KP and AS wrote and revised the manuscript. KP performed the experiments and the analysis. This work was supported by the National Institutes of Health (NIH) Career Transition Award (K22AI112816) to AS.

All experimental procedures were carried out at the University of California, Irvine.
1. Buffers and Solutions
<ol>
	<li>Prepare Lysogeny Broth (LB) in a 1 L glass bottle by adding 25 g of LB-Miller mix in 1 L of double-distilled water (ddH2O). For Petri dishes, add an additional 20 g of agar. Autoclave to sterilize. Pour 25 mL of molten agar medium into 10 cm-diameter Petri dishes and allow to solidify at room temperature. Store liquid media at room temperature and agar plates at 4 &#176;C.</li>
	<li>Prepare Glucose Yeast Peptone (GYP) plates in a 1 L glass bottle by mixing 1 g of D-glucose, 2 g of peptone, 0.25 g of yeast extract, 4.2 g of KH2PO4, 5.1 g of Na2HPO47H2O, and 25 g of agar in 1 L of ddH2O. Autoclave to sterilize. Pour 25 mL of molten agar into 10 cm-diameter Petri dishes and allow to solidify at room temperature. Store at 4 &#176;C.</li>
	<li>Prepare Peptone S (PS) medium in a 1 L glass bottle by mixing 10 g of peptone, 7 g of yeast extract, 15 g of D-glucose, 0.12 g of Na2HPO47H2O, 1.4 g of KH2PO4, 40 &#181;g of vitamin B12, and 80 &#181;g of Folic acid in 800 mL of ddH2O. Calibrate an electronic pH meter using pH 4 and pH 7 standards if the pH meter has not been used within a day.
	<ol>
		<li>Measure the pH of the PS medium using the pH meter. Add either 5 M KOH or 5 M H3PO4 to adjust the pH to 6.5. Adjust the final volume to 1 L using ddH2O. Filter sterilize using 0.22 &#181;m vacuum filter and store at 4 &#176;C. 
		NOTE: Proceed cautiously with the pH adjustments by wearing protective equipment including gloves, protective clothing, eye protection, and face protection.</li>
	</ol>
	</li>
	<li>Prepare 10x Development Buffer Prime (DB&#39;) buffer in a 1 L glass bottle by adding 3.4 g of KH2PO4, 13.4 g of Na2HPO4 7H2O, and ddH2O to a total volume of 800 mL. Calibrate an electronic pH meter using pH 4 and pH 7 standards if the pH meter has not been used within a day.
	<ol>
		<li>Measure the pH of the buffer using the electronic pH meter. Adjust the pH to 6.5 by gently adding either 5 M KOH or 5 M H3PO4 as needed. Adjust the final volume to 1 L using ddH2O and sterilize by autoclaving. Store at room temperature. 
		NOTE: Proceed cautiously with the pH adjustments by wearing protective equipment including gloves, protective clothing, eye protection, and face protection.</li>
	</ol>
	</li>
	<li>Make 1x Development buffer (DB) by mixing 100 mL of 10x DB&#39; buffer with 1 mL of sterilized 2 M MgCl2, and 1 mL of sterilized 1 M CaCl2. Adjust the final volume to 1 L in a 1 L glass bottle using an autoclaved sterilized ddH2O. Store at room temperature.</li>
	<li>Prepare PS:DB medium in a 1 L glass bottle by mixing 100 mL of sterilized PS medium, 100 mL of sterilized 10x DB&#39; buffer, and sterilized ddH2O to a total volume of 1 L. Supplement with 1 mL of sterilized 2 M MgCl2 and 1 mL of sterilized 1 M CaCl2. Store at 4 &#176;C.</li>
	<li>Prepare the calcein-AM stock solution by adding 50 &#181;L of DMSO to 50 &#181;g of calcein-AM in the plastic container supplied by the manufacturer. Store at -20 &#176;C.</li>
</ol>
2. Growth and Maintenance of Amoebae
<ol>
	<li>Grow E. coli strain B/r17 as a food source for amoebae during the initial growth period. Streak E. coli B/r from a frozen stock stored at -80 &#176;C onto an LB agar plate and incubate at 37 &#176;C overnight to obtain single colonies.</li>
	<li>Inoculate a single colony of E. coli B/r into 2 mL of LB medium and incubate in a roller drum or a shaker at 37 &#176;C overnight.</li>
	<li>Bring the overnight E. coli culture to the room temperature. Using a wooden stick, inoculate amoebae from a frozen stock kept at -80 &#176;C into 750 &#181;L of the overnight culture. 
	NOTE: Use D. discoideum strain AX3, which is capable of axenic growth18. This step does not require axenic growth, but subsequent steps will have his requirement.</li>
	<li>Immediately add 700 &#181;L of the amoebae-E. coli mixture onto a Glucose Yeast Peptone (GYP) plate. Spread the culture evenly on the surface of the plate by tilting it back-and-forth and side-to-side as needed. Avoid the use of a spreader.</li>
	<li>Place the GYP plate with agar facing up in a box that maintains high humidity. Use two disposable plastic containers (183 cm x 183 cm x 91 cm) stacked on the top of each other. Fill the bottom container with approximately 25 mL of water and place the top container with several 0.5 cm-diameter holes drilled through the flooring of the container to allow moisture to move from the water reservoir to the top container.</li>
	<li>Incubate the GYP plate and box at 22 &#176;C for 4 - 5&#160;days until amoeba spores are observed growing near the surface of the plate (Figure 2). Use a refrigerated incubator to incubate plates rather than incubation at room temperature. 
	&#8203;NOTE: After spores have grown, they remain viable on plates for several weeks when stored at 4 &#176;C. Store the plates with agar side facing up. After 3 - 4 days of incubation, zones of clearing within the bacterial lawn can be observed, which indicate the onset of amoeba sporulation.
	<ol>
		<li>Observe small amoebae spores above the plate surface after 4 - 5 days of incubation (Figure 2). 
		NOTE: Do not grow the amoebae for longer than a week, as the&#160;quality of the spores decreases beyond this time period.</li>
	</ol>
	</li>
	<li>Collect amoebae spores to prepare for the axenic growth of the amoebae by sweeping a sterile 1 mL pipette tip parallel to the surface of the plate. Collect spores from half of a 10 cm Petri dish. 
	NOTE: Avoid touching the tip to the surface of the agar plate as this motion will collect large numbers of E. coli.</li>
	<li>Resuspend the amoebae on the tip of the pipette by immersing the tip into 1 mL of PS medium and gently pipetting up and down.</li>
	<li>Transfer the inoculated mixture into a 250-mL flask containing 25 mL of the PS medium supplemented with the antibiotic-antimycotic solution at 0.25x concentration. 
	NOTE: Store the antibiotic-antimycotic solution as 250 &#181;L aliquots at -20 &#176;C. Avoid repeated cycles of thawing and freezing of this solution as this may decrease its efficacy.</li>
	<li>Grow amoebae axenically at 22 &#176;C in a rotating shaker at 100 rpm. For the virulence assay in step 5, grow cells to an optical density measured at 600 nm (OD600) of 0.2 to 0.5. 
	NOTE: This step requires 3 - 4 days of growth from the time of inoculation (step 2.7). If cells have grown to a higher density, dilute the culture back in PS medium to an OD600 of 0.05 or lower and grow back to an OD600 of 0.2 to 0.5.</li>
</ol>
3. The Growth of P. aeruginosa
<ol>
	<li>Pick a single colony of P. aeruginosa from an LB plate, inoculate it into a 2 mL PS:DB culture, and grow overnight at 37 &#176;C to saturation. 
	NOTE: Do not add the antibiotic-antimycotic solution to the PS:DB media.</li>
	<li>Dilute the overnight culture 1:100 or 1:1,000 into a 6 cm-diameter Petri dish using PS:DB. If the virulence of planktonic cells is solely being assayed, grow the cultures using conventional culture tubes instead. Shake the culture on a benchtop rotator set at 100 rpm at 37 &#176;C.</li>
	<li>Harvest cultures at specific time points to ensure reproducibility. At 30 min to 1 h prior to assessing virulence, prepare an agar pad as described in section 4. 
	NOTE: Virulence in P. aeruginosa is induced by approximately 8 h of growth on surfaces.</li>
	<li>To isolate the surface-attached cells, remove the liquid medium from the Petri dish, wash immediately with 1 mL of DB buffer to remove planktonic cells, and proceed immediately to step 5.1. 
	CAUTION: Do not wash longer than 30 s as this will perturb and detach surface-attached cells and may affect the virulence measurement.</li>
	<li>To isolate planktonic cells, transfer 10 &#181;L of the culture from the Petri dish to a clean Petri dish and proceed immediately to step 5.1.</li>
</ol>
4. Microscopy - Preparation of the Agar Pad
<ol>
	<li>Prepare agar pads about 30 min to 1 h before amoebae are ready to be mixed with P. aeruginosa.</li>
	<li>Microwave 50 mL of 1% (w/v) agar in DB buffer in a 250 mL flask. Mix every 20 s until clumps of agar disappear.</li>
	<li>Cool the molten agar by placing it in preheated 55 &#176;C water bath for 15 min.</li>
	<li>Add 15 &#181;L of the calcein-AM stock solution to 15 mL of the molten agar in a conical tube. Mix by gently inverting the tube 4 - 5 times and proceed immediately to the next step. 
	NOTE: Perform this step in a polypropylene 15 mL centrifuge tube. Do not use a thick glass container as this will cause the agar to solidify too quickly.</li>
	<li>Pour the molten agar mixture on top of a 12 cm x 10 cm glass plate and allow the agar to solidify for 10 minutes at room temperature. Cut the solidified agar pad into smaller 1.5 cm x 1.5 cm sections by placing the glass plate over a printed grid and cutting along the grid using a metal ruler.</li>
	<li>Keep the gel hydrated in a humid box until it is ready for use.</li>
</ol>
5. Microscopy - Preparation of the Bacteria-amoeba Sample for Imaging
<ol>
