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

Summary

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.

Abstract

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—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—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.

Introduction

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. aeruginosa^1,2,3,4.

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 others^{9,10,11,12,13,14,15,16}. 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 assays^17,18 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'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.

Protocol

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

1. Streak P. aeruginosa from a frozen stock onto an LB (Lysogeny Broth) agar plate. Incubate 16 to 24 h at 37 °C
   Note: Perform P. aeruginosa experiments inside of a BSL-2 biological safety cabinet to ensure sterility. Use appropriate safety precautions to minimize the chance of pathogenic infection or contamination.
2. Transfer this plate to 4 °C. This plate may be retained at 4 °C and used to inoculate cultures for up to one week. After one week, decontaminate and discard the plate and make a new LB streak plate.
   Note: P. aeruginosa virulence decreases after prolonged storage.
3. Two days prior to setting up assay plates, inoculate 3 - 5 mL of sterile LB broth with a single colony of P. aeruginosa from the plate made in 1.1. Incubate at 37 °C for 12 to 16 h.
   Note: Do not incubate longer than 16 h. In rich liquid cultures, P. aeruginosa PA14 begins to lyse.
4. Prepare sterile 10 cm plates with Slow Kill media (3 g NaCl, 3.5 g peptone, 1 mM CaCl₂, 1 mM MgSO₄, 25 mL of phosphate buffer (amount per liter: 132 mL of K₂HPO₄ (1M) and 868 mL of KH₂PO₄ (1M)) and 18 g agar/L).
   Note: Prepare plates ahead of time. They can be stored for up to 3 weeks in an airtight container at 4 °C.
5. Seed each 10 cm Slow Kill plate (SK plate) with 350 μL of P. aeruginosa from fresh overnight LB culture. Using a sterile bacterial spreader, spread bacteria evenly across surface of media and allow to dry in a BSL-2 biological safety cabinet.
6. Incubate plates at 37 °C for 24 h.

2. Preparation of RNAi Bacteria

1. Streak out or pin-transfer desired strains of RNAi-containing bacteria onto LB agar plates containing appropriate antibiotics (carbenicillin at 100 µg/mL and tetracycline at 15 µg/mL for Ahringer and Vidal libraries) from a frozen stock.
   Note: When working with nonpathogenic bacteria, use either a BSL-2 A/B biological safety cabinet or a BSL-1 laminar flow hood to ensure sterility of the cultures and to minimize the chance of cross-contamination of the library.
2. Incubate plates for 24 h at 37 °C.
3. Store the plates at 4 °C for up to two weeks.
   1. After two weeks, discard the plates and replace them with freshly made plates.
4. Test multiple RNAi strains in parallel by individually inoculating a single colony from each clone into 4 mL of carbenicillin-supplemented LB in a single well of a 24-well deep-well plate or into a sterile test tube.
   Note: Tetracycline has been reported to reduce the efficiency of RNAi. Do not use it in this media.
5. Place 24-well deep well plates into a shaking incubator optimized for multiwell plates and incubate at 37 °C for 16 h while shaking at 950 rpm.
   Note: It is hard to obtain uniform bacterial growth in multiwell plates using a conventional shaking incubator. The shaking incubator needs to be able to shake at a minimum of 750 rpm in order for the cultures to grow properly. In the absence of a specialized shaker, it is possible to grow each culture in an individual tube. Cultures can be transferred into the 24-well plate for centrifugation afterward, if desired.
6. Collect bacteria by centrifuging for 5 minutes at 2,000 x g.
7. Decant supernatant by inverting the plate and shaking vigorously. Resuspend RNAi bacteria in 100 µL of S Basal (5.85 g NaCl, 1 g K₂HPO₄, 6 g KH₂PO₄ dissolved into 1 L of ultrapure water and sterilized by autoclave).
8. Pipette resuspended bacteria into an appropriate number of wells of a multiwell NGM plate (Nematode Growth Media, 3 g NaCl, 2.5 g peptone, 1 mM CaCl₂, 1 mM MgSO₄, 25 mL of phosphate buffer (amount per liter: 132 mL of K₂HPO₄ (1M) and 868 mL of KH₂PO₄ (1M)), and 18 g agar per liter of water) supplemented with IPTG (1 mM final concentration) and carbenicillin (100 μg/mL final concentration).
   1. Use 3.5 mL of NGM media per well of 6-well plate, 1 mL per well of a 24-well plate, or 150 µL per well of 96-well plate.
   2. Use 100 μL/well of bacteria for 6-well plate; 50 μL/well for 24-well; or 20 μL/well for 96-well plate. Allow to dry.
      Note: Drying is normally performed in a sterile flow hood to promote rapid, uniform drying. Uniform drying is very important. Avoid over-drying, as cracks in the agar promote burrowing of worms. This leads to worm losses and potential clogging of liquid-handling systems.
9. Use the prepared plates immediately or store them at 4 °C for up to two weeks for later use.
   Note: To prevent plates from drying out, they can be sealed with plastic paraffin film or placed in a tightly-sealed container with damp paper towels.

