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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F., 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=Bactofencin+a,+a+new+type+of+cationic+bacteriocin+with+unusual+immunity.">Bactofencin a, a new type of cationic bacteriocin with unusual immunity.</a> 4 (6), 00498-00413 (2013).</li><li>Son, M. 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=Conserved+mutations+in+the+pneumococcal+bacteriocin+transporter+gene,+blpA,+result+in+a+complex+population+consisting+of+producers+and+cheaters.">Conserved mutations in the pneumococcal bacteriocin transporter gene, blpA, result in a complex population consisting of producers and cheaters.</a> MBio. 2 (5), (2011).</li><li>Kochan, T. 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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><tbody><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>Fisher</td><td>B21261X</td><td>For growth from freezer cultures</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C100-500mg</td><td>4,741 Units per plate</td></tr><tr><td>Todd Hewitt Broth + Yeast Extract (THY)</td><td>Fisher</td><td>DF0492-17-6&amp;nbsp;</td><td>30 g of THB, 5 g yeast extract, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>Trypticase Soy Broth + Agar plates (TSA)</td><td>Fisher</td><td>B11768&amp;nbsp;</td><td>30 g of TS, 15 g agar, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside (X-gal)</td><td>Sigma</td><td>B4252-50MG</td><td>Dissolve in dimethylformamide</td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Spectrophometer</td><td></td><td></td><td>Measures the OD&lt;sub&gt;620&lt;/sub&gt; of cultures</td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO2</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td></td><td></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

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

細菌におけるタンパク質-RNA結合を定量するためのアッセイ

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

遺伝学

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 ...

免疫・感染学

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

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

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

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

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

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更<em&gt;肺炎球菌</em&gt;蛍光染料を使用

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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 ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

細菌 - 宿主相互作用を特徴づけるために、バイオミメティック材料

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

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.

ソフト寒天オーバーレイ技術の使用は、細菌産生阻害する化合物をスクリーニングするために

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

高スループット赤痢菌-特定の殺菌試験

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用の調査のための高スループット共培養アッセイ

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. アフルギノーサ 抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

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

要約

さらに動画を探す

この動画の章

細菌におけるタンパク質-RNA結合を定量するためのアッセイ

T6SS媒介細菌競争を監視するには、Visualアッセイ

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更

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

細菌 - 宿主相互作用を特徴づけるために、バイオミメティック材料

ソフト寒天オーバーレイ技術の使用は、細菌産生阻害する化合物をスクリーニングするために

高スループット赤痢菌-特定の殺菌試験

微生物相互作用の調査のための高スループット共培養アッセイ

P. アフルギノーサ抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

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

要約

さらに動画を探す

この動画の章

細菌におけるタンパク質-RNA結合を定量するためのアッセイ

T6SS媒介細菌競争を監視するには、Visualアッセイ

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更

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

細菌 - 宿主相互作用を特徴づけるために、バイオミメティック材料

ソフト寒天オーバーレイ技術の使用は、細菌産生阻害する化合物をスクリーニングするために

高スループット赤痢菌-特定の殺菌試験

微生物相互作用の調査のための高スループット共培養アッセイ

P. アフルギノーサ 抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

P. アフルギノーサ抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー