私たちのラボでの研究は、マックス·フォン·ペッテンコーファー研究所、ルートヴィヒ·マクシミリアン大学、ミュンヘン、ドイツ研究協会（DFG、HI 1511/3-1）とBundesministeriumエリーゼBildungウントForschung（BMBF） &quot;医療感染ゲノム&quot;イニシアティブによってサポートされていました（ 0315834C）。

1。 LCVアイソレーションの準備<ol><li> レジオネラ出ストリークは、4日間の小型商用車の分離前に5μg/ mLのクロラムフェニコール（CAM）を使用したCYE寒天プレートにグリセロールストックからDsRedタンパク質-Expressの13,14を生成します 。 37細菌をインキュベート℃に</li><li> 1×10 のうち7種D. GFP-カルネキシンまたは75 cm 2の組織培養におけるRAW264.7マウスマクロファージを生産discoideumのは 1日前の感染をフラスコ。 D. 20μg/ mLのG418で10ミリリットルHL5培地を使用します。 23℃discoideumのとアメーバをインキュベートC、5％CO 2を RAW264.7マクロファージに対しては、10％FCS（熱不活性化）と1％グルタミンを添加した10ミリリットルRPMI 1640培地を使用して、37℃で細胞を成長させる°。感染症やサンプルごとに少なくとも3 75センチメートル2フラスコ（最小6×10 7細胞）を使用します。 </li><li> L.で一晩培養物を接種CYEプレートからレジオネラ 。 AYE培地3 mLで15 mLの試験管を取るおよび5μg/ mLのカム。 0.1のOD 600nmのを生成するために、細菌懸濁液の100μlで接種する。 21から22時間37℃でオーバーヘッド回転ホイールの一晩培養をインキュベートします。 </li></ol> 2。 LCVの分離<ol><li> D.の媒体を変更するdiscoideumの細胞には、次の感染を妨げる抗生物質を、削除します。 </li><li>一晩培養物のODを測定します。細菌は、2×10 9細菌/ mlに相当する≥3のOD 600nmで 、で、そのピークの感染に達している必要があります。 </li><li> L.の約500μlを添加して細胞を感染させる10ミリリットルHL5培地（D. discoideumの場合）または10ミリリットルRPMI 1640（マクロファージ）に増殖する細胞にpneumophilaの一晩培養。これは50の感染多重度（MOI）に対応しています。その後、感染は10分間500×gで細胞に細菌の遠心分離によって同期されます。遠心分離は続いているD.のインキュベーションにより、 25°Cと°C、5％CO 2、それぞれ37 RAW264.7マクロファージにおけるdiscoideumの細胞 。 1時間の感染時には、遠心分離工程が含まれています。 </li><li>感染後、培地を除去し、細胞外細菌を除去するために一回細胞を洗浄する。 D.、氷冷SorCバッファを使用タマホコリカビとRAW264.7マクロファージ、氷冷PBS。各フラスコにプロテアーゼ阻害剤（Roche社）を添加した3 mLのHSバッファを追加し、セルスクレイパーを用いて細胞を収集します。 15 mLの試験管内のプールに対応するサンプルを。 </li><li>サンプルの均質化のために3 mLのプラスチック製のルアーロックシリンジとステンレス鋼ボールホモジナイザーを使用しています。あなたは氷の上で動作することを確認してください。開始する前に、任意の洗剤汚染を避けるために、気泡を取り除くために、氷冷HSバッファを使用して、それをフラッシュするために、蒸留水でボールホモジナイザーを洗浄する。 8μmのクリアランスのボールを使用しています。 </li><li>シリンジUNDに最初の3 mLを埋める日にそれをマウント電子ホモジナイザー。ホモジナイザーを前後に9回のサンプルを押してください。均質化しない物質による汚染を避けるために、その後シリンジを交換します。収集し、プール均質化したサンプルを15 mLの試験管内および顕微鏡分析のために150μlのサンプルを取る。異なるサンプルを続行する前に、ボールホモジナイザーを解体し、洗浄してください。 </li><li> 4℃で氷の上でシェーカー上で30分間またはオーバーヘッドスピニングホイール（10〜20 rpm）で2％CSやFCSでホモジネートをブロック℃、 </li><li>アフィニティー精製SIDCに対する一次抗体（希釈1:3000）またはLCV膜への排他的に結合する他の細菌のマーカーに対してを使用してブロッキングした後。ホモジネートに追加する前に、渦抗体溶液。 4℃で氷の上でシェーカー上で1時間またはオーバーヘッドスピニングホイール（10〜20回転）でサンプルをインキュベート</li><li>その間に、Histodenz勾配を準備します。勾配あたり15 mLの試験管を使用して、一つのサンプルの3 75センチメートル2フラスコ。モミ聖、チューブに35％Histodenzの5.75 mLを加え、そして第二に、慎重の上に10パーセントHistodenz液5.75 mLを加える。その後、慎重に1時間水平にチューブを捨てる。 </li><li>ブロッキング試薬、一次抗体、4ペレット°Cの試料で15分間600×gで遠心したホモジネートのインキュベーション後に。上清を捨て、1.5 mLのHSバッファーでペレットを再懸濁します。新鮮な15 mLの試験管にサンプルを転送し、後に顕微鏡分析のために40μlのサンプルを取る。 </li><li>二次抗体を用いてペレットをインキュベートします - MACSヤギ抗ウサギIgGマイクロビーズ（希釈1:25） - シェーカー上で30分間に4℃でその間に、MACS磁気ホルダーに列を入れて、0.5 mLのHSバッファでそれらを平衡化します。 </li><li>その後、カラムにサンプルを適用します。フロースルー、その後の顕微鏡分析のための150μlを収集します。 0.5 mLのHSバッファーでカラムを3回洗浄します。 </li><li> tから列を削除潮吹き即座0.5 mLのHSバッファを持つ彼は、磁石と溶出液商用車。顕微鏡分析のために20μlのサンプルを取る。免疫磁気分離による精製工程は、1当たりの収量が期待される×10 7感染D. discoideumの細胞約1×10 6をそのまま小型商用車、約七倍のフロースルー13と比較して溶出液中に濃縮されています。 </li><li>垂直の位置に戻しHistodenz勾配管を入れて、慎重に上に溶出液をロードします。 4℃で1時間3350×gでチューブを遠心℃、 </li><li>遠心分離後の長いガラスパスツールピペットを用いて試験管の底から始めて1.5 mLの画分を取る。完全に8分画、チューブの底からそれぞれを収集します。目に見える層が連続Histodenz勾配で観察されていません。小型商用車の最高収率は通常、〜2の割合で分4で期待されているあたり1×10 5をそのまま小型商用車×10 7でアメーバ13ロード·レギュレーション小型商用車のマイナー量は、それぞれの画分3と5にも存在しています。したがって、精製された小型商用車の全体的な収率は〜1×10 6×10 7あたり6無傷の小型商用車が感染しているD. discoideumの細胞 13。小型商用車の回収は、室温（RT）で行い、小型商用車は数時間安定して見えることができます。 </li></ol> 3。収集されたサンプルの顕微鏡分析<ol><li>ポリ-L-リジンコートしたカバースリップを24ウェル平底組織培養プレートを準備します。 </li><li> 24ウェルプレートにすべてのLCVの精製時に採取したサンプルに加え、それぞれの密度勾配分画の150μlをピペット。高Histodenz濃度を希釈するために各ウェルに0.5mLのHSバッファを追加します。その後、4℃で10分間600×gでプレートを遠心分離℃に慎重に上清を除去し、室温で20分間、4％パラホルムアルデヒド（PFA）でサンプルを修正します。固定後のサンプルを2回洗浄する。私達D.の電子SorC RAW264.7マクロファージdiscoideumのとPBS。 </li><li><ol class="lalpha"><li> D.を発現している GFP-カルネキシンからのサンプルをマウントします。直接、例えばVectashieldを用いたガラススライド上にLCVの分離、中に別の手順で撮影discoideumの 、。 </li><li> RTで10分間ブロッキング溶液（1％PBSにBSA）で、LCVの分離中に別の手順で撮影したRAW264.7マクロファージから、サンプルをインキュベートします。 （;をブロッキングバッファーで1:1000アフィニティー精製ウサギ抗SIDC）商用車を染色し、室温で湿潤チャンバー内で1時間インキュベートし、抗SIDC一次抗体を使用しています。その後、PBSでサンプルを3回洗浄。室温でウェットと暗室で30分間、二次抗体（ブロッキング溶液で1:200例えば抗ウサギCy5）ででインキュベートします。最後に、PBSでサンプルを3回洗浄し、例えばVectashieldを用いたガラススライド上にマウントします。 </li></ol></li><li> GFP-カルネキシンから分離された小型商用車の形態、整合性と量が発現しているD. DIScoideumまたはRAW264.7マクロファージからは、適切なフィルタを搭載した蛍光顕微鏡で分析されています。 </li></ol> 4。代表的な結果免疫親和性分離と密度勾配遠心分離によりLCV浄化の品質と収率は、蛍光顕微鏡（ 図1）を続けることができます。サンプルの概要は、DからLCVの分離中に収集さdiscoideu mまたはマクロファージ（ 図2）が表示されます。 L.のホモジネートレジオネラ感染貪食細胞の破片や細胞外細菌の多くもそのまま小型商用車を示していますが、。サンプルをペレット化した後、画像は時々、ビット密度の高いホモジネートに似ています。無傷の小型商用車が列に固執する必要があるため、フロースルーは、主に細胞外細菌や細胞の破片を含んでいます。カラムからサンプルを溶出後、溶出液が大量にそのまま商用車が含まれています。私たちの手の中に、LCV purificati<html>には 、Dのためのより効果的であると思われるマクロファージよりdiscoideumの 。 D.でdiscoideumの病原体胞が丸く表示され、孤立した小型商用車の収率はマクロファージ（ 図3）と比べて10倍以上高くなっています。無傷のマクロファージでは、異なる抗体で染色されるLVCは、より不規則な形が表示されます。 <img alt="図1" src="/files/ftp_upload/4118/4118fig1.jpg" /> LCVの分離図1回路図の概要。 LCV膜に排他的にローカライズし、細菌のエフェクタータンパク質SIDCに対するアフィニティー精製抗体を用いた免疫磁気分離により、小型商用車のA）の単離。二次MACSマイクロビーズ結合抗体と磁石は、細胞破片から小型商用車を分離するために使用されています。 B）免疫磁気分離LCVをした後、さらにHistodenz密度勾配遠心により精製される。小型商用車は、画分4に濃縮されています。 upload/4118/4118fig2.jpg &quot;/&gt; 図2。LCVの精製中に収集したサンプル。ホモジネート、ペレット、D.からのフロースルーと溶出液からの画像タマホコリカビやマクロファージが描かれている。サンプルはLを示すD.でDsRedを·エクスプレス（赤）を製造するレジオネラ GFP-カルネキシン信号を生成するdiscoideumの （緑、上部のパネル）又は抗SIDC抗体（シアン、下部パネル）で染色したマクロファージインチバー、10μmである。 <img alt="図3" src="/files/ftp_upload/4118/4118fig3.jpg" /> 図3 DからLCVを精製したタマホコリカビやマクロファージ。サンプルは、LのHistodenz密度勾配遠心分離後、画分4を表示（A）D.でDsRedを·エクスプレス（赤）を製造するレジオネラdiscoideumのは抗SIDC抗体（シアン）で染色したマクロファージではGFP-カルネキシン信号（緑）または（B）を生成します。バー、10μmである。

