この作品は、ウェルカムトラスト（WT 0795880）によって賄われていた。

次のプロトコルは、S.の内皮細胞の浸潤の研究を紹介します黄色ブドウ球菌は、しかし、理論的には任意の培養可能な細菌による細胞侵入を研究するために使用することができます。 S.への特定の段階黄色ブドウ球菌と内皮細胞が示されている。 1。細菌の調製<ol><li>文化S. 37 10ミリリットルブレインハートインフュージョン（BHI）ブロス中で4月16日H（必要な成長段階に応じて）℃〜200 rpmで振盪しながら空気中のため黄色ブドウ球菌株。これらの成長条件は、Sの固有のものです黄色ブドウ球菌と他の細菌のために適切に調整する必要があります。 </li><li> DMEMに相当量の細菌のペレットの培養上清と再懸濁の除去、室温で遠心分離の代替ラウンドで（5,000 × g、10分）;ダルベッコ変法イーグル培地（Invitrogen DMEM）中の細菌を3回洗浄する。その後、必要に応じて調節することができる細菌の得られた懸濁液の光学密度を測定します。 S.用黄色ブドウ球菌は、我々は、〜10 9 CFU ml -1のに対応するOD 600 = 1、懸濁液を準備。 </li></ol> 2。内皮細胞培養<ol><li>文化ウシ胎児血清（FBS; 10％）を添加したDMEMで内皮細胞株EA.hy926 1 37時とL -グルタミン（2mMの）°、5％CO 2でCを。また、プールされた初代ヒト臍帯静脈内皮細胞（HUVECを）ロンザ（バーゼル、スイス）から購入し、37℃で2％FBS、ウシ脳抽出物（ヘパリンを含む）、ヒト内皮成長因子およびヒドロコルチゾンを添加した内皮基礎培地で培養することができます° C、5％CO 2、メーカーの指示（ロンザ）によると。これらの成長条件は、これらの細胞に特異的であり、他の宿主細胞の種類に適応する必要があります。 </li><li>目で検証、コンフルエントに完了するためにT75フラスコで内皮細胞を成長させる。 </li><li>カバースリップの挿入のための24ウェルプレートを準備します。ファイン鉗子は、（火炎滅菌）1つの不透明な一光沢のある表面を持ってカバースリップを、移動するために必要である。細胞接着を可能にするために上向きに直面している不透明な面を持つ24ウェルプレートのウェルに配置してカバースリップ。 </li><li> 3ミリリットルトリプシン- EDTA（0.25％）とT75フラスコから細胞を解放すると、関連する培地10mlに加える。 </li><li> Thermanoxのガラス製カバースリップを含む24ウェルプレートに再懸濁した細胞を500μlを添加します。細胞の一つT75コンフルエントフラスコは約5で得られた2 24ウェルプレート、× 1ウェルあたり10 5細胞（500μlの培地中で）のために十分な細胞を提供した。 </li><li>前述のように48時間プレートをインキュベートし、反転光顕微鏡によるコンフルエント100％のセルを確認してください。 </li><li>ディップ洗浄PBSでカバースリップをし、各ウェルに10％FBSを含む490μlのDMEMを含む新しい24ウェルプレートに加える。 </li><li>細胞の浸潤における特定の代謝過程の役割を調べるために、阻害剤は培養細胞にアッセイ中に維持細菌や濃度を添加する前に1時間を追加することができます。例えば、S.のアクチン再構成の役割を決定する内皮細胞の黄色ブドウ球菌の侵入、50μMのサイトカラシンDを追加することができる、またはカベオラの役割のために、5mMのメチル-β-シクロデキストリンを添加することができる。 </li></ol> 3。浸潤アッセイ<ol><li>よく、10％FBSを含む490μlのDMEMで内皮細胞のコンフルエントな層で洗浄したカバースリップを含む各洗浄細菌の10μlを（約2 × 10 7 CFU ml -1の 黄色ブドウ球菌での結果）を追加します。 </li><li> 37℃で15〜90分間インキュベート° Cを5％CO 2で。 </li><li>細胞（付着性と内部化）に関連する細菌の総数を測定するために、カバースリップをPBSで3回浸漬洗浄し、PBSで500μlの0.5パーセントトリトンX - 100を含有する新鮮なウェルに加える。細胞が完全にすべての内部化された細菌を溶解し、解放を確保するために、直接カバーグラスの表面でピペットの先端を指して、数回ピペッティング。 </li><li> TSAプレートの表面に液体懸濁液（またはこの必要なの希釈液）をめっきによって細菌を列挙する。 TX - 100は多くのグラム陰性菌を溶解するので、サポニンは、代わりに2を使用できます。 </li><li>内部化された細菌数を測定するために、各ウェルを含むバインドされていない細菌から培養上清を除去し、200μgのml -1のゲンタマイシンを添加した500μlのDMEM/10％FBSに交換してください。我々は日常的にそれが安価であるので、ここではなく、リソスタフィンよりゲンタマイシンを使用し、私たちは実験的なプロトコルを変更することなく、細菌の種類によって（ブドウ球菌とLactococci）を用いた実験を切り替えることができます。 </li><li> 37プレートをインキュベート℃、5％のC 60分間CO 2のすべての細胞外細菌を殺すために。 </li><li> 、カバースリップをPBSで3回洗浄する溶解し、上記の接着アッセイのために説明されているようにTSA上にめっきによって列挙する。 </li><li>いくつかのケースでは、視覚的に光学顕微鏡を用いて細菌の数をカウントすることが好ましいかもしれない。このインスタンス内の、および特定のS.へ黄色ブドウ球菌の細胞浸潤は、物理的に細胞外細菌を破壊する代わりに、ゲンタマイシンのリソスタフィン（10μgのml -1）を使用してください。 </li><li> 37℃20分のためのカバースリップをインキュベート· CO 2のCは、リソスタフィンのソリューションで、その後すすぎとCytopath（Cellpath）で固定。 </li><li> 5分間クリスタルバイオレット（0.5％、w / v）のとカバーグラスを覆います。 </li><li>空気乾燥、水に浸漬洗​​い、ガラススライド上にマウントします。コンフルエント内皮細胞の1mm 2あたりの細菌の数は、光学顕微鏡を用いて定量することができます。 </li></ol> 4。代表的な結果： S.の表面上のフィブロネクチン結合タンパク質の発現（FnBPAとFnBPB） 黄色ブドウ球菌は、内皮細胞に侵入する能力を付与する。最近の研究は、FnBPA 3のフィブロネクチン結合ドメインを再定義しています。野生型（WT）S.黄色ブドウ球菌 8325.4はFnBPAとB（ΔFNB）の両方を欠いている株が内部化の有意な減少レベル（図1A）を示した一方、高効率で血管内皮細胞に侵入する。をコードするプラスミドのFNBと変異体の相補マイナスフィブロネクチン結合ドメイン（pFnR0は）（図1A）の侵入を促進しなかった。これとは対照的に、コードするプラスミドと変異体の相補全体FNB遺伝子（pFnBA4）はWTのレベル（図1A）に侵略を復元。 内皮細胞の浸潤におけるFnBPAの役割は、異種発現宿主ラクトコッカスラクティスを使用して実証することができます。 L.のプラスミドにコードされたFnBPAの発現ラクティス （pRM9 9）有意にないFnBPA（CTL）を（オープンバー、図1B）を発現しない細菌と比較して内皮細胞への接着を増強しなかった。対照的に、FnBPA発現L.ラクティスは、非発現株（クローズドバー、図1B）に比べ有意に高いレベルでの内皮細胞に侵入した。 実験は、重複に4回行ったし、平均値±標準偏差が表示されます。 * WTまたはCTLの値から（P = &lt;0.05）統計的に有意に異なるデータを表す。 <img src="/files/ftp_upload/2693/2693fig1.jpg" alt="図1" /> 図1 EAの侵略。 S.のHy926内皮細胞黄色ブドウ球菌 （A）またはL.ラクティス （B）。

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<html>我々が記述するアッセイは、細菌による宿主細胞の浸潤を調べるために広く使用されているゲンタマイシンプロテクションアッセイに基づいています。細胞内細菌を殺すためにゲンタマイシンと他の抗生物質の無力の観測は、細菌4月8日から宿主細胞の浸潤の初期の研究に活用された。 24、48あるいは96ウェルプレートの使用は、短期間で比較的低コストで定量的、再現性の高い大量のデータを生成することができます。それは専門機器を必要としない、それが様々な細菌や細胞と動作するように調整することができる、と侵略9で細菌と宿主の両方の細胞プロセスの役割を研究するために使用できるため、アッセイはまた魅力的です。確かに、最古の実験以来、ゲンタマイシン保護アッセイは、広くさまざまな細菌や細菌10,11の偶数の組み合わせの範囲が宿主細胞の浸潤を測定するために採用されている。核となる原則は同じである一方、メソッド内の多くの微妙な変化が報告されている。この記事では、我々のバージョンを記載し、変更が他の細菌や宿主細胞のために必要となる場合がありますどこに示されている。このアプローチを使用して、宿主細胞の浸潤を測定する実験を設計する際に考慮すべき重要な点の数があります。 ゲンタマイシンに対する細菌感受性が。これは明らかに見えるかもしれませんが、それは研究されている細菌は、濃度、温度、使用する時間の長さ以上でゲンタマイシンの影響を受けやすいことを確認することが重要です。鈍感の場合には、それは他の抗生物質5を使用することも可能です。別のアプローチは、溶菌酵素（リソスタフィン、mutanolysin、リゾチーム）12を使用することができます。 インキュベーションと接種は。最初にこのアッセイを実行する場合には、使用される最適なインキュベーション時間と細菌の接種を確立することが重要です。したがって、初期の実験は、時間をかけて接着し、侵入（最大6時間まで5分）の両方を調べる必要があります。それは、侵略が感染の多重度に影響されるかを評価することも重要である（MOI、細胞当たりの細菌数）。一般的に、これは培養細胞への損傷を低減するので可能な細菌の少ない番号を使用するのが最適です。長期潜伏期間や大規模な接種が9,13を使用している場合は株の間の重要な相違点を見逃す可能性があります。 細胞の損傷が。使用前に細菌を洗浄することが重要です。しかし、細胞損傷は、毒素産生に限定されるものではありません。細菌の侵入、アポトーシスと正常な細胞機能の破壊の誘導は、すべての細胞の損傷を引き起こす可能性があります。これは内部化細菌を殺し、侵略が14を発生していないという印象を与え、細胞内へのゲンタマイシンの浸透につながることができます。 インキュベーション条件は、温度および培地の組成は、侵攻に大きな影響を与えることがあります。例えば、 化膿連鎖球菌によるHeLa細胞の浸潤は通常、血清15の存在する可溶性フィブロネクチンの存在に依存している。別の考慮事項は、実験の過程で培地中の細菌の増殖です。 文化と細胞内細菌の定量化。TX - 100は細胞内細菌を殺すしないかもしれないが、それは彼らの成長を阻害することがあります。そのため、界面活性剤の存在下で固体培地上に細菌の複製を確認することが重要です。影響を受ける場所、それは洗剤の低濃度を使用したり、サポニンなどの代替を使用することがあります。クリスタルバイオレット染色および顕微鏡アッセイ上ゲンタマイシンプロテクションアッセイの利点は、集中的に少ない労働力であるということです、そしてそれは内部化された細菌の亜集団の検出と分化することができます。例えば、S.黄色ブドウ球菌は通常のコロニーのタイプ（NCT）または個々の細菌細胞の顕微鏡による区別はないと小コロニーの変異体（SCV）16を 、生成することができます。ゲンタマイシンプロテクションアッセイ上クリスタルバイオレット染色および顕微鏡アッセイの利点は、細胞の凝集が考慮することができますし、&quot;実行可能な、非培養可能&quot;状態に入る細胞内細菌が17日に検出されることです。 細菌の侵入における宿主細胞のプロセスの役割を検討 。多くの研究は、アクチン再構成を妨げるサイトカラシンD、などの宿主細胞機能の阻害剤を用いている。このような研究はまた、標的細胞表面の受容体と特異的な遺伝子18のsiRNAのノックダウンその抗体を含めることができます。それは、これらの治療は、培養細胞の生存または添付ファイルに影響を与えたり、細菌の毒性を持っていないことを確認するために極めて重要です。 今後の方向性： ontent&quot;&gt;このアッセイは、通常、マルチウェル細胞培養プレート中で行われるため、それは潜在的な細胞機能の阻害剤のライブラリの検討を巻き込むようなハイスループットスクリーニングの操作、それを適応することは理論上可能です、抗感染薬または抗生物質細胞内細菌を標的とするように設計。また、そのようなアプローチは接着、浸潤や細胞生存に関与する遺伝子を同定するために変異体ライブラリーをスクリーニングするために使用することができる。いくつかのケースでは、モデルシステムでせん断力を（フロー）に含めることが望ましいかもしれない、より密接に生体環境における模倣する。フローセルとマイクロ流体細胞培養システムの設計および製造における現在の進歩は、せん断力を組み込む宿主-病原体のモデルにゲンタマイシン保護アッセイを採用する可能性を提供し、内皮細胞をモデル化するときなど。 要約すると、ここで説明するプロトコルは、長年にわたって使用される同じようなアプローチに基づいています。それは広く、培養細胞や細菌種の多くの種類に適用可能であり、迅速かつ比較的少ないコストで、大量のデータを生成することができます。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31885-023</td>
<td>&nbsp;</td>
<tr>
<td>Brain heart infusion</td>
<td>&nbsp;</td>
<td>BioChemika</td>
<td>53286</td>
<td>&nbsp;</td>
<tr>
<td>Foetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>HUVECs</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-2519</td>
<td>&nbsp;</td>
<tr>
<td>Endothelial basal medium</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3121</td>
<td>&nbsp;</td>
<tr>
<td>Bovine brain extract (including heparin)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092</td>
<td>&nbsp;</td>
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<td>Human endothelial growth factor</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4017C</td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4035C</td>
<td>&nbsp;</td>
<tr>
<td>GA-1000</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092C</td>
<td>&nbsp;</td>
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<td>Gentamicin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15710</td>
<td>&nbsp;</td>
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<td>Lysostaphin</td>
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<td>LSPN-50</td>
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<td>Thermo Fisher</td>
<td>TKT-210-330P</td>
<td>&nbsp;</td>
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<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
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<td>Cytopath</td>
<td>&nbsp;</td>
<td>TCS Biosciences</td>
<td>HC8595</td>
<td>&nbsp;</td>
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<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3886</td>
<td>&nbsp;</td>
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<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4049</td>
<td>&nbsp;</td>
<tr>
<td>T75 flasks</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>430641</td>
<td>&nbsp;</td>
<tr>
<td>Cytochalasin D</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2618</td>
<td>&nbsp;</td>
<tr>
<td>Methyl-B-cyclodextrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>332615</td>
<td>&nbsp;</td>		</tbody>
		</table>
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invasion of human cells by a bacterial pathogen

