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 Vækst Medium <ol><li> Gøre albumin dextrose og salt stamopløsning (ADS) ved at opløse 25 g bovint serumalbumin, 10,0 g dextrose og 4,05 g natriumchlorid i 460 ml deioniseret vand. Filter-sterilisere ADS og opbevares ved 4 ° C. </li><li> Gøre 7H9 bouillon ved tilsætning af 4,7 g af 7H9 pulver og 2 ml af glycerol til 900 ml renset vand. Autoklaver 7H9 bouillon ved 121 ° C i 10 min og lad den afkøle til stuetemperatur, før der fortsættes. Gøre 7H9ADST ved tilsætning af 100 ml ADS og 0,5 ...

<ol>
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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. 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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. 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Målet med denne undersøgelse var at skabe en enkel og omkostningseffektiv HTS fremgangsmåde under anvendelse af en human intracellulær infektion model for M. tuberculosis. Tuberkulose er en human sygdom karakteriseret ved infektion af alveolære makrofager af M. tuberculosis. På grund af biosikkerhed spørgsmål, har forskning med biologiske modeller af både bakterien og værten cellerne blevet anvendt i fortiden. Det har imidlertid vist sig, at brugen af surrogat bakterier og ikke-menneskelige m...

Mycobacterium tuberculosis, det forårsagende middel af tuberkulose (TB), er en førende årsag til sygelighed og dødelighed i hele verden. Medikamentfølsomme TB er en behandlelige sygdom, der kræver flere antibiotika i en periode på 6 måneder. Trods en behandlelige sygdom, blev TB dødelighed anslået til 1,5 millioner i 2015 en. I de seneste 10 år har der været stigende bekymring over forekomsten af resistente M. tuberculosis. Multiresistent tuberkulose (MDR-TB) er defineret som TB der er resistent overfor mindst Isoniazid (INH) og Rifampicin (RMP), og de fleste MDR-TB-stammer er også modstandsdygtige over for vælge second-line TB lægemidler, hvilket begrænser behandlingsmuligheder . Virkningerne af lægemiddelresistens skabe en større udfordring til behandling af patienter med samtidig infektion med humant immundefekt virus (HIV); 400.000 patienter på verdensplan døde af HIV-associeret tuberkulose i 2015 1. Skuffende, USA Food and Drug Administration har godkendt kun én ny TB lægemiddel mod MDR-TB, bedaquiline, i de seneste 40 år 2. Fremskridt i at finde bedre og kortere TB terapier er et presserende behov for at være på forkant i kampen mod tuberkulose og multiresistent tuberkulose. Traditionelt er TB lægemiddelscreeninger udført under in vitro vækstbetingelser i vækstmedium, hvorved forbindelser tilsættes til en voksende kultur og deres effektivitet til udryddelse mikroorganismerne bestemmes ved at tælle kolonidannende enheder (CFU) på fast medium. Da CFU tæller er arbejdskrævende, tidskrævende, og kostbar, har forskellige indirekte metoder blevet udviklet til at afhjælpe dette problem. Sådanne fremgangsmåder indbefatter Alamar Blue levedygtighedsassay 3, bestemmelse af fluorescens 4 fra grønt fluorescerende protein (GFP) eller luminescens 5 fra luciferase-udtrykkende bakterier, og skønnet over den samlede adenosintriphosphat (ATP) 6, 7. Typisk TB er kendetegnet ved en M. tuberculosis-infektion i lungen, hvor bakterierne opholde sig og replikere inde i fagosomer af alveolære makrofager 8. Den enkle i-bouillon fænotypisk skærm kan passe ekstracellulære patogener; men i historisk perspektiv, ramte forbindelser mod M. tuberculosis identificeret ved anvendelse af denne fremgangsmåde ofte undlader at leve op til forventningerne i nedstrøms valideringsrutiner infektionsmodeller. Vi foreslår, at TB medicin er bedst udføres i en intracellulær værtscelle infektion model. Ikke desto mindre er intracellulære modeller besidder mange teknologiske og biologiske barrierer for high-throughput screening (HTS) udvikling. En stor hindring er kompleksiteten af ​​infektionsprocessen, eksemplificeret ved talrige trin og den omfattende fjernelse af ekstracellulære bakterier ved i-mellem vask. En anden stor forhindring er den lange tid retingelserne, som vækst detektion, normalt ved CFU regner dyrkningsplader, er en proces der tager over 3 uger at gennemføre. En løsning til at erstatte CFU tællinger er blevet tilvejebragt ved automatiseret fluorescerende mikroskopi i kombination med fluorescerende bakterier. Men denne løsning kræver en indledende udstyr investering, der er uden for rækkevidde for mange forskningslaboratorier,. En enkel, billig, og sygdom-relevant HTS metode ville i høj grad øge lægemiddelopdagelsesprocessen. I denne undersøgelse rapporterer vi en ny, modulært HTS-system, der har til formål at give en hurtig og yderst skalerbar, men økonomisk, assay egnet til bestemmelse af aktiviteten af forbindelser mod intracellulær M. tuberculosis. Dette system består af tre moduler: (i) intracellulære, (ii) cytotoksicitet, og (iii) i-bouillon assays. Det kombinerede endelige resultat giver en omfattende beskrivelse af de sammensatte egenskaber, med yderligere information med hensyn til den potentielle virkemåde. Denne screening system er blevet anvendt i flere projekter med forskellige forbindelsesbiblioteker, der er målrettet virkemåde, herunder analyse af lægemiddelsynergi 9, stimulering af autophagy 10, og inhiberingen af M. tuberculosis -secreted virulensfaktor (ikke offentliggjort). Forbindelser med ukendt virkningsmekanisme er også blevet undersøgt 11. En modificeret version af denne metode blev også vedtaget af vores industrielle partner som den primære screeningsmetode til at identificere nye forbindelser mod 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 intracellulær screening under anvendelse af M. tuberculosis, der udtrykker luciferasegenet Figur 2A og Tabel 1 indeholder de rå data indsamlet af luminometeret, udtrykt i relative luminescerende enheder (RLU), der viser virkningen af en stigende koncentration af TB lægemiddel rifampicin på M. tuberculosis inde THP-1-celler. Figur 2A er et sca...

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 udviklet en modulær højkapacitetsscreening system til at opdage hidtil ukendte forbindelser mod Mycobacterium tuberculosis, målretning intracellulære og i-bouillon voksende bakterier samt cytotoksicitet mod pattedyrværtscelle.

System til Effekt og cytotoksicitet Screening af inhibitorer Målretning 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 ...

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

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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 eksperimentel model til at studere tuberkulose-Malaria coinfektion på Natural Transmission af 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

High-throughput Screening for Bredspektret Chemical hæmmere af 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

Demonstration af en multiresistent<em&gt; Mycobacterium tuberculosis</em&gt; Forstærkning Microarray

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

Hurtig screening af HIV revers transkriptase og Integrase hæmmere

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 Lung Tissue Model for Funktionelle Undersøgelser om<em&gt; Mycobacterium tuberculosis</em&gt; Infektion

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 at evaluere Drug Effekt mod makrofag passeres<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 af 18FDG PET/CT billeddannelse som et redskab til at studere Mycobacterium tuberculosis infektion 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 tuberkulose 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 ...