Name: Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa (Video) | JoVE Journal
Uploaded: 2018-07-12T12:30:00.000+00:00
Duration: 7 min 27 s
Description: Learn methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by Pseudomonas aeruginosa. This reliable protocol enables repeatable data generation for studying bacterial pathogenesis.

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This research was funded in parts by NIH grants HL66299 to TS, RB, and SL, HL60024 to TS, and HL136869 to MF.

All animal procedures were reviewed and approved by the Institutional and Animal Care Committee of the University of South Alabama and were performed in accordance with all federal, state, and local regulations. Primary cultures of rat pulmonary microvascular endothelial cells (PMVECs) were obtained from the Cell Culture Core Facility at the University of South Alabama&#8217;s Center for Lung Biology. Cells were prepared using previously described procedures16.
1. Generation of Cytotoxic Supernatants
Note: Here, we use two different strains of P. aeruginosa: PA103, which has an intact type III secretion system capable of transferring the exoenzymes ExoU and ExoT to target cells during infection, and &#8710;PcrV, which lacks a type III secretion system and is incapable of transferring exoenzymes to target cells following inoculation.
<ol>
	<li>Streak bacteria onto Vogel-Bonner (VB) agar plates17 and grow overnight at 37 &#176;C.
	<ol>
		<li>To prepare plates, make the following stock solution (Vogel-Bonner salts; 10x&#160;stock): measure 2 g MgSO4, 20 g citric acid (free acid), 100 g K2PO4, and 35 g NaH2PO4. Add ddH2O to 1 L and autoclave for 15 min with slow exhaust.</li>
		<li>Place 7.5 g agar in 450 mL ddH2O and autoclave as above.</li>
		<li>Following autoclaving, place both solutions into a 50 &#176;C water bath to cool.</li>
		<li>Add 50 mL of the 10x VB stock salt solution to the agar.</li>
		<li>Add carbenicillin to 400 &#181;g/mL (for the strains of P. aeruginosa being used) and mix well by swirling the flask.</li>
		<li>Place 5 mL of the VB agar solution into individual sterile culture dishes, allow the agar to solidify, and then store at 4 &#176;C until the day of bacterial seeding.</li>
		<li>On the day of bacterial seeding, pre-warm a plate to 37 &#176;C and then streak bacteria onto the plate using a sterile loop. Place the plate in a 37 &#176;C incubator overnight.</li>
	</ol>
	</li>
	<li>Verify PMVEC purity by positive staining with fluorescent Griffonia simplicifolia lectin and negative staining with fluorescent Helix pomatia lectin (a marker for arterial endothelial cells). Aliquot cells and freeze in liquid nitrogen.</li>
	<li>For experiments, maintain cells in Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) supplemented with 10% fetal bovine serum, and grow cells in a 37 &#176;C incubator containing 5% CO2. Use cells at passages 5 - 15.
	<ol>
		<li>For generation of cytotoxic supernatants, plate 2 x 106 cells into individual 150 cm dishes and grow for 3 - 4 days until confluence is achieved. Use two plates per experiment (see below).</li>
		<li>For analysis of cytotoxic supernatants, plate 1 x 105 cells into individual wells of a 6-well dish and grow for four days until confluence is achieved.</li>
	</ol>
	</li>
	<li>To produce supernatant, wash one of the two 150 cm dishes of PMVECs from step 1.3.1 with phosphate-buffered saline, trypsinize, and then count the cells (we use an automated cell counter and follow the manufacturer&#8217;s protocol; see Table of Materials). Wash the other dish of PMVECs with Hank&#8217;s Balanced Salt Solution (HBSS) and then infect with either strain of P. aeruginosa at a multiplicity of infection (MOI) of 20:1. This is achieved as follows:
	<ol>
		<li>For P. aeruginosa, OD540 0.25 = 2 x 108 CFUs/mL. To prepare this dilution, add 1 mL HBSS to a 1 mL disposable cuvette. Scrape enough bacteria off of the plate that was prepared in step 1.1 until an OD540 of 0.25 is achieved when measured using a spectrophotometer. The next step will provide a calculation of how much bacteria will be needed.</li>
		<li>Once the number of PMVECs in the culture dish is determined (step 1.4 above), determine the appropriate number of bacteria to be added to the second, non-trypsinized culture dish containing the PMVECs. For example, if it is determined that the culture dish of PMVECs contains 1.3 x 107 cells, then prepare 1.3 x 107 cells x 20 bacteria/cell x 1 mL/2 x 108 CFUs = 1.3 mL bacteria.</li>
		<li>Dilute the 1.3 mL of bacteria into HBSS to a final volume of 20 mL, so that the entire surface of a 150 cm dish can be covered with fluid, then add the diluted bacteria to the plate of PMVECs.</li>
		<li>Incubate the PMVECs inoculated with the bacteria at 37 &#176;C in a 5% CO2 incubator for 4 - 5 h, until gaps can be seen forming in the cell monolayer when the plate is observed microscopically (See Figure 1 for proper level of gap formation).</li>
		<li>Collect the supernatant, then centrifuge for 10 min at 2,000 x g in a tabletop centrifuge to remove cellular debris. 
		Note: Any table top centrifuge capable of generating sufficient g force to pellet unlysed cells and large cell fragments can be used for this step.</li>
		<li>Pour the supernatant into a syringe that has a 0.2 &#181;m filter attached to its end, then pass the supernatant through the filter to remove bacteria.</li>
