>Detection of Vn-binding at the bacterial surface using flow cytometry 
	NOTE: In flow cytometry, we used side scatter and forward scatter to gate positive events. To examine the interactions with Vn, Hif clinical isolates (n=10)7 were selected together with E. coli BL21 (DE3) as a negative control (Figure 1A).
	<ol>
		<li>Culture Hif clinical isolates in...

<ol>
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C., Hallstrom, B. M., Bernhard, S., Singh, B., Riesbeck, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+sequence+diversity+in+the+Moraxella+catarrhalis.+UspA2/UspA2H+head+domain+on+vitronectin+binding+and+antigenic+variation.">Impact of sequence diversity in the Moraxella catarrhalis. UspA2/UspA2H head domain on vitronectin binding and antigenic variation.</a> Micro. Infect. 15 (5), 375-387 (2013).</li><li>Singh, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fine-tuned+interaction+between+trimeric+autotransporter+haemophilus+surface+fibrils+and+vitronectin+leads+to+serum+resistance+and+adherence+to+respiratory+epithelial+cells.">A fine-tuned interaction between trimeric autotransporter haemophilus surface fibrils and vitronectin leads to serum resistance and adherence to respiratory epithelial cells.</a> Infect. Immun. 82 (6), 2378-2389 (2014).</li><li>Kohler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+vitronectin+and+Factor+H+to+Hic+contributes+to+immune+evasion+of+Streptococcus+pneumoniae.+serotype+3.">Binding of vitronectin and Factor H to Hic contributes to immune evasion of Streptococcus pneumoniae. serotype 3.</a> Thromb. haemostasis. 113 (1), 125-142 (2015).</li><li>Onelli, E., Citterio, S., O'Connor, J. 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The authors have no financial conflicts of interest.

Bacterial pathogens recruit Vn to the cell surface and utilize this complement regulator to prevent the deposition of complement factors and completion of the membrane attack complex2. Vn also functions as a bridge molecule between bacterial surface proteins and host cell surface receptors, thus enabling pathogens to adhere to the surface of epithelial cells and subsequently mediate internalization. In this study, we describe protocols that can be used to estimate i) binding of Vn to the surface o...

Vitronectin (Vn) is an important human glycoprotein involved in maintaining homeostasis via regulation of the fibrinolytic system. Vn also functions as a complement regulator by inhibiting the terminal complement pathway during C5b6-7 complex formation and C9 polymerization. Several bacterial pathogens have been shown to recruit Vn to the cell surface as a means of resisting complement deposition1,2,3. In addition, Vn functions as a &#34;sandwich&#34; molecule between bacteria and host epithelial cell receptors, thereby promoting adherence and internalization of pathogens2,4,5. Binding of Vn to the bacterial cell surface is mediated by other currently unidentified proteins. Fully elucidating the functional role of Vn-binding in evasion of the hose immune response will therefore require identification of Vn-recruiting proteins.
The initial step in identifying Vn-binding proteins is to test whether a pathogen of interest can bind purified Vn. Flow cytometry is a convenient and straightforward method to determine whether Vn is bound to pathogen cells. In this study, we assessed the binding of Vn to various Haemophilus influenzae type f (Hif) clinical isolates6. The method described herein is quantitative and can be used to distinguish the binding capacity of a wide variety of bacterial strains. In a previous study, we characterized protein H (PH) of Hif as a Vn-binding protein7. Therefore, in the present study, the Vn-binding potential of wild-type (WT) Hif and Hif M10&#916;lph mutants were compared using the described protocols.
Once it is determined that a pathogen binds Vn, the second step is to characterize the surface proteome in order to identify potential Vn-binding proteins. A variety of approaches can be used for this purpose8,9, but these methodologies are not described in this report. The method most suitable for examining protein-protein interactions is to recombinantly express selected bacterial surface proteins in E. coli and purify by affinity chromatography. Here, we use PH and its molecular interaction with Vn to illustrate the method. Interactions between recombinant PH and Vn were characterized using an enzyme-linked immunosorbent assay (ELISA)7 and a recently developed label-free technique known as bio-layer interferometry (BLI)10,11. Whereas ELISAs can be used to confirm protein-protein interactions, BLI provides detailed data regarding the kinetic parameters of the interactions.
To study the functional role of Vn in bacterial adherence, two different assays can be utilized. The first assay described here is direct measurement of bacterial adherence to Vn-coated glass surfaces, whereas the second assay examines adherence to the surface of epithelial cells. For the first assay, glass slides were coated with Vn, and the binding of WT or mutant Hif strains was assessed by Gram-stain and microscopy. This technique readily distinguishes bacteria based on the ability to bind Vn12. Bacterial adhesion to mammalian cells was then analyzed by adding cultured bacteria onto a monolayer of type II alveolar epithelial cells; bacterial attachment was assessed by counting the number of colony-forming units (CFUs). Adhered and internalized bacteria can be distinguished in the presence or absence of Vn4,13.
The role of Vn acquisition in bacterial serum resistance was evaluated using a serum killing assay (i.e., serum bactericidal activity). To assess the significance of Vn acquisition in serum resistance, the bactericidal activity of Vn-depleted serum (VDS) was compared with that of normal human serum (NHS). The method used readily distinguishes Vn-binding versus non-binding bacteria based on serum resistance. We used this method to study the role of Vn in the serum resistance of several bacterial pathogens4,12.
Numerous methods have been reported for studying host-pathogen interactions. Here, we describe a set of protocols that can be easily adapted to the study of any pathogen in order to assess the role of Vn in pathogenesis. We tested these protocols using various pathogens, and Hif was chosen as an example for this report.

