We thank Dr. Diane Baronas-Lowell for critical reading of the manuscript. This work was supported by an FAU Charles E. Schmidt College of Science Seed Grant and an Aging Scholarship from the Ellison Medical Foundation to K.J.

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.
<ol>
	<li>Weigh out 5.5 g XLD agar and resuspend in 5 ml deionized water.</li>
	<li>Mix thoroughly until all agar is wet. Add 95 ml deionized water until all lumps are gone and the medium is completely resuspended.</li>
	<li>Boil the medium ...

<ol>
	<li>Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans II. , (1997).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genetics+of+Caenorhabditis+elegans.">The Genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Aballay, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+host+for+the+study+of+host-pathogen+interactions.">Caenorhabditis elegans as a host for the study of host-pathogen interactions.</a> Curr Opin Microbiol. 5, 97-101 (2002).</li><li>Kurz, C. L., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans:+an+emerging+genetic+model+for+the+study+of+innate+immunity.">Caenorhabditis elegans: an emerging genetic model for the study of innate immunity.</a> Nat Rev Genet. 4, 380-390 (2003).</li><li>Mylonakis, E., Aballay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worms+and+flies+as+genetically+tractable+animal+models+to+study+host-pathogen+interactions.">Worms and flies as genetically tractable animal models to study host-pathogen interactions.</a> Infection and Immunity. 73, 3833-3841 (2005).</li><li>Ford, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+descriptive+study+of+human+Salmonella+serotype+typhimurium+infections+reported+in+Ontario+from+1990+to+1997.">A descriptive study of human Salmonella serotype typhimurium infections reported in Ontario from 1990 to 1997.</a> Can J Infect Dis. 14, 267-273 (2003).</li><li>Voetsch, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoodNet+estimate+of+the+burden+of+illness+caused+by+nontyphoidal+Salmonella+infections+in+the+United+States.">FoodNet estimate of the burden of illness caused by nontyphoidal Salmonella infections in the United States.</a> Clin Infect Dis. 38 Suppl 3, (2004).</li><li>Aballay, A., Yorgey, P., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+proliferates+and+establishes+a+persistent+infection+in+the+intestine+of+Caenorhabditis+elegans.">Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans.</a> Curr Biol. 10, 1539-1542 (2000).</li><li>Alegado, R. A., Tan, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antimicrobial+peptides+contributes+to+persistence+of+Salmonella+typhimurium+in+the+C.+elegans+intestine.">Resistance to antimicrobial peptides contributes to persistence of Salmonella typhimurium in the C. elegans intestine.</a> Cell Microbiol. 10, 1259-1273 (2008).</li><li>Jia, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+genes+protect+against+Salmonella+typhimurium+infection+and+mediate+insulin+signaling-regulated+pathogen+resistance.">Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 14564-14569 (2009).</li><li>Tenor, J. L., McCormick, B. A., Ausubel, F. M., Aballay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans-based+screen+identifies+Salmonella+virulence+factors+required+for+conserved+host-pathogen+interactions.">Caenorhabditis elegans-based screen identifies Salmonella virulence factors required for conserved host-pathogen interactions.</a> Curr Biol. 14, 1018-1024 (2004).</li><li>Levine, B., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+by+self-digestion:+molecular+mechanisms+and+biological+functions+of+autophagy.">Development by self-digestion: molecular mechanisms and biological functions of autophagy.</a> Developmental Cell. 6, 463-477 (2004).</li><li>Birmingham, C. L., Smith, A. C., Bakowski, M. A., Yoshimori, T., Brumell, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+controls+Salmonella+infection+in+response+to+damage+to+the+Salmonella-containing+vacuole.">Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole.</a> J Biol Chem. 281, 11374-11383 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+Sequencing+Consortium.+Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">The C. elegans Sequencing Consortium. Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282, 2012-2018 (1998).</li><li>Fire, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome Biol. 2, 1-10 (2001).</li><li>Liang, J., Xiong, S., Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNA-mediated+interference+feeding+strategy+to+screen+for+genes+involved+in+body+size+regulation+in+the+nematode+C+elegans.">Using RNA-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode C elegans.</a> J. Vis. Exp. (72), (2013).</li><li>Fraser, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+C.+elegans+chromosome+I+by+systematic+RNA+interference.">Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.</a> Nature. 408, 325-330 (2000).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: the online review of C elegans biology. , 1-11 (2006).</li><li>Aballay, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+mediated+by+ced-3+and+ced-4+protects+Caenorhabditis+elegans+from+Salmonella+typhimurium-mediated+killing.">Programmed cell death mediated by ced-3 and ced-4 protects Caenorhabditis elegans from Salmonella typhimurium-mediated killing.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 2735-2739 (2001).</li><li>Melendez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+genes+are+essential+for+dauer+development+and+lifespan+extension+in+C.+elegans.">Autophagy genes are essential for dauer development and lifespan extension in C. elegans.</a> Science. 301, 1387-1391 (2003).</li></ol>

