This work was supported by BC Lung Association and Mitacs.

1.菌株および増殖培地<ol><li>ウシ血清アルブミン、デキストロース10.0gを、脱イオン水の460 mLの塩化ナトリウムの4.05グラムの25グラムを溶解することによってアルブミンデキストロースおよび塩のストック溶液（ADS）を作ります。 4℃でのADS及びストアをフィルター滅菌します。 </li><li> 7H9粉末4.7gの精製水900 mLでのグリセロールの2ミリリットルを添加することにより、7H9ブロスを作ります。 10分間121℃で7H9ブロスをオートクレーブし、それが進行する前に、室温まで冷却することを可能にします。 ADS 100mLおよび7H9ブロス900mLのにTween80を0.5ミリリットルを添加することによって7H9ADSTを作ります。 4°Cで保存。 </li><li>硫酸カナマイシン50mgを秤量し、脱イオン水1mLに溶解します。最終濃度は50mg / mLです。フィルター滅菌し、-20℃で保存。 7H9ADSTの1リットル当たりのカナマイシンストック溶液0.5mlを加えます。 注：この培地はとてもボリュームを拡張適切培養サイズに応じて、新鮮されるべきです。 </li><li> 7Hに結核菌を育てます9ADSTは文化を立っ中でカナマイシンを補いました。毎日文化を振ると、OD600が凝集を避けるために、1.0に到達する前にそれを希釈します。 注：この方法の開発のために使用されるヒト結核菌株は、菌H37Rv pJAK2.Aプラスミド12で形質転換しました。 pJAK2.Aは、HSP60プロモーターからホタルルシフェラーゼ遺伝子の高レベル発現を可能にし、カナマイシンを使用して選択することができるpMV361ベクターに基づく組込みプラスミドです。 </li></ol> 2. THP-1培地とメンテナンス<ol><li> （FBSおよび2mMグルタミン約10％）RPMI不完全培地を作製するために、熱不活性化ウシ胎児血清（FBS）の50 mLおよびRPMI 1640を500mLの200mMのL-グルタミンを5mLを加えます。 </li><li>標準的プロトコール13に従ってTHP-1細胞の培養を維持します。 PASの間に0.2培地1mL当たり100万の細胞密度を維持しながら簡単に説明すると、RPMI不完全培地中のTHP-1細胞を増殖させます賢人。 </li></ol>ルシフェラーゼ発現ヒト結核菌H37Rvを用い3.ハイスループット細胞内スクリーニング<ol><li>波長600nmで分光光度計で活発に増殖する細菌懸濁液の光学密度を測定します。ミリリットル当たり0.1 OD 600 = 3×10 7細菌の換算係数を使用して、細菌密度を計算します。 </li><li>新しい遠心管に10：1の感染の多重度のために十分な細菌（MOI）を行うピペット。 10分間3,000×gでペレット化し、液体を吸引。 RPMI1640の450μLにヒト血清の50μLを追加します。実験の値を適切にするボリュームをスケール。 </li><li>細菌をオプソニン化するために、10％ヒト血清を含有するRPMI1640 500μLあたり1×10 8の細菌の密度でペレットを再懸濁します。混合物を30分間37℃でインキュベートすることを可能にします。血球計および反転microscoで計数することによりTHP-1細胞の培養密度を決定PE。 </li><li> 100×gでの滅菌遠心管中の細胞をペレット化し、10分間37°C。上清を吸引し、ミリリットル当たり100万個の細胞の密度で、不完全RPMI中に細胞を再懸濁。 40 / mlの最終濃度までホルボール-12-ミリステート-13-アセテート（PMA）を加えます。 注：これは分化ミックスと呼ぶことにします。 </li><li>オプソニン化結核菌は、10のMOIでのTHP-1分化混合物と結合：1、96ウェル平底白色プレートにウェル当たり100μLの最終ミックスのアリコート。定期的に均一性を確保するために、混合物を攪拌。分化および感染は5％CO 2を含む加湿インキュベーター中で37℃で一晩進行させます。 </li><li> RPMI各100μLでウェルを2回洗浄します。不完全RPMI中で所望の濃度に希釈した化合物を添加し、3日間インキュベートします。 </li><li>ウェルから培地を吸引除去します。各ウェルにルシフェラーゼアッセイ試薬の50μLを追加します。 Tでプレートをシールransparent接着プレートシーラー。 22℃でのインキュベーションの5分を許可した後、ウェルあたり1秒でルミノメーターで読み出しを得ます。 </li></ol> 3-（4,5-ジメチルチアゾール-2-イル）-2,5-ジフェニルテトラゾリウムブロミド（MTT）アッセイ14を使用4.細胞毒性分析<ol><li>不完全な透明な96ウェルプレートにPMAの40 / mlのを補充したRPMI中でTHP-1細胞に分化します。ウェル当たりミリリットル当たり100万、アリコート100μLの細胞密度を維持します。分化は、5％のCO 2を含む加湿インキュベーター中で37℃で一晩進行させます。 </li><li>ウェルに不完全なRPMIで希釈した化合物を加えるウェルから培地を吸引除去し、RPMI 1640で二回洗って。 3日間インキュベートします。 </li><li> 5mg / mlのストック溶液を作製するためにリン酸緩衝生理食塩水（PBS）100mLにMTT 0.5gを溶かします。離れた光から-20℃で滅菌フィルターとストア、。それは、このソリューションは、新鮮な作るのがベストです。 </li><li> 2.5時間beforE 3日間の潜伏期間の終わりには、各ウェルにMTT溶液25μLを追加し、潜伏期間を完了します。 </li><li> 50％のNを準備し、N個のジメチルホルムアミド（DMF）250mLの脱イオン水とDMFの250ミリリットルを混合することによって。 </li><li>次のようにMTT抽出緩衝液を調製：500mLの瓶にSDSを100gを秤量し、50％のDMF 300 mLを加え。 SDSを溶解させるために、低熱を適用します。純粋な酢酸10mlおよび1MのHCl 12.5 mLを加え。 50％のDMFと500mLのマークに埋めます。抽出緩衝液の最終組成は、50％DMF、20％SDS、2.5％酢酸、2.5％1 M塩酸です。 </li><li>処置期間の終わりに、各ウェルに（任意の結晶を溶解するために45℃に加温し）抽出緩衝液100μLを加えます。混合物を37で一晩インキュベートすることを許可します 5％CO 2を含む加湿インキュベーター内℃です。 570 nmの吸光度を読みます。 注：細胞毒性アッセイは、最良のセル内と並行して行われますウラル画面THP-1細胞の同じバッチと年齢を使用しました。 </li></ol>レサズリンアッセイ3を使用して、ブロス活性分析5. <ol><li> （ - 0.8 OD 600〜0.5）対数中期に7H9ADSTに結核菌を成長させます。 0.01 OD 600に7H9ADSTと文化を希釈します。各ウェルに各希釈した化合物の試験濃度及びアリコート100μLを2倍する7H9ADSTで化合物を希釈します。 </li><li>各ウェルに希釈した細菌懸濁液100μLを移します。プレートを5日間、加湿インキュベーター中、37℃でインキュベートすることを可能にします。脱イオン水および滅菌フィルタ100mLにレサズリン10mgを溶解します。 </li><li>レサズリン溶液を30μLを加え、48時間後に色の変化を監視します。細菌増殖はピンクに青から色変換によって示されます。 注：定量分析もまた530での励起と共に、590nmでの蛍光のいずれかを測定することによって行うことができる - 560nMもしくは570 nmの吸光度と600nmで15。 </li></ol>

