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. bakteriestamme og vekstmedium <ol><li> Gjør albumin dekstrose og saltløsning (ADS) ved å løse 25 g bovint serum-albumin, 10,0 g dekstrose, og 4,05 g natriumklorid i 460 ml deionisert vann. Filter-sterilisere ADS og oppbevar ved 4 ° C. </li><li> Gjør Brook 7H9 buljong ved tilsetning av 4,7 g Brook 7H9 pulver og 2 ml glycerol og 900 ml renset vann. Autoklaver Brook 7H9 kokes ved 121 ° C i 10 minutter, og la den avkjøles til romtemperatur før man går videre. Gjør 7H9ADST ved å tilsette 100 ml ADS og 0,5 ml...

<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>

Målet med denne studien var å skape en enkel og kostnadseffektiv HTS-metoden ved anvendelse av et humant intracellulær infeksjonsmodell for M. tuberculosis. Tuberkulose er en human sykdom karakterisert ved infeksjon av alveolære makrofager av M. tuberculosis. På grunn av biosikkerhet problemer, har forskning som involverer biologiske modeller av både bakterien og vertsceller blitt brukt i det siste. Imidlertid har det vist seg at bruken av surrogat bakterier og ikke-humane modeller er fattige pre...

Mycobacterium tuberculosis, det forårsakende middel for tuberkulose (TB), er en ledende årsak til morbiditet og dødelighet over hele verden. Medikament-sensitive TB er en behandle sykdom som krever flere antibiotika for en periode på 6 måneder. Til tross for å være en mulig å behandle sykdom, ble TB dødelighet beregnet til å være 1,5 millioner i 2015 1. I de siste 10 år har det vært økende bekymring over forekomsten av legemiddelresistent M. tuberculosis. Multidroge-resistente TB (MDR-TB) er definert som TB som er resistent overfor minst Isoniazid (INH) og Rifampicin (RMP), og de fleste MDR-TB stammer er også motstandsdyktig til å velge andre linjer TB medisiner, og begrenser således behandlingsmuligheter . Virkningene av medikamentresistens skape en større utfordring for behandling av pasienter som er infisert med humant immunsviktvirus (HIV); 400.000 pasienter over hele verden døde av HIV-assosiert TB i 2015 en. Skuffende, USA Food and Drug Administrasjon har godkjent bare en ny TB legemiddel mot MDR-TB, bedaquiline, de siste 40 år 2. Fremskritt i å finne bedre og kortere TB terapi er et presserende behov for å ligge i forkant i kampen mot tuberkulose og MDR-TB. Tradisjonelt er TB narkotika-skjermer utført under in vitro-vekstbetingelser i vekstmedium, hvorved forbindelser tilsettes til en voksende kultur og deres effektivitet i å utrydde mikroorganismer bestemmes ved å telle kolonidannende enheter (CFU) på fast medium. Som CFU teller er arbeidskrevende, tidkrevende og kostbart, har ulike indirekte metoder er utviklet for å lindre dette problemet. Slike metoder omfatter Alamar blå levedyktighet assay 3, bestemmelse av fluorescens 4 fra grønt fluorescerende protein (GFP) eller luminescens 5 fra luciferase-uttrykkende bakterier, og estimering av det totale adenosintrifosfat (ATP) 6, 7. Typisk TB er karakterisert ved en M. tuberculosis-infeksjon i lungen, hvor bakteriene befinner seg og replikere inne i phagosomes av alveolære makrofager 8. Den enkle i-buljong fenotypisk skjerm kan passe ekstracellulære patogener; imidlertid, i de historisk perspektiv, treffer forbindelsene overfor M. tuberculosis er identifisert ved hjelp av denne metoden ofte mislykkes i å leve opp til forventningene på nedstrømsvaliderings trinn i infeksjonsmodeller. Vi foreslår at TB stoffet er best utføres i en intracellulær vertscelle infeksjonsmodell. Ikke desto mindre, intracellulære modeller har mange tekniske og biologiske barrierer for high-throughput screening (HTS) utvikling. Et stort hinder er kompleksiteten av infeksjonsprosessen, eksemplifisert ved en rekke trinn, og den omstendelige fjernelse av ekstracellulære bakterier ved i mellom vasking. Et annet stort hinder er den lange tids remelsene, som vekst deteksjon, normalt ved å CFU telling på dyrkingsplater, er en prosess som tar over 3 uker å fullføre. En løsning for å erstatte CFU tellingene har blitt gitt av automatisert fluorescens mikroskopi i kombinasjon med fluoriserende bakterier. Dette krever imidlertid løsningen en innledende utstyr investering som er utenfor rekkevidde for mange forskningslaboratorier. En enkel, billig, og sykdomsrelevant HTS-metoden i stor grad vil forbedre drug discovery prosessen. I denne studien, rapporterer vi en ny, modul HTS system som tar sikte på å gi en hurtig, og skalerbar, men økonomisk, analyse egnet for å bestemme aktiviteten av forbindelsene overfor intracellulær M. tuberculosis. Dette system er sammensatt av tre moduler: (i) intracellulære, (ii) cytotoksisitet, og (III) i-buljong analyser. Den kombinerte endelige resultatet gir en fullstendig beskrivelse av de sammensatte egenskaper, sammen med ytterligere informasjon med hensyn til potensielle virkningsmåte. dette screening system har vært brukt i flere prosjekter med ulike sammensatte biblioteker som er rettet mot virkningsmåte, inkludert analyse av stoffet synergi 9, stimulering av autophagy 10, og inhibering av M. tuberculosis -secreted virulens faktor (upublisert). Forbindelser med ukjent virkningsmekanisme har også blitt studert 11. En modifisert versjon av denne metode ble også tatt i bruk av vår industripartner som den primære undersøkelsesmetode for å identifisere nye forbindelser for intracellulær M. tuberculosis 11.

<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 screening ved hjelp av intracellulær M. tuberculosis uttrykker luciferasegenet Figur 2A og tabell 1 inneholder de rå data samlet inn av luminometeret, uttrykt i relative enheter (RLU luminescens), som viser virkningen av en økende konsentrasjon av den TB medikament rifampicin på M. tuberculosis inne i THP-1-celler. Figur 2A er et spredningsdi...

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

Vi har utviklet en modulær high-throughput screening system for å oppdage nye forbindelser mot Mycobacterium tuberculosis, rettet mot intracellulær og i-buljong voksende bakterier, så vel som cytotoksisitet mot pattedyrvertscellen.

System for Effekt og Cytotoksisitet Screening av inhibitorer målretting Intracellulær<em&gt; Mycobacterium tuberculosis</em

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

The overall goal of this protocol is to provide multidimensional assessment of how compounds affect M.tuberculosis survival inside human macrophages. This method is centered on a Luciferase-based efficacy assay. When combined with cytotoxicity an MIC assay can provide useful information into modification of HIT compounds in TB drug development.The main advantage of our method is that it is time and labor saving. It is replacing a CFU colony forming unit assay with a Luciferase based assay. Generally individuals new to this technique will struggle with consistency with manual pipetting instead of automation.A consistent pipetting technique is crucial. Grow THP1 cells in RPMI in complete medium. While maintaining a cell density of 0.2 to one million cells per milliliter of medium between passages.Use a spectrophotometer to measure the OD600 of an actively growing bacterial suspension. Then calculate the bacterial density using the conversion factor of 0.1 OD600 equals three times 10 to the seventh bacteria per milliliter. Pipette out sufficient bacteria for an MOI of 10 to one into a new centrifuge tube.Then pellet the bacteria at 3, 000 times G for 10 minutes. To opsonize the bacteria, add 50 microliters of human serum to 450 microliters of RPMI 1640 and use it to resuspend the bacterial pellet at a density of one times 10 to the eighth bacteria per 500 microliters of medium. Allow the mixture to incubate at 37 degrees Celsius for 30 minutes.Next, using a hemocytometer and an inverted microscope, determine the THP1 cell culture density. Then, in sterile centrifuge tubes, pellet the cells at 100 times G in 37 degrees Celsius for 10 minutes. Aspirate the supernatant and resuspend the cells in RPMI in complete medium at a density of one million cells per milliliter.Add PMA to a final concentration of 40 nanograms per milliliter. This is differentiation mix. Combined opsonized M.tuberculosis with THP1 differentiation mix at an MOI of 10 to one.And aliquot the final mix at 100 microliters per well in a 96 well white flat bottom plate. Then allow the differentiation and infection to incubate at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide overnight. The following day, use 100 microliters of RPMI to wash the wells twice.Then add compounds diluted to the desired concentrations and RPMI in complete medium. Incubate the plates for three days. When using multichannel pipettes, remember to practice slow and steady plunger action and always land the pipette tips in the same spot inside each well to minimize disturbance to the THP1 cells.Following the incubation, aspirate the medium from the wells and add 50 microliters of luciferase assay reagent to each well. Then use transparent adhesive plate sealers to seal the plates. Incubate the plates at 22 degrees Celsius for five minutes, then use a luminometer to obtain a readout at one second per well.To differentiate THP1 cells, after preparing differentiation mix as demonstrated earlier in this video, dispense 100 microliters of the differentiation mix into each well of a clear, 96 well plate of cells. Incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide overnight. The following day, aspirate the medium from the wells and use RPMI 1640 to wash them twice.Add 100 microliters of compounds diluted in RPMI incomplete to the wells. Then, incubate the cells for three days. Dissolve 0.5 grams of MTT in 100 milliliters of PBS to make a stock solution of five milligrams per milliliter.Then filter sterilize the solution. Two and a half hours before the end of the three day incubation period, add 25 microliters of MTT solution to each well of cells and complete the incubation period. Prepare 50%NN dimethylformamide or DMF by mixing 250 milliliters of DMF with 250 milliliters of deionized water.Prepare MTT extraction buffer as follows. Lay 100 grams of SDS in a 500 milliliter bottle and add 300 milliliters of 50%DMF. Apply low heat to allow the SDS to dissolve.Add 10 milliliters of pure acetic acid and 12.5 milliliters of one molar hydrochloric acid. Then, use 50%DMF to fill up the bottle to the 500 milliliter mark. At the end of the treatment period, add 100 microliters of extraction buffer to each well.Incubate the mixture at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide overnight. The following day, read the absorbance at 570 nanometers. To carry out a resazurin assay, grow M.tuberculosis in 7H9 ADST to midlock phase.Dilute the culture with 7H9 ADST to an OD600 of 0.01. Use 7H9 ADST to dilute the compounds to two times the testing concentrations. And aliquot 100 microliters of each diluted compound into each well of a clear 96 well plate.Transfer 100 microliters of the diluted bacterial suspension into each well. Incubate the plates at 37 degrees Celsius in a humidified incubator for five days. Dissolve 10 milligrams of resazurin in 100 milliliters of deionized water.And filter sterilize the solution. Add 30 microliters of resazurin solution to the wells. Then incubate the cells for 48 hours.Bacterial growth is indicated by a color conversion from blue to pink. Carry out quantitative analysis according to the text protocol. This scatterplot shows the raw luminescence data measured in relative luminescence units for the treatment of various concentrations of rifampicin on M.tuberculosis and THP1 cells.In this plot, the percent reduction and luminescence in treated wells compared to untreated wells is shown. Rifampicin is capable of reducing 99.9%of the relative light units, or RLUs from intracellular M.tuberculosis at a concentration of 0.1 micrograms per milliliter. Similar to previously published results determined from CFU.In this figure, the cytotoxicity caused by increasing concentrations of G1-1H used in the MTT assay is illustrated. Concentrations of 10 and three micromolar are clearly below the IC 50 cutoff. This figure shows the effects of various concentrations of the antibiotic apramycin on resazurin conversion by M.tuberculosis.The in-broth MIC90 was between 2.5 and five micrograms per milliliter. This is slightly higher than the previously published value of 1.5 micrograms per milliliter. But it is still well within the acceptable range.As demonstrated here, evaporation becomes significant for all experiments with prolonged incubation times. So care must be taken to keep incubators well humidified to avoid inconsistencies. Once mastered, this technique can provide reliable intracellular data in as little as four days.While attempting this procedure it is important to remember to be gentle with THP1 cells, and be consistent with pipetting if attempting without automation. Following this procedure, other methods like high content screening can be used to determine if hit compounds are bacteriostatic or bactericidal. After its development, this protocol paved the way for researchers in the field of tuberculosis drug discovery to rapidly assess compound efficacy against MTB inside human macrophages.Don't forget that working with microbacterium tuberculosis is dangerous. Use precautions and proper equipment when handling this microorganism.

Research

JoVE Journal

Immunology and Infection

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

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

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) ...

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

Vekst av Mycobacterium tuberculosis Biofilm

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

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

En eksperimentell modell for å studere Tuberkulose-Malaria coinfection på Natural Overføring av Mycobacterium tuberculosis Og Plasmodium berghei</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

Høy gjennomstrømming Screening for Bredspektret Chemical hemmere av RNA-virus

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

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrerer en multiresistent<em&gt; Mycobacterium tuberculosis</em&gt; Amplification Mikromatrise

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

Rapid Screening av HIV revers transkriptase og integrasehemmere

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

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

En 3D Menneskelig lungevev Modell for funksjonelle studier på<em&gt; Mycobacterium tuberculosis</em&gt; Infeksjon

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

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

En High-throughput Kompatibel Assay å evaluere Drug Effekt mot macrophage passert<em&gt; Mycobacterium tuberculosis</em

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

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analyse av 18FDG PET/CT Imaging som et verktøy for studerer Mycobacterium tuberculosis infeksjon og behandling i ikke-menneskelige primater

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis i mus med Reporter enzym fluorescens

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