		<li>Use an aliquot of the sterile supernatant to test cytotoxicity (see 2.1) and freeze the rest of the supernatant at -80 &#176;C for future use.</li>
	</ol>
	</li>
</ol>
2. Analysis of Cytotoxic Supernatants
<ol>
	<li>Cytotoxicity Assay
	<ol>
		<li>Grow PMVECs in 6-well dishes until confluence. Wash wells once with HBSS, then add 1.5 mL of filter-sterilized supernatant (collected from either PA103- or &#8710;PcrV-inoculated cells) to individual wells. Add sterile HBSS to another well as a negative control.</li>
		<li>Place the plate into the CO2 incubator for 21 - 24 h, then observe for cell killing/cytotoxicity (see next step). Use supernatants that exhibit cytotoxicity for further experimentation, and discard those that do not show cell killing.</li>
		<li>Quantitation of cell killing
		<ol>
			<li>Measure lactate dehydrogenase (LDH) release from dead cells using commercially available LDH Assay Kits and manufacturer&#8217;s recommended procedures.</li>
			<li>Alternatively, quantify cell killing microscopically using ImageJ software. For this, record microscopic images and areas of the culture dish containing intact cells, then quantify regions lacking cells (indicative of cell death in a monolayer) using a custom ImageJ (National Institutes of Health) macro (see Supplementary Coding File). If desired, carry out the macro steps manually as follows:
			<ol>
				<li>Input images (RGB or Tiff format) into the macro and adjust contrast to 15% saturated pixels. Duplicate contrast-adjusted images and perform the &#8220;subtract background&#8221; command to obtain a high-contrast image of both cell and gap area within the field of view.</li>
				<li>Subtract this image from the original image and combine the resulting image with the original image using the image calculator &#8220;AND&#8221; function. Convert the resultant image to black (gaps) and white (cells) mask with the threshold function (minimum 0, maximum 5), and use the &#8220;binary erode&#8221; function to remove noise from the image.</li>
				<li>Measure the ratio of black to white pixels within the resultant image using the &#8220;area fraction&#8221; measurement. Plot and express fractional areas for each treatment time point as percent of maximal gap area.</li>
				<li>Calculate means and compare using one-way ANOVA with Tukey&#8217;s post hoc test, with p values less than 0.05 considered significant.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Use immunoblot analysis to establish the presence of amyloid, oligomeric tau, and A&#946; in culture supernatants. Perform immunoblot analysis using standard procedures10,11,12, except for concentrating culture supernatants at least 10-fold prior to electrophoresis.
		<ol>
			<li>To concentrate supernatant, place 1 - 2 mL of supernatant into a centrifugation filter unit whose membrane has a molecular weight cut-off of 10 kDa. Then, place the filter unit into a table top centrifuge and centrifuge at 2,000 x g until the required degree of concentration is achieved (usually 45 - 60 min).</li>
			<li>Determine the amount of protein in the concentrated supernatant11 and load equal amounts of samples into individual wells of the gel.</li>
			<li>Run gels, transfer the proteins to nitrocellulose, then probe blots with either A11 anti-amyloid antibody, T22 anti-oligomeric tau antibody, or MOAB2 anti-A&#946; antibody followed by appropriate secondary antibodies. Develop the blots using standard chemiluminescence procedures.</li>
		</ol>
		</li>
		<li>To do a thioflavin T Assay, use thioflavin T fluorescence (ThT) to quantify amyloids in supernatants. For these measurements, use filter-sterilized supernatants.
		<ol>
			<li>Prepare a stock solution (50x) of thioflavin T by suspending 8 mg thioflavin T (ThT) into 10 mL phosphate buffered saline (PBS). After mixing, filter the solution through a 0.22 &#181;m filter to remove particulates.</li>
			<li>For measurements, add 20 &#181;L of ThT stock to 1 mL of PBS in a 1 mL spectrophotometer cuvette. Place the diluted sample into a spectrofluorimeter.</li>
			<li>Measure the baseline fluorescence emission using 425 nm excitation and scanning the fluorescence emission from 450-575 nm in 2 nm increments.</li>
			<li>Perform a time-lapse scan using 425 nm excitation and 482 nm emission, with data acquired every 0.2 s for 60 s.</li>
			<li>The initial 20 s of the time-lapse scan measures fluorescence in the blank cuvette. At 20 s, pause the scan and add 10 &#181;L of the filter-sterilized supernatant to the cuvette. Mix the cuvette by inversion and then place back into the spectrofluorimeter.</li>
			<li>Resume the time-based scan, acquiring the final 40 s of data.</li>
			<li>Upon completion of the time-lapse scan, perform a final fluorescence emission spectrum scan using identical settings, as detailed in 2.1.5.3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Brancati, F. L., Chow, J. W., Wagener, M. M., Vacarello, S. J., Yu, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+pneumonia+really+the+old+man%27s+friend%3F+Two-year+prognosis+after+community-acquired+pneumonia%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Is pneumonia really the old man's friend? Two-year prognosis after community-acquired pneumonia.</a> Lancet. 342 (8862), 30-33 (1993).</li>
