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>

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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 characteristics of a prion12. The generation of cytotoxic prion molecules during pneumonia provides a potential explanation for the elevated death rates observed in pneumonia survivors following their hospital discharge. Experiments in progress are testing this possibility.
The key event for all of the described studies is the generation of the cytotoxic supernatant, and two important variables need to be considered. First, in the outlined experiments rat PMVECs were used for both the infection step and for the cytotoxicity assay. Whether other cell types could be used for production of cytotoxins has not been investigated in detail, and it is possible that other cells may be able to generate cytotoxic supernatants following treatment with various strains of P. aeruginosa. As shown, the cytotoxins are amyloid in nature and include oligomeric tau and A&#946;. It might be reasonable to expect that other cell types that express tau and/or beta amyloid protein might be induced to generate cytotoxins following bacterial inoculation, although this possibility remains to be tested. The outlined procedures could be used for examining whether other cell types can be induced to produce cytotoxic amyloids following different insults.
The second important variable in the generation of the cytotoxic supernatants is the duration of treatment with bacteria. It is essential that the incubation of the PMVECs be allowed to progress until gaps can be seen in the monolayer. The identification of gaps in the cell monolayer is an indication exoenzymes have been injected by the Pseudomonas bacteria into the host cell cytoplasm and that the exoenzymes are active, resulting in retraction of cell borders (Figure 1). Our experience is that the time point of the retraction of cell borders is a reliable indicator of cytotoxin release into the supernatant. It is tempting to speculate that the two events are related as one of the cytotoxic amyloids being released is oligomeric tau, and disruption of tau activity results in microtubule depolymerization which contributes to cell shape changes. In experiments where treatments have been less than the amount of time required to induce gap formation, little mature cytotoxic amyloid has been detected.
The cytotoxicity assay described in this manuscript is simple and reliable. Cell death can be observed easily by microscopic examination and quantified directly from recorded microscopic images. The methods that are described here have been used exclusively for assessing cytotoxin production following infection of rat pulmonary endothelial cells with P. aeruginosa. It seems likely that the procedures could be readily adapted to assess whether cytotoxic amyloids are produced following infection with other pneumonia causing agents, including both nosocomial-associated bacteria and bacteria and viruses responsible for community-acquired types of pneumonia. Moreover, the reported procedures have only been used to examine cytotoxic amyloid generation during infection of cultured cells. Cell culture is an artificial system, and the procedures outlined here most likely could be adapted to assess the generation of cytotoxins during pulmonary infection of both animal models and human patients. Collection of biological fluids, such as bronchoalveolar lavage, from either infected animals or patients could be used in the same types of assays reported here to assess whether cytotoxic amyloids are generated during infection processes in vivo. Obviously, the durations of incubation with bacteria might be quite different when inoculating into animals, and bronchoalveolar lavage would have to be collected at multiple time point to determine ultimate duration of treatment.
Immunoblot analyses have demonstrated that amyloids are present in the supernatants, including oligomeric tau and A&#946; (Figure 3). Although the immunoblot procedures are standard, there is a key step that must be performed prior to initiating immunoblot analyses. Specifically, it is critical that the supernatants be concentrated at least ten-fold prior to immunoblotting as the amount of amyloid in the un-concentrated supernatant is below the limit of detection by immunoblot analysis. As reported here, centrifugation using tubes equipped with a filter is the method routinely used for this concentration step, but it seems reasonable that other methods, such as TCA precipitation, would be equally as useful for this step.
The quantitation of cell killing using images represents an important advance. Presently available procedures for quantifying cell death rely on the purchase of fairly expensive kits that allow measurement of LDH release from dead cells. The assay described here involves no cost as ImageJ programs are free to download from the NIH website. The simple adaptation of ImageJ programs allows fairly accurate measurement of cell killing. However, it is worth pointing out that a couple of minor artifacts are encountered during performance of the outlined protocol. The major issue is that cell fragments, such as areas of focal contact, may remain associated with the culture dish as cells die. These residual cell fragments can be detected by the camera and give an artificial indication that cells remain on the dish. As such, it is rare that a value of 100% cell killing is obtained using this procedure.
A weakness of the reported assay system is that the cytotoxin amyloids in the preparation have never been completely defined. As such, quantitation is difficult. ELISA assays can be used to provide total levels of different amyloids, without providing any detailed information about the individual species in each preparation. Until more precise assays, such as mass-spec or in vitro synthesized amyloid oligomers, can be developed, the best that one can do at present is to start each experiment with the same number of cells, incubate for the same duration, and verify that cytotoxicity occurs at the same rates as previous preparations that have been used. Variation from any of the well-established norms introduces additional uncertainty.
In summary, simple in vitro methods are outlined for demonstrating the production of toxic amyloid molecules by pulmonary endothelial cells following infection with a pneumonia-causing agent. The assays are reliable and likely can be adapted for other organisms and for use using biological fluids obtained from infected animals and human patients. These methods will be useful for studies investigating the long-term consequences of pneumonia and for establishing the mechanisms leading to end-organ failure in patients following hospital discharge.