<table border="0" cellpadding="0" cellspacing="0" style="width:1013px;" width="1014">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1.5 mL thermomixter</td>
			<td style="width:288px;">Eppendorf</td>
			<td style="width:119px;">5355</td>
			<td style="width:272px;">dry block heating and cooling shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL polystyrene round-bottom tube&#160;</td>
			<td>BD Falcon</td>
			<td>60819-138</td>
			<td>12 &#215; 75 mm style</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">5% CO2 supplied incubator&#160;</td>
			<td style="width:288px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">BBD6220</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">6 mL polystyrene round-bottom tube&#160;</td>
			<td style="width:288px;">VWR</td>
			<td>89000-478</td>
			<td>12 &#215; 75 mm style with cap</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">24-well plates</td>
			<td style="width:288px;">BD Falcon</td>
			<td>08-772-1H</td>
			<td style="width:272px;">Cell culture grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">30% Hydrogen peroxide (H2O2) solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">H1009-100ML</td>
			<td>Laboratory analysis grade</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">75 cm2 tissue culture flask</td>
			<td style="width:288px;">BD Falcon</td>
			<td>BD353136</td>
			<td>Vented</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well black flat bottom plate</td>
			<td style="width:288px;">Greiner Bio-One</td>
			<td>655090</td>
			<td>Tissue culture treated &#181;Clear black plates</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">A549 Cell Line human</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">86012804-1VL</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Cell detachment enzyme (Accutase)&#160;</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>A6964-500ML</td>
			<td style="width:272px;">Cell Culture Grade</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:335px;">AR2G sensors</td>
			<td style="width:288px;">Pall Life Science</td>
			<td>18-5095</td>
			<td style="width:272px;">Sensor to immobilized protein by amino coupling&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Acetone</td>
			<td style="width:288px;">VWR</td>
			<td>97064-786</td>
			<td>Analysis grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Bovine Serum Albumins (BSA)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>A2058</td>
			<td style="width:272px;">Suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bibulous paper&#160;</td>
			<td style="width:288px;">VWR</td>
			<td>28511-007</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:335px;">Bio-layer interferometer</td>
			<td style="width:288px;">Pall Life Science</td>
			<td style="width:119px;">FB-50258</td>
			<td style="width:272px;">Bilayer interferometry measuring equipment</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Crystal violet solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>HT90132-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">C4BP (C4b binding protein)</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td style="width:119px;">A109</td>
			<td>Bought as Frozen liquid form</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">Calcium chloride (CaCl2)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>C5670-500G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Carbol-fuchsin solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>HT8018-250ML</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Citric acid</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>251275-500G</td>
			<td>American Chemical Society (ACS) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Decolorizing solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>75482-250ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">E. coli host (E. Coli BL21)</td>
			<td style="width:288px;">Novagen</td>
			<td>69450-3</td>
			<td>Protein expression host</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">F12 medium</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>D6421</td>
			<td style="width:272px;">Cell Culture Grade</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:335px;">Flow cytometer&#160;</td>
			<td style="width:288px;">BD Biosciences</td>
			<td style="width:119px;">651154</td>
			<td style="width:272px;">Cell analysis grade for research applications&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Fetal Calf Serum (FCS)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>12003C</td>
