Name: system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis
Uploaded: 2017-04-05T14:30:00
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. Bacterial Strain and Growth Medium
<ol>
	<li>Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of dextrose, and 4.05 g of sodium chloride in 460 mL of deionized water. Filter-sterilize the ADS and store at 4 &#176;C.</li>
	<li>Make 7H9 broth by adding 4.7 g of 7H9 powder and 2 mL of glycerol to 900 mL of purified water. Autoclave the 7H9 broth at 121 &#176;C for 10 min and allow it to cool to room temperature before proceeding. Make 7H9ADST by adding 100 mL of A...

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Global tuberculosis report 2015. , (2016).</li><li>Yajko, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+method+for+determining+MICs+of+antimicrobial+agents+for+Mycobacterium+tuberculosis.">Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis.</a> J Clin Microbiol. 33, 2324-2327 (1995).</li><li>Khare, G., Kumar, P., Tyagi, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+screening-based+identification+of+inhibitors+against+the+intraphagosomal+survival+of+Mycobacterium+tuberculosis.">Whole-cell screening-based identification of inhibitors against the intraphagosomal survival of Mycobacterium tuberculosis.</a> Antimicrob Agents Chemother. 57, 6372-6377 (2013).</li><li>Andreu, N., Fletcher, T., Krishnan, N., Wiles, S., Robertson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+antituberculosis+drug+activity+in+vitro+and+in+macrophages+using+bioluminescence.">Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.</a> J Antimicrob Chemother. 67, 404-414 (2012).</li><li>Mak, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+screen+to+identify+inhibitors+of+ATP+homeostasis+in+non-replicating+Mycobacterium+tuberculosis.">A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis.</a> ACS Chem Biol. 7, 1190-1197 (2012).</li><li>Pethe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+genetic+screen+in+Mycobacterium+tuberculosis+identifies+carbon-source-dependent+growth+inhibitors+devoid+of+in+vivo+efficacy.">A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.</a> Nat Commun. 1, 57 (2010).</li><li>Hmama, Z., Pena-Diaz, S., Joseph, S., Av-Gay, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoevasion+and+immunosuppression+of+the+macrophage+by+Mycobacterium+tuberculosis.">Immunoevasion and immunosuppression of the macrophage by Mycobacterium tuberculosis.</a> Immunol Rev. 264, 220-232 (2015).</li><li>Ramon-Garcia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+drug+combinations+for+tuberculosis+therapy+identified+by+a+novel+high-throughput+screen.">Synergistic drug combinations for tuberculosis therapy identified by a novel high-throughput screen.</a> Antimicrob Agents Chemother. 55, 3861-3869 (2011).</li><li>Lam, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitazoxanide+stimulates+autophagy+and+inhibits+mTORC1+signaling+and+intracellular+proliferation+of+Mycobacterium+tuberculosis.">Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.</a> PLoS Pathog. 8, e1002691 (2012).</li><li>Sorrentino, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Intracellular+Screen+for+New+Compounds+Able+To+Inhibit+Mycobacterium+tuberculosis+Growth+in+Human+Macrophages.">Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages.</a> Antimicrob Agents Chemother. 60, 640-645 (2015).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broad-range+of+recombination+cloning+vectors+in+mycobacteria.">A broad-range of recombination cloning vectors in mycobacteria.</a> Plasmid. 62, 158-165 (2009).</li><li>. THP-1 (ATCC TIB-202) Available from: <a target="_blank" href="https://www.atcc.org/products/all/TIB-202.aspx">https://www.atcc.org/products/all/TIB-202.aspx</a> (2016)</li><li>Hansen, M. B., Nielsen, S. E., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-examination+and+further+development+of+a+precise+and+rapid+dye+method+for+measuring+cell+growth/cell+kill.">Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.</a> J Immunol Methods. 119, 203-210 (1989).</li><li>Invitrogen. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AlamarBlue assay. , (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handbook+of+anti-tuberculosis+agents.+Introduction.">Handbook of anti-tuberculosis agents. Introduction.</a> Tuberculosis (Edinb). 88 (2), 85-86 (2008).</li><li>Riss, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Viability Assays. , (2004).</li><li>Meyer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+efficacy+of+apramycin+in+murine+infection+models.">In vivo efficacy of apramycin in murine infection models.</a> Antimicrob Agents Chemother. 58, 6938-6941 (2014).</li><li>Ballell, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fueling+open-source+drug+discovery:+177+small-molecule+leads+against+tuberculosis.">Fueling open-source drug discovery: 177 small-molecule leads against tuberculosis.</a> ChemMedChem. 8, 313-321 (2013).</li><li>Grundner, C., Cox, J. S., Alber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+tyrosine+phosphatase+PtpA+is+not+required+for+Mycobacterium+tuberculosis+growth+in+mice.">Protein tyrosine phosphatase PtpA is not required for Mycobacterium tuberculosis growth in mice.</a> FEMS Microbiol Lett. 287, 181-184 (2008).</li><li>Wong, D., Bach, H., Sun, J., Hmama, Z., Av-Gay, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+protein+tyrosine+phosphatase+(PtpA)+excludes+host+vacuolar-H+-ATPase+to+inhibit+phagosome+acidification.">Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification.</a> Proc Natl Acad Sci U S A. 108, 19371-19376 (2011).</li><li>Bach, H., Papavinasasundaram, K. G., Wong, D., Hmama, Z., Av-Gay, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+virulence+is+mediated+by+PtpA+dephosphorylation+of+human+vacuolar+protein+sorting+33B.">Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B.</a> Cell Host Microbe. 3, 316-322 (2008).</li><li>Chanput, W., Peters, V., Wichers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Impact of Food Bioactives on Health: in vitro and ex vivo models. , 147-159 (2015).</li><li>Maess, M. B., Wittig, B., Lorkowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+of+human+THP-1+macrophages+by+nucleofection.">Highly efficient transfection of human THP-1 macrophages by nucleofection.</a> J Vis Exp. , e51960 (2014).</li><li>Ferguson, J. S., Weis, J. J., Martin, J. L., Schlesinger, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+protein+C3+binding+to+Mycobacterium+tuberculosis+is+initiated+by+the+classical+pathway+in+human+bronchoalveolar+lavage+fluid.">Complement protein C3 binding to Mycobacterium tuberculosis is initiated by the classical pathway in human bronchoalveolar lavage fluid.</a> Infect Immun. 72, 2564-2573 (2004).</li><li>Andreu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+bioluminescent+reporters+for+use+with+mycobacteria.">Optimisation of bioluminescent reporters for use with mycobacteria.</a> PLoS One. 5, e10777 (2010).</li><li>Queval, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microscopic+phenotypic+assay+for+the+quantification+of+intracellular+mycobacteria+adapted+for+high-throughput/high-content+screening.">A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening.</a> J Vis Exp. , e51114 (2014).</li><li>Christophe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening+identifies+decaprenyl-phosphoribose+2'+epimerase+as+a+target+for+intracellular+antimycobacterial+inhibitors.">High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.</a> PLoS Pathog. 5, e1000645 (2009).</li><li>Pethe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+Q203,+a+potent+clinical+candidate+for+the+treatment+of+tuberculosis.">Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis.</a> Nat Med. 19, 1157-1160 (2013).</li></ol>

The goal of this study was to create a simple and cost-effective HTS method using a human intracellular infection model for M. tuberculosis. Tuberculosis is a human disease characterized by the infection of alveolar macrophages by M. tuberculosis. Due to biosafety issues, research involving biological models of both the bacterium and the host cells has been used in the past. However, it has been shown that the usage of surrogate bacteria and non-human models are poor predictors of hit-to-lead success in...

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. Drug-sensitive TB is a treatable disease that requires multiple antibiotics for a period of 6 months. Despite being a treatable disease, TB mortality was estimated to be 1.5 million in 20151. In the past 10 years, there have been increasing concerns over the prevalence of drug-resistant M. tuberculosis. Multidrug-resistant TB (MDR-TB) is defined as TB that is resistant to at least Isoniazid (INH) and Rifampicin (RMP), and most MDR-TB strains are also resistant to select second-line TB drugs, thus limiting treatment options. The effects of drug resistance create a greater challenge for treating patients co-infected with Human Immunodeficiency Virus (HIV); 400,000 patients worldwide died of HIV-associated TB in 20151. Disappointingly, the United States Food and Drug Administration has approved only one new TB drug against MDR-TB, bedaquiline, in the past 40 years2. Advances in finding better and shorter TB therapies are urgently needed in order to stay ahead in the fight against TB and MDR-TB.
Traditionally, TB drug screens are performed under in vitro growth conditions in growth medium, whereby compounds are added to a growing culture and their effectiveness in eradicating the microorganisms are determined by counting colony forming units (CFU) on solid medium. As CFU counts are labor intensive, time consuming, and costly, various indirect methods have been developed to alleviate this problem. Such methods include the Alamar Blue viability assay3, the determination of fluorescence4 from green fluorescent protein (GFP) or luminescence5 from luciferase-expressing bacteria, and the estimation of total adenosine triphosphate (ATP)6,7.
Typical TB is characterized by an M. tuberculosis infection of the lung, where the bacteria reside and replicate inside the phagosomes of alveolar macrophages8. The simple in-broth phenotypic screen may suit extracellular pathogens; however, in the historical perspective, hit compounds against M. tuberculosis identified using this method often fail to live up to expectations during downstream validation steps in infection models. We propose that TB drug is best performed in an intracellular host cell infection model. Nevertheless, intracellular models possess many technological and biological barriers to high-throughput screening (HTS) development. A big hurdle is the complexity of the infection process, exemplified by numerous steps and the elaborate removal of extracellular bacteria by in-between washing. A second major hurdle is the lengthy time requirements, as growth detection, normally done by CFU counting on culture plates, is a process that takes over 3 weeks to complete. One solution to replace CFU counts has been provided by automated fluorescent microscopy in combination with fluorescent bacteria. However, this solution requires an initial equipment investment that is out of reach for many research labs. A simple, low-cost, and disease-relevant HTS method would greatly enhance the drug discovery process.
In this study, we report a new, modular HTS system that is aimed at providing a rapid, and highly scalable, yet economical, assay suitable for determining the activity of compounds against intracellular M. tuberculosis. This system is composed of three modules: (i) intracellular, (ii) cytotoxicity, and (iii) in-broth assays. The combined final result provides a comprehensive description of the compound properties, with additional information as to the potential mode of action. This screening system has been used in several projects with various compound libraries that target mode of action, including the analysis of drug synergy9, the stimulation of autophagy10, and the inhibition of M. tuberculosis-secreted virulence factor (unpublished). Compounds of unknown mode of action have also been studied11. A modified version of this method was also adopted by our industrial partner as the primary screening method to identify new compounds against intracellular M. tuberculosis11.

<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.

High-throughput intracellular screening using M. tuberculosis expressing the luciferase gene
Figure 2A and Table 1 contain the raw data collected by the luminometer, expressed in relative luminescent units (RLU), showing the effect of an increasing concentration of the TB drug rifampicin on M. tuberculosis inside THP-1 cells. Figure 2A is a scatter plot of the raw...

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 (Video) | JoVE

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.

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 ...

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