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

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 killing is quantified.
Addition of P. aeruginosa to confluent layers of PMVECs induces formation of gaps between the cells. In vivo, gap formation leads to pulmonary edema and decreased pulmonary function. In the described assay, gap formation is used as the time-point for screening for cytotoxin release into cell culture supernatants and sufficient duration of incubation with PA103 is indicated by the presence of gaps that begin to form in the cell monolayer as shown in Figure 1. &#916;PcrV bacteria do not induce cell gaps under these incubation conditions. Once gaps are detected, the supernatant is collected, filter sterilized, and then added to na&#239;ve PMVECs to assess cytotoxic activity. The PMVECs in their respective supernatants then are placed in an incubator for 21 - 24 h, and then observed and photographed. As shown in Figure 2, supernatant collected from PMVECs that had been infected with P. aeruginosa strain PA103 contained cytotoxins that induced death of cultured cells by 21 h after the addition of the supernatant. In contrast, no cell death was observed in control cells treated with either HBSS medium or with supernatant collected from the &#916;PcrV strain of Pseudomonas. Immunoblot analysis demonstrated that amyloid molecules, including oligomeric tau and A&#946;, are generated during infection with the PA103 strain, while no amyloids are generated by the &#916;PcrV strain following inoculation of PMVECs (Figure 2). The role of amyloids as the cytotoxins in this assay has been confirmed by immunodepletion of amyloids from the supernatant prior to addition to cultured cells12. Although not shown here, immunodepletion of cytotoxic supernatant using A11 anti-amyloid antibody and T22 anti-tau oligomer antibody completely depletes cytotoxic activity from supernatants produced following PA103 infection (Balczon et al. 201712 and Balczon et al., in preparation).
A novel, inexpensive, and simple method of quantification of cell killing has been developed. For this assay, ImageJ software has been adapted to allow direct measurement of areas in a microscopic field that lack cells (indicative of cell killing). The reliability of this method has been verified by direct comparison to the well-established method of measuring cell killing, which is LDH release from cells. As shown in Figure 3, ImageJ software can be used to convert regions of a microscopic field that contain cells to white and regions that lack cells to black. Then, a simple ratio of white areas to black can be used to measure cell killing. When starting with a confluent monolayer of cells, all regions of the microscopic field are white. However, by 18 h after the addition of supernatants containing cytotoxins, gaps in the monolayer could be detected. The amount of area of the culture dish devoid of cells then increased in a linear manner until 36 h post-addition of the cytotoxic supernatant. When measuring LDH release, identical results were obtained, with LDH release being first detected at 18 h after the addition of cytotoxic supernatant. LDH release then increased in a linear manner until maximal cell killing was measured at 36 h after the addition of supernatant. Little cell death was measured in cells treated with supernatant collected from &#916;PcrV inoculated cells (Figure 3) or in cultures treated with HBSS for 36 h.
The amount of amyloid released from cells treated with bacteria can be quantified by measuring ThT fluorescence. Binding of amyloids to ThT causes a conformational shift in the molecule and an increase in fluorescence intensity. For this assay, thioflavin T is added to a cuvette and baseline fluorescence is measured. The fluorescence recording is then stopped while a small aliquot of sample is added. The cuvette then is returned to the fluorimeter and the change is fluorescence emission is recorded. As shown in Figure 4, supernatant collected from cells that were incubated with PA103 strain of P. aeruginosa contained amyloid as indicated by the increase in fluorescence emission. In contrast, supernatant from PMVECs inoculated with &#916;PcrV strain lacked amyloids, as indicated by an absence of increased fluorescence.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57447/57447fig1.jpg" /> 
Figure 1. Effects of P. aeruginosa inolculation on PMVEC morphology. A confluent monolayer of PMVECs before (A) and four hours after the addition (B) of PA103 strain of P. aeruginosa. Gaps can clearly be seen in the cell monolayer in the infected cells. Scale bar = 100 &#181;m. <a href="/files/ftp_upload/57447/57447fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57447/57447fig2.jpg" /> 
Figure 2. Analyses of cytotoxic supernatants. Part [I]. Representative images of control and treated cells. PMVECs were treated for 21 h with supernatant obtained from cells inoculated with either PA103 bacteria (B) or strain &#916;PcrV (C). Control cells were also incubated for 21 h with HBSS buffer (A). Scale bar = 100 &#181;m. Part [II]. Representative immunoblots of supernatants collected from either &#916;PcrV (A) or PA103 (B) infected PMVECs. Blots were probed with antibodies against amyloid (A11) or oligomeric tau (T22). Mr in kDa. <a href="/files/ftp_upload/57447/57447fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57447/57447fig3.jpg" /> 
Figure 3. Quantitation of cell killing. (A) Representative microscope fields from different time points (in h after supernatant addition) in which regions of the field containing and lacking cells were converted either to white or black, respectively, using ImageJ software. The same phase contrast image before conversion is also shown. Bar = 100 &#181;m. (B) Comparison of quantitation of cell killing using standard LDH release assay and the ImageJ assay. N = 4, **p &#60;0.05, ***p &#60;0.01. <a href="/files/ftp_upload/57447/57447fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57447/57447fig4v2.jpg" /> 
Figure 4. Representative ThT fluorescence assay data. Blank cuvettes containing ThT were excited at 425 nm and then emission fluorescence was measured at 482 nm. Background fluorescence was collected for 20 s, and then supernatant from either PA103- or &#916;PcrV-inoculated PMVECs was added to the cuvettes. Fluorescence emission was then recorded for 40 sec.&#160;

Watch this Scientific Journal Video about Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa at JoVE.com

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

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

methods-for-detecting-cytotoxic-amyloids-following-infection

Universidad San Sebastian

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JoVE Journal

Immunology and Infection

5.5K Views.  University of South Alabama. Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

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

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

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

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

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Biochemistry

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

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

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

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

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

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

Summary

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Chapters in this video

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

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Pseudomonas aeruginosa Induced Lung Injury Model

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

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

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells