Name: analysis of yersinia enterocolitica effector translocation into host cells using beta-lactamase effector fusions
Uploaded: 2015-10-13T15:00:00.000+00:00
Duration: 12 min 23 s
Description: Watch this Scientific Journal Video about Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions at JoVE.com

We thank Dr. Antonio Virgilio&#160;Failla for providing the FRET acquisition quantification algorithm and Erwin Bohn for providing the pMK-bla and pMK-ova constructs.

1.ピーク発光とクロストーク決意（また図2を参照してください） <ol><li>ドナー（クマリン誘導体のV450）およびアクセプター（フルオレセイン）色素の間の発光ピークとのクロストークの量を決定するために、405 nmの励起での個々のフルオロフォアの両方で標識された細胞のスペクトルスキャンを実行します。 </li><li> （ここでは455から465 nmおよび525から535 nm）のドナーとアクセプターチャネルに使用​​される各色素のピーク内に10 nmの帯域幅を決定します。波長以上の正規化強度をプロットし、アクセプターチャネルとその逆でドナー色素のクロストークの平均割合（ 図2）を計算します。 </li></ol> Y.の調製細胞培養の感染のエンテロコリチカ株<ol><li>以下の株を使用してください：WA-314 PMK-OVAが及び（WA-314は17 PMK-BLAとPMK-OVA 17、それぞれプラスミドで形質転換）WA-314ΔYopEPMK-BLA（WA-プラスミドPMK-BLA 17で形質転換314ΔYopE） </li><li>感染ストリークYの前に少なくとも2日WA-314 PMK-BLAとWA-314 PMK-OVAに必要な抗生物質（カナマイシンを含むLB寒天プレート上のクライオストックから株WA-314 PMK-OVA、WA-314 PMK-BLAとWA-314ΔEPMK-BLA エンテロコリチカ ;カナマイシンおよびWA-314ΔEPMK-BLAのためのテトラサイクリン）と27℃でO / Nを育てます。その後、4℃で最大1週間のプレートを保ちます。 </li><li>感染前のある日は、それぞれの株の1コロニーを、必要な抗生物質を含むLB培地の3ミリリットルを接種し、180rpmで振とう機で27℃でO / Nインキュベートします。 </li><li>感染の日に、抗生物質を含まない新鮮なLB培地40ml中にO / N培養物を2mlに接種し、III型分泌系の発現を誘導するために180rpmで振盪器中で37℃で90分間インキュベートします。 </li><li>適切なチューブに培養液を移し、氷の上または4℃で培養物を維持します今後。 5,000×gで、4℃で10分間培養液を遠心します。 </li><li>氷2〜3mlの冷PBS中で細菌ペレットを再懸濁します。分光光度計を使用して、8×10 8 CFU / mlの濃度の細菌懸濁液を希釈します。個別に対応する光学密度を決定します。 HeLa細胞に感染するまで氷上でこの懸濁液を保管してください。 </li></ol> 3.メッキHela細胞<ol><li>一日、96ウェルプレートのウェル中で、10％熱不活性化ウシ胎児血清（FCS）を含有する200μlのDMEMで感染シード1.5×10 4細胞 、HeLa細胞の前に、37℃で5％CO 2インキュベーター内で育てます。各株（三つの異なるインキュベーション時間）のための3つのウェルをシード。 </li></ol> 4. HeLa細胞の感染とCCF4を使用したロード<ol><li>培地に細菌懸濁液の3.5μLを追加することにより、HeLa細胞に感染し、そっと中を二回ピペッティングして混ぜます。細菌が上の沈降することを許可します細胞当たり約100バクテリアのMOI（感染の部分）の細胞です。時間経過の場合は-90分、-60分-30分で感染を開始し、一般的なエンドポイントを達成するために、0分まで、5％CO 2インキュベーター中、37℃でインキュベートします。 </li><li>インキュベーションが完了したときに、慎重にHeLa細胞を除去せずに培地を吸引し、CCF4の輸出を減らすためにエフェクターおよび2.5mMプロベネシドの発現を停止するために20μg/ mlのクロラムフェニコールを含有するPBSを50μl加えます。 </li><li>この時点で、顕微鏡ステージに96ウェルプレートに移し、各ウェルに、後の分析のために適切なスポットを定義します。 10分以内に、この手順を終了（取得の詳細については、セクション5.1を参照）。 </li><li>あたりがよく、20を含むCCF4 / AM負荷溶液（0.24μlの溶液A、1.2μlの溶液Bの70μL、18.56μlの溶液C（溶液A、B、及びCはCCF4 / AMローディングキット内に提供される）および50μlのPBSを（追加しますマルチPを使用して/ mlのクロラムフェニコールおよび2.5mMプロベネシド）ipetteを室温で5分間インキュベートすると。今から仕事の中断なし。 </li><li>ローディング溶液を吸引除去し、マルチピペットを用いて20μg/ mlのクロラムフェニコールおよび2.5mMプロベネシドを含む100μlのPBSを追加します。その後急速に顕微鏡ステージに96ウェルプレートを転送し、充填溶液の吸引から10分以内に取得を開始します。 </li></ol> 5.レーザー走査顕微鏡とデータ解析<ol><li>画像収集<ol><li>総光子数を最大化し、光毒性を最小限にするように撮影条件を設定し、光退色、クロス励起。 <ol><li>現代の共焦点レーザー走査型顕微鏡では、同時にアクティブなドナーおよびアクセプターチャネル、8ビット深度で高感度のGaAsP検出器を選択し、2.5エアリー単位（ すなわち、広い光学切片）に検出器のピンホールを開いて、20倍高い開口数の液浸対物レンズを使用し、〜750 nmの画素サイズ、3倍のフレーム平均とexcit405nmのダイオードレーザーの10％E。 </li><li>正確な定量化が同じ値に両チャンネルの検出器利得を設定確保し、いかなる飽和画素を回避すること。 注：ここでは、ゲインを150％に設定しました。 </li></ol></li><li> 1mlシリンジで例えば、ウェルの底に液浸媒体を拡散することによって、連続的な浸漬を確認し、任意の気泡の捕捉を避けます。 </li><li>透過光を有効にして、全てのウェル内の視野の代表フィールドを見つけます。正しく調整自動ステージを使用して、ソフトウェア内の位置をマークします。 </li><li>染色用のプレートを除去した後、（4.3節を参照）、プレートを再挿入し、焦点ずれを回避するために、上に重量を付加します。レーザ走査モードで保存された位置を確認し、適切にフォーカスし、横方向の位置を調整します。 </li><li> 2時間に、2分に期間を時間の経過を設定し、取得を開始します。 </li></ol></li><li>画像定量化（例としては、ビットプレーンSOFTWについて説明します）などIMARISの通りです<ol><li>ピクセル行列の算術計算を可能にするソフトウェアにデータセットをインポートします。ソフトウェアのアイコンをドナーとアクセプターチャネルの両方を含むソースファイルをドラッグ＆ドロップすることでこれを行います。 </li><li>同じオフセットを使用して、両方のチャネルでバックグラウンドを減算します。これを行うには、 メニューをクリックし、画像&gt;処理&gt;ベースライン減算をクリックします。各チャンネルを選択し、ベースライン値を入力します。 </li><li>予め決定された補正係数fとアクセプターチャネル（CH）にドナーチャネル（CH1）のブリードスルーを補正（1.1を参照）、新しいチャネルを作成します。 