著者は、彼らのサポートのためにエリソン医療財団に感謝します。私たちは、wormsorterを使用しての支援のためのアビーデルンブルク研究室のメンバーに感謝したいとも。

1。ヤングアダルト動物の同期化された文化の取得<ol><li> OP50-1 E.を接種し、いくつかのNGMプレート上でワームを育てる大腸菌の細菌（10×飽和文化からの濃縮）、多くのワームは、妊娠の段階に達するまで。 </li><li>同期された培養物（卵）を得るために卵プレップ液で妊娠動物を扱う。 </li><li>いくつかの60 mmのNGMプレート上のプレートの卵は、およそ150〜200の卵/プレートの密度で10倍に濃縮OP50-1を播種。 </li><li> （ - ヤングアダルト段階ワームは、L4に達するまで）2日間、25℃で培養する。 </li></ol> 2。 緑膿菌版の作製やワームに感染<ol><li>卵プレップ（ステップ1.1）の日に100μg/ mlの最終濃度王のB型メディアを含むリファンピシンの2ミリリットルにPA14 :: GFPの培養物に接種。攪拌O / Nで37℃で培養液を、 </li><li> PA14 :: GFP CULプレートアッセイに必要な数のスローキリングプレート（SKP）にTURE。各150ミリメートルSKPペトリ皿ピペットに75〜100飽和文化μlの滅菌ガラススプレッダーを使用して均一に広げる。 20〜24時間、37℃で培養する。 </li><li>インキュベーターからプレートを取り外し、それらを室温（〜20分）に冷却する。 M9緩衝液を使用して、PA14にステップ1.4からワームを洗っ:: GFPプレートを液体の最小容量の。 150mmプレートを使用する場合は、数百のワームは、単一のプレート上に配置することができる。 </li><li> 18〜21時間、25℃で培養する。 Pの緑膿菌に露出し、若い成虫は、これはnoncolonized動物VSコロニー形成の最適な分布を表示時間ウィンドウです。 </li></ol> 3。 Wormsorterの設定 サンプルの並べ替えを実行する前に、マシンが正常に機能していることを確認してください。詳細はhttp://www.unionbio.com/support/documents.aspx?id=43で得られた、と目することができますE基本的な手順は以下のとおりです。 <ol><li>約5 PSI（4.5〜5.5の範囲）にシースバルブの圧力設定を行ってください。 </li><li>そのシース流体流量が適切であることを確認ディスペンサーの下に15ミリリットルコニカルチューブを配置し、シースバルブをオンにし、ソーターバルブをオフにします。 60秒間、ディスペンサーの下にチューブを保持します。チューブ内のボリューム9-10 mlでなければならない。この範囲外である場合、それに応じてシース弁圧を調節する。 </li><li> （ステップ3.2と同様に設定）がシース液流量が正確な選別を可能にする確実40μmで調整粒子を使用してソート速度をテストする</li><li>シースバルブをオフにします。貯水池への制御粒子溶液約25ミリリットルを追加します。 </li><li>シースバルブ、サンプルバルブをオンにします。 </li><li>コンピュータソフトウェア上で、「制御粒子モード」に移動して取得]をクリックします。 </li><li>幅を近似するTOF（飛行時間）で、粒子流量は〜5-8粒子/秒であることを確認してください粒子の、40ミクロ​​ン。このようなTOFは、単一の40ミクロ​​ンのオブジェクトを一度に通過していることを示しています。値がこの範囲外である場合は、それに応じて（上記の指示に従う）設定を調整します。 </li></ol> 4。 Noncolonizedワーム対植民仕分け<ol><li> M9を使用して、15 mlのコニカルチューブにワームを洗う。成虫は、重力によって、チューブの底に沈む、上清を除去することができます。新鮮なM9でチューブを埋める。このプロセスを3〜4倍を繰り返します。これは幼虫の大部分だけでなく、余分な細菌を除去し、約10分かかります。 </li><li>優しく混合小さな攪拌棒付きwormsorterリザーバーにワームを追加します。 </li><li> 200に600にグリーンPMTを設定し、赤と黄色の光電子増倍管によって初期信号ゲインを調整します。 </li><li>設定を調整するためのデータの収集を開始。 </li><li>毎秒25〜30イベント（ワーム）のソート率を目指す。数字率が高すぎる場合、それに応じてM9と、リザーバ内のワームを希釈する。レートがある場合x低、M9の小さな体積でより多くのワームを追加します。 </li><li>実験はnoncolonizedワームVSコロニー形成の非常に厳しい分離を必要とする場合は、「純粋な」から「一致確認」に設定します。これは、イベント内の2つのオブジェクトが互いに接近しすぎ発生した場合、または同じドロップで、機械ではなく、それを集めるよりも、ドロップを拒否していることを保証します。 </li><li>関心の人口をソートするには、サイズ（成虫を得るために）と蛍光強度（noncolonizedのための低、コロニー形成のためのすなわち高い）を示す軸周りのゲートを描きます。蛍光ゲーの値は経験的に決定されなければならない。 </li><li>ワームの並べ替えを開始するためにソート弁を開放してワームを収集するために、マルチウェルプレートまたはディスペンサーの下にペトリ皿を置きます。 </li><li>ソートされた集団は、関心の集団であることを顕微​​鏡下で確認するために、動物の小集団を収集します。いない場合は、それに応じてゲーティングパラメータを調整し、サンプル収集を続行します。 </li></ol>

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著者らは、開示することは何もありません。

我々が説明する方法は、ワームとその環境の間の相互作用に従うこと、ワームの外実体の蛍光標識を利用しています。我々はこの場合、分離は、病原体のラベリングに基づいていたなし（または光）負荷のものとは重い病原体負荷でワームを分離するために用いられた。その後の遺伝子発現分析は、それらが病原体負荷とは無関係であったことを示唆している二つのグループの間の免疫応答?...

自動ワームのソートは、それがチューブ内に真っ直ぐに通過すると（通常はレポータータンパク質のトランスジェニック発現によって提供される）ワームに蛍光シグナルを測定することにより、オペレーティング·リダイレクション、コレクションチューブに/ウェルまたは廃棄物容器に応じてできるように、多くのFACSのようなものです研究者1が設定したパラメータをゲーティングする。 wormsorterは、多くの方法で研究を容易にすることができる。分析ツールとして、それを採用した例はほとんど千の遺伝子2プロモーター活性の時空間パターンを踏襲研究である。 しかしながら、wormsorterの主な用途は、標的遺伝子発現レベル又はウォーム3-5の軸に沿った蛍光タンパク質の局在化に続いて、遺伝子スクリーニングである。 ここでは、蛍光タグ付けされた病原体によるワームの定着を、以下に、wormsorterための新しいアプリケーションを記述します。これによりツールとして、我々はpathogとの関係に焦点を当てた植民地化/負荷と免疫応答備わり、ワームでの免疫応答の開始に責任のメカニズムに新たな洞察を得るために。 これまでに研究され、事実上すべての生物では、微生物病原体に対する自然免疫応答の開始は、病原体関連分子パターン（PAMP）の認識に依存し、かつ/または危険/損傷関連分子パターン（減衰させる）6,7。微生物の細胞壁の構成成分、その鞭毛、またはその脂質二重層6、最初に保存されている微生物の構造は、第二には、両方の放出された分子（ 例えば 、ATP 8）、改変されたタンパク質または改変された細胞プロセス9,10の他のマーカーが含まれています。両方のタイプの信号は、パターン分子の特異的結合の際に防御応答を導く一連の事象を活性化するパターン認識受容体（PRR）として指定されたタンパク質によって認識される。C.虫は tractaとして非常に有用であったBLEモデルは、宿主 - 病原体相互作用のさまざまな側面を分析するが、十分に理解されていない一つのことは、ワームに開始されるか免疫応答である。他の生物におけるパターン認識受容体（PRR）にオーソロガスである推定される受容体のどれものPAMPに結合することが示されておらず、他の生物における免疫応答のために極めて重要であるのPRRのオルソログの多くは、ワームの病原体応答に驚くほど限られた貢献度を示していると抵抗。例えば、グラム陽性病原体に抵抗するために不可欠であるショウジョウバエのToll受容体は、C言語で表現されているグラム陰性病原体ネズミチフス菌11からではなく、他のテストのグラム陰性からの保護に貢献する唯一のホモログ、TOL-1による線虫 、または陽性病原体11,12。これらの観察は、その免疫応答を示すデータと組み合わせる細胞のタンパク質翻訳hを破壊することによって誘導することができLEDなどのいくつかが提案することで虫は、主に減衰9,13,14を検出します。それにもかかわらず、免疫応答を誘導するために死んだ病原体の能力を記述するレポートは、PAMPの結合は、C.における病原体認識に重要な役割を有し得ることを示唆する虫 15,16。年齢に同期化された遺伝的に同一のCの免疫応答に焦点を当てたこれまでの研究虫集団は、細菌のグラム陰性病原体緑膿菌による腸のコロニー形成に大きな個体差を示した。 しかし、転写プロファイリング研究は、1事業体17,18として、これらの可変保菌集団を処理した。この変動を利用して、我々は、GFPを発現するPにさらさ線虫（Caenorhabditis elegans）の差別的植民集団を分離するための自動化されたwormsorterを中心にプロトコルを開発緑膿菌 。異なっ - 植民集団における遺伝子発現をFACI調べる病原体の負荷（および関連する損傷）との関係の評価と免疫応答をlitated とCに病原体認識についての新たな洞察を提供虫 19。下に我々はすべての蛍光標識された病原体に感染したワームのソートに適用することができるプロトコルを記述します。 潜在的なユーザーには、選別されることが必要虫数は、使用中のその後の分析およびプロトコルの性質に依存することに留意すべきである。例えば、マイクロアレイ遺伝子発現解析の場合には、&gt;千のワームは、標準的なプロトコルが使用される場合、十分なRNAを得るために必要とするが、増幅が用いられる場合約100ワームが十分である、材料の迅速な収集を可能にし、従って、ストレスを最小限にするワーム。

