私たちは、原稿の重要な読書のために博士ダイアンBaronas-ローウェルに感謝します。この作品は、FAUチャールズ·E.シュミット大学科学のシード·グラントとKJのエリソン医学財団からエージング奨学金によってサポートされていました

1。XLD（キシロースリジンデソキシコール）寒天プレート XLD寒天はXLD寒天プレート上で黒色コロニーとして現れるサルモネラ、のための選択的成長培地である。汚染の心配がない場合は、正規のLBプレートは、置換されていてもよい。 <ol><li> 5ミリリットルの脱イオン水に5.5グラムのXLD寒天再懸濁を量る。 </li><li>すべての寒天が濡れてまでよく混ぜる。すべての塊がなくなっているとメディアが完全に再懸濁されるまで脱イオン水を95ミリリットルを追加します。 </li><li> （オートクレーブしないでください）​​、完全に溶解する媒体が沸騰。 </li><li> 50℃の室温で培地を冷却</li><li>各95×15ミリメートル（直径×高さ）プレート（パラフィルムでシールしたプレートを1ヶ月まで4℃で保存することができる）中の25mlの寒天を注ぐ。 </li></ol> 2。線虫増殖培地（NGM）RNAiは、プレートの餌付け Cの準備エレガンス NGMプレートは、以前に記載されている19ここでの手順を簡単にするRNAi送り板を作るためにNGM培地に抗生物質アンピシリンおよびRNAi化学誘導物質イソプロピルβ-D-1-チオガラクトピラノシド（IPTG）を添加することが記載されている。 <ol><li> 3グラムのNaClおよび1 Lの脱イオン水に2.5グラムバクトペプトンを溶かす。 </li><li>メディアへの17グラムバクト寒天を追加します。 </li><li> 45分間オートクレーブし、メディアを水浴中で50℃に媒体を冷却する。 </li><li>を1mlのコレステロール（95％エタノール中5 mg / ml）を1 mlの1 MのCaCl 2、1 mlの1 MのMgSO 4、および25 mlの1 Mリン酸カリウム緩衝液（pH6.0）：以下の溶液を加える。よく混ぜる。 </li><li> 1ミリリットルの1M IPTGおよび500μlのアンピシリン（滅菌水に100ミリグラム/ ml）を追加します。 </li><li>よく溶液を混合し、滅菌手順に従ってピペットで援助と25ミリリットルの血清学的ピペットを用いて60×15ミリメートル（直径×高さ）ペトリ皿に注ぐ。 12ミリリットル寒天で各プレートを埋める。プレートを、1ヶ月まで4℃で保存することができる。 </li></ol> ベック-1オートファジー必須遺伝子はサルモネラ感染に対する防御における宿主遺伝子の機能を調べるために例として使用される。実験手順は、 図1および表1に示されている。感染についてのRNAi処理動物を調製するためのプロトコルは、括弧内に与えられた各実験手順の日に、続く。 <ol><li> BEC-1のRNAi摂食に接種し、100 mg / mlのアンピシリン（1日目 ）を補足した2ミリリットルのLB培地には-80℃で凍結した細菌フレークを配置することによって、空のベクターL4440のRNAi摂食細菌を制御します。新鮮なRNAiの細菌を持っている全体の実験中に週一回、この手順を繰り返します。使用しない場合は4℃の冷蔵庫に文化を保存してください。 </li><li> RNAiのプレート上で一晩のRNAi細菌培養の100ミリリットルをシード。 3 BEC-1 RNAiおよび3コントロールの空のVを準備RNAiのプレートをエクター。 37℃で一晩（2日目 ）でプレートをインキュベートする。 </li><li> 37℃のインキュベーターからのRNAiプレートを取り外し、それらをベンチに室温まで冷却する。よく葉L4野生型N2雌雄同体をピックアップし、RNAiを1にBECと空ベクターのRNAiプレートを制御するためにそれらを転送します。三連のプレートに、プレート当たり3ワームを配置します。ステップ3.2（3日目 ）に記載したのと同じ日に、RNAiのプレートを準備します。 </li><li> 36〜40時間、20℃のインキュベーター内のワームとのRNAiプレートをインキュベートし、ステップ3.3で作成新鮮対応のRNAiプレートにワームを転送します。ワームが転送された後、64時間（4日目 ）、20℃のインキュベーター中でプレートをインキュベートする。 </li></ol> 4。感染のサルモネラを準備<ol><li>ストリークサルモネラ -80℃で凍結した1 XLD寒天プレート上の在庫と一晩（5日目 、37℃でプレートをインキュベート</st栄&gt;）。 </li><li>よく孤立黒サルモネラコロニーを採取し、（6日目 ）で一晩振とうしながら37℃で2 mlのLB培地中で、それを接種。 </li><li> 1 Cの種子80ミリリットルサルモネラ一晩培養虫 60×15ミリメートル（直径×高さ）NGM寒天プレート、計6枚を準備。 6時間室温で培養する。細菌培養プレート（7日目 ）に芝生を乾燥させ、形成すべきである。 </li></ol> 5。 サルモネラとのRNAi治療ワームに感染<ol><li> BEC-1のRNAi処理を移し、 サルモネラプレートに空のベクターのRNAi治療L4のN2雌雄同体（ステップ3で設定したワームの子孫）を制御する。各グループの3プレートにプレート当たり40ワームを配置します。 48時（7日目 ）、20℃でワームのプレートをインキュベートする。 </li><li>ステップ3.1および3.2（7日目で説明したように、新鮮なRNAiプレートの1セットを準備し、 <stroNG&gt; 8日目）。 </li><li> 48時間の感染後、ステップ5.2で作成、対応するRNAiプレートにサルモネラ菌に感染したワームを転送し、20℃（9日目 ）でインキュベートする。 </li></ol> 6。生存アッセイ<ol><li>日々のワームの生存をスコアと産卵期間中に新鮮な対応のRNAiプレートにワームを転送します。ステップ3.1および3.2で説明したように前に各ワーム転送に新鮮なRNAiプレートのセットを準備します。エンド平坦化白金線で軽くワーム本体（頭部、中間部と尾）をタッチします。ワームの体の動きが全く観察されない場合にワームが死んだとして採点される。 </li><li>日々のワームの生存または一日おきのスコア、およびワームが産卵を停止した後に週二回、新鮮対応するRNAiプレートにワームを転送します。 </li><li>すべてのワームが死んだ後、1​​データセットとして三重プレートからの生存データをプールする。例えば、グラフパッドPなどの適切な統計ソフトウェアへの入力の各群の生存データを生存曲線を生成し、Kaplan-Meier生存分析を実行するRISM。全実験は結論を確認するために、少なくとも1回繰り返される。 </li></ol>

<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>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

線虫は、栄養源としてバクテリアを食べて、単純な遺伝的モデル生物である。従って、C.間の相互作用を調査するために、腸病原体にその正常細菌食品を代用することは容易である虫および選択した病原体。本明細書プロトコルは、 サルモネラ感染およびC.を組み合わせることが記載されているRNAiは、 サルモネラ感染に対する防御における宿?...

自由生活土壌線虫線虫（Caenorhabditis elegans）は、多くの生物学的な質問を研究するために使用されるシンプルかつ遺伝的に扱いやすいモデル生物である。C.虫が支配的自己施肥雌雄同体として存在する。雄配偶子形成を自発的に1,2の間にX染色体の非論理和によって生成される。豊富な食べ物、Cの存在下で虫は継続的に、成人に4幼虫の段階を経て発達する。温度も影響を与えるC.虫の開発;迅速な開発は、より高い温度で観察される。研究室では、C.エレガンスは、食品1,2播種し、細菌大腸菌 （菌株OP50）を含む寒天プレート上で20℃の標準温度で培養する。 過去10年間で、C.虫は宿主-病原体相互作用3-5を研究する無脊椎生物として浮上している。自然界では、C.虫は nutrienなどの細菌を食べるトン源1,2。その通常の細菌の実験室フード、OP50は、容易C.間の相互作用を調べるために他の病原体で置換することができる虫や任意の選択された病原体。これらの条件下で、腸の感染の主要部位である。実際に、細菌性病原体の広い範囲が致死的にC.に感染することが示されている虫 3-5。 グラム陰性菌のサルモネラは、世界中の6,7人の食中毒の原因となる胃腸病原体である。C.この細菌は、複製し、持続的な腸の感染症8月10日を示すもの虫はネズミチフス菌のための優れたモデルホストです。 でエレガンスは、新規の両方を識別するために使用されていると以前から知られているサルモネラ病原性は、11因子 。興味深いことに、C.虫の免疫系は正常にサルモネラの複製が制限されます。それはそのInhibが以前に報告されているオートファジー遺伝子のitionはCで増加サルモネラの複製をレンダリング虫 、感染したワーム10の早い死に至る。マクロオートファジーは、（本明細書オートファジーと呼ばれる）12分解のためにリソソームへの送達のための細胞質および細胞小器官を隔離する細胞内膜の転位を伴う動的プロセスである。オートファジーは、C.においてサルモネラの複製を制限することが報告されている虫や哺乳類10,13中。 C.虫のゲノムを配列決定し最初の多細胞真核生物ゲノムた。それは、RNAi治療14〜16に応答する。また、RNAiは、RNAiが16,17に供給として知られている標的遺伝子の二本鎖RNAを含む細菌を摂取するワームを施すことにより効果的に投与することができる。全ゲノムのRNAi送りライブラリは、ゲノムワイドなRNAiは16,18をスクリーニングするために生成されている。ここでは、 サルモネラ感染症のプロトコールは、RNAiがC.をテストできるように、給紙に結合されているサルモネラ感染に対して防御する能力について、目的の遺伝子エレガンス 。

<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

過去10年間で、C.虫は、グラム陰性菌、ネズミチフス菌に対する宿主防御を含め、ホストと病原体との相互作用を研究する無脊椎生物として浮上している。サルモネラはCの腸内で持続感染を確立虫や感染した動物の早期死亡をもたらす。免疫機構の数はCで同定されているサルモネラ感染に対して防御する虫 。オートファジー、進化的に保存されたリソソーム分解経路は、C.におけるサルモネラの複製を制限することが示されている虫と哺乳類における。ここで、プロトコルは、C.に感染することが記載されているヒトにおけるサルモネラ感染と同様のワームが、限られた時間のサルモネラに暴露されたネズミチフス菌 、 線虫と、 サルモネラ感染が著しくC.の寿命を短くする虫&lt;/ EM&gt;。一例として、必須の自食作用遺伝子ベック-1を使用して、我々はC.、この感染法を組み合わせたRNAiは、 線虫のアプローチを供給し、このプロトコルはC.の機能を調べるために使用することができる示しサルモネラ感染に対する防御において宿主遺伝子エレガンス 。 C.以来虫全ゲノムのRNAiライブラリが利用可能であり、このプロトコルは、包括的で物をスクリーニングすることが可能になるサルモネラおよびゲノムワイドのRNAiライブラリを使用して、他の腸内の病原体から守る遺伝子エレガンス 。

20℃では、野生型N2ワームの中央値は寿命が17日間（ 図2Aおよび表2）である。 サルモネラ感染が有意に10.5日（P = 0.0002、log-rank検定）にN2ワームの中央値の寿命を低下させる（ 図2（a） ）。 C.もし線虫遺伝子はサルモネラ感染に対する防御において重要な役割を果たし、それはその阻害はサルモネラ?...

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

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 。

感染するためのプロトコル<em&gt;線虫（Caenorhabditis elegans）</em&gt;と<em&gt;ネズミチフス菌</em

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

a-protocol-to-infect-caenorhabditis-elegans-with-salmonella

Research

JoVE Journal

Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

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 。 

ビデオ: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

識別のためのアレロタイプ化PCRサルモネラ血清型Enteritidisは、ハダル、ハイデルベルク、及びネズミチフス菌

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

免疫・感染学

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

表現型のハイスループットアッセイ<em&gt;サルモネラ菌</em&gt;ネズミチフス菌協会、侵略、およびマクロファージにおける複製

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

大腸菌Site-Specifically アセチル化蛋白質を生成する安易なプロトコル

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

病原性と線虫モデルにおけるアエロモナス感染症の病態の評価

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

マウス子牛に対する一貫したマッサージ様摂動の適用と、結果として生じる筋肉内圧変化のモニタリング

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

サルモネラおよび他の病原体に対する上皮細胞保護を研究・修飾するためのヒト誘導多能性幹細胞由来腸管オルガノイドの使用

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

慢性サルモネラ感染誘発性腸線維症

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

NF-&amp;#954;B-dependent Luciferase Activation and Quantification of Gene Expression in &lt;em&gt;Salmonella&lt;/em&gt; Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

サルモネラ感染組織培養細胞におけるNF-κB依存性ルシフェラーゼ活性化と遺伝子発現の定量

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...