ここで我々は細菌性病原体黄色ブドウ球菌によるヒト内皮細胞の浸潤を調べる方法について説明します。一般的なプロトコルは、事実上すべての培養可能な細菌が細胞の浸潤の研究に適用することができます。このようなアクチンの再編成やカベオラの役割としての侵略の特定の側面を検討されることができるステージが、、強調表示されます。宿主細胞はフラスコとするときに使用可能な状態で栽培されてThermanoxのカバースリップを含む24ウェルプレートに播種されています。カバースリップを使用すると、井戸からの細胞のその後の除去は、井戸（ 黄色ブドウ球菌は、添付する先）の側面に堆積した血清タンパク質からの干渉を低減することができます。細菌は、必要な密度まで増殖させ、任意の分泌タンパク質（例えば毒素）除去するために洗浄されています。内皮細胞のコンフルエントな層とカバースリップは、細菌を添加する前に新鮮な培養培地を含む新しい24ウェルプレートに転送されます。細菌や細胞を37℃5％CO 2で時間を必要な量のために一緒に℃でインキュベートする。 S.用黄色ブドウ球菌は、これは通常、15〜90分の間です。 Thermanoxカバースリップは、各ウェルから除去し、アタッチされていない細菌を除去するためにPBSで浸漬洗浄されています。合計られている細菌（付着と内部化されたが）定量化する場合は、カバースリップをPBSでX - 100 0.5％のトリトンを含む新鮮なもに配置されます。穏やかなピペッティングは、細胞溶解を完了すると、細菌は寒天培地段階希釈し、メッキによって列挙されているリード。細胞に侵入している細菌の数が必要な場合は、カバースリップはすべて外部の細菌を殺す1時間、継続ゲンタマイシンとインキュベーションを添加した500μlの組織培養の培地を含むウェルに追加されます。カバースリップをして洗浄す​​ることができる、細胞が溶解し、上記のような細菌は、寒天の上にメッキで列挙。実験は、直接可視化を必要とする場合、カバーガラスを固定して、染色、光蛍光または共焦点顕微鏡、あるいは電子顕微鏡用に調製することができる。

