Name: the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions
Uploaded: 2017-10-18T14:00:00
Description: Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

This work was performed at and supported by Worcester Polytechnic Institute.

1. Preparation of Nematode Growth Medium (NGM)
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Set media on stir plate, and allow to cool.
	<ol>
		<li>Once ...

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.
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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 apoptosis2,3,4, and Martin Chalfie for his work on green fluorescent protein5. Although this model organism has been traditionally used to study molecular and developmental biology, over the past 15 years, researchers have begun to use C. elegans to investigate the biology of various human pathogens including Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella enterica, and Serratia marcescens6,7,8,9,10. These studies revealed that many of the mechanisms involved in the human-pathogen interaction are conserved in nematodes, but also that there are some immunity mechanisms that are unique to this model organism11,12. In nature, C. elegans encounters a variety of threats from ingested pathogens present in the soil and this has provided a strong selective pressure to evolve and maintain a sophisticated innate immune system in its intestinal lumen. Many of the genes and mechanisms involved in the protection of intestinal lumen are orchestrated by highly-conserved elements that also exist in higher mammals11,13. C. elegans therefore represents a great model to study gastrointestinal pathogens like Salmonella enterica14, Shigella boydii15, or Vibrio cholera16.
Here we highlight the remarkable versatility of C. elegans as a model host to study infectious agents such as C. albicans. C. elegans as a model host allows for high throughput screening for virulence that is less expensive and time-consuming than a mouse model, which is commonly used to study candidiasis42.
In this study, we show that this model and the assosiated survival assay can be reliably used for studying host innate immune effectors important to counteract infections, pathogen determinants that drive virulence, and pharmacological compounds that can intervene in pathogenesis. Dissimilar to previously described assays, this method provides a means of studying exposure to a pathogen over the lifetime of the animal, from the larval stage to adulthood, rather than only adulthood to death43,44. In summary, our C. elegans - C. albicans model is a versatile and powerful tool that can be used not only to study the genetic bases that drive infection and immunity but also to identify new compounds for therapeutic intervention.

<table>
	<tbody>
		<tr>
			<td>Agar (granulated, bacterilogical grade)</td>
			<td>Apex BioResearch Products</td>
			<td>20-248</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Wire (95% Pt, 32 Gauge)</td>
			<td>Genesee Scientific</td>
			<td>59-1M32P</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovision Zeiss Inverted Microscope</td>
			<td>Axiovision Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Peptone</td>
			<td>Fisher BioReagants</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans strain Bli-3</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>Bli-3(e767) CB767</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP51-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol, Sigma Grade, minimum 99%</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Culture Tubes (20 x 150 mm)</td>
			<td>FIsherBrand</td>
			<td>14-961-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Microscope (NI-150 High Intensity Illuminator)</td>
			<td>Nikon Instrument Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism (Survival Curve Analysis Software)</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth (Miller&#39;s)</td>
			<td>Apex BioResearch Products</td>
			<td>11-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium Petri Dishes (35 X 10 mm)</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate monobasic</td>
			<td>Sigma</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wildtype C. albicans SC5314</td>
			<td>ATCC</td>
			<td>SC5314</td>
			<td></td>
		</tr>
		<tr>
			<td>Wildtype C. elegans</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions

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.

A pathogenesis assay (Figure 1) using C. albicans and C. elegans has previously been described by our lab17,18 and other labs19,20. 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...

Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

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

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

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

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

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