<li>Hedlund, J. U., Ortqvist, A. B., Kalin, M. E., Granath, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+of+importance+for+the+long-term+prognosis+after+hospital+treated+pneumonia%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">Factors of importance for the long-term prognosis after hospital treated pneumonia.</a> Thorax. 48 (8), 785-789 (1993).</li>
<li>Meier, C. R., Jick, S. S., Derby, L. E., Vasilakis, C., Jick, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+respiratory+tract+infections+and+risk+of+first-time+acute+myocardial+infarction%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Acute respiratory tract infections and risk of first-time acute myocardial infarction.</a> Lancet. 351 (9114), 1467-1471 (1998).</li>
<li>Waterer, G. W., Kessler, L. A., Wunderink, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Medium-term+survival+after+hospitalization+with+community-acquired+pneumonia%5BTitle%5D)+AND+%22Am+J+Resp+Crit+Care+Med%22%5BJournal%5D)">Medium-term survival after hospitalization with community-acquired pneumonia.</a> Am J Resp Crit Care Med. 169 (8), 910-914 (2003).</li>
<li>Yende, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+markers+at+hospital+discharge+predict+subsequent+mortality+after+pneumonia+and+sepsis%5BTitle%5D)+AND+%22Am+J+Resp+Crit+Care+Med%22%5BJournal%5D)">Inflammatory markers at hospital discharge predict subsequent mortality after pneumonia and sepsis.</a> Am J Resp Crit Care Med. 177 (11), 1242-1247 (2008).</li>
<li>Corrales-Medina, V. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+between+hospitalization+for+pneumonia+and+subsequent+risk+of+cardiovascular+disease%5BTitle%5D)+AND+%22J+Am+Med+Assoc%22%5BJournal%5D)">Association between hospitalization for pneumonia and subsequent risk of cardiovascular disease.</a> J Am Med Assoc. 313 (3), 264-274 (2015).</li>
<li>Frank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+exoenzyme+S+regulon+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">The exoenzyme S regulon of Pseudomonas aeruginosa.</a> Mol Microbiol. 26 (4), 621-629 (1997).</li>
<li>Engel, J., Balachandran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Pseudomonas+aeruginosa+type+III+effectors+in+disease%5BTitle%5D)+AND+%22Curr+Opin+Microbiol%22%5BJournal%5D)">Role of Pseudomonas aeruginosa type III effectors in disease.</a> Curr Opin Microbiol. 12 (1), 61-66 (2009).</li>
<li>Ochoa, C. D., Alexeyev, M., Pastukh, V., Balczon, R., Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+exotoxin+Y+is+a+promiscuous+cyclase+that+increases+endothelial+tau+phosphorylation+and+permeability%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability.</a> J. Biol. Chem. 287 (30), 25407-25418 (2012).</li>
<li>Balczon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+exotoxin+Y-mediated+tau+hyperphosphorylation+impairs+microtubule+assembly+in+pulmonary+microvascular+endothelial+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.</a> PLoS One. 8, e74343(2013).</li>
<li>Morrow, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+exoenzymes+U+and+Y+induce+a+transmissible+endothelial+proteinopathy%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Molec+Physiol%22%5BJournal%5D)">Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy.</a> Am J Physiol Lung Cell Molec Physiol. 310 (4), L337-L353 (2016).</li>
<li>Balczon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+infection+liberates+transmissible%2C+cytotoxic+prion+amyloids%5BTitle%5D)+AND+%22FASEB+Journal%22%5BJournal%5D)">Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.</a> FASEB Journal. 31 (7), 2785-2796 (2017).</li>
<li>Stevens, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Pseudomonas+aeruginosa+exoenzyme+Y+impairs+endothelial+cell+proliferation+and+vascular+repair+following+lung+injury%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Molec+Physiol%22%5BJournal%5D)">The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury.</a> Am J Physiol Lung Cell Molec Physiol. 306 (10), L915-L924 (2014).</li>
<li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creutzfeldt-Jakob+disease+and+scrapie+prions%5BTitle%5D)+AND+%22Alzheimer+Dis+Assoc+Disord%22%5BJournal%5D)">Creutzfeldt-Jakob disease and scrapie prions.</a> Alzheimer Dis Assoc Disord. 3 (1-2), 52-78 (1989).</li>
<li>Hunter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scrapie%3A+Uncertainties%2C+biology%2C+and+molecular+approaches%5BTitle%5D)+AND+%22Biochem.+Biophys.+Acta%22%5BJournal%5D)">Scrapie: Uncertainties, biology, and molecular approaches.</a> Biochem. Biophys. Acta. 1772 (6), 619-629 (2007).</li>
<li>King, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+functional+characteristics+of+lung+macro-+and+microvascular+endothelial+cell+phenotypes%5BTitle%5D)+AND+%22Microvasc+Res%22%5BJournal%5D)">Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes.</a> Microvasc Res. 67 (2), 139-151 (2004).</li>
<li>Marmont, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligomeric+lipoprotein+PelC+guides+Pel+polysaccharide+export+across+the+outer+membrane+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Oligomeric lipoprotein PelC guides Pel polysaccharide export across the outer membrane of Pseudomonas aeruginosa.</a> Proc Natl Acad Sci USA. 114 (11), 2892-2897 (2017).</li>
</ol>