	<li>To assay the virulence of surface-attached bacterial cells, add 10 &#181;L of amoeba cells from step 2.10 to surface-attached bacteria from step 3.4. 
	NOTE: This step describes the mixing of amoebae with bacteria.</li>
	<li>To assay the virulence of planktonic cells, mix 10 &#181;L of amoeba cells from step 2.10 with 10 &#181;L of planktonic bacteria from step 3.5. 
	NOTE: Do not wash the axenically-grown amoeba cells (at an OD600 of 0.2 to 0.5) before mixing with the bacteria. No E. coli growth will be observed in the amoeba culture due to the use of the antibiotic-antimycotic solution.</li>
	<li>Immediately place the 1.5 cm x 1.5 cm calcein-AM agar pad from step 4.5 on top of the bacteria-amoeba mixture on the Petri dish surface. 
	NOTE: Place the agar pad on top of the mixture using a quick smooth motion. Do not lower the pad slowly onto the mixture as this motion tends to push cells towards the periphery and away from the underside of the pad. This step will ensure that bacteria and amoebae are evenly mixed, immobilized and that both cell types are confined to the same plane.</li>
	<li>Remove excess liquid by gently pipetting out any remaining liquid surrounding the agar pad. Allow the Petri dish to dry for 20 min at room temperature.</li>
	<li>Cover with the Petri dish with a lid and incubate the immobilized amoeba-bacteria mixture at room temperature for an additional 40 min. Proceed to step 6.1. 
	NOTE: It is important to incubate all samples for the same amount of time, as the background fluorescence increases over time. An incubation time of 1 h is recommended. Incubations for longer than 1 h are less reproducible as amoebae cells may begin to burst.</li>
</ol>
6. Microscopy - Image Acquisition
<ol>
	<li>Image amoebae using a fluorescence microscope capable of acquiring brightfield and green fluorescence images. For green fluorescence, use green fluorescence protein (GFP) filters with 474/27 nm and 525/45 nm for excitation and emission, respectively. Use a 10X&#160;(&#8805;0.3 numerical aperture) objective to image the amoebae. 
	NOTE: The calcein-AM in the agar will cause dying amoebae to fluorescence in the green fluorescence channel. Healthy amoebae are otherwise not fluorescent.</li>
	<li>Adjust acquisition times for the phase contrast, GFP channels to limit phototoxicity, and optimize the dynamic range of the image intensities. Substitute the differential interference contrast (DIC) phase contrast if necessary. 
	NOTE: Use 100 - 200 ms exposures for phase contrast and GFP fluorescence if possible. Keep the exposure and instrument settings unchanged throughout the entire experiment. This is critical for computing the host killing index.</li>
	<li>Acquire images for at least 100 amoeba cells in order to obtain good statistics for calculating the host killing index.</li>
</ol>
7. Image Analysis and Host Killing Index
<ol>
	<li>Perform image analysis using ImageJ.</li>
	<li>Open the phase contrast image file in ImageJ by clicking on File | Open and selecting the image. Adjust the threshold by clicking on Image | Analysis | Threshold. Adjust the lower and upper threshold to select only the intensity peak (Figure 3A).</li>
	<li>Convert the image to binary by clicking Process | Binary | Make Binary. Fill holes by selecting Process | Binary | Fill Holes. 
	NOTE: Using the image in Figure 1 as the source, steps 7.1 - 7.2 will produce an image in which the amoebae and surface-attached bacteria appear as dark regions (Figure 3B).</li>
	<li>Ensure that the Area and Mean gray value boxes are checked in Analyze | Set Measurements. Measure the approximate size of the amoebae by selecting the oval tool on the main toolbar. Click Analyze | Measure and note the area of representative amoebae.</li>
	<li>Create masks for the amoebae by clicking Analyze | Analyze Particles. In the size box, enter the area that will include the amoebae but exclude the bacteria. Select Masks in the dropdown menu bar for the Show option. Ensure that the Add to Manager box is checked.</li>
	<li>Click OK and an image showing only the amoebae and an ROI Manager dialog will appear (Figure 3C).</li>
	<li>Open the fluorescence image using File | Open and selecting the appropriate file. Select all the regions in the ROI Manager by holding down the Shift Key and Clicking on each region in the list (Figure 3D).</li>
	<li>Click the Measure button in the ROI Manager. This will open a Results window displaying the mean intensity value of each amoeba.</li>
	<li>Copy the fluorescence values for each amoeba into a spreadsheet program and calculate the host killing index by taking the average of all fluorescence values.</li>
</ol>

