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

Summary

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage: sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Abstract

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 of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Introduction

Automatic worm sorting is much like FACS, operating by measuring a fluorescent signal in a worm (typically provided by transgenic expression of reporter proteins) as it passes straightened in a tube, allowing redirection either to a collection tube/well or to a waste container according to gating parameters set by the researcher¹. The wormsorter can facilitate research in many ways; an example of employing it as an analytical tool is a study that followed spatiotemporal patterns of promoter activity for almost 1,000 genes².
However, the major use of the wormsorter is in genetic screens, following target gene expression levels or localization of fluorescent protein along the axis of the worm^3-5.

Here, we describe a new application for the wormsorter, in following colonization of the worm by a fluorescently tagged pathogen. With this as a tool we focused on the relationship between pathogen colonization/load and the immune response, to gain new insights into the mechanisms responsible for initiation of immune responses in the worm.

In virtually all organisms studied to date, initiation of innate immune responses to microbial pathogens depends on recognition of pathogen-associated molecular patterns (PAMPs), and/or danger/damage-associated molecular patterns (DAMPs)^6,7. The first are conserved microbial structures that include components of the microbial cell wall, its flagellum, or its lipid bilayer⁶; the second, include both released molecules (e.g. ATP⁸), altered proteins or other markers of altered cellular processes^9,10. Both types of signals are recognized by proteins designated as pattern recognition receptors (PRRs), which upon specific binding of a pattern molecule activate a chain of events leading to a protective response. C. elegans has been extremely useful as a tractable model to dissect various aspects of host-pathogen interactions, but one thing that is not well understood is how immune responses are initiated in the worm. None of the putative receptors that are orthologous to pattern recognition receptors (PRRs) in other organisms have been shown to bind PAMPs, and many of the orthologs of PRRs that are pivotal for immune responses in other organisms show a surprisingly limited contribution to worm pathogen responses and resistance. For example, the Drosophila Toll receptor, which is essential for resisting Gram positive pathogens, is represented in C. elegans by a sole homolog, tol-1, which contributes to protection from the Gram negative pathogen Salmonella Typhimurium¹¹, but not from other tested Gram-negative, or -positive pathogens^11,12 . These observations, combined with data indicating that immune responses could be induced by disrupting cellular protein translation has led some to suggest that C. elegans primarily detects DAMPs^9,13,14. Nevertheless, reports describing ability of dead pathogens to induce immune responses suggest that PAMP binding may be have an important role in pathogen recognition in C. elegans^15,16. Previous work focusing on immune responses in age-synchronized genetically identical C. elegans populations, demonstrated large individual variability in intestinal colonization by the bacterial Gram-negative pathogen Pseudomonas aeruginosa.

However, transcriptional profiling studies treated these variably-colonized populations as one entity^17,18. Taking advantage of this variability, we developed a protocol focusing an automated wormsorter to separate differentially colonized populations of Caenorhabditis elegans exposed to GFP-expressing P. aeruginosa. Examining gene expression in differently-colonized populations facilitated assessment of the relationship between pathogen load (and the associated damage) and immune responses and provided new insights about pathogen recognition in C. elegans¹⁹. Below we describe the protocol, which could be applied to sort worms infected with any fluorescently labeled pathogen.

To potential users it should be noted that the number of worms required to be sorted out depends on the nature of the subsequent analyses and protocols in use. For example, in the case of microarray gene expression analysis, >1,000 worms will be required to obtain enough RNA, if standard protocols are used, but ~100 worms would suffice if amplification is employed, allowing fast collection of material and thus minimizing stress to the worms.

Protocol

1. Obtaining a Synchronized Culture of Young Adult Animals

1. Grow worms on several NGM plates seeded with OP50-1 E. coli bacteria (10x concentrated from a saturated culture) until many worms have reached the gravid stage.
2. Treat gravid animals with egg-prep solution to obtain a synchronized culture (eggs).
3. Plate eggs on several 60-mm NGM plates seeded with 10x concentrated OP50-1 at a density of roughly 150-200 eggs/plate.
4. Incubate plates at 25 °C for 2 days (until worms have reached the L4 - Young Adult stage).