None to report.

Here, simple in vitro methods are outlined which allow demonstration of the generation of cytotoxic amyloids during infection with a pneumonia causing organism. These methods include a cell culture cytotoxicity assay, immunoblotting, quantitation of cell killing using a novel microscopic method, and ThT binding. Analyses of the cytotoxic agents have demonstrated that they are amyloid in nature (Figure 2 and Figure 4) and exhibit charact...

Patients who survive pneumonia have elevated death rates in the months following hospital discharge1,2,3,4,5,6. In most cases, death occurs by some type of end-organ failure including renal, pulmonary, cardiac, or liver events, as well as stroke5,6. The reason for the elevated death rate in this patient population has never been established.
Pneumonia is classified as being either community-acquired or hospital-acquired (nosocomial), and agents that can cause pneumonia include bacteria, viruses, fungi, and chemicals. One of the major causes of nosocomial pneumonia is the bacterium Pseudomonas aeruginosa. P. aeruginosa is a gram-negative organism that uses a type III secretion system to transfer various effector molecules, termed exoenzymes, directly to the cytoplasm of target cells7,8. During infection of pulmonary endothelial cells, the exoenzymes target various intracellular proteins, including an endothelial form of the microtubule-associated protein tau9,10,11,12, leading to endothelial barrier breakdown resulting in severe pulmonary edema, decreased pulmonary function and, oftentimes, death.
As stated previously, patients who survive the initial pneumonia have elevated death rates in the first 12 months following hospital discharge. A potential mechanism for explaining this phenomenon is that some type of long-lived toxin is generated during the initial infection that leads to poor long-term outcome. Two observations support this possibility. First, cultured pulmonary endothelial cells that are treated initially with P. aeruginosa fail to proliferate for up to a week after the bacteria are killed by antibiotics13. Second, long-lived prions and agents with prion characteristics have been demonstrated in various human and animal diseases, particularly diseases associated with the nervous system14,15.
Methods for examining the potential production of long-lived cytotoxic agents during pulmonary infection have never been described. Here a series of simple in vitro assays are outlined that can be used for investigating cytotoxin production and activity following infection using a common pneumonia causing agent, P. aeruginosa. These assays should be readily adaptable to investigate possible cytotoxin induction following infection using other agents that cause pneumonia, and the supernatants that are generated also should be useful for investigating effects of the cytotoxins in whole organs or animals. Finally, the assays that are outlined here most likely will be adaptable to test animal and human biological fluids for the production of cytotoxins during pneumonia.

