P. Cossart研究室での研究は、パスツール研究所、研究所国立·デ·ラ·サンテら·デ·ラ·ルシェルシュMédicale、研究所国立·デ·ラ·ルシェルシュAgronomique、ERCアドバンスト·グラント（233348）、通信社国立·デ·ラ·ルシェルシュ（グラント三重でサポートされているSignRupVac）、ルイ·Jeantet財団と財団ル·ロッシュレMousquetaires。 AKは、パスツール、パリ大学国際博士課程/研究所カルノー病気Infectieusesから奨学金の受取人である。我々は、細胞のトランスフェクションプロトコルを最適化するためのジェイソンマーサーに感謝します。

1。携帯電話や細菌培養、トランスフェクションツールと一次抗体の調製<ol><li>個々のLを分離するために、新鮮な寒天プレートを準備します細菌グリセロールストック（50％glycerol/50％飽和菌液一晩培養）からのリステリアコロニーは-80℃で保存<ol><li>ブレインハートインフュージョン（BHI）寒天プレート上で凍結した細菌グリセロールストック、ストリーク細菌を輸送するために（-80℃で保存）、アルミラックを使用。 </li><li> 48時間（または個々の細菌コロニーを単離することができるまで）の間、37℃でプレートをインキュベートする。 </li><li> （それは液体培養を播種するために使用されます）1月の最大時間の間、4℃で、その後このワーキングプレートを保管してください。 </li><li>我々のプロトコルでは、L.を使用図1、Lに示すようにEGDe.PrfA *株1/2A血清型リステリアが、 P14.PrfA 4B リステリア血清型は、*株は、同様の結果が得られます。 </li></ol></li><li>彼ラATCC CCL2の細胞は、抗生物質の非存在下で、10％ウシ胎児血清（FBS）を補充したダルベッコ改変イーグル培地（DMEM）中で増殖させる。 </li><li>独立したスクランブルのプール（対照）および抗MetをsiRNAは、黒色384ウェル顕微鏡細胞培養プレートにロードされる。 <ol><li> RNaseフリー水500μlのsiRNAの160ピコモルを希釈し、ウェル当たり、この溶液5μl（最終濃度：1.6ピコモル）を追加します。 </li><li> 1年間の最大期間、-20℃で、このプレートを保管してください。 </li></ol></li><li>先に説明したように25 InlC-GST組換えタンパク質を用いて免疫化することによって得られたInlC細菌タンパク質に対するポリクローナルウサギ抗体。 </li></ol> 2。のsiRNA細胞トランスフェクションを逆転<ol><li>感染前の72時間は、各ウェル中で室温にRNaseフリー水5μlの1.6 pmolのsiRNAを含む黒色384ウェル顕微鏡細胞培養プレートをもたらす。 </li><li> 300で3分間プレートを遠心分離ウェルの壁に堆積されている可能性のsiRNAをダウンさせるために、室温でRCF。 </li><li> 0.4％リポフェクタミンRNAiMAXトランスを補った室温のDMEMを準備します。 </li><li> （siRNAのに追加する前に、より長い20分よりトランスフェクション培地をインキュベートしないでください）​​を各ウェルにDMEM / RNAiMAXトランスフェクション培地を25μLを加える。 </li><li>を形成するためのsiRNA-リポフェクタミン複合体を可能にするために室温で1時間プレートを保持次いで、トランスフェクション溶液でのsiRNAを混合するためにプレートを前後に移動させる。 </li><li>コンフルエント又は10ミリリットル37°C予め温めたPBSで1回、サブコンフルエント細胞培養フラスコからの洗浄HeLa細胞。 </li><li> 1ミリリットル37℃の細胞培養フラスコにトリプシンを予め温め、37℃で3〜5分間インキュベート添加することによってHeLa細胞をデタッチ</li><li> 10ミリリットル、37℃で再懸濁細胞を、16％FBSを補充したDMEMを予め温めた。 </li><li>細胞を計数し、補充したDMEM 1ml当たり12,000細胞の細胞懸濁液を調製16％FBS。 </li><li> 384ウェルプレートの各ウェルに細胞懸濁液50μlを加える。 </li><li>細胞を分配し、細胞を室温で10分間落ち着かせて前後に迅速にプレートを移動させる。 </li><li>パラフィルムでプレートを密封し、37℃で加湿した5％CO 2含有雰囲気中で72時間それを維持</li></ol> 3。携帯感染染色<ol><li>感染の前日には、Lの単一コロニーを取るBHI寒天プレートからのリステリアとを15mlポリスチレンチューブ内の液体のBHI培地5ml中に再懸濁する。 </li><li>細菌増殖を可能にするためにshacking装置中で37℃で一晩インキュベートする。 </li><li>感染日、一晩L. 1mlの洗浄し卓上型遠心機で10,600 RCFで2分間遠心分離することによって文化をリステリア 。 </li><li> （分泌された細胞毒素テリオOを含む）上清を捨て、1mlのPBSに再懸濁ペレット（repea）洗浄工程を3回以上トン。 </li><li> 600 nmでの細菌の光学濃度を読んで、細菌の数を（OD = 1は1E9細菌/ mlに相当します）を推定する。 </li><li>適切なLを用意する1％FBSを添加したDMEM中で希釈リステリア ：EGDe.PrfA *のような侵襲性の高い株を用いて、ウェル当たりの培地30μlに5E4の細菌の使用を（感染多重度が2000細胞について25と推定されている）を示唆している。 </li><li>各ウェルの細胞培養培地（80μL）を取り外し、Lの30μLを追加することによってそれを置き換える培地リステリア含有。 </li><li>感染プロセスを同期するために、室温で5分間、200 rcfでプレートを遠心分離する。 </li><li>予め温めておいたアルミブロック上、37℃で加湿した5％CO 2含有雰囲気中で1時間プレートをインキュベート。 </li><li> Lを削除する各ウェルからリステリア含有培地および10％FBSを添加したDMEM予め温め30μlのを追加し、細胞外Lを殺すために40μg/ mlのゲンタマイシンリステリア 。 </li><li>あらかじめ温めておいた金属ブロック上で37℃、5％のCO 2を含む雰囲気中で4時間インキュベートする。 </li><li> PBSの溶液を調製し（ホルムアルデヒドモノマーではなく、パラホルムアルデヒドポリマーが、使用されているように、この溶液を新たに調製すべきである）8％ホルムアルデヒドを補充した。 </li><li>細胞培養培地を廃棄せずに、8％のホルムアルデヒド（最終濃度：4％）を補充したPBSの30μlを添加し、室温で15分間インキュベートする。 </li><li>固定液を除去して（ウェルあたりのPBSを80μlの最終体積中に細胞を維持する）ウェル当たりのPBS 80μLで細胞を3回洗浄する。 </li><li>調製1:250のPBS中のウサギ抗InlC血清の希釈は、0.2％サポニンを補充。 </li><li> PBSを除去した後、各ウェルに一次抗体溶液10μlを添加し、室温で30分間インキュベートする。 </li><li>原発を捨てる抗体溶液とウェルあたり40μlのPBSで4回洗浄する。 </li><li> 0.2％サポニンを補充したPBSで二アレクサフルーア546結合抗ウサギ抗体（1:250）、DAPI溶液（1:1,500）およびファロイジン-DY647（1:150）に希釈する。 </li><li>各ウェルに、この二次染色溶液10μlを添加し、室温で30分間インキュベートする。 </li><li>二染色液を捨て、（各ウェルに40μLの最終容量を残して、プレートをシール）ウェルあたり40μlのPBSで4回洗浄する。 </li><li>プレートは直ちに画像化することができ、またはその後の分析のために、光から保護された4°C（アルミホイルで覆う）で保存することができる。 </li></ol> 4。画像収集と分析<ol><li> 10倍の対物レンズを使用して3の異なるチャネル（350ナノメートル、546ナノメートルと647ナノメートル）内の画像を取得する（ウェルあたり優先的に9画像を取得する）自動顕微鏡に搭載された。 </li><li> InlC信号は、画像を用いて測定することができるそれぞれ、DAPIおよびファロイジン染色を用いて、核および細胞体の自動セグメンテーションを可能にするCellProfiler等解析ソフトウェア。 </li></ol>

<ol>
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我々は、開示することは何もありません。

いくつかのパラメータがInlC信号の明確な検出を可能にする十分に大きな細胞質を表示する健康な細胞株の使用を含む、我々のInlC検出プロトコルの成功のために重要である。検定では、我々は、それらの細胞質ゾルのスペースの拡張に私たちの分析に特に適しているのHeLa CCL2細胞の使用を提案し、この記事で提示し、このようなヒーラ京都細胞のような他のHeLa細胞クローンは小さく、細胞質?...

A correction was made to <a href="http://www.jove.com/video/51043">Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes</a>. Three authors were omitted from the article at the time of publication. The acknowledgments section was also updated. The author list has been updated from:
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020
to
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Jason Mercer4, Mario Emmenlauer5, Christoph Dehio5, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute,2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Z&#252;rich 5Focal Area Infection Biology, Biozentrum, University of Basel
The acknowledgments where update from:
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We thank Jason Mercer for optimizing the cellular transfection protocol.
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Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We acknowledge support by grant 51RT 0_126008 for the Research and Technology Development (RTD) project InfectX in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology (to C.D.).

A correction to author list has been made for the article Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes. Three authors were omitted from the article at the time of publication.

Erratum: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

グラム陽性菌、リステリア·モノサイトゲネス 、宿主細胞に侵入するその内部移行液胞を破壊し、宿主細胞1の細胞質で複製する食品媒介病原体である。L.細胞および動物モデルで観察細菌の病原性形質の持続性に関連した研究室のコンテキスト（急速な成長、健康な人のための低毒性）での操作のしやすさをモノサイトゲネス （溶血作用、白血球増加は）のための主要なモデルとして、1960年の最初の使用は許可されて細胞内寄生の感染2に対する細胞性免疫の理論的基礎の確立のための研究。 1980年代後半から1990年代には、細菌の細胞内サイクル3だけでなく、最も重要な細菌の病原性の分子特性の解剖は4-7 Lの使用を好む要因操作とスタッドのための重要な分子ツールとしてのリステリア宿主細胞の機能のY。 リステリア属の非病原性の存在（ のL.イノキュア ）と病原性（ リステリア菌 ）種は、比較ゲノム研究の8のための道を開いたこと、一緒に完全なLの最近の設立にリステリアは、L.の進化の理解を増加している、9をトランスクリプトームヒトの病原体として、感染の研究の10のためのモデル系として、 リステリア 。 リステリア菌は、それらの宿主細胞受容体はそれぞれ、E-カドヘリンおよびMet、11月12日で、細菌の表面タンパク質INLAおよびinlBの相互作用によって宿主細胞にその内在化を誘導する。最初の候補ベースの研究はINLBクリティカルなエフェクターとしてINLA -侵入経路13のとホスホイノシチド3 -キナーゼ（PI 3-K）の重要な構成要素として、α/βカテニン-アクチンリンクが同定された依存侵入カスカ14〜15·デ·。プロテオミクスおよび機能に基づくアッセイは、その後、新規な細胞骨格要素16および宿主細胞の浸潤に必要な脂質セカンドメッセンジャー17の同定を可能にした。転写の研究18と質量分析に基づく定量的プロテオミクス19は最近、ホストシグナル伝達カスケードの活性化とLの間の免疫応答の抑制に関する新たな光を当てるしている感染をモノサイトゲネス 。低分子干渉RNA（siRNA）をサイレンシングによる遺伝子（kinomes、完全なゲノム）の大きなセットの不活性化に基づいて、システム生物学的アプローチは、最近、食作用を含む特定の細胞機能のコンテキストでグローバルなホストシグナル伝達カスケード、解析のための新たな道を開いていて、病原体の内在化20。ゲノム規模のsiRNAスクリーンは、以前L.の感染に必要な細胞のカスケードを調査するために行われている食細胞ショウジョウバエのリステリア </eM&gt; S2細胞21〜22が、この種の分析は、非食細胞中で行われていない、 生体内での感染のための重要な目標を表すものです。 我々はLで、感染の後期の顕微鏡検出のためのプロトコルを最適化しています上皮細胞内の細菌侵入のハイスループットのsiRNAの研究に適していネス 。我々のアッセイは、侵襲性の高いLのを利用していますprfAを点変異を提示するリステリア株、L.の主要な転写調節因子病原性は6因子 モノサイトゲネス ：（prfAを*という名前）は、この変異は23 prfAをが構成的に活性なレンダリング、したがって、そうでなければ不十分な感染非食細胞における細菌侵入を好む、侵入タンパク質INLAおよびinlBの発現増加につながる。感染のための私達の読み出しは、分泌された細菌タンパク質InlCの細胞質ゾル蓄積の検出に基づいている。この分子である細胞質内Lで優先的に発現される多面的エフェクターリステリア 9と参加し、細菌細胞間に24を広めるだけでなく、宿主の免疫応答25を変調するだけではなく。細胞内細菌によるInlC分泌の蛍光標識は、明らかに非感染細胞からの感染を区別することを可能にするだけでなく、その後の別工程で感染を分析するために使用することができ、エンドポイントの読み出しを表していない場合のみ：エントリー、液胞の脱出、細胞質性細菌を増殖および細胞から細胞への広がり。この顕微鏡ベースのプロトコルは、したがって、L.による宿主細胞の感染に関与する細胞経路を研究するためのsiRNAスクリーニングに結合することができるリステリア 。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td>For liquid BHI preparation</td>
		 </tr>
		 <tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>Supplement to liquid BHI for BHI agar plates </td>
		 </tr>
		 <tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51830-500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamicin</td>
			<td>Sigma </td>
			<td>G1397-10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Formaldehyde (16%)</td>
			<td>EMS</td>
			<td>15710</td>
			<td>Prepare fresh before each experiment</td>
		 </tr>
		 <tr>
			<td>Anti-Rabbit Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A-11035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D-1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phalloidin Dy647</td>
			<td>Dyomics</td>
			<td>647-33</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Scramble</td>
			<td>Dharmacon</td>
			<td>D-001810-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Met</td>
			<td>Dharmacon</td>
			<td>L-003156-00-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 384-well microscopy cell culture plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AxioObserver Z1 microscope</td>
			<td>Zeiss</td>
			<td>431007 9901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Neo</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Metamorph analysis software</td>
			<td>Molecular Devices</td>
			<td>4000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CellProfiler analysis software</td>
			<td>Broad Institute</td>
			<td>Public software available at <a href="http://www.cellprofiler.org/" target="_blank">http://www.cellprofiler.org/</a></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes

細菌の細胞内病原体は絶妙に操作し、宿主の標的組織への感染に必要な細胞機能を破壊するそれらの能力に起因する細胞のシグナル伝達カスケードを分析するための分子ツールとして考えることができる。これらの細菌の病原体の中では、 リステリア菌は、細胞性免疫応答の特徴づけに細胞内寄生のためのパラダイムとして使用されており、その細胞骨格と膜人身売買のダイナミクスを制御する分子経路の発見に尽力役割を果たしてきましたグラム陽性微生物である。この記事では、我々はLの遅い携帯感染の段階を検出するための堅牢な顕微鏡アッセイを記述リステリア InlC、感染細胞の細胞質に蓄積し、分泌された細菌タンパク質の蛍光標識をもとにして、このアッセイは、char型にするために、自動化ハイスループット低分子干渉RNAの画面に結合することができる感染のアップまたはダウンレギュレーションに関与する細胞シグナル伝達経路をacterize。

細胞質InlCの蛍光標識は、L.により細胞感染のための堅牢な読み出しを提供していますリステリア 、 図1に示されるように：それは位相コントラスト画像（矢印、 図1A）で観察することができ、それがどこにDAPI信号によって確認されるように、顕微鏡写真の中央のセルは非常株P14.PrfA * 23に感染している個々の細菌は、明らかに（ 図1B）を</s...

Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

リステリア菌は、頻繁に、細胞内寄生の研究のための主要なモデルとして用い、グラム陽性細菌の病原体である。後半L.イメージング低分子干渉RNA画面のコンテキスト内感染段モノサイトゲネスは、標的宿主細胞の細菌感染に必要な細胞経路をグローバル研究を可能にする。

細菌性病原体による細胞の感染を調査するためにイメージングInlC分泌<em&gt;リステリア菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

imaging-inlc-secretion-to-investigate-cellular-infection-bacterial

Research

JoVE Journal

Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

9.8K ビュー.  Pasteur Institute.  リステリア菌は、頻繁に、細胞内寄生の研究のための主要なモデルとして用い、グラム陽性細菌の病原体である。後半L.イメージング低分子干渉RNA画面のコンテキスト内感染段モノサイトゲネスは、標的宿主細胞の細菌感染に必要な細胞経路をグローバル研究を可能にする。 

ビデオ: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

免疫・感染学

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

細菌性病原体によるヒト細胞の浸潤

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

昆虫<em&gt;ハチノスツヅリガ</em&gt;細菌性病因を調査するための強力な感染モデルとして

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

の口頭伝達<em&gt;リステリア菌</em&gt;汚染された食品の摂取を介したマウスにおける

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

の自動分離<em&gt; C.虫</em&gt;可変細菌性病原体の植民地

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

リステリア単球遺伝子を用いた感染マウスにおける培養および心臓コロニー形成における心臓細胞の細菌浸潤の評価

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA精製は、細胞内から成長します<em&gt;リステリア菌</em&gt;マクロファージ細胞で

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

と実験感染<em&gt;リステリア菌</em&gt;ホストインターフェロンγ応答を研究するためのモデルとして、

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.