This research was supported by the Agriculture and Food Research Initiative competitive grant 2015-67012-22773 from the USDA National Institute of Food and Agriculture to K.L.H.

1. Preparation of Supernatant to Be Tested for Activity
<ol>
	<li>Prepare a 0.5 mg/ml stock solution of mitomycin C by dissolving the appropriate amount into sterile 0.1 M MgSO4 buffer (e.g. 1 mg mitomycin C/2 ml buffer). Store stock at 4 &#176;C in a light protected container (mitomycin C is light sensitive).</li>
	<li>Inoculate a single colony of P. syringae into 3 ml of King&#39;s medium B (KB) broth14. Incubate over night with shaking (200 rpm) at room temperature (21-25 &#176;C).</li>
	<li>The following morning, dilute the broth culture 1/100 into 3 ml of fresh KB broth. Incubate for 3-4 hr with shaking at room temperature. Add mitomycin C (0.5 &#181;g/ml, final concentration). Incubate the culture over night with shaking at room temperature. 
	NOTE: Mitomycin C causes double stranded DNA breaks, thus stimulating the cell&#39;s SOS response, leading to the production of both bacteriophage and bacteriocins.</li>
	<li>Pellet 1-2 ml mitomycin C induced cultures by centrifugation at 20,000 x g for 5 min in a bench top microcentrifuge.</li>
	<li>Remove and sterilize culture supernatants either by passing the supernatant through a 0.22 &#181;m pore size filter or by treating the supernatant with chloroform (100 &#181;l chloroform per 1 ml supernatant).
	<ol>
		<li>If using chloroform, vortex the mixture for 15 sec and let incubate at room temperature for 1 hr. Following incubation, centrifuge the mixture at 20,000 x g for 5 min. 
		NOTE: Chloroform efficiently kills cells by solubilizing their membranes. The benefit of using chloroform over filter sterilization is that it is cheaper and easier to scale-up if processing many (&#62;20) samples at a time. There may be a possibility that a given killing activity is lost following chloroform treatment as a result of partitioning into the organic phase.</li>
		<li>Remove the upper, aqueous phase using a 1 ml pipet to a fresh, sterile 1.5 or 2.0 ml microfuge tube. Be careful not to transfer any of the lower chloroform layer (it is better to remove less of the aqueous phase to avoid carry-over).</li>
		<li>Incubate the transferred supernatant uncapped in a fume hood to allow the residual chloroform to evaporate (several hours). Store supernatants at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Separation and Concentration of High Molecular Weight Bactericidal Compounds by Polyethylene Glycol (PEG) Precipitation
<ol>
	<li>To the sterile supernatant, add NaCl and PEG 8000 to 1 M and 10% final concentrations, respectively. Repeatedly invert the sample until both the NaCl and PEG are completely dissolved. Incubate samples in an ice bath for 1 hr or overnight at 4 &#176;C.</li>
	<li>Centrifuge samples at 16,000 x g for 30 min at 4 &#176;C. A pellet should form at the bottom of the centrifuge tube. Decant the supernatant and resuspend the pellet in desired volume (such as 1/10 or 1/100 of the original supernatant volume) of buffer (10 mM Tris, 10 mM MgSO4, pH 7.0) by repeated pipetting.</li>
	<li>Remove residual PEG by two sequential extractions with an equal volume of chloroform.
	<ol>
		<li>Combine chloroform with the resuspended pellet and vortex for 10 to 15 sec, then centrifuge the mixture at 20,000 x g for 5 min. Transfer the upper, aqueous phase to a fresh microfuge tube. Repeat this extraction until no white interface between the aqueous and organic phase is visible (usually 2 extractions total). Allow residual chloroform to evaporate from extracted supernatants in a fume hood.</li>
	</ol>
	</li>
</ol>
3. Preparation of Overlay and Testing Supernatants for Activity
<ol>
	<li>Inoculate a single colony of a P. syringae strain to be tested for sensitivity into 3 ml of KB, incubate over night with shaking at room temperature.</li>
	<li>The following morning, back dilute the culture 1/100 into fresh KB. Incubate 3-4 hr with shaking at room temperature.</li>
	<li>Prepare sterilized water agar by autoclaving a suspension 0.35-0.7% (w/v) agar in ultrapure water. 
	NOTE: A stock of soft water agar can be melted in a microwave and reused repeatedly, fresh overlay does not need to be prepared for each experiment. If water agar is reused, it is critical to ensure it is completely melted following microwaving. If not, the overlay will have a grainy texture upon solidifying that will make interpretation difficult. If this occurs, melt the agar for several minutes longer in the microwave than done previously.</li>
	<li>Maintain the molten soft agar in a 60 &#176;C water bath prior to use. Using a sterile serological pipet, transfer 3 ml of soft agar to a sterile culture tube. Return the culture tube to water bath to maintain in a molten state.</li>
	<li>To pour the overlay, first allow the molten agar to cool (it should feel warm but not hot to touch), but do not allow to solidify.</li>
	<li>In a sterile hood, inoculate 100 &#181;l of the tester strain culture into the soft agar and vortex to mix. Rotate culture by hand for 10-15 sec, then pour onto a bottom agar (KB agar). Tilt the plate in all directions to ensure the soft agar evenly covers the bottom agar. 
	NOTE: The bottom agar can be any solidified (1.5% agar) medium on which the test strain grows vigorously. For a standard 100 mm diameter Petri dish, use ~20 ml melted medium. The bottom agar can be prepared several weeks ahead of time (if maintained at 4 &#176;C without drying) or can be prepared the same day. If prepared the same day as performing the overlay, it is best to do so prior to step 3.4.</li>
	<li>Cover the plate and allow it 20-30 min to solidify. Be careful not to disturb the plate while solidifying.</li>
	<li>Once solidified, spot 2-5 &#181;l of supernatant (generated in sections 1 and 2, above) onto the overlay. Allow the plates to incubate over night at room temperature. Observe and record results the following morning. 
	NOTE: It may be useful to perform and spot from a serial dilution of the supernatant. This will allow researchers to distinguish between bacteriophage and bacteriocin clearing activity. In this case, it is recommended to perform 1:5 or 1:10 dilutions.</li>
</ol>

<ol>
	<li>Cavera, V. L., Arthur, T. D., Kashtanov, D., Chikindas, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriocins+and+their+position+in+the+next+wave+of+conventional+antibiotics.">Bacteriocins and their position in the next wave of conventional antibiotics.</a> Int J of Antimicrob Agents. 46 (5), 494-501 (2015).</li><li>Blaser, M. J., Cardon, Z. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+Predictive+Understanding+of+Earth's+Microbiomes+to+Address+21st+Century+Challenges.">Toward a Predictive Understanding of Earth's Microbiomes to Address 21st Century Challenges.</a> MBio. 7 (3), e00714-e00716 (2016).</li><li>Hibbing, M. E., Fuqua, C., Parsek, M. R., Peterson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+competition:+surviving+and+thriving+in+the+microbial+jungle.">Bacterial competition: surviving and thriving in the microbial jungle.</a> Nat Rev Microbiol. 8 (1), 15-25 (2010).</li><li>Michel-Briand, Y., Baysse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pyocins+of+Pseudomonas+aeruginosa.">The pyocins of Pseudomonas aeruginosa.</a> Biochimie. 84 (5-6), 499-510 (2002).</li><li>Ghequire, M. G. K., De Mot, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomally+encoded+antibacterial+proteins+and+peptides+from+Pseudomonas.">Ribosomally encoded antibacterial proteins and peptides from Pseudomonas.</a> FEMS Microbiol Rev. 38 (4), 523-568 (2014).</li><li>Cascales, E., Buchanan, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colicin+Biology.">Colicin Biology.</a> Microbiol and Mol Biol Rev. 71 (1), 158-229 (2007).</li><li>Brown, S. P., Le Chat, L., De Paepe, M., Taddei, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+Microbial+Invasions:+Amplification+Allows+Virus+Carriers+to+Invade+More+Rapidly+When+Rare.">Ecology of Microbial Invasions: Amplification Allows Virus Carriers to Invade More Rapidly When Rare.</a> Curr Biol. 16 (20), 2048-2052 (2006).</li><li>Grinter, R., Roszak, A. W., Cogdell, R. J., Milner, J. J., Walker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Crystal+Structure+of+the+Lipid+II-degrading+Bacteriocin+Syringacin+M+Suggests+Unexpected+Evolutionary+Relationships+between+Colicin+M-like+Bacteriocins.">The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins.</a> J Biol Chem. 287 (46), 38876-38888 (2012).</li><li>Hockett, K. L., Renner, T., Baltrus, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Co-Option+of+a+Tailed+Bacteriophage+into+a+Killing+Complex+in+Pseudomonas.">Independent Co-Option of a Tailed Bacteriophage into a Killing Complex in Pseudomonas.</a> MBio. 6 (4), (2015).</li><li>Iacobellis, N. S., Lavermicocca, P., Grgurina, I., Simmaco, M., Ballio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytotoxic+properties+of+Pseudomonas+syringae+pv.+syringae+toxins.">Phytotoxic properties of Pseudomonas syringae pv. syringae toxins.</a> Physiol Mol Plant Pathol. 40 (2), 107-116 (1992).</li><li>Baltrus, D. A., Hendry, T. A., Hockett, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+Genomics+of+Pseudomonas+syringae.">Ecological Genomics of Pseudomonas syringae.</a> Genomics of Plant-Associated Bacteria. , 59-77 (2014).</li><li>Gratia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+Relationships+between+Lysogenic+Bacteria+and+Particles+of+Bacteriophage.">Numerical Relationships between Lysogenic Bacteria and Particles of Bacteriophage.</a> Ann Inst Pasteur. 57, 652 (1936).</li><li>King, E. O., Ward, M. K., Raney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+simple+media+for+the+demonstration+of+pyocyanin+and+fluorescin.">Two simple media for the demonstration of pyocyanin and fluorescin.</a> J Lab Clin Med. 44, 301-307 (1954).</li><li>Heng, N. C. K., Wescombe, P. A., Burton, J. P., Jack, R. W., Tagg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Diversity+of+Bacteriocins+in+Gram-Positive+Bacteria+.">The Diversity of Bacteriocins in Gram-Positive Bacteria .</a> Bacteriocins. (Chapter 4), 45-92 (2007).</li><li>Kawai, Y., Saito, T., Uemura, J., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Detection+Method+for+Bacteriocin+and+Distribution+of+Bacteriocin-producing+Strains+in+Lactobacillus+acidophilusGroup+Lactic+Acid+Bacteria+Isolated+from+Human+Feces.">Rapid Detection Method for Bacteriocin and Distribution of Bacteriocin-producing Strains in Lactobacillus acidophilusGroup Lactic Acid Bacteria Isolated from Human Feces.</a> Biosci Biotechnol Biochem. 61 (1), 179-182 (2014).</li></ol>

The authors have nothing to disclose.

The soft-agar overlay technique described here has been widely applied for many decades by researchers interested in bacteriophages or bacteriocins. The main benefits of this approach are that it is simple, cheap and relatively easy to interpret. By combining the soft-agar overlay with PEG precipitation and serial dilution, antimicrobial agents can be separated into high vs. low molecular weight agents, and replicative vs. non-replicative agents (i.e. bacteriophage vs. tailocin, respectively). Finally, incorporation of targeted genetic manipulation (deletion and complementation) of putative bacteriocin or prophage loci can firmly establish the identity of a given antagonistic compound. We have recently used this method to describe a new bacteriophage-derived bacteriocin prevalent among Pseudomonas syringae strains9.
The growth media and conditions indicated in this protocol work well for Pseudomonas syringae and would likely suffice for other Gram-negative bacteria that grow vigorously under these conditions. However, researchers using this method will want to optimize the timing, culture medium, induction method, and incubation temperature for their system. The critical parameters include inducing the producing culture while in the logarithmic growth phase, as well as seeding the soft-agar overlay with sufficient logarithmic phase culture to ensure a uniform bacterial distribution, but not excessive culture, which will obscure potential clearing zones. There are many bacteriocins that are not induced as part of the SOS response, but rather are induced through peptide-based quorum sensing systems (particularly Gram-positive bacteriocins15), thus, a researcher may want to screen several different induction methods (such as modifying nutrient content of inducing medium, or allowing for extended incubation of the producing strain).
When using this technique, the soft-agar overlay must be thoroughly melted and maintained at 55-60 &#176;C prior to the addition of the seeding strain. We have had several experiments where the soft-agar was not thoroughly melted (though visually it appeared to be) and resulted in an overlay with a grainy appearance. Results from a grainy overlay can still be generally interpretable, however, this is dependent on the strength of the inhibition (weaker inhibition will be more difficult to observe). A final, confounding variable to consider when using this technique is that the temperature of the molten agar is allowed sufficient time to cool prior to inoculation with the seeding strain, so as to avoid killing the seeding strain. To avoid this issue, we ensure the soft-agar is warm, but not hot, to touch. Along these lines, we have also observed that if we give inadequate time for a seeding culture to acclimate to the molten agar, prior to pouring the overlay, we recover generally poor bacterial growth, which makes interpretation of specific inhibition difficult or impossible to interpret. This is why we allow 10-15 additional seconds of incubation between vortexing and pouring the overlay. The trade-off between maintaining the agar in a completely molten state (hotter is better) but not lethal to the seeding strain (hotter is not better) is one that will likely need to be determined by a researcher through trial and error.
If a PEG precipitation step is used, it would be beneficial to include a negative control where a sterile broth medium is processed identically to the culture supernatant. This will ensure that killing activity is not the result of residual PEG or chloroform within the sample supernatant.
As both bacteriophages and bacteriocins tend to be highly specific, it is not uncommon to encounter no apparent killing activity with a given combination of strains. This can result from a variety of strain-specific resistance mechanisms, one being that the tester strain either lacks or has an unrecognized version of the receptor needed for targeting by a given bacteriocin or bacteriophage.
An alternative method, bacteriocin screening method has been described by Kawai et al., but suffers from drawbacks and has not been widely adopted16. Specifically, this technique requires observing broth samples at time intervals that can be burdensome, and does not allow for the discrimination between bacteriophage-derived and bacteriocin-derived killing activities.
The main limitations of this technique is that it is not well suited for organisms that do not grow robustly (and thus will lack easily discernable clearing zones) or that harbor prophages or bacteriocins that are not robustly produced under laboratory conditions. Adaption of this technique to detect bacteriocins produced in environmental samples would both expand the utility of this technique and facilitate greater insight into the role of bacteriocins within natural settings.

