The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Summary

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

Abstract

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

Introduction

C. elegans has been used as a powerful model organism for more than 50 years. In the 1960s, South African biologist Sydney Brenner pioneered the use of C. elegans to study neuronal development, paving the way for a long lineage of scientists to study various aspects of cell and animal biology in nematodes. This lineage includes Nobel Prize laureates Craig Mello and Andrew Fire for their RNAi work¹, Robert Horvitz and John Sulston for their work on organ development and apoptosis^2,3,4, and Martin Chalfie for his work on green fluoresce....

Protocol

1. Preparation of Nematode Growth Medium (NGM)

1. For 1 L of media, combine 20 g agar, 2.5 g organic nitrogen source (e.g., bacto-peptone), and 3 g sodium chloride in a 2 L flask. Add 975 mL of sterile water.
   1. Add in a sterile stir bar. If using an automatic media pourer, autoclave tubing and media for 15 min; media should be autoclaved for longer if a higher volume is made.
2. Set media on stir plate, and allow to cool.
   1. Once media is warm to touch (approximately 60 °C) aseptically add 1 mL of 1 M MgSO₄● H₂O, 1 mL of 1M CaCl₂, 25 ....

Results

A pathogenesis assay (Figure 1) using C. albicans and C. elegans has previously been described by our lab^17,18 and other labs^19,20. We demonstrate the amenability of using C. elegans to study C. albicans virulence showing that C. albicans cells are quickly ingested by the worms and accumulate in the.......

Discussion

The methods for assaying C. elegans infection and survival over lifetime exposure to C. albicans that we have described can be modified to test another pathogen. Liquid cultures of another bacteria or fungus may be made and fed to C. elegans in a similar manner. Additionally, serial infections may be assayed by first exposing the larva to one pathogen as described, and then transferring the animals onto a new plate containing a separate pathogen after reaching adulthood.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar (granulated, bacterilogical grade)	Apex BioResearch Products	20-248
Aluminum Wire (95% Pt, 32 Gauge)	Genesee Scientific	59-1M32P
Axiovision Zeiss Inverted Microscope	Axiovision Zeiss
Bacto-Peptone	Fisher BioReagants	BP1420-500
C. elegans strain Bli-3	Caenorhabditis Genetics Center	Bli-3(e767) CB767
Calcium Chloride	Fisher Scientific	BP51-250
Cholesterol, Sigma Grade, minimum 99%	Sigma	C8667-25G
Disposable Culture Tubes (20 x 150 mm)	FIsherBrand	14-961-33
Dissection Microscope (NI-150 High Intensity Illuminator)	Nikon Instrument Inc.
E. coli	Caenorhabditis Genetics Center	OP50
GraphPad Prism (Survival Curve Analysis Software)	GraphPad Software
LB Broth (Miller's)	Apex BioResearch Products	11-120
Magnesium Sulfate	Fisher Scientific	10034-99-8
Medium Petri Dishes (35 X 10 mm)	Falcon	353001
Potassium Phosphate monobasic	Sigma	P0662-500G
Sodium Chloride	Fisher Scientific	BP358-1
Sodium Phosphate	Fisher Scientific	BP332-500
Wildtype C. albicans SC5314	ATCC	SC5314
Wildtype C. elegans	Caenorhabditis Genetics Center	N2