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The authors have nothing to disclose.

This protocol describes a rapid and quantitative method to assay virulence in P. aeruginosa. This protocol may be tested with other bacteria. However, it is important to keep in mind that the growth medium should be compatible with the amoeba growth conditions. In particular, we have optimized the protocol using PS:DB as the bacterial growth medium. If other media are used, it may be necessary to perform a growth media-only control in which there are no bacterial cells present to verify that the medium is compat...

Bacterial infection is one of the leading causes of mortality in human and animals1,2. The ability to measure virulence of bacteria in cultures or biofilms is important in healthcare and research settings. Here, we describe a versatile, rapid, and relatively simple method to quantify bacterial virulence. The eukaryotic organism Dictyostelium discoideum (amoeba) is used as the model host organism. D. discoideum has been used as a host to identify virulence factors in Pseudomonas aeruginosa (P. aeruginosa)3,4,5 and other bacteria6,7,8 and is susceptible to largely the same virulence factors that kill mammalian cells including type III secretion9,10. Previous virulence assays using D. discoideum have involved prolonged exposure of bacteria with D. discoideum cells over the course of hours3,4,5. The protocol, here, presents a rapid method of determining virulence using this amoeba. This protocol (Figure 1) describes how to: (1) grow the amoebae axenically (in the absence of bacteria), (2) grow bacteria for the assay, (3) prepare bacteria and host cells for microscopy, (4) perform epifluorescence microscopy, and (5) analyze amoeba fluorescence.
Amoebae are initially streaked out from frozen stocks and grown on a lawn of Escherichia coli (E. coli), where the amoebae produce spores. These spores are picked and inoculated into an enriched medium for axenic growth. The amoebae are maintained through axenic growth in nutrient-rich conditions until they are ready to be mixed with bacteria for the assessment of bacterial virulence. The survival or the death of the amoebae is quantified by measuring the fluorescence of calcein-acetoxymethyl (calcein-AM), which is cleaved by intracellular esterases and, thereby, activated for fluorescence11,12. Live amoebae exhibit little or no fluorescence whereas stressed and dying cells fluoresce intensely. This result is due to a little or no incorporation of calcein-AM into healthy amoebae and incorporation and cleavage of the substrate in stressed amoebae13. This behavior is notably distinct from calcein-AM fluorescence in mammalian cells11,14,15,16.
Bacteria that will be assessed for virulence are grown separately. Here, we describe how to measure the virulence of the opportunistic pathogen P. aeruginosa and detail how to quantify the virulence of planktonic (swimming) and surface-attached sub-populations. This protocol may be adapted to test the virulence of other bacteria. In the Representative Results section, we show that virulence is activated in surface-attached cells and is low in planktonic cells, which was reported previously13. Virulence-activated surface-attached P. aeruginosa kills amoebae while non-virulent planktonic cells are consumed by the amoebae. If the virulence of planktonic bacteria is solely being assayed, bacteria can be cultured in ordinary culture tubes rather than using Petri dishes as described in the protocol.
The growth of amoebae and P. aeruginosa cultures must be coordinated such that P. aeruginosa cultures reach the intended growth phase while the amoebae are growing at steady state in nutrient-rich conditions. This condition typically requires amoebae cultures to be diluted at least 1 day prior to when they are mixed with bacteria. Amoebae and bacteria are immobilized using agar pads, are co-incubated for 1 h, and imaged using a low resolution (10X, numerical aperture 0.3) objective, green fluorescence protein (GFP) filters, and an imaging camera. Analysis can be performed using freely-available ImageJ software or customized image analysis software. Our analysis was performed using our own software written using a scientific analysis package13. The software should create a mask using the phase contrast image and extract fluorescence values from the masked areas in the fluorescence image. Fluorescence values are averaged over at least 100 cells, resulting in a numerical host killing index.