2. Preparing Pseudomonas aeruginosa Plates and Infecting Worms

1. On the day of the egg-prep (step 1.1) inoculate a PA14::GFP culture into 2 ml of King’s B media containing rifampicin at a final concentration of 100 μg/ml. Incubate the culture at 37 °C with agitation O/N.
2. Plate the PA14::GFP culture onto as many Slow Killing Plates (SKP) as needed for the assay. On each 150 mm SKP Petri dish pipette 75-100 μl of saturated culture and spread evenly using a sterile glass spreader. Incubate the plates at 37 °C for 20-24 hr.
3. Remove plates from incubator and allow them to cool to RT (~20 min). Using M9 buffer, wash worms from step 1.4 onto PA14::GFP plates in a minimal volume of liquid. When using 150 mm plates, several hundred worms can be placed on a single plate.
4. Incubate plates at 25 °C for 18-21 hr. For P. aeruginosa-exposed young adult worms, this is the time window displaying the optimal distribution of colonized vs noncolonized animals.

3. Setting up the Wormsorter

Before performing the sample sort, ensure that the machine is functioning properly. Details can be obtained at http://www.unionbio.com/support/documents.aspx?id=43, and the essential steps are outlined below.

1. Set the sheath valve pressure to approximately 5 psi (4.5-5.5 range).
2. To verify that sheath fluid flow rate is appropriate, place a 15 ml conical tube beneath the dispenser, turn on the sheath valve, and turn off the sorter valve. Hold the tube beneath the dispenser for 60 sec. The volume in the tube should be 9-10 ml. If it is outside this range, adjust the sheath valve pressure accordingly.
3. To ensure that sheath fluid flow rate (set as in step 3.2) will allow accurate sorting, test the sort rate using 40 μm control particles:
4. Turn off the sheath valve. Add about 25 ml of control particle solution to the reservoir.
5. Turn on the sheath valve, and the sample valve.
6. On the computer software, navigate to the "control particle mode" and click acquire.
7. Make sure the particle flow rate is ~5-8 particles/sec, with a TOF (Time Of Flight) approximating the width of the particle, 40 μm. Such a TOF indicates that only a single 40 μm object is passing through at a time. If values are outside of this range, adjust the settings (following directions above) accordingly.

4. Sorting Colonized vs. Noncolonized Worms

1. Using M9, wash worms into 15-ml conical tubes. Allow adult worms to sink to the bottom of the tube by gravity and remove the supernatant. Fill the tube with fresh M9. Repeat this process 3-4x. This removes a large proportion of larvae as well as excess bacteria and takes about 10 min.
2. Add worms to the wormsorter reservoir with a gently-mixing small stir bar.
3. Adjust initial signal gain by setting the Green PMT to 600 and the Red and Yellow PMT to 200.
4. Begin acquiring data to adjust settings.
5. Aim for a sort rate of 25-30 events (worms) per sec. If the number rate is too high, dilute the worms in the reservoir with M9 accordingly. If the rate is too low, add more worms in a small volume of M9.
6. If the experiment requires a very stringent separation of colonized vs noncolonized worms, set the "coincidence check" to "pure". This ensures that in the event two objects occur too close to each other or in the same drop, the machine rejects the drop rather than collecting it.
7. To sort the population of interest, draw gates around the axes indicating size (to obtain adult worms) and fluorescence intensity (i.e. high for colonized, low for noncolonized). The values for fluorescence gating must be determined empirically.
8. Place a multi-well plate or a Petri dish underneath the dispenser to collect worms then open the sort valve to begin sorting worms.
9. Collect a small population of animals to verify under the microscope that the sorted population is the population of interest. If not, adjust the gating parameters accordingly and continue with sample collection.

Results

When age-matched, genetically identical C. elegans are exposed to P. aeruginosa, a wide distribution is observed in levels of colonization (Figure 1A). With the help of the protocol described here efficient separation of noncolonized from colonized worms can be achieved (Figure 1B). Unlike noncolonized worms, colonized worms show signs of damage, such as sluggishness and reduced defecation¹⁹. The latter may be a reason why worms colonized by P. aeruginosa...

Discussion

The method we describe takes advantage of fluorescent labeling of entities outside of the worm, to follow interactions between the worm and its environment. In the case we present, separation was based on labeling of a pathogen and was employed to separate worms with heavy pathogen load from those with no (or light) load. Subsequent gene expression analysis found no difference in immune responses between the two groups suggesting that they were independent of pathogen load. The signal that initiates the response was show...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
M9 Buffer	prepared in house		Recipe at wormbook.org
Rifampicin	Sigma	R3501
Egg prep solution	prepared in house		50 ml water; 40 ml bleach; 10 ml of 10 N sodium hydroxide
NGM plates	prepared in house		Recipe at wormbook.org
SKP plates	prepared in house		Recipe same as NGM only 0.35% peptone instead of 0.25%
Control test particles	Union Biometrica	310-5071-001