A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

Summary

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Abstract

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

Introduction

The free-living soil nematode Caenorhabditis elegans is a simple and genetically tractable model organism used to study many biological questions. C. elegans dominantly exists as self-fertilizing hermaphrodites. Males are spontaneously generated by non-disjunction of the X chromosome during gametogenesis^1,2. In the presence of abundant food, C. elegans continuously develop through four larval stages to adult. Temperature also influences C. elegans development; faster development is observed at higher temperatures. In the laboratory, C. elegans is cultured at a standard temperature of 20 °C on agar plates wit....

Protocol

1. XLD (Xylose Lysine Desoxycholate) Agar Plates

XLD agar is a selective growth medium for Salmonella, which appears as black colonies on XLD agar plates. However, if there are no concerns of contamination, a regular LB plate can be substituted.

1. Weigh out 5.5 g XLD agar and resuspend in 5 ml deionized water.
2. Mix thoroughly until all agar is wet. Add 95 ml deionized water until all lumps are gone and the medium is completely resuspended.
3. Boil the medium to dissolve completely (do not autoclave).
4. Cool the medium at room temperature to 50 °C.
5. Pour 25 ml agar in each 95 x 15 mm (....

Results

At 20 °C, the median lifespan of wild type N2 worms is 17 days (Figure 2A and Table 2). Salmonella infection significantly decreases the median lifespan of N2 worms to 10.5 days (p = 0.0002, log-rank test) (Figure 2A).

If a C. elegans gene plays an important role in defense against Salmonella infection, it is predicted that its inhibition will impart susceptibility to Salmonella infection. In fact, compared .......

Discussion

C. elegans is a simple genetic model organism that eats bacteria as its nutrient source. Thus, it is easy to substitute its normal bacterial food with an intestinal pathogen to investigate the interactions between C. elegans and the chosen pathogen. Herein a protocol is described to combine Salmonella infection and C. elegans RNAi feeding treatment to examine the role of host genes in defense against Salmonella infection. Previous infection protocols expose C. elegans.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB Broth	Fisher	BP9723-500
XLD agar	EMD Chemicals	1.05287.0500
Bacto Agar	Fisher	DF0140-01-0
Peptone	Fisher	BP1420-500
Sodium Chloride	Fisher	S671-500
Calcium Chloride	Fisher	C69-500
Magnesium Sulfate	Fisher	M65-500
IPTG	Gold Biotechnology	12481C50
Cholesterol	Sigma	C8667-25G
Ampicillin	Fisher	BP1760-25
Salmonella typhimurium	ATCC	ATCC14028
Petri Dish 95 x 15 mm	Fisher	FB0875714G
Petri Dish 60 x 15 mm	Fisher	08-757-13A
Falcon Serological pipet	Fisher	13-668-2
Falcon Express Pipet-Aid	Fisher	13-675-42
MaxQ6000 shaking incubator	Thermo Scientific	SHKE6000-7
Incubator	Percival	I-36DL