This research was supported by the Department of Veterans Affairs Career Development Award 1IK2BX001701 and the CTSA award UL1TR000445 from the National Center for Advancing Translational Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health. Scanning electron microscopy experiments were performed in part through the use of the VUMC Cell Imaging Shared Resource, supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126.

1. H.ヒトの胃上皮細胞と鉄可用性と共培養の様々な条件でピロリ菌の成長<ol><li> H.を選択これらの研究のためのピロリ菌株のPMSS1それが無傷のCAG病原性島を有しており、機能しているIV型分泌系を表現しているため。また、ネガティブコントロールとして同質遺伝子ケージ変異体（PMSSIΔCAGE）を利用する。 5％CO 2存在下、37℃で24時間、5％ヒツジ血液（血液寒天プレート）を補充したTSAプレート上で細菌を成長させる。 注：試薬を準備し、材料および装置の表で概説したような材料を組み立てる。各試薬の最終的な濃度は、括弧内に記載されています。 </li><li>滅菌綿棒を用いてプレートからの細菌をこすりや部品から製造された改質ブルセラブロス中に接種し（材料および機器の表を参照）およびコレステロールを補った。 37で一晩培養液をインキュベート毎分（rpm）200回転で振とうしながら、5％CO 2を補充した室内空気中のC°。 NOTE：コレステロール起因血清が複雑であるという事実のために、ウシ胎児血清の代わりに使用されている。結果のばらつきの原因となるようなヘムなどの栄養素の鉄の多数の供給源を含む。 </li><li>細菌培養の日に、12ウェルプレート（ウェル当たり約1×10 5細胞）でポリ-D-リシン処理したカバースリップ上にAGSヒト胃上皮細胞（ATCC CRL-1739）をシードする。 </li><li>次の日、100μMのFeCl 3を 、200μMのジピリジルの（合成鉄キレート剤）、または200μMのジピリジルプラス250μMのFeCl 3を補充しただけでは修正されたブルセラブロス中0.3のOD600以降に細菌培養を希釈。 200rpmで振盪しながら、5％CO 2を補充した室内空気中、37℃で4時間、これらの培養物をインキュベートする。 </li><li>千×gで遠心した細菌は、細胞を収集し、私を再懸濁する前に、上清を削除するにはnは新鮮修正ブルセラブロス等量のは、コレステロールを補った。文化の尺度OD600（×10 OD600 = 1.0 = 5.5 8細胞/ ml）及び20の感染多重度（MOI）の上皮細胞への細菌を追加：1。細菌細胞の生存率を評価するために、血液寒天プレート上に連続希釈し、プレート細菌細胞を実行する。 </li><li> Hをインキュベート走査電子顕微鏡（SEM）試料調製を行う前に、静的条件下で、5％CO 2の存在下、37℃で4時間ピロリおよびAGSヒト胃上皮細胞の共培養。 </li></ol> 2. SEM Hを可視化するためにサンプル調製ピロリ CAG-T4SS線毛<ol><li> Hを削除するピロリ菌とインキュベーターとデカント培養上清からのAGS共培養（IL-8 ELISAのために保存）。 0.05Mのカコジル酸ナトリウム緩衝液（pH7.4）で穏やかに3回洗浄する。 2.0％パラホルムアルデヒド溶液中で、室温で2.5〜4時間のサンプルを修正％グルタルアルデヒド、および0.05Mのカコジル酸ナトリウム緩衝液。一次固定した後、試料を0.05 Mカコジル酸ナトリウム緩衝液で3回洗浄する。 </li><li>つの連続10分間の固定ステップで0.05 Mカコジル酸ナトリウム緩衝液中の0.1％四酸化オスミウムを用いた二次固定を行う。二次固定した後、試料を0.05 Mカコジル酸ナトリウム緩衝液で3回洗浄する。 </li><li>一次および二次固定した後、 表1の通りのエタノール濃度を増加させて、順次洗浄することにより試料を脱水する。 </li><li>エタノール脱水後、液体二酸化炭素の3つの連続回の洗浄を行う。 1073 PSIの31.1ºCの温度及び圧力で臨界点乾燥機を用いて、臨界点でのドライサンプル。 </li><li>マウントは、アルミニウムのSEM試料スタブ上にカバースリップとの適切な接地を実現し、SEM画像中に充電を回避するために、サンプルエッジでコロイド銀の細い線を描く。 </li><li>コー​​トサンプルを5nmと試料の二次電子信号とエッジ効果を高めるためにスパッタコート機を用いて金 - パラジウム。 </li></ol>高解像度のSEM分析3.イメージングパラメータ<ol><li>電界放射銃走査電子顕微鏡（FEG-SEM）を持つビュー·サンプル。 </li><li> 5〜10ミリメートルの作動距離で高真空モードでの画像。 </li><li> 5 kVの加速電圧に設定してください。 </li><li> 2-2.5にスポットサイズを設定します。 </li><li>宿主 - 病原体インターフェースのより良いビューを達成するために、15〜25度の間の試料を傾けて。 </li><li>宿主 - 病原体相互作用のこれらの領域は線毛形成細菌のために濃縮されているように、高倍率の視聴のための上皮細胞のエッジに付着細菌を画像化する優先順位をつける。 </li></ol> Piliの数量化を評価するために4.統計分析<ol><li> H.を分析線毛/セルならびにpiliated脱pheを示す細胞のパーセント数を定量化するためにImageJソフトウェアを使用してピロリ CAG-T4SS線毛下記の指示に従ってNOTYPE。 </li><li> ImageJソフトウェアで開く顕微鏡写真ファイル。均一な幅（10-13 nm）を、長さ（60〜150 nm）を用いて、細菌細胞と宿主細胞との間に形成された構造として線毛を識別する。 注：線毛構造が主要なATPアーゼ大国IV型分泌線毛アセンブリをコードする遺伝子における不活性化を抱くようなΔ ケージ変異株として同質遺伝子株は、上のWTの細菌株上に存在するが、存在しない。 </li><li>直線ツールで線を引くと、次に「分析」タブをクリックすることにより、測定ツールを校正。ドロップダウンメニューから「セットスケール」を選択し、「既知の距離」というラベルの付いたボックスに倍率バーの値を入力し、適切な設定（ナノメートル）の長さの単位を設定します。 「OK」をクリックします。 </li><li>線毛の長さや幅を上に描画するために直線ツールを使用して、CAG-T4SS線毛を測定し、各線毛を測定するためには、 &quot;Ctrl-M」を押してください。測定された値を持つダイアログボックスを守ってください。これらの値を記録したりコピーし、表計算プログラムに貼り付けます。 </li><li>長さを使用し、幅のパラメータは、以前にCAG-T4SSのプロファイルに適合しない24、無視機能を設立しました。その後、10〜13 nmの幅と60〜150 nmの長さのカットオフ内にある値に基づいて線毛の数を定量化する。 </li><li>を、GraphPad Prismソフトウェアを使用して両側スチューデントt検定によって統計的に線毛の定量分析を行う。 </li></ol> 5.オプション：IL-8分泌の評価は、線毛の生産と相関する<ol><li>ステップ2.1から上清を用い、キットを製造業者の指示（材料および装置の表を参照）ごとに、IL-8のELISAを行う。 </li><li> IL-8は、宿主細胞によって分泌を分析し、培地単独中で成長WT PMSS1に対するパーセントのIL-8誘導を計算する。これを、GraphPad Prismを使用して、両側スチューデントt検定を用いて統計分析を実行するftware。 </li></ol>