Watch this Scientific Journal Video about Invasion of Human Cells by a Bacterial Pathogen at JoVE.com

Invasion of Human Cells by a Bacterial Pathogen

に焦点を当てて細菌の病原体による宿主細胞の浸潤の研究のための一般的なプロトコル、<em&gt;黄色ブドウ球菌</em&gt;とヒト内皮細胞。

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 ...

invasion-of-human-cells-by-a-bacterial-pathogen

Research

JoVE Journal

Immunology and Infection

34.6K ビュー.  University of Bath. に焦点を当てて細菌の病原体による宿主細胞の浸潤の研究のための一般的なプロトコル、黄色ブドウ球菌とヒト内皮細胞。

ビデオ: Invasion of Human Cells by a Bacterial Pathogen

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

M細胞による細菌の取り込みを評価するパイエル板腸管ループアッセイを連結したマウスのアプリケーション

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

免疫・感染学

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

血液培養から細菌性病原体の検出および抗菌剤耐性テストのための一日のワークフロースキーム

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

腸管上皮細胞の腸内細菌の侵入<em&gt;インビトロ</em&gt;劇的垂直拡散室モデルを用いた強化され

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

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

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

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 ...

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 the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

酵母ツーハイブリッドスクリーンを用いた非耕作植物病原体から細菌のエフェクターの機能をUnravelling

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.