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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>N,N-Dimethylformamide (DMF)</td>
			<td>Fisher Scientific</td>
			<td>D131</td>
			<td></td>
		</tr>
		<tr>
			<td>1M 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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1</td>
			<td>American Type Culture Collection</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>M. tuberculosis H37Rv</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></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></td>
			<td></td>
			<td></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

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 ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

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 ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

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.

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

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