<ol>
	<li>Cover, T. L., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+in+health+and+disease.">Helicobacter pylori in health and disease.</a> Gastroenterology. (6), 1863-1873 (2009).</li><li>Sycuro, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+crosslinking+relaxation+promotes+Helicobacter+pylori's+helical+shape+and+stomach+colonization.">Peptidoglycan crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach colonization.</a> Cell. 141 (5), 822-833 (2010).</li><li>Kodaman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+Helicobacter+pylori+coevolution+shapes+the+risk+of+gastric+disease.">Human and Helicobacter pylori coevolution shapes the risk of gastric disease.</a> Proc. Natl. Acad. Sci. U.S.A. 111 (4), 1455-1460 (2014).</li><li>Tegtmeyer, N., Wessler, S., Backert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+cag-pathogenicity+island+encoded+type+IV+secretion+system+in+Helicobacter+pylori+pathogenesis.">Role of the cag-pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis.</a> FEBS J. 278 (8), 1190-1202 (2011).</li><li>Fischer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+molecular+mode+of+action+of+the+Helicobacter+pylori+Cag+type+IV+secretion+apparatus.">Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus.</a> FEBS J. 278 (8), 1203-1212 (2011).</li><li>Odenbreit, S., Puls, J., Sedlmaier, B., Gerland, E., Fischer, W., Haas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+Helicobacter+pylori+CagA+into+gastric+epithelial+cells+by+type+IV+secretion.">Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion.</a> Science. 287 (5457), 1497-1500 (2000).</li><li>Bourzac, K., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori-host+cell+interactions+mediated+by+type+IV+secretion.">Helicobacter pylori-host cell interactions mediated by type IV secretion.</a> Cell Microbiol. 7 (7), 911-919 (2005).</li><li>Bronte-Tinkew, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+cytoxin-associated+gene+A+activates+the+signal+transducer+and+activator+of+transcription+3+pathway+in+vitro+and+in+vivo.">Helicobacter pylori cytoxin-associated gene A activates the signal transducer and activator of transcription 3 pathway in vitro and in vivo.</a> Cancer Res. 69 (2), 632-639 (2009).</li><li>Johnson, E. M., Gaddy, J. A., Cover, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+Helicobacter+pylori+triggered+by+contact+with+gastric+epithelial+cells.">Alterations in Helicobacter pylori triggered by contact with gastric epithelial cells.</a> Front. Cell. Infect. Microbiol. (2), 17 (2012).</li><li>Rohde, M., Puls, J., Buhrdorf, R., Fischer, W., Haas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+sheathed+surface+organelle+of+the+Helicobacter+pylori+cag+type+IV+secretion+system.">A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system.</a> Mol. Microbiol. 49 (1), 219-234 (2003).</li><li>Cover, T. L., Peek, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet,+microbial+virulence+and+Helicobacter+pylori+induced+gastric+cancer.">Diet, microbial virulence and Helicobacter pylori induced gastric cancer.</a> Gut Microbes. 4 (6), 482-493 (2013).</li><li>Queiroz, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+gastric+IL-1&#946;+concentration+and+iron+deficiency+parameters+in+Helicobacter+pylori+infected+children.">Increased gastric IL-1&#946; concentration and iron deficiency parameters in Helicobacter pylori infected children.</a> PLoS One. 8 (2), 57420 (2013).</li><li>Harris, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori-associated+hypochlorhydria+and+development+of+iron+deficiency.">Helicobacter pylori-associated hypochlorhydria and development of iron deficiency.</a> J. Clin. Pathol. 66 (4), 343-347 (2013).</li><li>Noto, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+deficiency+accelerates+Helicobacter+pylori-induced+carcinogenesis+in+rodents+and+humans.">Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans.</a> J. Clin. Invest. 123 (1), 479-492 (2013).</li><li>Tan, S., Noto, J. M., Romero-Gallo, J., Peek, R. M., Amieva, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+perturbs+iron+trafficking+in+the+epithelium+to+grow+on+the+cell+surface.">Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface.</a> PLoS Pathog. 7 (5), (2011).</li><li>Danielli, A., Roncarati, D., Delany, I., Chiarini, V., Rappouli, R., Scarlato, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dissection+of+the+Helicobacter+pylori+Fur+regulatory+circuit+by+genome-wide+location+analysis.">In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis.</a> J. Bacteriol. 188 (13), 4654-4662 (2006).</li><li>Pich, O. Q., Carpenter, B. M., Gilbreath, J. J., Merrell, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+analysis+of+Helicobacter+pylori+Fur-regulated+promoters+reveals+a+Fur+box+core+sequence+and+novel+Fur-regulated+genes.">Detailed analysis of Helicobacter pylori Fur-regulated promoters reveals a Fur box core sequence and novel Fur-regulated genes.</a> Mol. Microbiol. 84 (5), 921-941 (2012).</li><li>Cassat, J. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+in+infection+and+immunity.">Iron in infection and immunity.</a> Cell Host Microbe. 13 (5), 509-519 (2013).</li><li>Nielubowicz, G. R., Mobley, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+in+urinary+tract+infection.">Host-pathogen interactions in urinary tract infection.</a> Nat. Rev. Urol. 7 (8), 430-441 (2010).</li><li>Brickman, T. J., Cummings, C. A., Liew, S. Y., Relman, D. A., Armstrong, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+iron+starvation+response+in+Bordatella+pertussis+provides+new+insights+into+siderophore+utilization+and+virulence+gene+expression.">Transcriptional profiling of the iron starvation response in Bordatella pertussis provides new insights into siderophore utilization and virulence gene expression.</a> J. Bacteriol. 193 (18), 4798-4812 (2011).</li><li>Mobley, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+factors+associated+with+disease+development.">Helicobacter pylori factors associated with disease development.</a> Gastroenterology. 113 (6), 21-28 (1997).</li><li>Testerman, T. L., Conn, P. B., Mobley, H. L., McGee, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+requirements+and+antibiotic+resistance+patterns+of+Helicobacter+species+in+chemically+defined.">Nutritional requirements and antibiotic resistance patterns of Helicobacter species in chemically defined.</a> J. Clin. Microbiol. 44 (5), 1650-1658 (2006).</li><li>Senkovich, O., Ceaser, S., McGee, D. J., Testerman, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+host+iron+utilization+mechanisms+of+Helicobacter+pylori+revealed+with+iron-deficient+chemically+defined+media.">Unique host iron utilization mechanisms of Helicobacter pylori revealed with iron-deficient chemically defined media.</a> Infect. Immun. 78 (5), 1841-1849 (2010).</li><li>Shaffer, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+exploits+a+unique+repertoire+of+type+IV+secretion+system+components+for+pilus+assembly+at+the+bacteria-host+cell+interface.">Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.</a> PLoS Pathog. 7 (9), (2011).</li><li>Barrozo, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+plasticity+in+the+type+IV+secretion+system+of+Helicobacter+pylori.">Functional plasticity in the type IV secretion system of Helicobacter pylori.</a> PLoS Pathog. 9 (2), (2013).</li></ol>

The authors declare that they have no competing financial interests.

鉄は、細菌性病原体を含む人生のほとんどの形態、必須の微量栄養素​​である。侵入微生物の生存を制限するための努力では、脊椎動物のホストは「栄養免疫」18として知られるプロセスで栄養鉄を隔離する。これに応答して、細菌性病原体は、その周囲を感知し、そのような鉄獲得システム、毒素および毒素分泌機構19,20などの病原性機能の精緻化を調節するためにグローバルなシグナル伝達分子として鉄を使用するように進化してきた。 ピロリ菌は、鉄、亜鉛、および22を成長させるようなヘム、ヘモグロビン、トランスフェリン、ラクトフェリン23として代替鉄源を使用することができるマグネシウムなどの微量栄養素を必要とする。H.ピロリ菌は 、最も成功した病原体のように、それは、ホスト21からの栄養素の鉄を除去を助けるために病原性因子の幅広いレパートリーを進化してきました。一つの毒性因子、CagAは、エフェクターmolecuルCAG-T4SSのは、それが容易に栄養鉄源として細菌細胞によって使用され、複製15を促進することができる頂端表面に偏光された細胞の基底外側からトランスフェリンを再ルーティングに関与している。これにより、低鉄利用の条件のCagAタンパク質の分泌を促進するための直感的な進化戦略と思われる。 H.ピロリ CAG-T4SSは誘発炎症促進性宿主細胞応答のために重要な細菌の細胞小器官である。 CAG-T4SSの効果が十分に共培養し、感染の動物モデルの両方において研究されている。しかし、この毒素分泌系の高分子構造および組成の研究は、高解像度イメージングプロトコルの欠如によって妨げられてきた。 CAG-T4SSを研究我々の以前に公表された報告は、高分解能電子顕微鏡による細菌細胞あたり4 CAG-T4SS線毛の平均が24を分析して報告している。私たちの改善された方法を提供堅牢なイメージング技術は、細菌細胞の表面上のこの特徴の産生を促進する培養技術を用いてCAG-T4SSを可視化した。高分解能電界放出銃の走査型電子顕微鏡と組み合わせて培養技術の利用は、この撮影プロトコルのために重要である。 CAG-T4SS表面特徴の二倍の濃縮は、免疫組織化学を行うことに興味の研究者のための有意な増加はさらにCAG-T4SSの細胞内位置または組成物を特徴づけるためにそのような免疫蛍光法または免疫沈降技術として分析される。 CAG-T4SSの高分解能電子顕微鏡像が習得された後は、CAG-T4SS線毛25に対する細菌タンパク質の位置を決定するために、そのような免疫金標識のような他のアプリケーションを開発することが可能である。 我々の技術の一つの制限は、研究者がまだUSIに制限されているという事実であるCAG-T4SSの可視化のために共培養系ngの。 Hの単一のフィールドでは、サブセットのみ通常の培養条件下でのピロリ菌は、これらの構造（約50〜70％が、図3のパネルBを参照）を形成する。だから、フィールド内の多数の細胞は、非piliatedになります。これらの亜集団は、同じ場所に存在理由は不明であるが、これらの細胞は常に表面上の線毛を持つ細胞の線毛/セルとパーセントの両方の定量内会計処理されている。 CAG-T4SSのみホスト細菌の界面で観察されるので、SEMの試料調製物内の一次及び二次固定の手順は、タンパク質の構造及び真核生物および原核生物細胞の膜の両方の保存のために重要である。しかし、固定ステップが概説さよりも長く行われている場合、または溶液を新たに調製していない場合、過固定起こり得る。過剰な固定が発生した場合は、SEM下で観察し、CAG-T4SSこのによってマスクされたときに、サンプルが石の外観を持つことになります。 troublへeshoot、固定剤の存在下でインキュベートした低濃度の固定剤および/またはより短い時間を使用して、SEMの試料調製を繰り返す。 共培養系はまた、宿主タンパク質の大量の汚染に面倒な（例えば、質量分析法分析など）の生化学的特徴をレンダリングする。しかし、この細菌の分泌系の産生を調節する分子メカニズムを理解することは、最終的には、宿主細胞の非存在下でCAG-T4SS線毛の形成をトリガーする新しい技術につながる可能性があります。これは大幅な生化学的アッセイのための単離および精製技術を改善するであろう。 結論として、我々はHの増産で制限された鉄の可用性結果の状況を報告高分解能走査電子顕微鏡法によって可視化することができるピロリ CAG-T4SS。我々はまた、宿主 - 病原体interfa時にこの表面特徴の鉄依存抑制を報告CE。このアッセイは、最適化され、鉄調節毒素分泌物を形成し、宿主 - 微生物相互作用の様々に広く適用可能である多数の細菌性病原体のために利用することができる。

H. pylori infection is a significant risk factor for gastric cancer1. However, disease outcomes vary and depend on numerous factors such as host genetics, genetic diversity of H. pylori strains, and environmental elements such as host diet11. Previous reports have established that a correlation exists between H. pylori infection, iron deficiency (as measured by decreased blood ferritin and hemoglobin concentrations), and increased proinflammatory cytokine production, including IL-8 secretion, which ultimately leads to increased gastric disease progression12. Acute H. pylori infection is also associated with hypochlorhydria which impairs the host&#8217;s ability to absorb nutrient iron, and ultimately leads to changes in iron homeostasis13. These clinical findings suggest that iron availability within the gastic niche could be an important factor in disease outcome. In fact, animal models of H. pylori infection have demonstrated that low dietary iron consumption exacerbates gastric disease14. The reduced iron levels in these animals necessitate that H. pylori induce an iron-acquisition response in order to obtain the iron needed for bacterial replication. H. pylori has the capacity to perturb iron trafficking within host cells to facilitate bacterial replication in a CagA-dependent fashion15. Interestingly, the cag-pathogenicity island has been shown to be regulated by the iron-responsive transcription factor Fur16,17. Furthermore, Cag+ strains are associated with increased inflammation and gastric diseases such as cancer1. These findings support a model whereby H. pylori alters Cag-T4SS expression in an effort to obtain iron from host cells that reside in an iron deplete environment resulting in exacerbated disease outcomes. 
Two factors that increase inflammation and morbidity are Cag expression and low dietary iron intake. These facts support the hypothesis that reduced iron availability increases the production of Cag-T4SS pili at the host pathogen interface resulting in worse gastric disease11-14. The goal of the method provided in this manuscript is to establish the role of the micronutrient iron in the regulation of the Cag-T4SS pilus biogenesis. In previous work, we utilized two approaches to observe an iron-dependent increase in Cag-T4SS expression. First, output strains from animals maintained on high and low iron diets were analyzed and revealed that low-iron diet output strains produced more Cag-T4SS pili than high-iron diet strains14. Second, growing the H. pylori 7.13 strain in vitro in iron replete conditions resulted in reduced pili formation while cells grown in the presence of an iron chelator produced significantly more pili. 
We have continued to investigate the iron-dependent regulation of Cag-T4SS pili phenotype and offer the following optimized protocol and representative results performed with an additional Helicobacter pylori strain, PMSS1. The rationale behind the development of this technique was to correlate increased Cag-T4SS activity in conditions of iron-limitation with increased Cag-T4SS pilus formation. The broader implication and use of this technique will provide optimized culture conditions that result in elevated production of the Cag-T4SS pili. This assay will be useful to researchers seeking to determine the composition and architecture of the Cag-T4SS by enriching for this important bacterial surface feature. The sample preparation and visualization by field-emission gun electron microscopy has numerous advantages over alternative techniques such as light-microscopy methods to visualize the Cag-T4SS and will be appropriate to investigators interested in studying the regulation of this organelle10.

