このプロトコルの開発への貢献のためにサマンサオーチャード、キンバリーコールズ、エリンハーバート·チャン、グレッグ·リチャーズ、ミーガンメナード、とヨンジンパーク：筆者らはグッドリッチ·ブレアラボの過去のメンバーに感謝したいと思います。この作品は、全米科学財団の助成金、IOS-0950873と健康NRSAフェローシップFAI084441Zの国立研究所によって資金を供給された。

1。虫卵の殺菌と飼育<ol><li> 900-1,000 mlのH 2 Oで提供寒天の最初のオートクレーブ15グラムで食事を準備します直後によく研究室ブレンダーで166グラムの小麦胚芽食とブレンドしたオートクレーブ、ミックス。 4でアルミ箔にダイエットを転送し、冷却するための皿（または皿）に注ぎ、しっかりとラップし、℃で保存</li><li>到着時には、Mを殺菌する時々かき混ぜながら90ミリメートルろ紙ガラスフィルターホルダー及び真空フラスコ装置では、2〜3分間0.6％漂白剤溶液の250mlでsextaの卵。 </li><li>洗浄あたり250ミリリットル滅菌蒸留H 2 Oで3-4回漂白剤溶液を排出し、卵を洗うために真空をオンにします。 </li><li>ペトリ皿のふたに入れ新鮮な90ミリメートルろ紙に卵を移し、それらを一緒に彼らはもはや棒になるまで乾燥させます（約20〜30分）。 </li><li>各コンテナに約40個の卵を置いて、昆虫食とプラスチック容器の底に卵を移す（ 図1A）。卵は湿気によって真菌汚染を防ぐために食事から分離されています。 8時間暗の光周期：16時間明、26で卵℃を維持する。卵が孵化する前に、黄白色に明るくなります。 </li><li>孵化が完了したら、慎重に底のダイエット（ 図1B）を使用して新しい容器にそれぞれ約25幼虫を移す。その壊れやすい初期の発達段階の間にそれらを傷つけることを避けるためにピンセットを使用して、長い黒のホーンによって各幼虫をピックアップすることが重要です。上記のように2日間インキュベートする。幼虫の死は、この段階では珍しいですが、いくつか死んでいるか、可能性が高く、発達発育を妨げられた幼虫が観察されることがあります。このような昆虫は、さらなる研究から除外され、凍結によって犠牲にされるべきである。 </li><li>共食い行動を回避するために、食品の小さな断片（ 図1C）を使用して個々の容器に幼虫を移し、上記のようにインキュベートする。昆虫の開発を監視し、食品を交換し、洗浄糞は、OUコンテナのトン隔日彼らは第三幼虫の脱皮を（通常は孵化後6-9日）を受けるまで。これは昆虫の体（ 図1D）に沿って各腹脚とストライプの増加目立つ上に黒いフック状crochetsの外観が特徴、 第 4齢幼虫の段階である。幼虫は大きさにかなりばらつきがあるが、典型的には、 第 4齢期に入ったときに0.15〜0.4グラムの重量を量ることができる。同様の大きさの幼虫が注射のために選択されるべきである。 </li></ol> 2。注射用細菌の調製<ol><li>テストしたい成長期に標準的な手順に従って、テストする細菌株（s）を育てる。 </li><li>各菌株のペレットを500μlの室温で13,000 rpmで遠心機で2分間回転させて試験する。 </li><li> 1ミリリットル滅菌1Xリンに再懸濁した細胞は、上記のように、再び緩衝生理食塩水（PBS）とペレット。 </li><li> 0.5ミリリットル滅菌1×PBSおよびMEAで細胞を再懸濁懸濁液の光学密度（OD）を確認してください。必要であれば、同じODにすべての懸濁液を希釈します。必要に応じて滅菌増殖培地はまた、1X PBSの代わりにセルの再懸濁および希釈に使用することができる。 </li></ol> 3。 第 4齢幼虫の注入<ol><li>各希釈のための新鮮なピペットチップ（ 図2A）を使用して滅菌1X PBSで96ウェルマイクロタイタープレートの6ウェルの最初の菌株の連続10倍希釈液を準備します。各ウェル中の細胞懸濁液の最終容量は注入（昆虫あたり10μl）を加えたプレートに20μlの接種（ステップ3.2および3.7を参照）に必要な量を超えている必要があります。 </li><li>適切な増殖培地を含むペトリプレートの上部に沿ってスポット10μlを各5段階希釈（10 -6 10〜-2）用。スポットが中心（ 図2B）に広がるように、板を傾けてください。 </li><li>氷の上の容器における昆虫食と糞便や場所の昆虫を削除約5分。 </li><li>滅菌H 2 O（ 図2C）の1管に続いて70から100パーセントのエタノールの2管で3回ずつ洗浄することによって注射針を消毒する。液体の体積は水没針に十分でなければならない。 </li><li>血球計数器を用いて顕微鏡下で希釈細胞懸濁液を定量する。接種希望に応じて、適切なマイクロタイターウェル（ 図2D）から10μlを描く。 </li><li>綿棒は、昆虫の95％エタノールで表面、および腹部prolegsの一つ後ろに角度（45°未満）で昆虫に10mlの細胞懸濁液を注入します。穿刺腸をしないように注意するとちょうど表皮層（ 図2E）の下に内容を注入します。体液の一部喪失（透明、緑、青の液体）が正常であるが、注射部位から出現する粒子状物質と黄色の液体が腸内穿刺の指標である。この問題が発生した場合は、昆虫を犠牲にして再新しい幼虫を得るそれを置く。 </li><li>最初の注射グループ内の残りの各昆虫のためのステップ3.6を繰り返します。針の滅菌は、汚染が発生しない限り、細菌の同じ希釈率とひずみを注入し、各昆虫の間で必要ではありません。あるいは、繰り返しディスペンサーはサンプル間の注入量でより一貫性のために使用することができる。 </li><li>細菌の最初の株でそれぞれ昆虫を注入した後、今回はプレートの中央に細胞懸濁液をスポッティングし、それらの底部（ 図2F）に向かって流れさせる、ステップ3.2を繰り返します。適切な温度でプレートをインキュベートし、接種を列挙するためにコロニーをカウントします。この第二のめっき工程では、これが第一及び第二のめっきの間に異種のコロニー数をもたらすような試料は、注入工程中に汚染されていないことを確認するために使用されます。 </li><li>繰り返し実験における細菌の各株の3.8を通じて3.1を繰り返します。最後に、マスターを使用して滅菌した1×PBSで3月5日虫を注入陰性対照群としてのILE針。 8時間暗の光周期：16時間明、26℃で昆虫をインキュベートする。 </li><li>注入後の時間をかけて昆虫の生存を監視します。昆虫死が刺激されると随意運動の欠如として特徴付けられる。昆虫はしばしば死亡前に感染中の食欲不振、下痢（水様、黄色の排泄物）、および/または水の損失を（ &quot;発汗&quot;）を示すが、これらの特性は、同様に注目に値するかもしれません。 </li></ol>

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特別な利害関係は宣言されません。

Mの直接注入ここで説明するように昆虫病原細菌とsextaの幼虫は、細菌の病原性を解析するためのシンプルで効果的な手段として機能します。この方法はまた、別の実験対象および/または条件に合わせて高度に適応可能である。細菌は、注射前に、種々の方法で調製する​​ことができる。 Xの場合にはnematophila、中間対数期に栄養豊富なルリア-ベルターニ（LB）培?...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> 90ミリメートルろ紙</td><td>ワットマン</td><td> 1001 090 </td><td></td></tr><tr><td>ガラスフィルターホルダー</td><td>ミリポア</td><td> XX1004700 </td><td></td></tr><tr><td> たばこスズメガ卵</td><td>カロライナ生物サプライ</td><td> 143880 </td><td></td></tr><tr><td>マイマイガダイエット+寒天</td><td> MPバイオメディカル</td><td> 0296029301 </td><td></td></tr><tr><td> 5.5オンスプラスチック容器と蓋</td><td>ソロカップカンパニー</td><td> URC55-0090 PL4-0090 </td><td></td></tr><tr><td> 1オンスプラスチック容器と蓋</td><td> DARTコンテナ株式会社</td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1X PBS </td><td></td&gt; <td></td><td> 137mMのNaCl、2.7mMのKCl、8mMののNa 2 HPO 4、1.46 mMのKH 2 PO 4、pH7.4の</td></tr><tr><td>注射器</td><td>ハミルトン</td><td> 80208 </td><td> 30ゲージ、0.375 &quot;長さ、ポイントスタイル2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

一般的にタバコイモムシとして知られているたばこスズメガは 、タバコやトマトなど、ナス科の植物を食べ、重要な農業害虫と見なされます。 Mの感受性昆虫病原性細菌種の多様1-5、ならびに昆虫の免疫系6から8に関して入手可能な情報の富、および保留中のゲノム配列9〜sextaの幼虫は宿主-微生物相互作用を研究に使用するための優れたモデル生物作る発病時。さらに、M. sextaの幼虫は他の感受性の昆虫種に実験室での相対的に操作し、維持するために比較的大きいと簡単です。それらの大きなサイズも、感染に対する宿主応答の解析のための効率的な組織/体液抽出が容易になります。 ここで紹介する方法は、Mの血体腔内細菌の直接注入（血液空洞）を記述するsextaの幼虫。このアプローチ単に注射後に昆虫の死までの時間を監視することによって、様々な細菌種、株、または変異体の毒性の特性を分析し、比較するために使用できます。このメソッドは、通常、昆虫へのエントリを取得するための手段として、線虫ベクトルと関連付けるXenorhabdusとPhotorhabdus種の病原性を研究するために開発されました。昆虫病原性線虫は、通常、天然の消化器や呼吸器の開口部を介して幼虫に感染し、昆虫の体液（血液）、その後すぐに10にその共生細菌の内容物を放出する。ここで説明した注入法は、このように昆虫に細菌や線虫の影響を脱共役、線虫のベクトルの必要を回避します。この方法は、ニッキング11と経口毒性アッセイ12を含むentomopathogenesisを分析するための他の既存の方法を使用しては不可能である種菌内感染性物質の正確な列挙型（細胞やタンパク質）を可能に<e直接噴射方式は全細胞接種の病原性に対処する一方で、mは&gt;また、経口毒性アッセイは、幼虫の消化器系に導入され、分泌された毒素の病原性に対処します。 ここで説明したように直接注入法の有用性は、昆虫死亡率を監視することにより、細菌の病原性を解析することである。しかし、この方法は簡単にMに感染の影響を研究に使用するために拡張することができますsexta免疫システム。昆虫は体液性および細胞性応答の両方を介して感染に応答します。体液性応答は細菌関連パターンの認識や各種抗菌ペプチドのその後の生産7を含み、これらのペプチドをコードする遺伝子の発現をRNA抽出と定量PCR 13を介して直接感染後に監視することができます。感染に対する細胞応答は根粒形成、カプセル化、および血球6による感染性病原体の食作用を伴う</sup&gt;。これらの応答を解析するために、注入された昆虫を切開し、顕微鏡13,14により可視化することができます。

昆虫死亡率アッセイの代表例を図3に示します。この実験では、昆虫は野生型（ATCC19061）または中間対数期（株当たりn = 6虫）まで成長Xenorhabdus nematophilaの弱毒変異株（LRP 13）のいずれかの約50コロニー形成単位（CFU）を注射した。昆虫は約72時間観察し、各時点でまだ生きて注入された昆虫のパーセントを記録した。このケースでは、弱毒株は、昆虫殺害でク...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

ここで説明する方法は、血体腔に昆虫病原細菌の直接注入を利用<em&gt;たばこスズメガ</em&gt;昆虫の幼虫。<em&gt; M sexta</em&gt;市販されており、よく研究された昆虫です。したがって、この方法は、1つまたは両方のパートナーの視点からホスト細菌の相互作用を分析する簡単な方法を表しています。

の飼育とインジェクション<em&gt;たばこスズメガ</em細菌毒性を評価するための&gt;幼虫

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

rearing-injection-manduca-sexta-larvae-to-assess-bacterial

Research

JoVE Journal

Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K ビュー.  University of Wisconsin-Madison. ここで説明する方法は、血体腔に昆虫病原細菌の直接注入を利用たばこスズメガ昆虫の幼虫。 M sexta市販されており、よく研究された昆虫です。したがって、この方法は、1つまたは両方のパートナーの視点からホスト細菌の相互作用を分析する簡単な方法を表しています。

ビデオ: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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 ...

免疫・感染学

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

誘導発現系を適用すると、細胞内シグナル伝達と細菌の病原性因子の干渉を研究するために、

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

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 ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

貪食細胞における細胞内のレプリケーションの結核菌 abscessusの病原性マーカーの同定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

病原性と線虫モデルにおけるアエロモナス感染症の病態の評価

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

マウスのモデルを評価する生得の免疫反応黄色ブドウ球菌感染症

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

病原性細菌に対する免疫学的応答を評価するための Opsonophagocytic 殺害アッセイ

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...