Name: a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis
Uploaded: 2017-03-24T15:00:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

注意:作为结核分枝杆菌 MC 2 6206是无毒菌株17，18，在该协议的所有工作可在生物安全等级2设施（BSL-2）来进行。 1.培养条件的绿色荧光蛋白表达结核分枝杆菌 MC 2 6206（MTB-GFP） 注: 结核分枝杆菌H37Rv衍生营养缺陷型菌株MC 2 6206（ΔpanCD，ΔleuCD）与表达质粒pMN437 GFP的是整个协议16使用转化。它是可能与非表达GFP的野生型菌株，以使应变为研究人员更容易访问性取代的MTB-GFP菌株。但是，GFP的表达是希望的，以使吞噬作用和形成的Mtb /巨噬细胞的聚集体的视觉确认。对于LON摹长期储存， 结核杆菌 -GFP在-80被冻结°，辅以20％甘油完整7H9媒体（步骤1.1中所述）C。 <ol><li>通过用10％的Middlebrook OADC，0.02％四丁酚醛，24微克/毫升D-泛酸，50微克/毫升L-亮氨酸和50微克/毫升潮霉素B.补充7H9培养基制备完整的Mtb -GFP介质（7H9-C）的</li><li>解冻的Mtb -GFP一小瓶，并在一个30毫升正方形底部PETG瓶中加入10毫升7H9-C的。 </li><li>孵育在37℃下在旋转振荡器（50rpm下）。以光密度测量（OD 600）每隔几天，直到OD 600达到1.0（约5-8天）。 </li><li>根据需要保持的OD 0.2-1.0之间600稀释并传代培养。 </li><li>感染中，使用的Mtb-GFP的既定培养对数生长期（OD的0.6-1 600）内。为了避免块状文化，超声处理所述的Mtb培养瓶在水浴（130瓦; 3×5秒脉冲）ONCe或每周两次</li></ol> 2.培养条件 THP-1细胞<ol><li>用10％的热灭活胎牛血清，2mM的L-谷氨酰胺和10mM HEPES的补充的RPMI 1640培养基中制备完整的THP-1细胞培养基（RPMI-C）。 </li><li>孵育在T-75烧瓶中的细胞在37℃在5％CO 2的潮湿气氛中。 </li><li>使用血球计数细胞或流式细胞隔日和保持THP-1细胞在0.1至60万每毫升的密度。 </li></ol> 3.感染议定书生成山地车 /巨噬细胞聚集结构<ol><li> THP-1细胞制剂（每96孔板） <ol><li>离心机7×10 6 THP-1细胞在250×g离心5分钟。悬浮在7毫升的RPMI-C。 </li></ol></li><li> MTB-GFP准备 <ol><li>离心机2.8×10 8 - GFP 结核杆菌 3,200 XG 5分钟在吊桶式离心机ù唱的OD 600 1.0 = 3×10 8个细菌/ mL的转换。 </li><li>用RPMI-C和离心机洗一次，如步骤3.2.1。 </li><li>悬浮在7毫升RPMI-C，涡旋10秒。 </li></ol></li><li> 感染 注:参见图1模板。 <ol><li>通过在排A和H和列加入无菌水200μL的1和12用于水轮缘，以防止培养基的蒸发制备的96孔板中。 </li><li> 200μLRPMI-C加入了后台控制（空白）2列（B2至G2）。 </li><li>感染，添加的Mtb-GFP的悬浮液（步骤3.2），以THP-1细胞悬浮液（步骤3.1）和吹打混匀。最终THP-1细胞密度为5×10 5个每毫升和感染的对应多样性是40。 </li><li>倒入THP-1 / 结核杆菌 -GFP悬浮到25mL的贮存器。 </li><li>添加（通过G11 B3）THP-1 / 结核杆菌 - GFP停牌所有剩余的96口井的200微升全光照GA多道移液器。 注意:如果使用多个96孔板工作，定期重新悬浮剩余THP-1 / 结核杆菌悬浮液在贮存以确保均匀混合物加入到每个孔中。 </li><li>在37℃下孵育，用5％CO 2中7-10天。 </li><li>通过缓慢除去100改变介质每2天微升用过的培养基从每个的顶部孔，轻轻加入使用多通道移液管100微升预热的RPMI-C。不重悬井和扰乱在孔的底部的MTB /巨噬细胞的聚集体。 </li><li>用肉眼检验由每日荧光显微镜（4-10X目标）的孔中，服用的Mtb /巨噬细胞的聚集体的尺寸的纸币。 7-10天， 结核杆菌 /巨噬细胞的聚集体应足够大（参照图2，以供参考）下进行药效试验（第4节）。 </li><li>如果需要的话，使用自动细胞成像系统中的96孔的捕获图像嵌合与明场和GFP过滤器设置文件山地车 /巨噬细胞聚集形成。 注意:如果用户没有访问到自动成像系统，可以使用带GFP和明场能力的任何适当的显微镜可采取代表井的图像。 </li></ol></li></ol> 4.生长抑制试验，以评估药物疗效对结核杆菌从山地车 /巨噬细胞聚集派生<ol><li> 药物制剂（对于两种药物一式三份的条件） 注:参见图1A模板。 <ol><li>在一个独立的96孔板，通过对G10添加7H9-C媒体125微升到B2。 </li><li>制备两种药物以两倍所需的最高终浓度在1mL 7H9-C的占在步骤4.2.4稀释。 </li><li>添加每种药品的250微升至井B11-C11-D11和E11-F11-G11，分别为一式三份的治疗。 </li><li>使用多通道移液管，串联稀尤特试验药物通过移动从B11-G11 125微升到B10-G10两折。通过在每个步骤吹打5次混合。 </li><li>继续往前，从列125微升跨越板柱（从右到左），和第4列后停止。 </li><li>混合列4后，弃去125微升到废物容器中。列2和3不包含任何药物，以允许使用作为背景（空白）和正增长的控制。 注:此外，遵循模板图1B用于测试药品库。每个96孔板可在单一浓度容纳58的药物。制备使用双所需的终浓度，因为它会在半在步骤4.2.4稀释这种药物板。 </li></ol></li><li> 药物治疗: <ol><li>检索包含的Mtb的96孔板感染的巨噬细胞（ 结核杆菌 /巨噬细胞的聚集体）。 </li><li>小心倒出所有相关孔（通过G11 B2）与多通道移液管。在一个两步骤的方式执行这样的:先除去150μL的不倾斜的板，然后通过倾斜板和插入枪头到井的底部边缘去除剩余的介质（〜50μl）。作为结核杆菌 /巨噬细胞聚集体粘附到井的底部，没有任何材料应丢失。 </li><li>轻轻添加100μL的7H9-C媒体来含有的Mtb /巨噬细胞的聚集体板的所有相关的孔中（通过G11 B2）。 </li><li>使用多通道移液管，从含有96孔板（步骤4.1），以感染板的相应孔中的药物转移100μL。 </li><li>在密封袋的地方，并在37°C孵育3天。 </li></ol></li></ol> 5.药物疗效的量化使用刃天青酶标分析<ol><li>制备刃天青以0.8毫克的终浓度储备溶液/ mL的H 2 O通过0.22微米孔径的PVDF膜进行灭菌过滤。</li><li>制备刃天青通过在2混合刃天青原液，H 2 O和吐温80（H中2 O 20％溶液）工作溶液:1:1的比例。最终浓度是0.4毫克/毫升刃天青和5％吐温80。 </li><li>使用板阅读器，设置一个程序在530nm激发和590nm发射波长，在37℃，每30分钟24小时读取荧光。预暖板读取器至37℃。 </li><li>加入20微升的刃天青工作溶液到使用多通道移液管药物处理板（步骤4.11）的所有相关的孔（B2至G11）。 </li><li>放置在板读数器板和启动该程序，在步骤5.3设置。 注意:为方便高容量测定板的处理中，它是足够的开发在37℃培养箱的测定和执行单荧光读取每隔24小时。这也适用于不具有与动能和孵化能力访问酶标仪谁用户。 </li></ol>

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The authors have nothing to disclose.