			<td style="width:272px;">Suitable for cell culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Normal human serum (NHS)</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td>NHS</td>
			<td style="width:272px;">Pooled human serum</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">FITC-conjugated donkey anti-sheep antibodies&#160;</td>
			<td style="width:288px;">AbD Serotec</td>
			<td>STAR88F</td>
			<td style="width:272px;">Polyclonal</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Gentamicin</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>G1397</td>
			<td style="width:272px;">Cell culture grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glucose</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Gelatin</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>G9391</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:288px;">Marienfeld</td>
			<td>640210</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">HRP-conjugated anti-His tag antibodies</td>
			<td style="width:288px;">Abcam</td>
			<td style="width:119px;">ab1269</td>
			<td style="width:272px;">Polyclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Human factor H</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td style="width:119px;">A137</td>
			<td>Bought as Frozen liquid form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">C4BP</td>
			<td style="width:288px;">Complement Technology, Inc.</td>
			<td style="width:119px;">A109</td>
			<td>Frozen solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Human serum albumin</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1653-10G</td>
			<td>lyophilized powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histidine affinity resin column (HisTrap HP)</td>
			<td style="width:288px;">GE Health Care Life Science</td>
			<td style="width:119px;">17-5247-01</td>
			<td>Columns prepacked with Ni Sepharose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">His-tagged PH</td>
			<td style="width:288px;"></td>
			<td style="width:119px;"></td>
			<td>Recombinantly expressed and purified in our lab</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Iodine solution</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>HT902-8FOZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Methanol</td>
			<td style="width:288px;">VWR</td>
			<td>BDH1135-1LP</td>
			<td>Analysis grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">&#160;Microscope</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:119px;">IX73</td>
			<td style="width:272px;">Inverted microscope</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Microscope slides</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>S8902</td>
			<td style="width:272px;">plain, size 25 mm &#215; 75 mm&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">Magnesium chloride (MgCl2)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>M8266-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid containg C terminal 6x His-tag on the backbone (pET26(b))</td>
			<td style="width:288px;">Novagen</td>
			<td>69862-3</td>
			<td>DNA vector</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Polysorb microtitre plates&#160;</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>M9410</td>
			<td style="width:272px;">For ELISA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Potassium hydroxide (KOH)</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td style="width:119px;">6009</td>
			<td>American Chemical Society (ACS) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Sheep anti-human Vn antibodies</td>
			<td style="width:288px;">AbD Serotec</td>
			<td>AHP396</td>
			<td style="width:272px;">Polyclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Shaker&#160;</td>
			<td style="width:288px;">Stuart Scientific</td>
			<td style="width:119px;">STR6</td>
			<td>Platform shaker</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:335px;">Tissue culture flask</td>
			<td style="width:288px;">BD Falcon</td>
			<td style="width:119px;">3175167</td>
			<td style="width:272px;">75 cm2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">&#160;Thermomixer&#160;</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>T3317</td>
			<td>Dry block heating and cooling shaker</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Tetramethylbenzidine</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>860336-100MG</td>
			<td>ELISA grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Vitronectin (Vn) from human plasma</td>
			<td style="width:288px;">Sigma-Aldrich</td>
			<td>V8379-50UG</td>
			<td>cell culture grade</td>
		</tr>
	</tbody>
</table>