3. Maintenance and Preparation of C. elegans

Note: Before initiating experiments, generate a synchronized population of gravid, adult hermaphroditic worms as follows.

1. Wash gravid adults (i.e., with multiple embryos visible within the gonad) from 4 - 6 10-cm NGM plates into a 15 mL conical tube by adding 4 mL of S Basal to plate, swirling gently, and then pouring into a 15 mL conical tube.
2. Pellet worms via centrifugation at 1,000 x g for 30 seconds.
3. Aspirate supernatant, taking care not to disrupt the worm pellet.
4. Add 3.5 mL of worm bleach solution (final concentration 0.5M NaOH, 1% sodium hypochlorite, in sterile water) to the worm pellet, and mix by inverting for 1 minute.
5. Briefly vortex and centrifuge at 1,000 x g for 30 seconds.
6. Aspirate or decant the supernatant, taking care not to disturb the worm pellet.
7. Add 3.5 mL of fresh worm bleach solution to the worm pellet.
8. Vigorously mix worms in bleach solution. Periodically (approximately every ten seconds) visually inspect worms under a dissecting microscope. Watch for worm cuticles to break, releasing eggs. Once most of the adult worms have been dissolved (~95%), dilute bleach to 15 mL with S Basal to stop digestion.
   Note: Egg shells are more resistant to bleach than adult tissues, but they can be dissolved during prolonged exposure. To avoid egg dissolution, do not expose eggs to worm bleach solution for longer than 7 minutes. If egg shells are excessively damaged, the embryos will die.
9. Pellet embryos at 1,000 x g for 30 seconds and aspirate or decant the supernatant without removing the pellet of embryos.
10. Resuspend worms in S basal to a final volume of 15 mL to dilute remaining bleach solution.
11. Repeat the washing steps (3.9 - 3.10) three more times for a total of 4 consecutive washes.
12. After the fourth wash, aspirate supernatant and add up to 5 or 6 mL of sterile S Basal into the 15 mL conical tube. The goal is to resuspend embryos to a concentration of ~50 worms per µL.
    Note: The amount of S Basal depends upon the size of the egg pellet; the larger the pellet, the more S Basal is necessary.
13. Incubate the conical tube overnight on a table-top rotator at room temperature or 48 h at 15 °C. Larvae will hatch, but larval development will arrest at the L1 stage (L1 diapause) due to nutrient deprivation.
14. Examine the embryos under a dissecting microscope to verify that most of them have hatched and arrested at the L1 stage of development.
15. Determine the worm count by taking 20 µL of worm prep and diluting it with 80 µL of S Basal. Pipette 10 µL of diluted worm solution directly onto a blank NGM plate. Repeat 2 more times.
    1. Count the larvae in each spot and determine the average number. Divide this average by 2 to obtain an approximate count of worms per µL in the worm prep. Worm preps can then be used for propagation plates or for experiments.
       Note: After 4 - 5 days, starved larvae will begin to die; discard the prep at this point.
16. For propagating the strain, pipette an appropriate number of L1 worms (5,000 - 6,000) onto 3 - 4 10-cm NGM plates spotted with concentrated OP50 E. coli – “superfood” (E. coli OP50 spotted at 25X concentration (i.e., 1 L of overnight OP50 culture grown in LB yields 40 mL of 25X OP50 superfood); 2 mL of this concentrated bacterial suspension are spotted onto each 10 cm NGM plate).
17. To prepare plates for experiments, do one of the following (for “Basic Protocol” use 3.17.1 setup, for the “RNAi screen set up” use steps 3.17.2 - 3.17.4 as appropriate):
    1. Pipette ~ 5,000 worms/10 cm plate seeded with RNAi or OP50 superfood.
    2. Pipette ~ 500 worms/well in a 6-well plate seeded with RNAi.
    3. Pipette ~300 worms/well in a 24-well plate seeded with RNAi.
    4. Pipette ~100 worms/well in a 96-well plate seeded with RNAi.
       Note: For instructions on how RNAi was seeded please refer to steps 2.7 - 2.9 in Preparation of RNAi Bacteria.
18. If a fertile strain of worms is being used, incubate worms at 25 °C for ~44 - 48 h. Use these worms as quickly as possible after the population has reached the appropriate age. This minimizes the impact of egg hatching within the worms during the assay.
19. If the strain being used is temperature-sterile (e.g., fer-15(b26); fem-1(hc17) or glp-4(bn2)), incubate worms at 15 °C for ~16 h and then transfer to 25 °C for 44 h to complete development and prevent embryogenesis.