这里，我们已经详细适于药物功效测试的替代的Mtb感染模型说明。该模型考虑到应在早期结核病药物开发过程被给予更多的考虑的两个关键因素:生理相关障碍药物渗透和感染期间的Mtb的代谢变化的存在。虽然我们先前已经显示了感染模型的好处，并提出扩大感染吞吐量兼容性16的可能性，这里，我们证明，这是通过直接调整系统到96孔板形式确实可以实现的。产生<e...

<html>结核病（TB），尽管依然抗结核化疗方案超过40年1可用性一个严重的全球健康威胁。这是部分原因是由于使用多种药物组合为6个月以上长期治疗期的要求，从而导致病人不遵守2。耐药结核病近年来的出现进一步加剧的一个领域的问题，其中的临床批准的药物研制成功是几乎不存在3。事实上，尽管详尽的抗结核药物研发，只有一个单一的药物已被FDA批准用于在过去40年4临床使用。因此，迫切需要抗结核药物的新世代来解决这个问题。 结核病药物研发的一个关键问题是缺乏从化合物在体外活性在临床上成功转让给疗效="外部参照"&gt; 5，6，7。最初，基于目标方法被用于筛选抗Mtb的药物5，其未能转化为整个细菌细胞。即使使用的Mtb的细胞时，常常使用肉汤生长的培养物，不准确预测在体内 8,9药效进行。这些问题已被确认，并根据包含的Mtb或潜的Mtb的巨噬细胞的药物筛选测定法已成功地建立了8个 ，10个 ，11个 ，12个 。然而，即使是这些更先进的分析不该药物在非血管肺部病变遇到的渗透屏障给予充分的考虑，并在感染部位的坏死灶。确实，即使对于一线TB药利福平，亚最佳剂量已被质疑，由于体内组织和脑脊髓液（CSF） 中不足渗透13，14，15以及功效抗胞内结核杆菌 8,9下降。因此，新的模型和试验，将在早期的领先开发过程中考虑到这些参数，无疑会提高结核病药物研发努力。 为了满足这一需求，我们最近成立了一个廉价，快速，BSL-2兼容的替代感染结核分枝杆菌的药物疗效测试16的模型。产生密集的大巨噬细胞聚集结构内挤满结核杆菌这种感染模型，概括生理学相关蜂窝普及率障碍和产生的巨噬细胞传代<eM&gt;的Mtb具有改变的生理状况相似潜结核杆菌 。从这种感染模型导出结核杆菌与刃天青酶标法（REMA）相结合，以评估药物疗效，其产生的结果与其他细胞感染模型一致并与之共同结核病药物，以达到相对的血药浓度高CSF浓度的报道能力很好的相关性16。 在这里，我们详细描述的Mtb /巨噬细胞聚集体结构的产生，以产生巨噬细胞传代的Mtb适于使用REMA药敏试验。特别是，我们表明这种感染系统如何适合于96孔格式用于与候选抗结核药物通量筛选相容性。