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.

Vn-binding to the surface of bacteria was determined by flow cytometry. All Hif clinical isolates tested in this study recruited Vn to the cell surface. No interaction of Vn with the cell surface was observed for the E. coli negative control strain (Figure 1A). As shown in Figure 1B, PH is a major Vn-binding protein on the surface of Hif cells. Binding of Vn by the WT Hif strain M10 caus...

Watch this Scientific Journal Video about Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance at JoVE.com

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

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 ...

This method can help answer key questions in host-pathogen interaction field such as how bacterial pathogens cause disease in humans by utilizing human proteins. The main advantage of this technique is it can answer if any pathogen can utilize Vitronectin for establishing infection of the host. We will focus upon Vitronectin, a host protein that takes part in the extracellular matrix, but also functions as a complement inhibitor in determinant pathway of complement activation.These facts are very important and highly attractive for pathogens causing disease in humans. To prepare for flow cytometry, resuspend the bacterial pellets with 50 microliters of blocking buffer containing 250 nanomolar Vitronectin. Then, incubate the samples for one hour at room temperature without shaking.After incubation, pellet the bacteria by centrifugation at 3, 500 g for five minutes. Then, wash the pellets three times using PBS and similar centrifugation steps. Following wash, add 50 microliters of primary sheep antihuman Vitronectin polyclonal antibodies to the bacterial pellet at a one to 100 dilution in PBS BSA.Incubate the suspension for one hour at room temperature. Then, wash the bacteria three times with PBS to remove unbound antibodies. Next, add 50 microliters of PBS BSA containing fluorescein isothiocyanate conjugated donkey anti-sheep polyclonal antibodies to the pellet.Incubate at room temperature for one hour in the dark. Finally, after three washing steps, resuspend the bacterial pellet with 300 microliters of PBS and analyze by flow cytometry. To prepare for ELISA, dispense 100 microliters of protein solution into each well of a PolySorp microtiter plate.Close the plate with the lid and store at four degrees Celsius overnight to facilitate the immobilization of protein onto microtiter plate wells. Discard the solution from the microtiter plate by tilting upside down over the sink. Then, wash the wells three times with 300 microliters of PBS per well.Block the coated wells for one hour at room temperature with PBS containing 2.5 weight volume percent BSA. After removing the blocking solution, wash the wells three times with 300 microliters of PBST per well. Now, add 100 microliters of 50 nanomolar recombinant his-tagged PH to each sample.In the control wells, add only 100 microliters of PBS BSA. Incubate the plate for one hour at room temperature. Following incubation, discard the protein solution and remove the unbound proteins by washing the wells three times with 300 microliters of PBST per well.Then, add 100 microliters of PBS BSA containing horse radish peroxidase conjugated anti-his polyclonal antibodies. After incubating for one hour at room temperature, wash the wells three times with 300 microliters of PBST per well. Detect the antigen-antibody complexes by adding 100 microliters of ELISA detection reagent to each well.Then, add 50 microliters of one molar sulfuric acid per well to stop the reaction. Measure the optical density of the wells at 450 nanometers using a microplate reader. Immobilize human Vitronectin on amine reactive sensors using the amine coupling method according to the manufacturer's guidelines.Using PBS, serially dilute the recombinant PH ligand from zero to four micromolar and transfer the resulting solutions to a 96-well black flat-bottom microtiter plate. Run the experiment at 30 degrees Celsius using a bio-layer interferometry instrument. Pipette 10 microliters of a two microgram per milliliter solution of Vitronectin in PBS onto a glass microscope slide as a single drop.Allow the drop to dry on the slide for 30 minutes at room temperature. Wash the protein-coated glass slides three times by dipping them in a beaker containing PBS for two seconds to remove excess uncoated protein. Now, add 10 milliliters of a fresh Hif culture into a sterile plastic Petri dish and submerge the coated glass slides in the culture medium.Incubate the dishes at 37 degrees Celsius for one hour with shaking at 20 RPM. After incubation, remove any unbound bacteria by submerging the slides three times in a beaker filled with PBS before visualizing the bound bacteria by Gram staining as described in the text protocol. Culture A549 epithelial cells as described in the text