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...

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 gametogenesis1,2. 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 &#176;C on agar plates with seeded bacterium Escherichia coli (strain OP50) as food1,2.
In the last decade, C. elegans has emerged as an invertebrate organism to study host-pathogen interactions3-5. In nature, C. elegans eats bacteria as its nutrient source1,2. Its normal bacterial laboratory food, OP50, can be easily substituted with other pathogens to examine the interactions between C. elegans and any chosen pathogen. Under these conditions, the intestine is the primary site of the infection. Indeed, a wide range of bacterial pathogens has been shown to lethally infect C. elegans3-5.
The gram-negative bacterium Salmonella is a gastrointestinal pathogen that causes human food-borne illness worldwide6,7. C. elegans is a good model host for Salmonella typhimurium as this bacterium replicates and exhibits persistent intestinal infections8-10. C. elegans has been used to identify both novel and previously known Salmonella virulence factors11. Interestingly, the C. elegans immune system successfully limits Salmonella replication. It has been reported previously that inhibition of autophagy genes renders increased Salmonella replication in C. elegans, resulting in early death of infected worms10. Macroautophagy (herein referred to as autophagy) is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome for degradation12. Autophagy has been reported to limit the Salmonella replication in C. elegans and in mammals10,13.
The C. elegans genome was the first multicellular eukaryotic genome sequenced; it is responsive to RNAi treatment14-16. Moreover, RNAi can be administrated effectively by subjecting worms to ingest bacteria containing the double-stranded RNA of the target gene, known as RNAi feeding16,17. Whole genome RNAi feeding libraries have been generated for genome-wide RNAi screening16,18. Herein, a Salmonella infection protocol is coupled with RNAi feeding to allow testing C. elegans genes of interest for their ability to protect against Salmonella infection.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">BP9723-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XLD agar</td>
			<td>EMD Chemicals</td>
			<td>1.05287.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Fisher</td>
			<td>DF0140-01-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Fisher</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Fisher</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Fisher</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td>Gold Biotechnology</td>
			<td>12481C50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Fisher&#160;</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 95 x 15mm</td>
			<td>Fisher</td>
			<td>FB0875714G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 60 x 15mm&#160;</td>
			<td>Fisher</td>
			<td>08-757-13A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Serological pipet</td>
			<td>Fisher</td>
			<td>13-668-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Express Pipet-Aid</td>
			<td>Fisher</td>
			<td>13-675-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ6000 shaking incubator&#160;</td>
			<td>Thermo Scientific</td>
			<td>SHKE6000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Percival</td>
			<td>I-36DL</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

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.

At 20 &#176;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, comp...

Watch this Scientific Journal Video about A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium at JoVE.com

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

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.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

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Immunology and Infection

11.5K ビュー.  Florida Atlantic University.  線虫は、宿主-病原体相互作用を研究するための新しい遺伝モデルとして登場しました。ここでは、 温度を感染させるためのプロトコルを記述サルモネラ感染に対する防御における宿主遺伝子の役割を調べるために二重鎖のRNAi干渉法と結合ネズミチフス菌とエレガンス 。 