<table><tbody><tr><td>7H9</td><td>BD Difco</td><td>271310</td><td>Follow manufacturer&#039;s recommendations</td></tr><tr><td>Middlebrook OADC</td><td>BD Biosciences</td><td>212351</td><td></td></tr><tr><td>Tyloxapol</td><td>Sigma</td><td>T8761</td><td>Prepare 20% stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>D-Pantothenic acid hemicalcium salt</td><td>Sigma</td><td>P5710</td><td>Prepare 24 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>L-leucine</td><td>MP Biomedicals</td><td>194694</td><td>Prepare 50 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Hygromycin B</td><td>EMD Millipore</td><td>400051</td><td>Prepare 200 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Nalgene Square PETG media bottle</td><td>Thermo Fisher</td><td>2019-0030</td><td></td></tr><tr><td>RPMI 1640 media</td><td>Hyclone</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S12450H</td><td></td></tr><tr><td>L-glutamine</td><td>Corning</td><td>MT25005CI</td><td></td></tr><tr><td>HEPES</td><td>Hyclone</td><td>SH30237.01</td><td></td></tr><tr><td>Cytation 3 plate reader</td><td>Biotek</td><td></td><td>Interchangable with any fluorescent plate reader and microscope</td></tr><tr><td>Gen5 Software</td><td>Biotek</td><td></td><td>Recording and analysis of rezasurin coversion</td></tr><tr><td>Rifampicin&amp;nbsp;</td><td>Fisher Scientific</td><td>BP2679250</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Moxifloxacin Hydrochloride</td><td>Acros Organics</td><td>457960010</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>Prepare 800 &amp;mu;g/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Tween-80</td><td>Fisher Scientific</td><td>T164500</td><td>Prepare 20% stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr></tbody></table>

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.

以确认适应这种感染模型以96孔板格式的鲁棒性，我们在这里根据图中给出的模板检测结核杆菌的药物敏感性，从我们的96孔衍生适于感染模型利福平（RIF）和莫西沙星（磨溪） 1A。我们表明， 结核杆菌 /巨噬细胞聚集体结构的关键该测定的产生可以在96孔板格式（ 图2）被可靠地产生，由此使得可以通过兼容性（ ?...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

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 ...

a-high-throughput-compatible-assay-to-evaluate-drug-efficacy-against

Research

JoVE Journal

Immunology and Infection

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

7.7K 次观看.  University of Alabama at Birmingham. 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. 

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

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

科研

免疫与感染

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

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.

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

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

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 ...

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. ...

Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic strains and genetically engineered cells. Here, an alternative method to detect biochemical interaction between the macrophage microbial sensor SLAMF1 and Mycobacterium tuberculosis is described. Two technical approaches employing flow cytometry and fluorescence microscopy were developed. Total cell protein extracts from human macrophages were generated, then incubated with whole cells of M. tuberculosis (WCMtb)&#160;or M. tuberculosis antigens (Mtb Ags) overnight at 4 &#176;C and finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidyl succinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry was detected with a PE-specific anti-SLAMF1 antibody. The existence of interaction by fluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Ags to poly-D-lysine coated slides, which were incubated with the total protein extract from monocyte-derived macrophages. After cross-linking treatment, SLAMF1 was visualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies. The assays provided a strong biochemical tool to measure pathogen-immunoreceptor interactions, overcoming the difficulties associated with transgenic cell lines and protein gene expression modulation experiments.

This study provides a protocol for evaluating the interaction of Mycobacterium tuberculosis with the SLAMF1 microbial sensor. The assays were conducted on human monocyte-derived macrophages using flow cytometry and fluorescence microscopy. The described tools are relevant for studying interactions between pathogens and immunoreceptors.

用于研究 结核分枝杆菌 与免疫受体 SLAMF1 相互作用的荧光检测

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic ...

高通量兼容检测，以评估对巨噬细胞传代药物疗效

摘要

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此视频中的章节

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

增长结核分枝杆菌生物膜

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫

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

系统抑制剂靶向细胞内的疗效和毒性筛选

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

用于研究结核分枝杆菌与免疫受体 SLAMF1 相互作用的荧光检测

高通量兼容检测，以评估对巨噬细胞传代药物疗效

摘要

探索更多视频

此视频中的章节

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

增长结核分枝杆菌生物膜

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫

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

系统抑制剂靶向细胞内的疗效和毒性筛选

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

用于研究 结核分枝杆菌 与免疫受体 SLAMF1 相互作用的荧光检测

用于研究结核分枝杆菌与免疫受体 SLAMF1 相互作用的荧光检测