<table border="0" cellpadding="0" cellspacing="0" width="927">
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		<tr height="21">
			<td height="21" style="height:21px;width:261px;">M9 Buffer</td>
			<td style="width:139px;">Prepared in house</td>
			<td style="width:527px;">Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin&#160;</td>
			<td>Sigma</td>
			<td>R3501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg prep solution&#160;</td>
			<td>Prepared in house</td>
			<td>50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGM plates&#160;</td>
			<td>Prepared in house</td>
			<td>Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKP plates</td>
			<td>Prepared in house</td>
			<td>Recipe same as NGM only 0.35% peptone instead of 0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control test particles&#160;</td>
			<td>Union Biometrica&#160;</td>
			<td>310-5071-001</td>
		</tr>
	</tbody>
</table>

automated separation of c. elegans variably colonized by a bacterial pathogen

wormsorterは蛍光レポーターの発現に基づいてワームを並べ替えることが一般的には、 線虫（Caenorhabditis elegans）の研究で使用されているFACS装置に類似した楽器です。ここでは、GFPを発現病原体によるコロニー形成の程度に応じてワームをソートするために、本器の代替使用状況を強調表示します。この新しい使用は、私たちは、ワームの腸や免疫応答の誘導の植民地との関係に対処することができました。一方、C.異なる病原体への虫の免疫応答は、それらを開始し何まだ不明ですが、文書化されている。 （相互に排他的ではない）2主な可能性が病原体関連分子パターンの認識、感染によって引き起こされた損傷の検出である。 2つの可能性を区別するために、病原体への暴露は、それが引き起こす損傷から解離されなければならない。グラムNによって広く保菌されたワームのwormsorter有効に分離植民同様に暴露されたワームの可能性が高い病原体の負荷による損傷はなく、またはわずかに、とegative病原緑膿菌 、。これらの異なる集団は、病原体負荷および転写免疫応答の誘導との間の関係を評価した。結果は、2つの病原体認識の可能性を支持し、解離していることを示唆している。

年齢をマッチさせた場合には、遺伝的に同一で虫はPにさらされている緑膿菌は 、広い分布がコロニー形成（ 図1A）のレベルで観察される。植民ワームからnoncolonizedの効率的な分離が（ 図1B）を達成することができ、ここで説明されたプロトコルの助けを借りて。 noncolonizedワームとは異なり、植民ワームは、低迷して減少し、排便19などの損傷?...

Watch this Scientific Journal Video about Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen at JoVE.com

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

wormsorterは蛍光レポーターの発現に応じてワームをソートすることによって線虫（Caenorhabditis elegans）における遺伝子スクリーニングを容易にします。 GFPを発現病原体によるコロニー形成に応じて仕分けし、我々は、免疫応答を開始における病原体認識のよくわかっていない役割を調べるためにそれを利用する：ここでは、新たな使用方法を説明します。

の自動分離<em&gt; C.虫</em&gt;可変細菌性病原体の植民地

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

automated-separation-c-elegans-variably-colonized-bacterial

Research

JoVE Journal

Immunology and Infection

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

8.3K ビュー.  University of California, Berkeley.  wormsorterは蛍光レポーターの発現に応じてワームをソートすることによって線虫（Caenorhabditis elegans）における遺伝子スクリーニングを容易にします。 GFPを発現病原体によるコロニー形成に応じて仕分けし、我々は、免疫応答を開始における病原体認識のよくわかっていない役割を調べるためにそれを利用する：ここでは、新たな使用方法を説明し?...

ビデオ: Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

細菌性病原体によるヒト細胞の浸潤

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

免疫・感染学

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

血液培養から細菌性病原体の検出および抗菌剤耐性テストのための一日のワークフロースキーム

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

ライブセル蛍光顕微鏡を用いた細菌毒素誘導される応答の可視化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

細菌性病原体による細胞の感染を調査するためにイメージングInlC分泌<em&gt;リステリア菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

酵母ツーハイブリッドスクリーンを用いた非耕作植物病原体から細菌のエフェクターの機能をUnravelling

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

細菌のリポタンパク質と質量分析法による構造決定のための N 末端 Lipopeptides 準備の充実

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

高スループット、高コンテンツ、液体ベースのc. の elegans病態システム

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

自動行動分析の大規模なc. の elegans集団による視野が広い追跡プラットフォーム

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...