<table border="0" cellpadding="0" cellspacing="0" width="837">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:259px;"></td>
			<td style="width:196px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:475px;">Modified brucella broth</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Peptone from casein (10g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">70172</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Peptic digest of animal tissue (10g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">70174</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Yeast extract (2g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">92144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dextrose (1g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">D9434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium chloride (5g/L)</td>
			<td style="width:259px;">Thermo Fisher</td>
			<td style="width:196px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cholesterol (250X) (4mL/L)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:196px;">12531018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ferric chloride (100 or 250 uM)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">157740-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dipyridyl (200 uM)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">D216305-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:259px;"></td>
			<td style="width:196px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;width:475px;">Modified RPMI</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RPMI+HEPES (1X)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:196px;">22400-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Fetal bovine serum (100 mL/L)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:196px;">10438-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:259px;"></td>
			<td style="width:196px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;width:475px;">Electron Microscopy Preparation</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Paraformaldehyde (2.0% aqueous)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">15713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gluteraldehyde (2.5% aqueous)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">16220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium cacodylate (0.05 M)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">12300</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Osmium tetroxide (0.1% aqueous)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">19150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ethanol (absolute)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Colloidal silver paint</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">12630</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SEM sample stubs</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">75220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:259px;">Thermo Fisher</td>
			<td style="width:196px;">08-774-383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:475px;">IL-8 Secretion Evaluation</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Quantikine IL-8 ELISA kit</td>
			<td style="width:259px;">R&#38;D Systems</td>
			<td style="width:196px;">D8000C</td>
			<td></td>
		</tr>
	</tbody>
</table>

high resolution electron microscopy of the helicobacter pylori cag type iv secretion system pili produced in varying conditions of iron availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

本稿では、鉄利用能を変化させる条件はH.を調節する能力を有することが実証されているホスト病原体の界面でのピロリ菌 CAG-T4SS線毛生合成。 、H。単独の培地で培養した場合ピロリ菌は 3線毛/セルの平均を形成している。ときにH.ピロリ菌は、鉄で栽培されて枯渇細菌の増殖（ 図1）サブ阻害性である（合成キレート剤ジピリジルを使用）の条件は、細菌が作り出す数々のCAG-T4SS線毛（〜7線毛/セル）とき、人間の胃の細胞と共培養（ 図2）。栄養鉄の外因性源が存在する場合には逆に、CAG-T4SS線毛の形成が抑制される（FE試料中の1未満線毛/細胞、1未満線毛/ FE +ディップサンプル中の細胞）（ 図2）。宿主-病原体界面での線毛の定量化は、鉄制限、Hの状態であることを明らかにしピロリ菌は、CAG-T4SS線毛た（p &lt;0.000の2倍の増加を示す<html> 1）培地のみで増殖した細胞と比較して11％（p &lt;0.05）にパーセントpiliated細胞が増加した（ 図3）。興味深いことに、CAG-T4SSの活性は、ホストIL-8分泌によって測定されるように、鉄制限条件下で107パーセントを強化し、培地のみ（p = 0.03、およびpと比較して過剰鉄利用能の条件下で49％抑制される&lt; SEMデータをサポート0.001、それぞれ）の結果。しかし、線毛の寸法は鉄の可用性（ 図4）のすべての条件のうち一貫性を維持。また、鉄利用能がケージ変異体の表面上に線毛の生合成を変化させず、それは実際には、実証された構造であるという仮説を支持し、宿主細胞からのIL-8応答を誘導するこの同質遺伝子誘導体の能力を変更しない、 CAG-T4SSとされていない追加の無関係な表面特徴。 N &quot;SRC =&quot; /ファイル/ ftp_upload / 52122 / 52122fig1highres.jpg &quot;幅=&quot; 500 &quot;/&gt; 図1：鉄の可用性の様々な条件で決定された細菌の生存能力H.。ピロリ菌だけでは修正されたブルセラブロス（培地のみ）で培養した、または、100μMのFeCl 3（FE）、200100μMのジピリジル（DIP）、または200100μMのジピリジルプラス、250μMのFeCl 3（FE +ディップ）を補充した。細菌は、5％CO 2の存在下、37℃で2日間インキュベーション前に連続希釈し、細菌学的培地上にプレーティングした。コロニーを計数し、コロニー形成単位/ mLの（CFU / ml）を算出した。バーは、3回の独立した実験の平均値+/- SEMを示す。 DIP、FE、またはFE +ディップを用いた治療は大幅に細菌の生存能力は変更されません。 <img alt="図2" fo:content-width="5in" src="/files/ftp_upload/52122/52122fig2highres.jpg" width="500" /> 図2：高RH.のesolution走査型電子顕微鏡分析ピロリ CAG-T4SS線毛。細菌は200μMのジピリジル、またはDを補った、100μMのFeCl 3、C）培地を補っA）培地のみ、B）培地中で増殖させた）、200μMのジピリジルプラス250μMのFeCl 3を添加した培地AGS人間との共培養の前に胃の細胞。矢印は、宿主 - 病原体の界面に形成されたCAG-T4SS線毛を示している。サンプルは、CAG-T4SS生合成を評価するために、高分解能走査型電子顕微鏡により分析した。過剰利用可能な栄養鉄の条件は、CAG-T4SS線毛の形成を抑制しつつ、低鉄利用の条件は、CAG-T4SS線毛形成の有病率を増加させる。 <img alt="図3" fo:content-width="5in" src="/files/ftp_upload/52122/52122fig3highres.jpg" width="500" /> 図3：様々な中で培養線毛/セルおよびパーセントpiliated細胞の定量化鉄利用の条件。細菌は、単独（培地のみ）修正されたブルセラブロス中で増殖させた、または媒体は、100μMのFeCl 3（FE）、200100μMのジピリジル（DIP）、または200100μMのジピリジルプラス、250μMのFeCl 3（FE +ディップ）コ·前に補充した人間の胃上皮細胞と培養する。サンプルは、ImageJソフトウェアを使用して列挙された高分解能走査電子顕微鏡および線毛によって分析した。 A）は、培地単独と比較して細胞あたりの線毛の散布た（* p &lt;0.0001）、および独立した3から派生培養条件あたり60-112細胞由来の培地のみに比べpiliated細胞（* P &lt;0.05）のB）のバーグラフ描いパーセント生物学的な実験。 <img alt="図4" fo:content-width="5in" src="/files/ftp_upload/52122/52122fig4highres.jpg" width="500" /> 図4：鉄の可用性の様々な条件で培養した細胞から測定線毛寸法。 BACTERIAコ·前に修正されたブルセラ単独のブロス（培地のみ）、または中、100μMのFeCl 3（FE）を補充し、200100μMのジピリジル（DIP）、または200100μMのジピリジルプラス、250μMのFeCl 3（FE +ディップ）で増殖させた人間の胃上皮細胞と培養する。試料は、高分解能走査型電子顕微鏡で分析し、線毛寸法はImageJソフトウェアを用いて測定した。 A）線毛幅は、すべての条件のうち13.1 +/- 2.4程度の平均値である。 B）線毛の長さは、すべての条件のうち75.8 +/- 16.0ナノメートルの平均である。線毛寸法の有意差は鉄利用の様々な条件で見られない。 補足図1：高分解能走査型電子顕微鏡での分析H.鉄の可用性の様々な条件でのピロリ菌ケージ変異体。細菌はFeCl 3を 、100μMで補充A）培地で増殖させた、B）培地D、200μMのジピリジル（補充したIP）またはC）、200μMのジピリジル（DIP）を添加した培地を加え、250μMのFeCl 3 AGSヒト胃細胞と共培養する前に。サンプルは、CAG-T4SS生合成を評価するために、高分解能走査型電子顕微鏡により分析した。低または高鉄の条件は図2に可視化された構造は、CAG-T4SS線毛であるという仮説を支持し、あらゆるCAG-独立の線毛生合成を調節しない。 補足図2：鉄の可用性の条件を変化させることで成長させた細菌に応答したホストのIL-8分泌のELISA分析。野生型（白棒）またはケージ変異体（灰色のバー）細菌は、100μMのFeCl 3（FE）、200100μMのジピリジル（DIP）、または200μMのジピリジルを補った修正されたブルセラ単独のブロス（培地のみ）、または培地中で増殖させたプラス250 uMのFeCl 3を （FE +ディップ）は、ヒトの胃上皮細胞との共培養の前に。増加したILの鉄制限の結果-8、CAG-T4SS活動に依存しているホストの応答。バーは、3つの別個の生物学的な実験から得られ、平均+/- SEMを示す。 （* P &lt;0.05、培地のみで増殖したPMSS1と比較して）。

Watch this Scientific Journal Video about High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability at JoVE.c

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

の高分解能電子顕微鏡<em&gt;ヘリコバクター·ピロリ</em鉄の可用性の変化する条件で生産&gt; CAG IV型分泌系線毛

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

high-resolution-electron-microscopy-helicobacter-pylori-cag-type-iv

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

15.0K ビュー.  Vanderbilt University School of Medicine. Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

ビデオ: High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

低温電子断層撮影と自動化されたサブ断層アベレージングを使用して、HIVエンベロープ糖タンパク質の分子構造の決定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

免疫・感染学

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

マイクロ波は、透過型電子顕微鏡による植物ウイルス病の迅速診断をアシスト

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

プロデュース上皮損傷の解析<em&gt;赤痢アメーバ</em&gt;感染症

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

開発<em&gt; in vitroで</emとの相互作用を研究するための&gt;モデル系<em&gt;エクウス</em&gt;<em&gt; caballus</emその高親和性受容体FcεRIとした&gt;のIgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

マラリアに一塩基多型のジェノタイピングを溶融高解像度の感度を上げる方法

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

感染症のためのモデルとしてのオルガノイド：人間の文化とマウス胃オルガノイドとピロリ菌のマイクロインジェクション

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

ワンステップの負のクロマトグラフィー精製<em&gt;ヘリコバクター・ピロリ</emで過剰発現&gt;好中球活性化タンパク質<em&gt;エシェリヒア・コリ</em&gt;バッチモードでの

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

研究のための方法論<em&gt; B。枯草菌</em小分子バイオフィルム阻害剤を特徴付けるためのモデルとしての&gt;バイオフィルム

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

ウイルスから離れた高収率細胞外小胞製剤の精製

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

の高分解能電子顕微鏡

要約

さらに動画を探す

この動画の章

低温電子断層撮影と自動化されたサブ断層アベレージングを使用して、HIVエンベロープ糖タンパク質の分子構造の決定

マイクロ波は、透過型電子顕微鏡による植物ウイルス病の迅速診断をアシスト

新生児と大人の中枢神経系から培養ミクログリア

プロデュース上皮損傷の解析

開発

マラリアに一塩基多型のジェノタイピングを溶融高解像度の感度を上げる方法

感染症のためのモデルとしてのオルガノイド：人間の文化とマウス胃オルガノイドとピロリ菌のマイクロインジェクション

ワンステップの負のクロマトグラフィー精製

研究のための方法論

ウイルスから離れた高収率細胞外小胞製剤の精製