Name: a high-throughput, high-content, liquid-based c. elegans pathosystem
Uploaded: 2018-07-01T15:00:00
Description: Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

This study was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) Award RR150044, Welch Foundation Research Grant C-1930, and by the National Institutes of Health K22 AI110552 awarded to NVK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Caution: P. aeruginosa and E. faecalis are Biosafety Level 2 pathogens, and proper safety precautions must be taken to prevent accidental infection and to minimize contamination of surfaces. All media and materials that come into contact with pathogens must be sterilized and/or discarded. Further guidelines are available from the CDC publication Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition.
1. Preparation and Maintenance of P. aeruginosa...

<ol>
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This assay (or similar assays where other pathogens are substituted for P. aeruginosa or E. faecalis) is useful for a variety of purposes, including drug discovery. It is also useful for addressing fundamental biological questions, such as identifying virulence factors, the elucidation of host defense pathways, and determining the regulatory machinery involved in the host-pathogen interaction.
Although the P. aeruginosa Liquid Killing assay is robust, there are sever...

The identification and development of effective, broad-spectrum antibiotics, now almost a century ago, led to a watershed moment in public health where there was a wide-spread belief that infectious disease would be a scourge of the past. Within a few short decades, this optimism began to wane, as pathogen after pathogen developed resistance mechanisms that limited these once miraculous treatments. For some time, the arms race between drug discovery efforts and the pathogens seemed balanced. However, the misuse of antimicrobials has recently culminated in the emergence of pan-drug resistant strains of Klebsiella pneumoniae, Acinetobacter baumanii, Serratia marcescens, and P. aeruginosa1,2,3,4.
P. aeruginosa is an opportunistic, gram negative, multi-host pathogen that is a severe threat to patients with severe burns, those who are immunocompromised, or have cystic fibrosis. It is also increasingly identified as a causative agent in severe nosocomial infections, particularly due to its ongoing acquisition of antimicrobial resistance. To begin to address this threat, we have used the well-documented C. elegans-P. aeruginosa infection system5. Our lab has leveraged this system to develop a liquid-based, high-throughput, high-content screening platform to identify novel compounds that limit the ability of the pathogen to kill the host6. Intriguingly, these compounds seem to belong to at least three general categories, including antimicrobials7 and virulence inhibitors8. Other high-content drug discovery assays in C. elegans have been reported for Mycobacterium tuberculosum, Chlamydia trachomatis, Yersinia pestis, Listeria monocytogenes, Francisella tularensis, Staphylococcus aureus, Candida albicans, and Enterococcus faecalis, among others9,10,11,12,13,14,15,16. These types of assays have several well-recognized advantages, such as limiting false positive hits that may be toxic to both the host and the pathogen, increased likelihood of bioavailability compared to a chemical screen, and the ability to identify hits beyond simply limiting microbial growth, such as anti-virulents, immune stimulatory molecules, or compounds that otherwise tilt the balance of the host-pathogen interaction in favor of the former. Additionally, the compounds discovered in these screens are often effective in mammalian hosts.
It is worth noting that at least two other assays17,18 are available to carry out high-throughput screens in C. elegans in liquid. However, each of these assays is a modification that allows the prototypical intestinal-colonization assay, known as slow-killing, to be performed in liquid, increasing throughput and allowing compounds to be more readily screened. Careful characterization has conclusively demonstrated that the mechanisms of bacterial virulence are different between these assays and our liquid-based screen7. Since both types of virulence are observed in mammalian systems, it is important to consider which virulence determinant is most relevant for the experimenter&#39;s interests prior to assay selection.
Here we demonstrate an optimized version of the liquid-based C. elegans-P. aeruginosa assay. We also report the adaptation of our liquid-based assay method to accommodate the gram-positive bacterial pathogen Enterococcus faecalis. Like P. aeruginosa, E. faecalis is increasingly identified as a serious nosocomial threat with a growing armament of antimicrobial resistance pathways1. Although a previous method for high-throughput screening of E. faecalis exists14, it requires preinfection with the pathogen, which complicates the procedure and increases the likelihood of contaminating equipment like the COPAS FlowSort. Our protocol eliminates the need for pre-infection, improving the safety profile. Finally, we report a means by which either of these assays can be combined with feeding RNAi, allowing the user to search for host factors that play a role in the establishment of, or resistance to, infection.

<table>
	<tbody>
		<tr>
			<td>COPAS FP BioSorter</td>
			<td>Union Biometrica</td>
			<td></td>
			<td>Large object flow cytometer/worm sorter</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EL406 Washer Dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron Pro</td>
			<td>Infors HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 Deep-Well RB Block</td>
			<td>Thermo Fisher Scientific</td>
			<td>CS15124</td>
			<td></td>
		</tr>
		<tr>
			<td>384-Well plate</td>
			<td>Greiner Bio-One</td>
			<td>MPG-781091</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Media (NGM)</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) plates</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) media</td>
			<td></td>
			<td></td>
			<td>Amount per liter:&#160; 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>USBiological Life Sciences</td>
			<td>L1520</td>
			<td></td>
		</tr>
		<tr>
			<td>Brian Heart Infusion broth (BHI)</td>
			<td>Research Products International Corp</td>
			<td>50-488-526</td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Bleach Solution</td>
			<td></td>
			<td></td>
			<td>Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water</td>
		</tr>
		<tr>
			<td>S Basal</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 5.85 grams NaCl, 6 grams KH2PO4, 1 gram K2HPO4, and 1 Liter of milli-Q water</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>USBiological Life Sciences</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>USBiological Life Sciences</td>
			<td>S5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>USBiological Life Sciences</td>
			<td>P3300</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>USBiological Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffer</td>
			<td></td>
			<td></td>
			<td>amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Acros Organics</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>USBiological Life Sciences</td>
			<td>P5100</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Sodium Hypochlorite Solution</td>
			<td>BICCA</td>
			<td>7495.5-32</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution</td>
			<td>Fisher Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-easy</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Nucleic Acid Stain</td>
			<td>Fisher Scientific</td>
			<td>S11368</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P. aeruginosa (PA14)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. faecalis(OG1RF)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli superfood (OP50)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAi expressing bacteria (HT115)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput, high-content, liquid-based c. elegans pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

Important parameters for assay performance
A proper understanding of the biology underlying this assay is necessary for troubleshooting and optimizing the assay. To that end, we refer first to several key papers elucidating the mechanisms of pathogenesis of P. aeruginosa-mediated killing in liquid7,20. Provided that the steps outlined above are followed...

Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

This method can answer questions in host pathogen interaction and drug discovery, such as the the importance of various virulence factors and the ability of small molecules to ameliorate with the genesis. The main advantage of this technique is that it takes place in a liquid format with small volumes. To begin, while working inside a BSL 2 biosafety cabinet, streak P.aeruginosa from a frozen stock onto an LB Agar plate.Incubate the plate at 37 degrees C for 16 to 24 hours, then store the plate at four degrees Celsius for up to one week. Two days prior to setting up assay plates, use a single colony from the plate to inoculate three to five milliliters of sterile LB broth. Incubate the culture at 37 degrees Celsius for 12 to 16 hours.After preparing the slow kill or SK media according to the text protocol, with 350 milliliters of p. Aeruginosa from the fresh overnight LB culture, seed each 10 centimeter SK plate. Use a sterile bacterial spreader to evenly spread the bacteria, and allow the plates to dry in the biosafety cabinet.Then, incubate the plates at 37 degrees Celsius for 24 hours. To test multiple RNAi bacterial strains in parallel in a single well of a 24 well deep well plate, inoculate a single colony from each clone of previously prepared RNAi containing bacteria into four milliliters of carbenicillin supplemented LB.Place the plates into a shaking incubator optimized for multi-well plates at 37 degrees Celsius and 950 rpm for 16 hours, then collect the bacteria by centrifugation at 2, 000 g for 5 minutes. Decant the supernatant by inverting the plate and vigorously shaking it.Using 100 microliters of S Basal resuspend the RNAi bacteria. Pipette the resuspended bacteria into an appropriate number of wells of a multi-well NGM plate supplemented with IPTG and carbenicillin and allow the plate to dry. After preparing L1 worms according to the text protocol, prepare plates for basic experiments by pipetting approximately 5, 000 worms per 10 centimeter plate, seeded with RNAi or OP50 superfood.To set up for an RNAi screen, pipette approximately 300 worms per well in a 24 well plate seeded with RNAi. If the strain being used is temperature-sterile, incubate the worms at 15 degrees Celsius for approximately 16 hours and then transfer them to 25 degrees Celsius for 44 hours to complete development and prevent embryogenesis. To carry out a liquid killing assay use a cell scraper to remove the P.aeruginosa from an SK plateand resuspend the bacteria in approximately 5 milliliters of S.Basal.Using a spectrophotometer meaure the OD 600 of the bacterial suspension. Prepare 24 milliliters of diluted stock of P.Aeruginosa in S.Basal at an OD 600 equal to 09, then add 21 milliliters of liquid SK media, and, using a multichannel pipette, transfer 45 microliters of bacteria and media to each well of a 384 well plate. Wash the worms from their source into a 50 milliliter conical tube, and allow them to settle under gravitational force.Aspirate the supernatant to 5 milliliters, then use a total of 50 milliliters of S.Basal to resuspend the worms, and repeat the wash twice more. Using a worm sorter, sort approximately 22 worms into each well of a 384 well plate, according to the text protocol, then use a gas-permeable film to seal the plate and incubate it at 25 degrees Celsius for 24 to 48 hours. At the desired time, use a microplate washer and S.Basal to wash the 384 well plate a total of 5 times.One of the easiest mistakes to make is to aspirate the 384 well plate before worms have completely settled. It takes practice to accurately see whether the worms have settled completely. After the final wash, aspirate the supernatant down to 20 microliters.Then, add 50 microliters of 98 micromolar nucleic acid stain per well, for a final concentration of 0.7 micromolar. Incubate the plate at room temperature for 12 to 16 hours. After the desired incubation period, use the microplate washer to wash the plates a minimum of three times.For a data acquisition use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence. Add one milliliter of S Basal per well of a 24 well plate containing 300 worms per well. Gently agitate the worms by shaking the plate, then transfer worms to an empty, sterile 24 deep well plate.Allow the worms to settle gravitationally, about five minutes. Aspirate the supernatant, leaving approximately one milliliter per well, Then add 7 milliliters of S Basal to each well. Repeat the wash two more times, and then after the final wash aspirate all but approximately 400 microliters of supernatant.After pipetting the bacteria into 384 well plates to avoid starvation, using the resampler function of the worm sorter sort 22 worms into each well of a 384 well plate. Finally, following sorting, add small molecules or other experiment specific materials. As illustrated in this graph, when the steps outlined in this video are followed, a time dependent killing of C.elegans will be observed only in the presence of P.aeruginosa.In contrast, in the absence of key bacterial nutritional supplements, little to no killing will be observed. As shown here, the two step incubation of P.aeruginosa at 37 degrees Celsius and then 25 degrees Celsius, which is critical for conventional slow killing assays, and was originally implemented in liquid killing, is dispensable in this assay. The protocol tolerates a wide range of initial bacterial concentrations, from as low as an OD 600 equal to 0025, and still exhibits time and concentration dependent killing, although the timing does shift.In addition, as few as four wells is frequently sufficient to obtain statistically significant data. An example of the utility of processing is shown here when the signal-to-noise ratio is high, as in this example, analysis is simple and discriminating between positive and negative conditions is trivial. In these cases, even weak hits can be readily identified.Once mastered, this technique can be performed in about 60 to 75 minutes of hands on time per 384 well plate, not including sorting. While attempting this procedure, it's critical to remember to add all the components to your media or virulence will be compromised. This assay simplifies the application of whole organism phenotype based screening to drug discovery in host-pathogen research.After watching this video, you should have a good understanding of how to carry out liquid-based C elegans pathogenesis assays. This procedure can be modified by substituting with other pathogens, such as E.Faecalis for P.Aeruginosa, facilitating the search for treatments for other bacterial infections. Don't forget that working with infectious bacteria like P.Aeruginosa or E.Faecalis can be dangerous.Appropriate precautions, such as proper training, good personal protective equipment, and proper technique, should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...