This work has been supported by Elizabeth E. Kennedy Research Award.

産生株の作製<ol><li>ストリーク肺炎球菌株は、-80°Cの冷凍庫ストックから5％ヒツジ血液をトリプシン大豆寒天プレート上バクテリオシン産生について試験される。 NOTE：阻害について試験されるべき株の初期成長のための血液プレートの使用が大幅アッセイの再現性を増大させる。成長条件は、特定の生物のために修正する必要があるかもしれないが、また、バクテリオシンを産生する他の非肺炎球菌株を使用することができる。私たちは、成功したオーバーレイアッセイでストレプトコッカス·ミティス及びラクトコッカス·ラクティスを使用している。 </li><li> 5％CO 2で37℃で一晩プレート（O / N）でインキュベートする。 </li><li>プロデューサー株のインキュベーション後、TSAの25ミリリットルを含有するプレート上にカタラーゼの3,000〜4,000台をピペッティングすることによりトリプシン大豆寒天（TSA）プレートを用意し、滅菌ガラスビーズを用いてカタラーゼ液を広げた。下に約10分間、プレートの乾燥をしましょう生物学的安全キャビネット。 </li><li>ピペットチップ上に細菌の可視量を収集する。乾燥したTSAプレートにピペットチップを刺す。 注：複数の産生株は、単一のTSAプレートに刺したことができます。結果のハローが重ならないように、それはスタブアウトスペースに重要です。 <ol><li>可能であれば、陽性対照及び陰性対照としてオーバーレイで使用される菌株として知られているバクテリオシンプロデューサーが含まれています。 注：正および負の対照は、以前に公開されているで、リクエスト10を承ります。 </li></ol></li><li> 6時間、5％のCO 2、37℃で、複数のスタブを含むTSAプレートをインキュベートする。 </li></ol> 2オーバーレイ株のグリセロールストックを作る<ol><li>オーバーレイアッセイの日に先立って（グリセロールを添加した後、中期指数期まで増殖させ、その後、-80℃で保存した培養物）グリセロールストックを準備します。肺炎球菌strの5-10コロニーを接種するアインは、0.5％酵母抽出物（THY）とトッドヒューイット培地中の感受性またはフェロモン分泌について試験される。 注：BLPC誘導性レポーターの構築は、以前に2に記載されていると株はリクエストを承ります。 </li><li>振盪せずに37℃の水浴中に密閉蓋つきTHY 5mlでガラス管中5-10コロニーをインキュベートする。 </li><li>彼らは0.5にOD 0.3の620に到達するまで37℃で培養をインキュベートする。 注：これは使用した株に依存して、約4〜5時間かかります。 <ol><li>静かにチューブを外径を測定する前に、数回転倒。 </li></ol></li><li>実験は、同じ日に行うことができないか、株はグリセロールストックを作成し、後日実験を再開、将来的には複数回使用する場合。グリセロールストックの場合、20％の最終濃度に培養液にグリセロールを追加します。 -80℃で1.5 mlマイクロチューブやストアに分注し培養。注：サンプルは、sすることができ-80℃で数ヶ月の​​ためにtored。グリセロールストックをしていない場合は、オーバーレイアッセイに直接サンプルを使用。 </li></ol> 3オーバーレイアッセイ<ol><li>直前に、アプリケーションをオーバーレイオーバーレイ成分を混合する。 15ミリリットルコニカルチューブでは、THYの5ミリリットル、カタラーゼ3,000-4,000 U、およびいずれかその日または室温で解凍し、培養のグリセロールストックを増殖させたオーバーレイ株ブロス培養液200μlを加える。レポーター株を使用する場合は、オーバーレイ混合物にX-galを（40 mg / ml）を50μlを加える。プレートごとに1つの円錐管を使用してください。 </li><li>前の溶融TSAの添加、室温でオーバーレイ混合物を平衡化する。 </li><li>インキュベーターから刺した株で、TSAプレートを取り外します。電子レンジで固体TSA溶融状態で使用可能になるまで55℃の水浴させること。 1.5％寒天で溶融し、TSAの3ミリリットルを追加し、混合物をオーバーレイする55℃に冷却した。 <ol><li>ピペッティングの直前にオーバーレイ混合物を準備し、混合物は非常に固化し始めますすぐに。注：オーバーレイは、めっき前に固化し始めた場合、結果は解釈不可能になります。 </li></ol></li><li>ピペッティングによって、5mlのピペットが混合物中に気泡を導入しないように注意しながら数回上下オーバーレイ混合物を混ぜる。ゆっくりと刺したTSAプレート上に直接オーバーレイの混合物をピペット。寒天を硬化させること数分間ベンチトップ上でプレートを保管してください。 <ol><li>これはオーバーレイに刺した文化からの成長を引っ張ってくる、オーバーレイ混合物を配布するプレートを回転させないでください。 </li></ol></li><li>慎重に、5％CO 2、O ​​/ N（約16〜18時間）を含む37℃のインキュベーター内にオーバーレイして、TSAプレートを配置。 </li><li>一晩成長させた後、プレートを観察します。プレートは、阻害またはフェロモンシグナルが発生した場所以外のオーバーレイ株の増殖から不透明に表示されます。これらはそれぞれ、刺さ歪み周りクリアまたは青のゾーンのいずれかとして表示されます。 注：なお、MEASUR青色またはクリアリングの直径のements定量的評価のために決定することができ、直径の差は、プレートに刺された細菌の量に依存して変化し得る。オーバーレイアッセイは、任意の定量的評価は慎重に解釈されるべきで、定性的な分析である。陰性対照と比較して目に見えるクリアが活性とみなされる。 </li></ol>