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bacto agar, dehydrated</td><td>BD Difco</td><td>214010</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Life Technologies</td><td>15240062</td><td>Aliquot &amp;lt; 1 mL and store at -20 &amp;deg;C</td></tr><tr><td>Calcein-acetoxymethyl ester (calcein-AM)</td><td>Life Technologies</td><td>C34852</td><td>Calcein Acetoxymethyl (AM)</td></tr><tr><td>Calcium chloride, anhydrous</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Chemical</td><td>D16500</td><td>Dextrose</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Folic acid</td><td>Sigma-Aldrich</td><td>F8758</td><td></td></tr><tr><td>LB-Miller</td><td>BD Difco</td><td>244620</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Yeast extract</td><td>Oxoid</td><td>LP0021</td><td></td></tr><tr><td>Special peptone</td><td>Oxoid</td><td>LP0072</td><td></td></tr><tr><td>Potassium hyroxide</td><td>Fisher Chemical</td><td>P250</td><td></td></tr><tr><td>Potassium phosphate monobasic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Fisher Chemical</td><td>S373</td><td></td></tr><tr><td>Vitamin B12</td><td>Sigma-Aldrich</td><td>V2876</td><td></td></tr><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dictyostelium discoideum</td><td>Siryaporn lab</td><td>Strain AX3&lt;sup&gt;18&lt;/sup&gt;</td><td></td></tr><tr><td>Escherichia coli</td><td>Siryaporn lab</td><td>Strain B/r&lt;sup&gt;17&lt;/sup&gt;</td><td></td></tr><tr><td>Pseudomonas aeruginosa</td><td>Siryaporn lab</td><td>PA14</td><td>PA14 strain&lt;sup&gt;19&lt;/sup&gt;</td></tr><tr><td>Pseudomonas aeruginosa &amp;Delta;lasR</td><td>Siryaporn lab</td><td>AFS20.1</td><td>PA14-derived strain&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;micro;m filter&amp;nbsp;</td><td>Millipore</td><td>SCGPT01RE</td><td>For filter sterilization</td></tr><tr><td>Conical tube, 15 mL</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>Glass storage bottles</td><td>Pyrex</td><td>13951L</td><td>250 mL, 500 mL, 1000 mL</td></tr><tr><td>Petri dish, 6 cm diameter</td><td>Corning</td><td>351007</td><td>60 x 15 mm polystyrene plates</td></tr><tr><td>Petri dish, 10 cm diameter</td><td>Fisher</td><td>FB0875712</td><td>100 x 15 mm polystyrene plates</td></tr><tr><td>Plastic containers with lid</td><td>Ziploc</td><td>2.57E+09</td><td>Square 3-cup containers</td></tr><tr><td>Glass plates</td><td>Bio-Rad</td><td>1653308</td><td>For preparing agar pads. Other glass plates may be used with similar dimensions.</td></tr><tr><td>Wooden sticks</td><td>Fisher</td><td>23-400-102&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Eclipse Ti-E microscope&amp;nbsp;</td><td>Nikon</td><td>MEA53100</td><td>Microscope setup</td></tr><tr><td>10X Plan Fluor Ph1 objective&amp;nbsp; 0.3 NA</td><td>Nikon</td><td>MRH20101</td><td>Microscope setup</td></tr><tr><td>Fluorescence excitation source</td><td>Lumencor</td><td>Sola light engine</td><td>Microscope setup</td></tr><tr><td>Fluorescence filter set</td><td>Semrock</td><td>LED-DA/FI/TX-3X3M-A-000&amp;nbsp;</td><td>Microscope setup</td></tr><tr><td>Orca Flash 4.0 V2 Camera</td><td>Hamamatsu</td><td>77054098</td><td>Microscope setup</td></tr><tr><td>ImageJ</td><td>NIH</td><td>v. 1.49</td><td>Software for image analysis</td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>R2013</td><td>Software for image analysis</td></tr><tr><td>Orbital shaker incubator</td><td>VWR</td><td>89032-092</td><td>For growth of bacteria at 37 &amp;deg;C</td></tr><tr><td>Platform shaker</td><td>Fisher</td><td>13-687-700</td><td>For growth of amoebae at 22 &amp;deg;C</td></tr><tr><td>Spectrophotometer</td><td>Biochrom</td><td>Ultrospec 10</td><td></td></tr><tr><td>Undercounter refrigerated incubator</td><td>Fisher</td><td>97990E</td><td>For growth of amoebae at 22 &amp;deg;C</td></tr><tr><td>Isotemp waterbath&amp;nbsp;</td><td>Fisher</td><td>15-462-21Q</td><td>For cooling media to 55 &amp;deg;C</td></tr></tbody></table>

