Name: system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis
Uploaded: 2017-04-05T14:30:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

This work was supported by BC Lung Association and Mitacs.

1.细菌菌株和生长培养基<ol><li>通过溶解25克牛血清白蛋白的10.0克右旋糖，并在460毫升去离子水4.05克的氯化钠使白蛋白右旋糖和盐储备溶液（ADS）。在4℃下过滤灭菌的ADS和存储。 </li><li>通过添加4.7克7H9粉末和2mL甘油至900毫升的精制水使7H9肉汤。高压釜中在121℃的7H9肉汤10分钟，并允许其继续之前冷却到室温。通过加入100mL的ADS和0.5mL吐温80至900毫升7H9肉汤使7H9ADST。储存于4℃。 </li><li>称取50毫克卡那霉素硫酸盐的和在1mL去离子水中溶解;的最终浓度为50毫克/毫升。过滤灭菌，并在-20℃下存储。添加卡那霉素每1L 7H9ADST的储备溶液的0.5毫升。 注:本媒体应新鲜，所以要根据文化尺寸适当缩放卷。 </li><li>生长在7H 结核分枝杆菌9ADST在稳定培养补充卡那霉素。每天摇动文化和OD600达到1.0之前，避免结块稀释。 注意:用于此方法的发展中的结核分枝杆菌菌株H37Rv的被转化有质粒pJAK2.A 12。 pJAK2.A是基于所述pMV361载体，其允许从HSP60启动子的萤火虫荧光素酶基因的高水平表达，并且可以使用卡那霉素进行选择的整合质粒。 </li></ol> 2. THP-1培养基和维护<ol><li>添加热灭活的胎牛血清（FBS）和5毫升200mM的L-谷氨酰胺至500毫升的RPMI 1640中的50毫升，使不完全的RPMI培养基（约10％FBS和2mM谷氨酰胺）。 </li><li>根据标准协议13保持THP-1细胞培养物。简言之，将生长THP-1细胞在RPMI培养基不完全，同时维持0.2的细胞密度至100万每毫升介质的PAS之间先贤。 </li></ol> 3.高通量筛选细胞内使用荧光素酶表达结核分枝杆菌H37Rv <ol><li>测量在600nm的波长在分光光度计中活跃生长的细菌悬浮液的光密度。使用每毫升0.1 OD 600 = 3×10 7细菌的换算系数计算该细菌密度。 </li><li>吸取出足够的细菌用于感染复数10（MOI）的:1到一个新的离心管中。沉淀在3000×g离心10分钟，吸出液体。添加人血清的50μL到RPMI1640 450μL。缩放到合适的值用于实验的音量。 </li><li>到调理细菌，重悬沉淀以每500μL1×10 8个细菌RPMI1640含有10％人血清的密度。使混合物在37℃下温育30分钟。通过用血细胞计数器和反相microsco计数确定THP-1细胞培养物密度PE。 </li><li>沉淀在无菌离心管将细胞在100×g下和37℃下10分钟。吸出上清液并重新悬浮在RPMI细胞不完全以每毫升1个百万细胞的密度。佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）添加至40毫微克/毫升的最终浓度。 注意:这将是分化组合被称为。 </li><li>结合调理结核分枝杆菌与THP-1分化混合物以10的MOI:1和等分以每孔100μL的最终混合物在96孔平底白色板。定期搅拌混合物，以确保均匀性。允许分化和感染在37℃下在含有5％CO 2的加湿培养箱中进行过夜。 </li><li>用100微升的各RPMI的洗涤各孔两次。添加在不完全RPMI稀释至所需浓度的化合物孵育3天。 </li><li>吸从孔中的培养基。添加荧光素酶测定试剂50μL的各孔中。密封用叔板ransparent粘合剂板密封剂。允许5分钟的温育在22℃，然后在1秒获得光度计读出每孔。 </li></ol> 4.细胞毒性分析使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）测定14 <ol><li>分化的THP-1细胞在RPMI在干净的96孔板中不完全补充有PMA的40毫微克/毫升。维持100万每毫升的细胞密度和每等份孔100μL。允许分化为在37℃下在含有5％CO 2的加湿培养箱中进行过夜。 </li><li>从抽吸孔中的培养基并用RPMI 1640洗涤两次他们不完全添加到孔中在RPMI中稀释的化合物。孵育3天。 </li><li>溶解0.5克MTT在100ml磷酸盐缓冲盐水（PBS），使5 mg / mL的储备溶液。无菌过滤器，并储存在-20℃下，避光;最好是使这种解决方案新鲜。 </li><li> 2.5小时beforE中的3天孵育期结束时，加入MTT溶液25μL至每个孔，并完成潜伏期。 </li><li>制备50％的N，N-二甲基甲酰胺（DMF）通过混合250毫升DMF用250mL去离子水。 </li><li>制备MTT提取缓冲液如下:在500毫升瓶中称取将100g SDS，并添加50％DMF的300mL的。涂抹低热量，使SDS溶解。加入10 mL的纯乙酸和12.5毫升的1M HCl。填充到500毫升标记，用50％DMF;提取缓冲液的最终组成为50％DMF，20％SDS，2.5％乙酸，和2.5％1M盐酸。 </li><li>在治疗期结束时，加入100μL提取缓冲液（加温到45℃以溶解任何晶体）到每个孔中。使混合物在37温育过夜 在含有5％CO 2的潮湿培养箱°℃。阅读吸光度在570纳米。 注:细胞毒性测定最好在平行于一个小区内进行的使用THP-1细胞的相同的分批和年龄ular屏幕。 </li></ol> 5.在肉汤活性分析使用刃天青试验3 <ol><li>生长在7H9ADST 结核分枝杆菌对数中期（〜0.5 - 0.8 OD 600）。稀释文化与7H9ADST 0.01 OD 600。稀释在7H9ADST化合物到每个稀释的化合物的测试浓度和分装100μL的2倍到每个孔中。 </li><li>传送稀释的细菌悬浮液的100μL到每个孔中。将平板在37℃下在湿润的培养箱孵育5天。溶解10mg的刃天青的在100毫升的去离子水和无菌过滤器。 </li><li>加入30μL的刃天青溶液并监测48个小时后的颜色变化;细菌的生长是通过从蓝色的颜色转换到粉红色指示。 注意:也可以通过在含有530激发590nm处测量时，要么的荧光进行定量分析 - 560纳米或在570nm的吸光度和600nm处15。 </li></ol>

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The authors declare no competing financial interests for this work.

这项研究的目标是创建使用结核分枝杆菌的人的细胞内感染模型的简单和具有成本效益的方法HTS。结核病是人类疾病，其特征感染肺泡巨噬细胞的由结核分枝杆菌 。由于生物安全问题，包括细菌和宿主细胞两者的生物模型研究已经在过去使用。然而，已经表明，替代细菌和非人体模型的使用是在药物开发的命中对铅的成功不能预测，表明药物筛选最好用结核分枝杆菌感染<sup c...

结核分枝杆菌 ，结核病（TB）的病原体，是全世界发病率和死亡率的主要原因。药物敏感的TB是可治疗的疾病，需要一段6个月多种抗生素。尽管是一种可治疗的疾病，结核病的死亡率，估计是在2015年1 150万。在过去的10年来，一直在增加了耐药结核分枝杆菌的发病率的担忧。多药耐药TB（MDR-TB）被定义为TB即至少对异烟肼（INH）和利福平（RMP），最MDR-TB菌株也耐选择二线结核病药物，从而限制了治疗选择耐。耐药性的作用创造了治疗患者共感染人类免疫缺陷病毒（HIV），一个更大的挑战;全球40万例，到2015年，死亡1例HIV相关结核。令人失望的是，美国食品和药物ADMINIST比已批准对MDR-TB，bedaquiline只有一个新的抗结核药物，在过去40年中2。在找到更好的和更短的结核病治疗方法的进步，迫切需要以对抗结核病和耐多药结核病的斗争中保持领先地位。 传统上，TB药物屏幕在生长培养基中体外生长条件，从而被添加到生长的培养物的化合物和它们在消灭微生物效力是由固体培养基上计数菌落形成单位（CFU）测定条件下进行。作为CFU计数是劳动密集，耗时且昂贵，各种间接的方法已经被开发来缓解这个问题。这样的方法包括阿尔玛蓝活力试验3，荧光4的从绿色荧光蛋白（GFP），或从荧光素酶表达菌发光5的测定，总三磷酸腺苷的估计（ATP）6,7。 典型TB的特征是肺，其中所述细菌驻留和复制肺泡巨噬细胞8的吞噬体内部的结核分枝杆菌感染。简单的在肉汤表型屏幕可能适合胞外病原体;然而，在历史的角度看，打使用这种方法鉴定的针对结核分枝杆菌的化合物往往不能期间感染模型下游验证步骤，以达到预期。我们建议，抗结核药物在细胞内宿主细胞感染模型表现最好。然而，细胞内的机型具备高通量筛选（HTS）开发的许多技术和生物屏障。一大障碍是感染过程的复杂性，由许多步骤和由精心制作的洗涤除去在中间外细菌的举例说明。第二个主要障碍是漫长的时间再quirements，生长检测，通常由CFU上培养板计数完成，是一个过程，需要超过3周才能完成。一种解决方案，以取代CFU计数已经提供通过自动化荧光显微镜结合荧光的细菌。然而，这种解决方案需要一个初始设备投资是遥不可及的许多研究实验室。一个简单的，低成本的，与疾病相关的HTS方法将大大提高药物发现过程。 在这项研究中，我们报告一个新的，模块化的HTS系统，其目的在于提供适合于确定针对细胞内结核分枝杆菌的化合物的酶活性的快速，高度可扩展的，而经济，测定。该系统由三个模块组成:（ⅰ）细胞内，（ⅱ）细胞毒性，和（iii）中的发酵液测定法。将合并的最终结果提供化合物性质的全面描述，以作为动作的电位模式的附加信息。这SCreening系统已经结合靶向的作用模式不同的化合物库，包括药物的协同作用9的分析多个项目被使用，自噬10的刺激，和结核分枝杆菌的抑制-secreted毒力因子（未发表）。的未知作用模式化合物也已研究了11。该方法的修改版本也通过了我们的工业伙伴作为初筛方法来标识对细胞内结核分枝杆菌 11的新化合物。

<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.

使用结核分枝杆菌表达荧光素酶基因的高通量筛选的细胞内 图2A和表1包含由光度计收集的原始数据，以相对发光单位（RLU）来表示，表现出对结核分枝杆菌的TB药利福平THP-1细胞内的浓度的增加的效果。 图2A是在RLU测定各种浓度的利福平的原始发光的散点图。误差棒表示平均值?...

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

我们已经开发了模块化高通量筛选系统，用于发现新化合物对结核分枝杆菌 ，靶向细胞内和细胞在肉汤中生长的细菌以及细胞毒性对哺乳动物宿主细胞。

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</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 次观看.  University of British Columbia. 我们已经开发了模块化高通量筛选系统，用于发现新化合物对结核分枝杆菌 ，靶向细胞内和细胞在肉汤中生长的细菌以及细胞毒性对哺乳动物宿主细胞。 

视频: 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.

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

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

科研

免疫与感染

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.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

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.

木炭琼脂刃天青分析的可视化分枝杆菌的半定量，中等吞吐量枚举

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.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

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.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</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.

用于宿主靶向治疗结核病临床前检测的自动化培养系统

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>使用双报告基因的 脓肿分枝 杆菌高通量筛选测定

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>作者聚焦:用于高效发现 脓肿分枝杆菌 治疗的可扩展药物筛选方案

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.

分枝杆菌高通量药物筛选的分析开发

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

使用 Micro-CFU 方法加速结核病测定

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.

作者聚焦:优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

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.

用于结核病疫苗功效评估的微集落形成单位测定

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 ...

系统抑制剂靶向细胞内的疗效和毒性筛选

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样品制备

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

木炭琼脂刃天青分析的可视化分枝杆菌的半定量，中等吞吐量枚举

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

高通量兼容检测，以评估对巨噬细胞传代药物疗效

用于宿主靶向治疗结核病临床前检测的自动化培养系统

分枝杆菌高通量药物筛选的分析开发

分枝杆菌高通量药物筛选的分析开发

分枝杆菌高通量药物筛选的分析开发

用于结核病疫苗功效评估的微集落形成单位测定