Ch1のF * - = CH 2 CH 2」 注：アクセプターのブリードスルードナーチャンネルにはノイズのレベルを超える検出されませんでした。 <ol><li>メニュー&gt;画像処理&gt;チャネル算数をクリックして、ABS（CH 2 - （CH1 * 0.33））を入力します。その後メニュー&gt; [編集]&gt; [チャンネルを削除]をクリックしてチャンネル2を削除します。ていることを確認してくださいブリードにより補正アクセプターチャネルとして残る新しいチャンネル2.オプション：[メニュー]&gt; [編集]&gt; [画像のプロパティに行くことによって、元の色にグレーのチャンネルカラーを変更します。カラー&gt;ベースカラーをマッピングされた&gt;チャネル4を選択し、緑などに変更します。 </li></ol></li><li>相対FRET係数を決定するために、次の操作で新しいチャネルを作成します。 CH3 = CH 2 &#39;/（CH1 + Ch2の&#39;） <ol><li> ./メニュー&gt;画像処理&gt;チャネル算数に移動して、CH2を入力（CH1 + CH2） </li></ol></li><li> 8ビット画像でのFRETチャンネルを適切に視覚化するために、第四のチャネルを作成します。 CH4 = 255 * Ch3の<ol><li>メニュー&gt;画像処理&gt;チャネル算数に移動し、255 *のCH3を入力します。[メニュー]&gt; [編集]&gt; [画像のプロパティに行くことによって、チャネルの色を変更します。チャンネル4&gt;マップされたカラー&gt;カラーテーブルファイル&gt;ジェットを選択します。 </li></ol></li><li>チャネルCH3およびCh4を股関節に3×3のメディアンフィルタを適用します。tは、画像のノイズを低減します。これを行うには、メニュー&gt;画像処理&gt;スムージング&gt;メジアンフィルタにクリックします。両方のチャンネルを選択し、フィルタサイズ「3x3x1」を適用します。 </li><li>細胞のシグナルの定量のために、適切にオフセットが小さすぎる構造を除外するように大きさの構造体をフィルタを設定することにより、Ch4を内のセルを検出します。 <ol><li> ビューを突破し、「 新しいサーフェスの追加」アイコンをクリックしてください]を選択します。表面ウィンドウで検出アルゴリズムのパラメータを定義します。より高速な処理のために、2つのチェックボックス「セグメント関心領域のみ」と「最終的にはプロセス全体のイメージを &quot;有効にします。その寸法を編集して、関心領域を定義します。 </li><li>ソース・チャンネルとしてチャンネル4を選択し、しきい値設定&gt;背景差分（局所的なコントラスト）によりしきい値を設定し、10μmの直径を設定します。最大intensiに手動で0に最小強度しきい値と最大しきい値を設定しますTY。地域によって構造を濾過し、 例えば、187μm²に最小値を設定します。表面検出を終了します。 </li></ol></li><li>携帯エンティティにおけるドナー蛍光強度の平均値（CH1）及び相対FRET係数（CH 3）とその標準偏差を抽出します。これを行うには、表面特性にアクセスしてください。サーフェススタイル/品質&gt;サーフェスを選択することにより、構造物の外観を変更します。フィルタ]タブをクリックすることで、すべての構造を選択します。選択&gt;統一を処理するために行くことによって検出された構造を統一。ファイルにすべての統計情報をエクスポート&gt;統計]タブをクリックして、MS Excelに表面の統計情報をエクスポートします。 </li><li>時間をかけてこれらの値をプロットします。転位置エフェクターの量を表すために合理的なパラメータとして、各条件のためのドナーチャンネル蛍光増加の最大傾斜の10分の間隔を指定します。 M =Δドナー蛍光強度/Δ時間（分）などの傾きを計算します。 </li></ol></lI&gt; </ol>