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J., Dawid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HtrA+protease+of+Streptococcus+pneumoniae+controls+density-dependent+stimulation+of+the+bacteriocin+blp+locus+via+disruption+of+pheromone+secretion.">The HtrA protease of Streptococcus pneumoniae controls density-dependent stimulation of the bacteriocin blp locus via disruption of pheromone secretion.</a> J Bacteriol. 195 (7), 1561-1572 (2013).</li><li>Lux, T., Nuhn, M., Hakenbeck, R., Reichmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+bacteriocins+and+activity+spectrum+in+Streptococcus+pneumoniae.">Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae.</a> J Bacteriol. 189 (21), 7741-7751 (2007).</li><li>Reichmann, P., Hakenbeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+a+peptide-inducible+two-component+system+of+Streptococcus+pneumoniae.">Allelic variation in a peptide-inducible two-component system of Streptococcus pneumoniae.</a> FEMS Microbiol Lett. 190 (2), 231-236 (2000).</li><li>Saizieu, A., 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=Microarray-based+identification+of+a+novel+Streptococcus+pneumoniae+regulon+controlled+by+an+autoinduced+peptide.">Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide.</a> J Bacteriol. 182 (17), 4696-4703 (2000).</li><li>Barbour, A., Philip, K., Muniandy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+production,+purification,+characterization+and+mechanism+of+action+of+salivaricin+9+lantibiotic+produced+by+Streptococcus+salivarius+NU10.">Enhanced production, purification, characterization and mechanism of action of salivaricin 9 lantibiotic produced by Streptococcus salivarius NU10.</a> PLoS One. 8 (10), 77751 (2013).</li><li>Wladyka, B., 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=Isolation,+biochemical+characterization,+and+cloning+of+a+bacteriocin+from+the+poultry-associated+Staphylococcus+aureus+strain+CH-91.">Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91.</a> Appl Microbiol Biotechnol. 97 (16), 7229-7239 (2013).</li><li>Shea, E. 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The authors have nothing to disclose.

このオーバーレイアッセイは、バクテリオシンの生産者のための活動の範囲を決定し、肺炎球菌株の間で発生する可能性があり、競争力の相互作用を実証するために迅速な方法です。オーバーレイアッセイは、単一のプレート上にバクテリオシン産生のための複数の株をスクリーニングするために使用するように適合させることができる。私たちは、成功した96ウェルプレートの半分からSBA?...

肺炎球菌 、鼻咽頭の多菌コミュニティの共通の植民は、細菌によって生産抗菌ペプチド（バクテリオシン）の産生を介して他の肺炎球菌や近縁種と競合することができます。今日までに調べすべて完全に配列決定肺炎球菌株は、B acteriocin- リットル IKE のp eptide座、BLPのバージョンが含まれています。肺炎球菌によるバクテリオシン産生は、 インビトロおよびインビボの競争1-4 の両方の結果に重要であることが実証されている。競争のダイナミクスは、特定のペプチドフェロモンに反応して同族の免疫タンパク質とともに多様なバクテリオシンの発現により影響を受ける。 BLP座の誘導は、ペプチドフェロモン、BLPCは、センサーキナーゼ、BlpHに結合し、その結果シグナル伝達カスケードを開始するクオラムセンシングシステムによって制御されているBLP軌跡5,6の転写。 BLPCの4対立遺伝子変異その同族BlpH 6にそれぞれ結合することがあります。細胞がそれ自身のバクテリオシンの効果を生き残るためには、同族の免疫タンパク質をコードする遺伝子は、バクテリオシン遺伝子のそれぞれと同じオペロン上に転写される。株は保護のために必要な特定の免疫遺伝子を欠いている場合は、競合他社の株は、外因性フェロモンに反応してBLP軌跡を上方制御することができない場合、または殺害が発生します。トランスポーター複合体、BlpABは、ペプチドフェロモンとバクテリオシンペプチド2,4の分泌および処理するために必要です。最近では、肺炎球菌株の大部分は、彼らは彼らができないフェロモン及びバクテリオ1,2を分泌するレンダリングblpA遺伝子における保存された変異を有する意味する「詐欺師」であることが示されている。これらの株は、いななきが分泌する外因BLPC 2に応答することができる免疫タンパク質の産生を可能にする退屈な菌株。 清からのバクテリオシンの、粗調製物は、純度を達成するために、生化学的方法のステップを使用して産生株から調製することができるが、この手法は、単離物の大規模なコレクションをスクリーニングするために有用ではないとバクテリオシンの発現のレベルは、ブロス増殖させた培養物において低い場合2,7- 9。オーバーレイアッセイは、インビトロで存在し得る菌株間の競合力学を調査するために迅速な方法として使用される。これを行うには、肺炎球菌株は、予め接種寒天プレートの上およびBLP座の誘導に十分なローカルの細菌濃度を達成するように成長させている。第二株を、次いで、感度をテストするための事前接種菌株の上に塗布される溶融ソフトアガー溶液中に接種する。 BLP軌跡（阻害活性とは無関係）の活性のために評価するために、株は番目の一連の重ねることができるリーは以前BLPCのレポーター株を記載した。これらの株は肺炎球菌により分泌される最も一般的な3つのBLPCフェロモンに反応する三つの異なるblpH対立遺伝子のいずれかを含むように構築された。レポーター株はBlpH依存性プロモーターの制御下にあり、自己刺激10,11を防止BLPC遺伝子における欠失を運ぶ統合されたlacZ遺伝子を有している。

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:351px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Trypticase Soy Agar with 5% Sheep Blood</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B21261X&#160;</td>
			<td>For growth from freezer cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Catalase</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C100-500mg</td>
			<td>4,741 Units per plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Todd Hewitt Broth + Yeast Extract (THY)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">DF0492-17-6&#160;</td>
			<td>30 g of THB, 5 g yeast extract, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Trypticase Soy Broth + Agar plates (TSA)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B11768&#160;</td>
			<td>30 g of TS, 15 g agar, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">5-Bromo-4-chloro-3-indolyl &#946;-D-galactopyranoside (X-gal)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">B4252-50MG</td>
			<td>Dissolve in dimethylformamide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Petri dishes</td>
			<td style="width:71px;">Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Spectrophometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Measures the OD620 of cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C incubator with 5% CO2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">15 ml conical tube</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

オーバーレイアッセイで2の可能な結果があります。それは、一晩のインキュベーション後に刺した株の増殖を見ることが可能なはずである。 図1Aに、オーバーレイ株が産生されるバクテリオシンに感受性である。クリアランスのゾーンは刺さ歪みを取り巻く可視化することができる。また、オーバーレイ混合物に刺した菌株の旋回が可能であり、 図1Aに見ることが?...

Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

定性的にオーバーレイアッセイを使用すると、肺炎球菌バクテリオシンに対する細菌の産生と感度を測定します

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

using-overlay-assay-to-qualitatively-measure-bacterial-production

Research

JoVE Journal

Immunology and Infection

13.4K ビュー.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. 

ビデオ: Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

マウスでイメージ細菌感染にルシフェラーゼを用いた

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

免疫・感染学

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

BCGまたはM. TB（BCGタグレポーター遺伝子を用いて、抗酸菌の生存率を測定するための機能全血アッセイルクス / M. Tbをルクス）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS媒介細菌競争を監視するには、Visualアッセイ

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用肺炎球菌植民に対するプロバイオティクスの影響を調査<em&gt;体外</em&gt;接着アッセイ

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

の莢血清型<em&gt;肺炎球菌</em&gt;膨張反応を使用して、

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

の莢膜血清型<em&gt;肺炎球菌</em&gt;ラテックス凝集による

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

細菌Pyomelanin産生を阻害し、酸化ストレスに対する感受性の増加に対応を決定するための方法

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.