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 l....

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 Pl....

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