4. Liquid Killing Assay Setup (Basic Protocol)

Note: This is a protocol for one bacterial strain and one source of worms.

1. Using a cell scraper, remove the P. aeruginosa from an SK plate and resuspend in ~ 5 mL of S Basal. Scale up if needed. Measure the optical density of the bacterial suspension using a spectrophotometer (OD₆₀₀)
2. Prepare 24 mL of diluted stock of P. aeruginosa in S Basal at OD₆₀₀ ≈ 0.09 (3X final concentration). See 4.6.2 for final content per well, and scale volume of bacterial dilution accordingly.
3. Add 21 mL of liquid Slow Kill media (3 g of NaCl, 3.5 g of peptone/L supplemented with CaCl₂ and MgSO₄ to a final concentration of 1 mM). Using a multichannel pipette, transfer 45 µL of bacteria and media to each well of a 384-well plate.
4. Wash worms from their source (i.e., Step 3.17.1) into a 50 mL conical tube and allow worms to settle under gravitational force. Aspirate supernatant to 5 mL. Resuspend in a total of 50 mL S basal.
5. Repeat step 4.4 twice.
6. Using a worm sorter, sort approximately 22 worms into each well of the 384-well plate.
   Note: The setup drops each worm in ~1.1 µL of liquid. 22 worms will amount to 25 µL, bringing total assay volume to 70 µL/well.
   1. The final composition of each well consists of 70 µL. 45 µL of this volume is added as bacterial medium (0.03 OD₆₀₀ bacteria in 24 µL S Basal, and 21 µL SK supplemented with CaCl₂ and MgSO₄). The other 25 µL is added with the 22 worms.
   2. If the sorter used here (see Table of Materials) is unavailable, worms can be diluted to a final concentration of 1 worm/µL in S Basal and pipetted 25 µL/well using a multichannel pipette. However, results may exhibit increased variability. Alternatively, it is possible to use a peristaltic liquid dispenser, as described by Leung and colleagues¹⁹.
7. After worms have been sorted into the 384-well plate, seal the plate with a gas-permeable film and incubate plates at 25 °C for 24 - 48 h.
8. At the desired time, use a microplate washer to wash the 384-well plate with S Basal a total of 5 times.
   1. After the second wash, aspirate most of the media, leaving ~20 µL. Vigorously shake plates using a microplate vortexer for at least 30 seconds to loosen any debris from the bottom of the wells).
9. After the final wash, aspirate supernatant down to 20 µL. Add 50 µL of 0.98 µM nucleic acid stain (see Table of Materials)/well of the 384-well plate, for a final concentration of 0.7 µM.
10. Incubate at room temperature for 12 - 16 h. This dye will only stain dead worms. After the desired incubation period, wash plates using the microplate washer to remove any excess stain (a minimum of 3 washes).
11. For data acquisition, use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence (531 nm excitation and 593 emission).
    1. Use a low magnification objective to allow an image of the whole well to be captured.
    2. Alternatively, use flow vermimetry. This technique uses a large object flow cytometer as a worm fluorimeter, measuring the size and fluorescence of whole organism (i.e., C. elegans) in a fashion that is strongly similar to flow cytometry.
12. Use automated image analysis software (such as CellProfiler, a free image analysis studio based on MatLab http://cellprofiler.org/) to calculate the fluorescent and total worm areas per well in an unbiased fashion.
    Note: The quotient of these numbers represents the fraction death for each well. If one well had 7 stained worms out of 20 total, then the fraction death comprises 0.35 or 35%.
    1. Prior to first use, the automated image processing pipeline must be validated by manual scoring. This is done by comparing results from the automated pipeline to scores obtained by visually inspecting images and recording fractions of dead worms for each of them. The validation process ensures that the parameters for the automated imaging processing are accurate and represent the data correctly.

5. Adaptation for Screening Multiple C. elegans Strains or Knockdowns (RNAi Screen Setup)

Note: This is an RNAi screen described for a 24-well plate setup.

1. Add 1 mL of S Basal per well of a 24-well plate (see step 3.17.3). Gently agitate worms by shaking the plate, then transfer worms to an empty, sterile 24 deep-well plate. Allow worms to settle gravitationally (~5 minutes).
2. Aspirate supernatant, leaving approximately 1 mL. Add 7 mL of S Basal to each well.
3. Repeat steps 5.1 - 5.2 a minimum of 2 more times. After final wash, aspirate supernatant, leaving approximately 400 µL.
4. Using the Resampler function of the worm sorter, sort 22 worms from the 24-well plate worm suspension into each well of a 384-well plate (each well of a 24-well plate yields 8 - 12 wells of a 384-well plate).
   1. To avoid starvation, pipette bacteria into 384-well plates (either a strain that serves solely as food, such as OP50, or a pathogenic strain) prior to sorting.
      Note: Pathogens can be added by following steps 2.3 - 2.5 or 6.4 - 6.5, and food source bacteria can be diluted and added by following the same scheme as for P. aeruginosa.
5. After worms have been sorted into the 384-well plate, add small molecules or other experiment-specific materials.

6. Adaptation for E. faecalis

Note: Only the differences from P. aeruginosa assay are described.

1. Prepare a fresh source of E. faecalis by streaking bacteria from a frozen stock onto a BHI (Brain Heart Infusion) agar plate and incubating for 16 to 24 h at 37 °C.
   Note: Perform E. faecalis experiments inside of a BSL-2 biological safety cabinet to ensure sterility. Use appropriate safety precautions to minimize the chance of pathogenic infection or contamination.
2. Plates can be stored at 4 °C for up to one week. After this period, replace with a fresh plate.
3. The night before sorting worms, inoculate 3 - 5 mL sterile BHI with a single colony of E. faecalis. Incubate 12 - 16 h at 37 °C with agitation.
4. Add bacteria to the wells as for P. aeruginosa (3x bacteria in S Basal, OD₆₀₀≈0.09).
   Note: For E. faecalis, the final well composition is: E. faecalis (OD₆₀₀≈0.03), BHI = 10% v/v, with the remaining volume comprised of S basal. Remember that 25 µL of S basal will be delivered with worms.
   Note: E. faecalis produces thick biofilms that absorb the fluorescent stain, causing blurred images. Extensive washing may be insufficient to remove this biofilm. In this case, worms can be transferred to an empty plate using a multichannel pipette. To facilitate transfer, Tween 20 can be added to S Basal to a final concentration of 0.1% (v/v) Tween 20 in S Basal. This aids in removing the worms from the biofilm.

Results

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 liquid^7,20. Provided that the steps outlined above are followed...

Discussion

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...

Materials

List of materials used in this article

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