a rapid image-based bacterial virulence assay using amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

We grew wild-type P. aeruginosa strain PA1419 or a &#916;lasR strain20 in the same PA14 background in 6 cm-diameter Petri dishes and assayed the virulence of planktonic and surface-attached cells. Cultures were inoculated from single-colonies into PS:DB cultures, grown overnight in culture tubes in a roller drum at 37 &#176;C to saturation, diluted 1:100 into PS:DB, grown for 8 h in 6 cm-diameter Petri dishes shaking at 100...

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A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

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Research

JoVE Journal

Bioengineering

8.2K Views.  University of California. Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Video: A Rapid Image-based Bacterial Virulence Assay Using Amoeba

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Immunology and Infection

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 ...

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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.

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.

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

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Long-Term Tracking and Quantification of Acanthamoeba Species Motility

Quantification of Acanthamoeba spp. Motility

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential contamination of contact lenses after water exposure, the natural behavior of this pathogen remains elusive. Understanding Acanthamoeba's movement patterns can inform us about how it colonizes contact lenses and contaminates the patient's cornea. It is fortunate that Acanthamoeba spp. are visible via brightfield microscopy starting at 4x magnification. Previous techniques have been developed to quantify Acanthamoeba motility in regard to cytopathic effects or under-exposure to the electric field. Here, we describe a method to track and quantify Acanthamoeba spp. motility long-term (hours to days), which is a protocol applicable to multiple amoeba strains, surfaces, and nutritional status of amoeba. This procedure is germane to determining many core motility quantifications, such as distance, speed, confinement, and directionality, which are necessary to monitor different stages of infection, proliferation, or behavioral change.

Author Spotlight: Studying Behavior of Acanthamoeba to Develop Targeted Strategies for Preventing Acanthamoeba Keratitis

This procedure describes how to visualize, track, and quantify Acanthamoeba spp. motility.

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential ...

Biology

Assessment of Leishmania Virulence within Cultured Macrophages

Source: Sarkar, A. et al., <a href="https://app.jove.com/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of&#160;Leishmania&#160;Parasites.</a>&#160;J. Vis. Exp.&#160;(2018)
This video investigates Leishmania&#39;s virulence by treating macrophages with metacyclic promastigotes, resulting in the formation of intracellular amastigotes. The confirmation of Leishmania&#39;s virulence is achieved through nucleic acid fluorescent dye staining. This insight can contribute to the development of new therapies for leishmaniasis.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

Source: Smail, Y., et al. <a href="https://app.jove.com/v/57728">Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for detecting local and global calcium ion response upon Shigella infection of human epithelial cells. Shigella invades host cells and releases invasion effectors, impacting intracellular calcium levels. The amplitude and frequency of the local and global cytosolic calcium are studied using a fluorescent indicator.

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Summary

Explore More Videos

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

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

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Quantification of Acanthamoeba spp. Motility

Assessment of Leishmania Virulence within Cultured Macrophages

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells