私たちは、優れた技術支援のためにエリザベートギュメ、クリストフ·ビュイソンとルドビクBridouxに感謝したいと思います。我々は、システムの初期設定のためのシルヴィーSalamitouとSINDA Fedhilaに大いに世話になっています。

1。昆虫飼育卵から終齢幼虫への全サイクルは、25℃で約5週間です1つまたは2つの追加の週は、大人の蝶を得るために必要とされている。 <ol><li>少なくとも100蛹または新しくマージされた大人のGを置く5リットルの金網ケージ内mellonella蝶 。男性蝶は10〜15 mmを測定します。成人男性蛾がかすかな光と闇のマーキングとベージュです。 20ミリメートルの周りの女性の蝶対策。雌は茶色/グレー色で、男性よりも暗くなります。 </li><li>産卵用ケージに4層の紙の2つのパックを一時停止します。 2日後、成人女性は、論文間の端に卵を産むでしょう。 </li><li>週2回、花粉や蜜ろうを含んだ空気が循環するようにグリッドを使用して、新しいプラスチックの飼育箱で卵の紙パックを置く。卵は約3日で孵化し、小さな幼虫は、ワックスや花粉を食べて開発を開始。あるいは、幼虫は人工飼料consistin上に配置することができます500グラムの液体蜂蜜のミックスのグラム、400グラムグリセリン、100グラム、乾燥ビール酵母、250グラムの小麦粉、200グラム脱脂粉乳と400グラムポレンタ。幼虫当たりの食品の適切な比率を提供するために、定期的に新しい箱に幼虫を分離し、2日おきに餌を補給してください。 </li><li>幼虫は幼虫スタジアムの6段階で飼育されています。各スタジアムは幼虫の頭部カプセルの滑りによって特徴づけられる。幼虫は蛹化前の最後の段階に到達するとき、彼らは摂食を停止し、光絹繭の構築を開始。 </li><li>その保護繭では、幼虫は休もうと蛹に変身。ワックスワームは、成人蛾に出てくるまで1〜2週間の蛹の段階で残っています。大人の蛾は飲みも食べません。合が発生し、ワックスワームのライフサイクルが再び開始されます。 </li><li>病理アッセイを標準化するために、最後の幼虫期幼虫を取る。この段階では、彼らが供給を停止し、飼育箱のカバーに移動して、絹の生産を開始。このsタジさんは5日程度かかります。 </li></ol> 2。感染症のための昆虫セレクション<ol><li> 2〜3センチ、重量が180から250ミリグラムアール終齢​​幼虫を選択感染前24時間およびそれらを飢えさせる空のボックスに入れます。 </li><li>幼虫の周りに生まれたばかりのシルクの繭を削除します。 </li></ol> 3。細菌の準備<ol><li>経口摂取について10 4コロニー形成単位（cfu）から/ mlのイントラhaemocoelic注射用10 8 cfu / mlにとcfu / mlで3×10 6〜1×10 8 cfu / mlの範囲の最終濃度（取得するCFUを取得するためにバチルス菌懸濁液を調製LD 50）は、細菌種に依存しています。我々の場合、B.でセレウス菌は、ルリア-ベルターニブロス培地で栽培されている（LB、10g / lのトリプトン、5 g / l酵母エキス、10g / lのNaCl）で37℃で攪拌下になるまでの成長の湾曲に対応した固定相への参入、カーブ。 OD 600 nmを測定する（Bの1と2の間セレウス菌 ）と以前に作成した滴定曲線を用いて、細菌濃度を評価するためにそれを使用します。室温とリン酸に再懸濁し、10分間5000×gで遠心分離によって収穫細菌は（：1MのKH 2 PO 4、1M K 2 HPO 4、5MのNaCl、pH7.2のPBS）緩衝生理食塩水。各幼虫は所望の濃度で細菌懸濁液10μlを受け取ることになります。 </li><li>代わりに、胞子懸濁液が感染して調製することができる。胞子形成培地HCT（5g / Lのトリプトン、2g / Lのカゼイン加水分解物、12.5 mMのK 2 HPO 4、12.5 mMの硫酸マグネシウム 、0.05mMののMnSO 4、1.2mMのZnSO 4を 、1.2mMのFeの中の細菌を培養して胞子を得る30℃2（SO 4）3、0.5％H 2 SO 4、25mMのCaCl 2）を20℃で3日間。収穫遠心分離により胞子（10,000×gで、15分間）し、滅菌蒸留水で2回洗浄する。胞子PELを再懸濁し°Cの残りの栄養型細菌を除去するために78℃で15分間滅菌蒸留水と熱でみましょう。 LB寒天プレート上で連続希釈液をメッキすることにより懸濁液を数え上げる。各幼虫は所望の濃度で胞子懸濁液10μlを受け取ることになります。 </li></ol> 4。毒素製剤を叫ぶ<ol><li> BからCry1C毒素を準備チューリンゲンシス菌株407をコードする遺伝子を有するプラスミドCry1C pHTF31C 21で形質転換された。 30℃で100mlのHCTの培地で培養菌株°と72時間Cフル胞子形成、培養上清中の毒素結晶製造と解放を可能にする（エリスロマイシン10mg/mlの）抗生物質。 </li><li> 72パーセント-79％ショ糖勾配上清から結晶を精製する。最初の40 mlチューブの79％ショ糖の17ミリリットルを追加します。 79％ショ糖の上に72％のスクロースを注意深く層17ミリリットル。最後に、層10倍濃縮胞子形成培養の5-6 mlおよびチューブを閉じます。 20,00で14時間チューブを遠心0×gで、4℃、胞子から結晶を分離する。勾配界面で結晶を集める。それを洗うために25ミリリットル冷滅菌水で結晶ペレットを再懸濁します。 20分間8000 xgで遠心分離します。このステップは、結晶に付着するすべてのスクロースを削除するには、3回繰り返されます。 5ミリリットル滅菌水の結晶を再懸濁し、純度を評価するために、顕微鏡下で観察する。 </li><li> 50 mMのNaOHにpresolubilized結晶ソリューション上の古典的なブラッドフォード染色によって毒素タンパク質濃度を評価する。毒素結晶は使用するまで-20℃で保存することができます。 </li></ol> 5。インジェクターの準備<ol><li>正確な注入量を制御するには、1μL/秒（ 図1A）に設定速度で自動化されたシリンジポンプ（ 例えば 、KD科学KDS 100）を使用します。 </li><li> 300μlの水、または20から25の幼虫の感染の菌液（条件当たり1シリンジ）を1mlの皮下注射器を埋める。慎重TAによって気泡を除去シリンジpping、その後シリンジに0.45×12 mmの針を取り付けます。 </li><li>注射器で注射器を置きます。 </li><li>注入量を制御するために、空のチューブに10μlのブランク注入を実行します。 </li></ol> 6。昆虫Intrahaemocoelicインジェクション<ol><li>親指と人​​差し指の間に手動で昆虫を置きます。その後幼虫皮膚におけるインジェクタ（キューティクル）（ 図1C）で固定針を挿入します。 </li><li> 10μLの菌液を（胞子または栄養細菌）を注入しながら所定の位置に昆虫を保つ。 </li><li>慎重に針から昆虫を削除します。 </li><li>小さなペトリ皿（直径5cm）、皿あたり5幼虫に感染した昆虫を置きます。 </li><li>各実験条件のために少なくとも20の幼虫に感染します。 </li><li> 48時間インキュベーター内で37℃で皿を置きます。 </li></ol> 7。昆虫強制給餌（摂取） <ol><li> 2ミリリットルエッペンドルフチュー​​ブに、0.2μgの/＆mを混ぜるU; 3×10 4〜10μlあたり1×10 7の範囲の最終濃度を得るためにいずれかの胞子又は栄養細胞懸濁液とCry1C毒素のリットル。 </li><li> 20から25までの幼虫の感染の細菌Cry1C毒素溶液300μlで、30Gと25mmの皮下注射針を1mlシリンジを埋める。シリンジから気泡を除去する。 </li><li>手動ので、その口はインジェクタ（ 図1D）で固定針を入力すると、自動化されたシリンジポンプを用いて細菌溶液10μlとそれを強制的に食べさせることが虫を置きます。 </li><li>各実験条件のために少なくとも20の幼虫に感染します。 </li><li>小5センチメートルペトリ皿（シャーレ当たり5幼虫）に感染した昆虫を置きます。 48時間インキュベーター内で37℃で皿を置きます。 </li></ol> 8。記録昆虫死亡<ol><li>強制給餌又は注射の実験の後、25℃フードなしでシャーレ内の幼虫を保つ℃または37℃幼虫のmoをチェック48時間の期間にわたって定期的にrtality。死んだ幼虫は不活性であり、通常は黒を（ 図1B）を入れます。 </li><li>死亡率のデータは、log-プロビット·プログラム14を用いて分析することができる。このプログラムは、線量の死亡曲線の直線性をテストし、致死量（LD 50）を提供しています。あるいは、生存または死亡データを分析するようなプリズム（グラフパッド）などの他のプログラムを使用することができる。 </li></ol>

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昆虫、特に幼虫期の使用は、いくつかの病原体の感染モデルとして、頻繁になってきています。いくつかの側面のための選択のモデルは、大人と幼虫期の1,2の両方として使用ショウジョウバエ （フライモデル）です。鱗翅目昆虫G. mellonellaも主に注射することにより細菌の病原性を検定するために使用されています。哺乳類の病原体が研究されている場合ショ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>ワックスと花粉</td><td>ラ·ルーシャRoanaise </td><td> 303000 </td><td>任意の蜂蜜生産者</td></tr><tr><td>自動化されたシリンジポンプ</td><td> KDサイエンティフィック</td><td> KDS 100 </td><td></td></tr><tr><td>注射器1ミリリットル</td><td>テルモ</td><td> BS 01T </td><td></td></tr><tr><td>針0.45×12ミリメートル</td><td>テルモ</td><td> NN 2613R </td><td></td></tr><tr><td>シャーレ5センチメートル</td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td>ニードル30G、25mmの皮下注射</td><td> Burkard製作所株式会社</td><td> PDE0005 </td><td></td></tr></tbody></table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

細菌の病原性の研究は、多くの場合、適切な動物モデルを必要とします。感染の哺乳動物モデルは高価であり、倫理的な問題が発生することがあります。感染モデルとして昆虫の使用は貴重な代替手段を提供します。このような線虫のような他の非脊椎動物モデルのホストに比べ、昆虫は、抗菌防御の比較的高度なシステムを持って、その結果、より多くの哺乳類の感染過程に関連する情報を生成する可能性があります。哺乳類と同様に、昆虫は、複雑な自然免疫系1を所有しています 。体液中の細胞は、微生物の侵略を貪食または封入することが可能であり、かつ体液性応答はリゾチームと小さな抗菌ペプチド2,3の誘導生産を含む。また、アナロジーは昆虫幼虫中腸および哺乳類消化器系の腸細胞の上皮細胞間に発見されています。このような細胞接着などの細菌感染過程に不可欠な最後に、いくつかの基本的なコンポーネントへの抵抗抗菌ペプチド、組織の劣化や酸化ストレスへの適応は、昆虫や哺乳類1の両方で重要であると思われる。このように、昆虫は哺乳類の感染症に関与微生物の病原因子の同定および特徴付けのための多価ツールです。 大きいワックス蛾ハチノスツヅリガの幼虫は、 黄色ブドウ球菌 、 プロテウス·ブルガリなどの哺乳動物の真菌（ フザリウム·オキシスポルム 、 アスペルギルスフミガタ 、 カンジダ·アルビカンス ）および細菌性病原体を含む微生物感染症の広い範囲の病因に有用な洞察を提供することが示されている、 セラチア、緑膿菌 、 リステリア菌や腸球菌 4-7。かかわらず、細菌種の、キューティクルを通して直接注入することにより感染ガレリア幼虫を用いて得られた結果は、一貫してシミのそれらと相関LAR哺乳類研究：哺乳動物モデルで減衰された細菌株は、 ガレリアの低い病原性を示すもので、重篤なヒトへの感染を引き起こす株は、 ガレリアモデル8-11の強毒性である。 ガレリアの経口感染が化合あまり使用され、追加され、特定の毒素のように、死亡率に到達するために必要とされています。 G. mellonella幼虫存在するいくつかの技術的な利点：彼らはこのように細菌の定義された用量の注入を可能にする、（蛹化前の終齢幼虫が250ミリグラム約2cm長いとウェイトです）比較的大である、彼らは、様々な温度（20℃で飼育することができる30℃〜C）と感染症研究は、哺乳類の環境を模倣した実験を可能にする、37℃12,13の上に15℃の間で行うことができる。また、昆虫の飼育は簡単で、比較的安価です。幼虫の感染症はいくつかの手段、includinによって細菌の病原性を監視することができますLD 50 14グラムの計算では、細菌の生存15,16および感染過程17の検査の測定。ここでは、Gのすべてのライフステージをカバーする、昆虫の飼育を記述mellonella。口腔内およびhaemocoelic：我々は、接種の2つのルートによる感染の詳細なプロトコルを提供します。このプロトコルに使用される細菌のモデルは、 セレウス菌 、胃腸にだけでなく、他の重篤な局所または全身性日和見感染18,19に関与グラム陽性病原体である。

G.に細菌のイントラhaemocoelic注入mellonellaは、いくつかのヒト病原体の自然免疫因子に対する組織損傷および抵抗を扱う多くの病原因子の同定のために非常に有用であることが証明されています。例として、 図2A は Bの様々な用量の注射後、昆虫の死亡率を表すセレウス菌 （野生型と変異株）22は、 図2Bは、Gの感染後の細菌の...

Watch this Scientific Journal Video about The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis at JoVE.com

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

大きいワックス蛾の幼虫の経口およびイントラhaemocolic感染<em&gt;ハチノスツヅリガ</em&gt;記載されている。この昆虫は昆虫だけでなく、哺乳類の日和見菌の病原因子を研究するために使用することができます。昆虫の飼育、感染との実施例の方法<em生体内で&gt;</em&gt;分析が記載されている。

昆虫<em&gt;ハチノスツヅリガ</em&gt;細菌性病因を調査するための強力な感染モデルとして

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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JoVE Journal

Immunology and Infection

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

31.6K ビュー.  INRA, Micalis UMR1319, France. 大きいワックス蛾の幼虫の経口およびイントラhaemocolic感染ハチノスツヅリガ記載されている。この昆虫は昆虫だけでなく、哺乳類の日和見菌の病原因子を研究するために使用することができます。昆虫の飼育、感染との実施例の方法分析が記載されている。

ビデオ: The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8ウェルチャンバースライドを用いたin vitro のバイオフィルム形成における

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

免疫・感染学

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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

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 ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;ハチノスツヅリガ</em&gt;モデル生物として研究する<em&gt;レジオネラ·ニューモフィラ</em&gt;感染症

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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 ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

亜硝酸塩の催奇形性を評価するためのモデルとしてのゼブラフィッシュ

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

病原性、病態、および薬の有効性を調査するため、代替の非哺乳類動物モデルとしてゴキブリをヒス マダガスカル

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

伝染性ファブリキウス嚢病ウイルスの病原性を研究するためのEx Vivo鶏ファブリキウス嚢細胞培養モデル

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...