Recently, there has been significant interest in deepening our understanding of microbial ecology (particularly the microbiomes of various environments), as well as new antibacterial compounds to use in combating antibiotic resistant pathogens1,2. An interconnecting theme between these interests is understanding antagonistic interactions among bacterial strains in their natural environment. There are numerous ways in which bacteria antagonize competitors3. Bacteriocins, a diverse group of proteinaceous, antibacterial compounds, have long been studied for their role in mediating interbacterial antagonism, with two of the most studied species being Pseudomonas aeruginosa4,5 and Escherichia coli6, important human pathogens. In addition to bacteriocins, induced prophages can also act as anticompetitor agents, allowing a strain to invade into a niche that is already colonized7. Pseudomonas syringae is a plant pathogen known to produce an array of antimicrobial agents, including single protein bacteriocins8, bacteriophage tail-derived bacteriocins9 (termed tailocins), as well as non-proteinaceous secondary metabolites10. Recently there has been interest in understanding how these antimicrobials influence the ecology of this organism, as well as how they can be harnessed to control plant disease11.
A widely utilized method for studying both bacteriocins and bacteriophage is the soft-agar overlay technique. This method was first described by Gratia in 1936 to aid in enumerating bacteriophage12,13.
Here we describe the application of the soft-agar overlay method, in combination with targeted genetic manipulation and polyethylene glycol (PEG) precipitation, in distinguishing among three different antimicrobial agents (a bacteriophage, a high molecular weight bacteriocin, and a low molecular weight bacteriocin) produced by a single bacterial strain. The benefit of this approach is that it is relatively simple and cheap, which is why, despite it being decidedly &#39;low-tech&#39;, it is still widely utilized.

<table border="0" cellpadding="0" cellspacing="0" width="717">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Mitomycin C</td>
			<td style="width:179px;">Santa Cruz Biotechnology</td>
			<td style="width:140px;">sc-3514</td>
			<td style="width:167px;">Carcinogenic. use appropriate PPE (i.e. gloves, eye protection, and face mask), particularly when preparing the stock solution.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>34854</td>
			<td>Acutely toxic, respiratory hazard. Use appropriate PPE (glove and eye shield), handle open containers within a chemical fume hood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene Glycol (PEG)</td>
			<td>Amresco</td>
			<td>0159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacteriological grade agar</td>
			<td>Genesee Scientific</td>
			<td>20-274</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of the soft-agar overlay technique to screen for bacterially produced inhibitory compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

The combination of the soft agar overlay and PEG precipitation can be used to identify and characterize different antimicrobial agents produced by the same strain. Figure 1 shows two strains of P. syringae (A and B) that are inhibited by a third strain of the same species. The two strains, however, are inhibited by different bacteriocins. The clearing zone on strain A exhibits a sharp edge, whereas the clearing zone on strain B is larger, and exhibits a non-sharp border. Genetic manipulations within the producing strain that specifically inactivate either the tailocin (tailocin deficient) or low molecular weight bacteriocin (bacteriocin deficient) confirm that strains A and B are sensitive to distinct bacteriocins. Figure 2 shows that bacteriophage-mediated killing can be distinguished from other non-replicative antimicrobials by comparing a dilution series. Because both bacteriocins and prophage can be induced by mitomycin C treatment, activities of these two agents can closely resemble each other (particularly if the activated phage is abundant). In the case of bacteriophage-mediated clearing, dilution of the supernatant will resolve into individual plaques (clearing zones) while bacteriocin-mediated clearing will not resolve into such plaques.
<img alt="Figure 1" src="/files/ftp_upload/55064/55064fig1.jpg" /> 
Figure 1: Phenotypic distinction of multiple antimicrobial agents produced by the same strain. Strain A is sensitive to a high molecular weight bacteriocin (tailocin) but not an alternative low molecular weight bacteriocin, as evidenced by lack of clearing in the tailocin deficient strain, but not the bacteriocin deficient strain. Conversely, strain B is insensitive to the tailocin, but is sensitive to the low molecular weight bacteriocin. The tailocin is efficiently recovered following PEG precipitation, while lower molecular weight bacteriocins are not. WT = wild type. <a href="http://cloudflare.jove.com/files/ftp_upload/55064/55064fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55064/55064fig2.jpg" /> 
Figure 2: Distinguishing between non-replicative antimicrobials and bacteriophages. (A) Dilution of a high titer of bacteriophages results in individual plaques that become less numerous (high titer top, low titer bottom). (B) Dilution of bacteriocins results in uniformly less clearing that does not resolve into individual plaques. <a href="http://cloudflare.jove.com/files/ftp_upload/55064/55064fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds at JoVE.com

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

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32.0K Views.  University of Arizona. We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria. 

Video: Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...