<table><tbody><tr><td>Rabbit anti beta amyloid</td><td>Thermo</td><td>71-5800</td><td></td></tr><tr><td>A11 amyloid oligomer antibody</td><td>StressMarq</td><td>SPC-506D</td><td></td></tr><tr><td>T22 anti-tau oligomer antibody</td><td>EMD Millipore</td><td>ABN454</td><td></td></tr><tr><td>Thioflavin T</td><td>Sigma Aldrich</td><td>T3516</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>0.22 micron syringe filters</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>HRP Goat antirabbit IgG</td><td>Abcam</td><td>ab6721-1</td><td></td></tr><tr><td>Strains PA103 and &amp;Delta;PcrV</td><td></td><td></td><td>These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI</td></tr><tr><td>FITC Griffonia lectin</td><td>Sigma Aldrich</td><td>L9381</td><td></td></tr><tr><td>TRITC Helix pomatia lectin</td><td>Sigma Aldrich</td><td>L1261</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>0.22 micron nitrocellulose</td><td>BioRad</td><td>162-0112</td><td></td></tr><tr><td>Type 2 collagenase</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30898.03IH</td><td></td></tr><tr><td>PenStrep</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Carbenicillin Disodium salt</td><td>Sigma&amp;nbsp;</td><td>C1389</td><td></td></tr><tr><td>Microcetrifugation concentrators- 10,000 MW cut-off</td><td>Millipore</td><td>UFC801008</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma&amp;nbsp;</td><td>795496-500G</td><td></td></tr><tr><td>Magnesium phosphate heptahydrate</td><td>MP Biomedicals</td><td>MP021914221</td><td></td></tr><tr><td>Citric Acid</td><td>G Biosciences</td><td>50-103-5801</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma</td><td>S9506-500G</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10277</td><td></td></tr></tbody></table>

methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by pseudomonas aeruginosa

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

A simple in vitro assay has been developed to assay for the presence of cytotoxins in supernatants of cells infected with the bacterium P. aeruginosa. Basically, culture medium from infected cells is collected 4 h after bacterial addition, the bacteria are removed by filter sterilization of the culture supernatant, and then the sterile supernatant is added to a new population of cells. The cells are then observed 21 - 24 h after the addition of supernatant and cell killi...

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

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Research

JoVE Journal

Immunology and Infection

Source: Balczon, R., et al. Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa. J. Vis. Exp. (2018)This video demonstrates the protocol to quantify amyloids in culture supernatants from pulmonary endothelial cells infected with virulent bacteria, using thioflavin T, a fluorescent dye that binds to amyloid beta-sheet structures. An increase in fluorescence compared to baseline indicates the presence of amyloids in the culture...

Quantifying Bacterial Infection-Induced Amyloids Using Thioflavin T Fluorescence

Source: Balczon, R., et al. Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa. J. Vis. Exp. (2018)This video demonstrates the procedure for generating cytotoxic supernatant from pulmonary endothelial cells following infection with Pseudomonas aeruginosa. It outlines how bacterial effector proteins disrupt host cell structure and induce the release of cytotoxic amyloid oligomers into the supernatant.

Obtaining Cytotoxic Amyloids Following Bacterial Infection of Rat Pulmonary Endothelial Cells

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Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

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Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

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P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

Antibiotic resistance amongst adherent-invasive Escherichia coli strains poses challenges for assessing bacterial invasion using the traditional gentamicin protection assay. An improved method to evaluate bacterial invasion quantitatively by enumerating colony-forming units and qualitatively through visualization of infected cells by immunofluorescence confocal microscopy is described.

Amikacin Protection Assay for Quantification and Visualization of Escherichia coli Cell Invasion

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Learn methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by Pseudomonas aeruginosa. This reliable protocol enables repeatable data generation for studying bacterial pathogenesis.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

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