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The authors declare that they have no competing financial interests.

ここでは、正常Yでエフェクター転座の定量分析のために、TEM-1ベータラクタマーゼレポーターベースのアッセイを適用しましたエンテロコリチカ 。この敏感な特定の比較的簡単な技術の多くの異なる変形例が文献に記載されています。この研究では、レーザ走査顕微鏡は、蛍光シグナルの最も高感度で正確な検出を行いました。具体的にドナーとアクセプター色素と個別に調整...

III型分泌系を直接標的真核細胞中にコードされた細菌のエフェクタータンパク質を提供するために、グラム陰性菌の異なる属が利用する特殊なタンパク質輸出機です。分泌機構自体が高度に保存されている一方で、エフェクタータンパク質の特殊なセットは、細胞シグナル伝達経路を操作し、特定の細菌の病原性戦略1を容易にするために、異なる細菌種間で進化してきました。 エルシニアの場合には、7エフェクタータンパク質、いわゆるYops（ エルシニア外側タンパク質）まで、宿主細胞の接触の際に転位などすなわち食作用およびサイトカイン産生、免疫細胞応答を破壊するために一緒に作用され、細菌の細胞外の生存を可能にします2-4。トランスロケーションのプロセスをしっかりと異なる段階5で制御されています。これは、T3SSの主要な活性化は、ターゲットCEとの接触によって誘発されることが確立されていますLL 6。しかし、この開始の正確なメカニズムはまだ解明されています。 エルシニアで転座のいわゆる微調整の第二のレベルは、細胞のRho GTP結合タンパク質Rac1のまたはRhoAのアップまたはダウンレギュレーション活動によって達成されます。転位しYopEのGAP（GTPアーゼ活性化タンパク質）関数は、Rac1活性をダウンレギュレートし、それに応じて負帰還型によって転位を減少するベーまたは細胞傷害性壊死因子Y（CNF-Y）でのRac1 などの活性化は、増加した転座7-9につながります機構10,11の。 有効かつ正確な方法は、転座は、 エルシニア 、宿主細胞の相互作用の間に調節する方法を調査するための前提条件です。多くの異なるシステムは、特定の利点と欠点を有するそれぞれ、この目的のために使用されてきました。いくつかのアプローチは、ウェスタンブロット分析を行った異なる界面活性剤によって感染した細胞の溶解ではなく、細菌に依存しています。目これらの方法の電子共通の欠点は、マイナーが、避けられない細菌溶解は、潜在的に細菌に関連するエフェクタータンパク質と細胞溶解液を汚染することです。しかし、細胞外エフェクタータンパク質および真核細胞の選択的溶解のためのジギトニンのその後の使用を分解するプロテイナーゼKでの細胞の処理は、この問題12を最小化するために提案されました。重要なことは、これらのアッセイは、決定的にほとんどが市販されていない高品質の抗エフェクター抗体に依存。転座を監視するために、エフェクタータンパク質とGFPなどの蛍光タンパク質の翻訳融合を使用しようとすると、おそらく蛍光タンパク質の球状三次構造と分泌13前にそれらを展開するために分泌装置のできないことに成功しませんでした。しかしCYAのような、いくつかの異なるレポータータグ（カルモジュリン依存性アデニル酸シクラーゼ） 百日咳菌毒素 cyclolysin 14またはフラッシュのドメインタグが正常に転座を分析するために使用されました。フラッシュタグ、非常に短いテトラ（4Cys）モチーフタグは、分泌のプロセスを妨げることなく、二ヒ素色素のFlashでの標識を可能にしながら、元のアッセイではCYAの酵素活性は、細胞内の融合タンパク質の信号を増幅するために使用されます15。 ここで適用されるアプローチは、によってシャルパンティエらは 、初めて報告され、転エフェクターTEM-1β-ラクタマーゼ融合物16（ 図1A）によって蛍光共鳴エネルギー移動（FRET）色素CCF4の細胞内変換に基づいています。 CCF4 / AMは、クマリン誘導体（ドナー）およびフルオレセイン部分（アクセプタ）はセファロスポリンのコアによって連結された細胞透過性化合物です。真核生物の細胞内へのパッシブエントリー時には、非蛍光性エステル化CCF4 / AM化合物を仕込み、蛍光CCF4に携帯エステラーゼにより処理され、それによって捕捉され、セル内の。 530 nmの緑色の蛍光シグナルを発するフルオレセイン部分へのFRETの405 nmの結果でのクマリン部分の励起。 β-ラクタマーゼFRETによってセファロスポリンのコアの切断後に破壊され、クマリン部分の励起は、NM at460青色の蛍光発光につながります。この方法の別の用途は、その汎用性を強調し、文献に記載されています。この方法は 、インビトロで転座の分析を可能にし、 また 、インビボで、例えば、この技術は、インビボ 17-19 に転座の標的白血球集団を同定するために、マウスの感染モデルに使用しました。信号の読み出しは、プレートリーダー、FACS分析または蛍光顕微鏡を用いて行うことができます。注目すべきは、この方法は、感染プロセス20,21の間に、生細胞の顕微鏡検査により、リアルタイムで移動を監視する可能性を提供します。ここで、レーザ走査型蛍光顕微鏡に適用したreadou蛍光シグナルのトンそれは最高の感度と精度を提供していました。具体的には、高感度の検出器と組み合わせて、ナノメートル精度で放射窓を調整する能力は、最適化された蛍光検出と最小限にクロストークを容易にします。また、この顕微鏡の設定は、転座のリアルタイムモニタリングのために適合することができ、潜在的に細胞レベルでの宿主 - 病原体相互作用の同時分析のために可能になります。 Yのこの研究転速度でエンテロコリチカ野生型株とhypertranslocator表現型10,11を示す YopE欠失変異体は、例示的に分析しました。

<table><tbody><tr><td>LB-Agar</td><td>Roth</td><td>X969.2</td><td></td></tr><tr><td>LB-Medium</td><td>Roth</td><td>X968.2</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8662-500ML</td><td></td></tr><tr><td>96-well plate (black, clear bottom)</td><td>Greiner Bio One</td><td>655087</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td><td>Gibco/Life Technologies</td><td>31966-047</td><td></td></tr><tr><td>Probenecid</td><td>Sigma-Aldrich</td><td>P8761-25G</td><td></td></tr><tr><td>LiveBLAzer FRET-B/G Loading Kit with CCF4-AM</td><td>Life Technologies</td><td>K1095</td><td></td></tr><tr><td>Lectin from Triticum vulgaris (wheat) FITC conjugate</td><td>Sigma-Aldrich</td><td>L4895</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>V450 Rat anti-Mouse CD8a</td><td>BD Bioscience</td><td>560469</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>Immersol W immersion oil&amp;nbsp;</td><td>Zeiss</td><td>444969-0000-000</td><td>Refractive index = 1.3339 @ 23 &amp;deg;C</td></tr><tr><td>TCS SP5 II confocal laser scanning microscope</td><td>Leica microsystems</td><td></td><td>2x GaAsP-Hybrid detectors, 4 channel spectrometer, acusto optical beam spliter, motorized XY stage, adjustable pinhole, objective 20X HC PL APO CS IMM/CORR, 405&amp;#160;nm diode laser 50&amp;#160;mW</td></tr><tr><td>Imaris 7.6 software</td><td>Bitplane</td><td></td><td>Plugins included ImarisXT and MeasurementPro</td></tr><tr><td>MatLab compiler runtime</td><td>MathWorks</td><td></td><td></td></tr><tr><td>Prism 5</td><td>GraphPad software</td><td></td><td></td></tr></tbody></table>

analysis of yersinia enterocolitica effector translocation into host cells using beta-lactamase effector fusions

病原性エルシニア属を含む多くのグラム陰性菌。真核生物の標的細胞へのエフェクタータンパク質を転位するIII型分泌系を使用します。宿主細胞の内部にエフェクタータンパク質は、細菌の利益のために細胞機能を操作します。より良い転座を測定するために、宿主細胞の相互作用、敏感かつ正確なアッセイ中のIII型分泌の制御を理解するために必要とされます。ここでは、 腸炎エルシニアエフェクタータンパク質断片（エルシニア外側タンパク質; YopE）の融合に基づくアッセイのアプリケーションを記述している。転座の定量分析のためのTEM-1β-ラクタマーゼとのアッセイは、細胞の切断に依存している浸透性のFRET染料（CCF4 / AM）転座β-ラクタマーゼ融合による。 β-ラクタマーゼによってCCF4のセファロスポリンのコアの切断後、フルオレセインへクマリンからのFRETが中断され、クマリン部分の励起は、青色蛍光発光につながります。この方法の別の用途は、その汎用性を強調し、文献に記載されています。この方法は、マウスモデルでは、例えば、インビトロでもインビボで転座の分析を可能にします。蛍光信号の検出は、FACS分析または蛍光顕微鏡は、プレートリーダーを用いて行うことができます。ここで説明するセットアップでは、異なるエルシニア変異体によるHeLa細胞へのエフェクター融合物のインビトロ転位はレーザー走査顕微鏡によってモニターされます。リアルタイムでβ-ラクタマーゼエフェクター融合によるFRETレポーターの細胞内変換を記録する堅牢な定量的な結果を提供します。ここでは、Yで増加転座を実証する、典型的なデータを示し、 エンテロコリチカ YopE変異野生型株と比較。

定量的に、標的細胞へのエフェクターの移動を分析するために記載された方法の能力を実証するために、種々の転動態を有する2つのエルシニア菌株を試験した：Y.野生型株WA-314 エンテロコリチカ （血清群O8、病原性プラスミドpYVO8 22宿す）及びその誘導体WA-314ΔYopE（pYVO8ΔYopE23を保有する WA-314）。初期の作品は、ことが示されたY.機能YopEの展示に有?...

Watch this Scientific Journal Video about Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions at JoVE.com

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

解析<em&gt;腸炎エルシニア</em&gt;エフェクター転β-ラクタマーゼエフェクター融合を用いて宿主細胞に

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

analysis-yersinia-enterocolitica-effector-translocation-into-host

Research

JoVE Journal

Immunology and Infection

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

8.5K ビュー.  University Medical Center Hamburg-Eppendorf. Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

ビデオ: Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

免疫・感染学

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

酵母毒性およびサプレッサースクリーンを用いた細菌エフェクタータンパク質を標的とする宿主経路の同定

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

遺伝学

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

哺乳動物細胞への機能性細菌エフェクターのエレクトロポレーション

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

酵母の菌タイプIIIエフェクターの発現により誘導される増殖阻害表現型の同定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

生物学

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用<em&gt; Q熱リケッチア</em&gt;のsiRNAサイレンシング中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

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 ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

の世代<em&gt;エンテロバクター</em&gt;エスピー。 YSU要求株にはトランスポゾン突然変異誘発を用いて

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

緑の蛍光蛋白質感染時に細菌から配信されるエフェクターを視覚化するシステムを分割します。

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

遺伝暗号拡張を用いた細菌分泌タンパク質の超解像イメージング

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

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