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The authors declare no competing financial interests for this work.

本研究の目標は、 結核菌のための人間の細胞内感染モデルを使用して、シンプルでコスト効率のHTS方法を作成することでした。結核は、 結核菌によって肺胞マクロファージの感染によって特徴付けられるヒト疾患です。バイオセーフティ問題に、細菌と宿主細胞の両方の生物学的モデルを含む研究は過去に使用されてきました。しかし、サロゲート細菌および非ヒトモデルの?...

結核菌 、結核（TB）の原因物質は、世界中の罹患率と死亡率の主要な原因です。薬物感受性TBは、6ヶ月の期間にわたって複数の抗生物質を必要とする治療可能な疾患です。治療可能な疾患であるにもかかわらず、結核死亡率は、2015年1における150万と推定されました。過去10年間で、薬剤耐性結核の有病率に対する懸念が高まっています。多剤耐性結核（MDR-TB）は、少なくともイソニアジド（INH）、及びリファンピシン（RMP）、最もMDR-TB株は、このように治療の選択肢を制限する、第二のラインTB薬を選択することも耐性があるに耐性のあるTBのように定義されます。薬剤耐性の効果は、患者のヒト免疫不全ウイルス（HIV）で同時感染を治療するために大きな課題を作成します。 40万患者は世界中で2015 1でHIV関連結核で死亡しました。残念なことに、米国食品医薬品Administ配給は、過去40年間2に、MDR-TB、bedaquilineに対する唯一の新しい結核治療薬を承認しました。より良いと短いTBの治療法を見つけるの進歩が緊急TBとMDR-TBとの戦いに先に滞在するために必要とされています。 伝統的に、TB薬物スクリーニングは、化合物が成長している培養物に添加し、微生物を根絶におけるそれらの有効性は、固体培地上のコロニー形成単位（CFU）をカウントすることによって決定されることにより、増殖培地中でインビトロ増殖条件下で行われます。 CFU数は労働集約的、時間がかかり、かつ高価であるように、様々な間接的な方法は、この問題を軽減するために開発されています。そのような方法は、アラマーブルー生存率アッセイ3、緑色蛍光タンパク質（GFP）またはルシフェラーゼを発現する細菌からの発光5からの蛍光4の決意、および全アデノシン三リン酸の推定（Aを含みますTP）6、7。 典型的なTBは、細菌は肺胞マクロファージ8のファゴソーム内部に存在し、複製肺の結核菌の感染によって特徴付けられます。シンプルな中ブロス表現型の画面は、細胞外病原体に合うこと。しかし、歴史的な観点では、この方法を用いて同定された結核菌に対する化合物は、多くの場合、感染モデルにおける下流の検証ステップの間、期待に応えるに失敗ヒット。私たちは、結核治療薬は、最高の細胞内宿主細胞の感染モデルで実行されていることを提案します。それにも関わらず、細胞内のモデルは、ハイスループットスクリーニング（HTS）の開発に多くの技術および生物学的障壁を保有します。大きなハードルは多くの工程と洗浄の間で-による細胞外細菌の精巧な除去によって例示される、感染プロセスの複雑さです。第二の主要なハードルは、長い時間の再ですquirementsは、通常CFU培養プレート上でカウントすることによって行わ増殖検出として、完了するまでに3週間以上を要するプロセスです。 CFUカウントを交換する1つの解決策は、蛍光細菌との組み合わせで自動化された蛍光顕微鏡によって提供されています。しかし、この解決策は、多くの研究室のための手の届かないところにある初期の設備投資が必要となります。シンプル、低コスト、および疾患関連HTS方法を大幅に創薬プロセスを高めるであろう。 本研究では、細胞内の結核菌に対する化合物の活性を決定するのに適した、迅速、かつ拡張性の高い、まだ経済的、アッセイを提供することを目的とする新しい、モジュラーHTSシステムを報告しています。 （I）細胞内、（ii）の細胞毒性、および（iii）中のブロスアッセイ：このシステムは、三つのモジュールから構成されています。合わせた最終的な結果は、作用の潜在的なモードなどの追加情報と、化合物の特性の包括的な説明を提供します。このSCreeningシステムは、薬物相乗作用9の分析、オートファジー10の刺激、および結核菌 -secreted毒性因子（未発表）の阻害を含む作用機序を標的とする様々な化合物ライブラリー、といくつかのプロジェクトで使用されてきました。アクションの未知のモードの化合物はまた、11を研究されています。この方法の修正版は、細胞内結核菌 11に対して新規な化合物を同定するための一次スクリーニング法としての産業パートナーによって採択されました。