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利害の衝突が宣言されません。

以前に公開された方法とは対照的に、このプロトコルは、2つのステップは、最初の免疫磁気アプローチによる小型商用車の分離、そして第二に、密度勾配遠心分離により小型商用車の精製に基づいています。 LCVの分離が容易に蛍光標識した細菌やGFP産生細胞または抗体染色のいずれかが使用されている蛍光顕微鏡、続くことができます。ここで説明するプロトコルは、高純度の小型商用車?...

purification of pathogen vacuoles from legionella-infected phagocytes

日和見病原体レジオネラはまた、このように重症肺炎&quot;レジオネラ症&quot; 1の原因となる肺胞マクロファージで複製アメーバ耐性菌です。原生動物や哺乳類の食細胞で、L.レジオネラは 、特定の複製許容コンパートメント、 &quot; レジオネラ菌を含む液胞&quot;（LCV）を形成するために保存されたメカニズムを採用しています。 LCVの形成は、宿主細胞に、多くの275 &quot;エフェクター&quot;のタンパク質を移行し、細菌ICM /ドットIV型分泌系（T4SS）が必要です。エフェクターは宿主タンパク質と同様に脂質を操作し、分泌、エンドソームやミトコンドリアの細胞内小器官2から4と ​​通信します。 小型商用車の形成は、還元の方法で把握することが困難であり、複雑な堅牢かつ冗長なプロセスを表します。統合的なアプローチは、パスのグローバルな分析を含む、包括的にLCVの形成を理解する必要がありますogen - 宿主因子の相互作用とそれらの時間的および空間的ダイナミクス。この目標に向けた第一歩として、無傷の商用車は、プロテオミクスとリピドミクスで精製し、分析されます。 組成や病原体を含有する液胞の形成は、液体クロマトグラフィーや質量分析に結合された2-Dゲル電気泳動を用いたプロテオーム解析により検討されています。社会的な土壌アメーバキイロタマホコリカビや哺乳類の食細胞のいずれかから単離された小胞は、 リーシュマニア 5、 リステリア菌 6 菌 7、 ロドコッカス 8、 サルモネラ 9またはレジオネラ属菌をかくまった10。彼らは、例えば電子顕微鏡は、LCVの形態、整合性と純度を分析する必要がしかし、これらの研究で用い精製プロトコルは、時間のかかる退屈な作業です。さらに、これらのプロトコルは、専用のために病原体胞の特定の機能を利用しないrichment。 ここで紹介する方法は、Dを用いることにより、これらの制限を克服ホスファチジルイノシトール4 -リン酸（PtdIns（4）P）3,11に結合することにより、蛍光LCVマーカーを生産し、細菌のエフェクタータンパク質SIDCを対象に、どのLCV膜に選択的にアンカーでdiscoideumの 。小型商用車は、古典的Histodenz密度勾配遠心分離12,13（ 図1）第二段階に続いて磁気ビーズに結合されたSIDCと二次抗体に対するアフィニティー精製一次抗体を使用して、免疫磁気分離による最初のステップで濃縮され。 D.から単離された小型商用車のプロテオーム研究discoideumのは 13前にLCVの形成に関与していない食胞、ミトコンドリア、小胞体とゴルジ体と同様に、いくつかの低分子量GTPase、関連付けられたタンパク質を含む560以上の宿主細胞タンパク質を、明らかにした。小型商用車はで濃縮および精製プロトコルは、さらに顕微鏡（免疫蛍光法、電子顕微鏡）、生化学的方法（ウエスタンブロット）およびプロテオミクスまたはlipidomicアプローチによって分析することができますここで概説した。

Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

この資料では、無傷の単離および精製する方法を説明します<em&gt;レジオネラ菌</emアメーバやマクロファージからの&gt;を含む液胞（商用車）。 2ステップのプロトコルは、密度勾配遠心法により細菌のLCVマーカー、さらに精製することに対する抗体を用いた免疫磁気分離による濃縮LCVで構成されています。

病原体胞の精製から<em&gt;レジオネラ菌</em&gt;感染した食細胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

Research

JoVE Journal

Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K ビュー.  Ludwig-Maximilians-Universität. この資料では、無傷の単離および精製する方法を説明しますレジオネラ菌を含む液胞（商用車）。 2ステップのプロトコルは、密度勾配遠心法により細菌のLCVマーカー、さらに精製することに対する抗体を用いた免疫磁気分離による濃縮LCVで構成されています。

ビデオ: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

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 ...

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. ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

真菌病原体の食作用の生細胞ビデオ顕微鏡

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

の種特異的検出のためのLAMP法（LAMP）アッセイ<em&gt;アイメリア</em&gt;鶏に感染する

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

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. ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

ヒト好中球由来の巨大食細胞の新規亜集団の開発および同定<em&gt;インビトロ</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

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 ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.