protocol.After one wash and trypsinization of the cells at 37 degrees Celsius, dilute the cell suspension to 5, 000 cells per milliliter using complete medium containing five micrograms per milliliter of gentamycin. Prior to bacterial infection, replace the medium in the wells with F12 medium and incubate overnight at 37 degrees Celsius. Wash the cell monolayer three times with one milliliter of PBS at room temperature.Then, place the plate on ice and 200 microliters of pre-chilled F12 medium containing 10 micrograms per milliliter of Vitronectin. After incubating the plate at four degrees Celsius for one hour, discard the solution by pipetting and wash the cell layer twice with one milliliter of PBS at room temperature. Add 100 microliters of freshly grown Hif wild type in F12 medium to each well.Incubate the plate for two hours at 37 degrees Celsius. Then, aspirate the medium with a pipette and wash the A549 epithelial cells three times with PBS. Add 50 microliters per well of cell detachment solution and follow with a five-minute incubation at 37 degrees Celsius.Next, add 50 microliters of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells from each well to a six milliliter glass tube containing four glass beads. Lyse the cells at room temperature by vortexing for two minutes.After diluting the cell lysate 100-fold, plate 10 microliters of the diluted sample onto a chocolate agar plate. Count the colonies following an overnight incubation at 37 degrees Celsius. To perform the analysis of Vitronectin-dependent resistance to the bactericidal activity of human serum, first culture and pellet the bacteria as described in the text protocol.Following centrifugation, resuspend the bacterial pellet with one volume of Dextrose-Gelatin-Veronal buffer. Now, add 1, 500 CFU of bacteria to 100 microliters of DGVB plus plus containing 5%serum. Incubate the sample at 37 degrees Celsius for 15 minutes with shaking at 300 RPM.Remove a 10 microliter aliquot from the reaction mixture at zero minutes and 15 minutes. Place the aliquot on chocolate agar and incubate the plate at 37 degrees Celsius overnight. After incubation of the chocolate agar plates, count the colonies appearing on the plates and calculate the percentage of bacteria killed as described in the text protocol.All Hif clinical isolates tested in this study recruited Vitronectin to the cell surface as determined by flow cytometry. Binding of Vitronectin by the wild type Hif strain caused a shift in the population whereas the mutant did not bind Vitronectin. Protein-protein interactions between major Vitronectin-binding protein PH and Vitronectin were estimated by ELISA.The results indicate a significant interaction between PH and both Vitronectin and the positive control relative to the negative control. Interaction between PH and Vitronectin were estimated in real-time using bio-layer interferometry to have an affinity of 2.2 micromolar. Furthermore, bacterial lacking expression of PH on the cell surface exhibited reduced adherence to Vitronectin-coated glass surfaces in comparison to wild type cells.In addition, the presence of Vitronectin on the surface of epithelial cells significantly enhanced the adherence of Hif. To estimate the complement inhibiting activity of Vitronectin, serum-mediated killing was examined. Wild type Hif cells exhibited higher serum resistance than mutant cells.No bacteria were killed in the presence of heat-inactivated serum. Interestingly, wild type Hif exhibited reduced survival in Vitronectin-depleted serum and replenishment of Vitronectin increased bacterial survival. However, the mutant strain did not respond to Vitronectin depletion because it could not recruit Vitronectin to the cell surface.Once mastered, this technique it can be done in three to four days for any pathogen and remember to use the fresh culture of the bacterial pathogens. After watching this video, you should have good understanding on how to analyze the Vitronectin binding to a pathogen. In addition to this technique, Vitronectin binding surface proteins of bacteria can be identified by using proteomics and western blotting.This technique is important for the researchers in the field of host-pathogen interaction and also to explore the mechanism of virulence in bacteria.

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Research

JoVE Journal

Immunology and Infection

8.0K Views.  Lund University. This method can help answer key questions in host-pathogen interaction field such as how bacterial pathogens cause disease in humans by utilizing human proteins. The main advantage of this technique is it can answer if any pathogen can utilize Vitronectin for establishing infection of the host. We will focus upon Vitronectin, a host protein that takes part in the extracellular matrix, but also functions as a complement inhibitor in determinant pathway of complement activation.These fac...