<table><tbody><tr><td>RPMI 1640</td><td>Sigma-Aldrich</td><td>R5886</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>12483020</td><td></td></tr><tr><td>Middlebrook 7H9</td><td>Becton, Dickinson and Company</td><td>271210</td><td></td></tr><tr><td>Tween80</td><td>Fisher Scientific</td><td>T164</td><td></td></tr><tr><td>Albumin, Bovine pH7</td><td>Affymetrix</td><td>10857</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>BP350</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>kanamycin sulfate</td><td>Fisher Scientific</td><td>BP906</td><td></td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M2128</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide (DMF)</td><td>Fisher Scientific</td><td>D131</td><td></td></tr><tr><td>1 M Hydrocholoric acid (HCl)</td><td>Fisher Scientific</td><td>351279212</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>351269</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP166</td><td></td></tr><tr><td>Resazurin</td><td>Alfa Aesar</td><td>B21187</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229</td><td></td></tr><tr><td>THP-1</td><td>American Type Culture Collection</td><td>TIB-202</td><td></td></tr><tr><td>&lt;em&gt;M. tuberculosis&lt;/em&gt; H37Rv</td><td></td><td></td><td></td></tr><tr><td>96-well flat bottom white plate</td><td>Corning</td><td>3917</td><td></td></tr><tr><td>95-well flat bottom clear plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Transparent plate sealer</td><td>Thermo Fisher Scientific</td><td>AB-0580</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Biomate 3</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Biotek</td><td>Epoch</td><td></td></tr><tr><td>luminometer</td><td>Applied Biosystems</td><td>Tropix TR717</td><td></td></tr></tbody></table>

system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

ルシフェラーゼ遺伝子を発現結核菌を用いた高スループットの細胞内スクリーニング 図2Aと表1はルミノメーターによって収集された生データを含む、THP-1細胞内の結核菌に対するTB薬物リファンピシンの濃度の増加の効果を示し、相対発光単位（RLU）で表しました。 図2Aは、</stron...

Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

我々は、細胞内および細胞内のブロス増殖する細菌、ならびに哺乳動物宿主細胞に対する細胞毒性を標的、 結核菌に対する新規化合物を発見するためのモジュラーハイスループットスクリーニングシステムを開発しました。

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

system-for-efficacy-cytotoxicity-screening-inhibitors-targeting

Research

JoVE Journal

Immunology and Infection

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

8.5K ビュー.  University of British Columbia. 我々は、細胞内および細胞内のブロス増殖する細菌、ならびに哺乳動物宿主細胞に対する細胞毒性を標的、 結核菌に対する新規化合物を発見するためのモジュラーハイスループットスクリーニングシステムを開発しました。 

ビデオ: System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

のサンプル調製<em&gt;結核菌</em核磁気共鳴代謝研究のための&gt;抽出

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

免疫・感染学

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

高スループット/高含有スクリーニングに適応した細胞内マイコバクテリアの定量化のための顕微鏡表現型アッセイ

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

マイコバクテリアの半定量的、ミディアムスループット列挙のためのチャコール寒天レサズリンアッセイの可視化

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高スループット、リアルタイム、細胞内抗菌活性のデュアル読み出しテストおよび真核細胞の細胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性<em&gt;結核菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

結核の宿主向け治療の前臨床試験に使用する自動培養システム

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

<meta charset="utf8"></head><body>デュアルレポーターを用いた Mycobacterium abscessus のハイスループットスクリーニングアッセイ

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

<meta charset="utf8"></head><body>著者スポットライト: M. abscessus 治療の効率的な発見のためのスケーラブルな薬物スクリーニングプロトコル

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Micro-CFU法による結核判定の迅速化

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

著者スポットライト:結核ワクチンの有効性と抗原提示分析の効率的な評価のためのCFU決定の最適化 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

結核ワクチンの有効性評価のためのマイクロコロニー形成ユニットアッセイ

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム

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高スループット/高含有スクリーニングに適応した細胞内マイコバクテリアの定量化のための顕微鏡表現型アッセイ

マイコバクテリアの半定量的、ミディアムスループット列挙のためのチャコール寒天レサズリンアッセイの可視化

高スループット、リアルタイム、細胞内抗菌活性のデュアル読み出しテストおよび真核細胞の細胞毒性

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性

結核の宿主向け治療の前臨床試験に使用する自動培養システム

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

結核ワクチンの有効性評価のためのマイクロコロニー形成ユニットアッセイ