Name: a high-throughput, high-content, liquid-based c. elegans pathosystem
Uploaded: 2018-07-01T15:00:00
Description: Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

Denne studien ble støttet av kreftforebygging og Research Institute of Texas (CPRIT) Award RR150044, Welch Foundation Research Grant C-1930, og den nasjonale institutter for helse K22 AI110552 til NVK. Fond ikke hadde noen rolle i studien design, innsamling og analyse, beslutningen om å publisere eller utarbeidelse av manuskriptet.

Forsiktig: P. aeruginosa og E. faecalis er grad 2 patogener, og riktige sikkerhetsforanstaltninger må tas for å hindre utilsiktet infeksjon og for å redusere forurensning av overflater. Alle medier og materialer som kommer i kontakt med patogener må bli sterilisert og/eller forkastet. Ytterligere retningslinjene er tilgjengelig fra CDC publikasjonen biosikkerhet mikrobiologisk og biomedisinsk Laboratories (BMBL), 5th edition.
1. forberedelse og vedlikehold av P. aerugin...

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I., 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-throughput+screen+for+novel+antimicrobials+using+a+whole+animal+infection+model.">High-throughput screen for novel antimicrobials using a whole animal infection model.</a> ACS Chemical Biology. 4 (7), 527-533 (2009).</li><li>Rajamuthiah, R., 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=Whole+animal+automated+platform+for+drug+discovery+against+multi-drug+resistant+Staphylococcus+aureus.">Whole animal automated platform for drug discovery against multi-drug resistant Staphylococcus aureus.</a> PLoS One. 9 (2), e89189 (2014).</li><li>Breger, 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=Antifungal+chemical+compounds+identified+using+a+C.+elegans+pathogenicity+assay.">Antifungal chemical compounds identified using a C. elegans pathogenicity assay.</a> PLoS Pathogens. 3 (2), e18 (2007).</li><li>Garvis, 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=Caenorhabditis+elegans+semi-automated+liquid+screen+reveals+a+specialized+role+for+the+chemotaxis+gene+cheB2+in+Pseudomonas+aeruginosa+virulence.">Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis gene cheB2 in Pseudomonas aeruginosa virulence.</a> PLoS Pathogens. 5 (8), e1000540 (2009).</li><li>Zhou, Y. 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=An+efficient+and+novel+screening+model+for+assessing+the+bioactivity+of+extracts+against+multidrug-resistant+Pseudomonas+aeruginosa+using+Caenorhabditis+elegans.">An efficient and novel screening model for assessing the bioactivity of extracts against multidrug-resistant Pseudomonas aeruginosa using Caenorhabditis elegans.</a> Bioscience, Biotechnology, and Biochemistry. 75 (9), 1746-1751 (2011).</li><li>Leung, C. K., Deonarine, A., Strange, K., Choe, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+biosensing+with+fluorescent+C.+elegans+strains.">High-throughput screening and biosensing with fluorescent C. elegans strains.</a> Journal of Visual Experiments. (51), (2011).</li><li>Kirienko, N. V., Ausubel, F. M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+confers+resistance+to+siderophore-mediated+killing+by+Pseudomonas+aeruginosa.">Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (6), 1821-1826 (2015).</li><li>Kirienko, N. V., Cezairliyan, B. O., Ausubel, F. M., Powell, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+PA14+pathogenesis+in+Caenorhabditis+elegans.">Pseudomonas aeruginosa PA14 pathogenesis in Caenorhabditis elegans.</a> Methods in Molecular Biology. 1149, 653-669 (2014).</li><li>Kang, D., Kirienko, D. R., Webster, P., Fisher, A. L., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyoverdine,+a+siderophore+from+Pseudomonas+aeruginosa,+translocates+into+C.+elegans,+removes+iron,+and+activates+a+distinct+host+response.">Pyoverdine, a siderophore from Pseudomonas aeruginosa, translocates into C. elegans, removes iron, and activates a distinct host response.</a> Virulence. , 1-41 (2018).</li><li>Garsin, D. 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=Long-lived+C.+elegans+daf-2+mutants+are+resistant+to+bacterial+pathogens.">Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens.</a> Science. 300 (5627), 1921 (2003).</li><li>Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M., Troemel, E. 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Denne analysen (eller lignende analyser der andre patogener ut P. aeruginosa eller E. faecalis) er nyttig for ulike formål, herunder medisiner. Det er også nyttig for å ta opp grunnleggende biologisk spørsmål, for eksempel identifisere virulens faktorer, utviklingen av vert forsvar trasé, og bestemme regulatoriske maskineriet involvert i interaksjonen som vert-patogen.
Selv om P. aeruginosa flytende drapet analysen er robust, finnes det flere punkter hvor forsi...

Identifisering og utviklingen av effektive, bredspektret antibiotika, nå nesten et århundre siden, førte til et vannskille øyeblikk i folkehelsen der det var en utbredt tro at smittsomme sykdommer ville være en svøpe fortiden. I noen få korte tiår begynte optimismen å avta, som patogen etter patogen utviklet resistens som begrenset disse en gang mirakuløse behandlinger. For noen tid syntes våpenkappløp mellom drug discovery innsats og patogener balansert. Men har misbruk av poesi aksent nylig kulminerte i fremveksten av pan-resistente stammer av Klebsiella pneumoniae, Acinetobacter baumanii, Serratia marcescensog P. aeruginosa1, 2,3,4.
P. aeruginosa er en opportunistisk, gram negativ, multi-Host patogen som er en alvorlig trussel for pasienter med alvorlige forbrenninger, som er nedsatt immunforsvar, eller har cystisk fibrose. Det er også stadig identifisert som en utløsende agent i alvorlig nosokomiale infeksjoner, spesielt på grunn av pågående oppkjøpet av resistensutvikling. For å begynne å ta denne trusselen, har vi brukt den veldokumenterte C. elegans-P. aeruginosa infeksjon system5. Våre lab har utnyttet dette systemet for å utvikle en vannbasert, høy gjennomstrømning og høyt innhold screening plattform for å identifisere romanen forbindelser som begrenser patogen å drepe vert6. Intriguingly, synes disse forbindelsene å tilhøre minst tre generelle kategorier, inkludert poesi aksent7 og virulens hemmere8. Andre høyt innhold drug discovery analyser i C. elegans er rapportert for Mycobacterium tuberculosum, Chlamydia trachomatis, blodlegemer, Listeria monocytogenes, Francisella tularensis, Staphylococcus aureus, Candida albicans, og Enterococcus faecalis, blant annet9,10,11,12,13,14,15,16. Disse typer analyser har flere anerkjente fordeler, for eksempel begrense falske positive treff som kan være giftig for både verten og patogen, økt sannsynlighet for biotilgjengelighet sammenlignet med en kjemisk skjerm, og evnen til å identifisere treff utover bare begrense mikrobiell vekst, som anti-virulents, immun stimulerende molekyler eller forbindelser som ellers vipper vert-patogen samhandlingen for den tidligere balansen. I tillegg er forbindelser i disse skjermene ofte effektive i pattedyr verter.
Det er verdt å merke seg at minst to andre analyser17,18 er tilgjengelig til å utføre høy gjennomstrømming skjermer i C. elegans i væske. Hver av disse analyser er imidlertid en endring som lar prototypiske intestinal kolonisering analysen, kjent som sakte-drapet, utføres i væske, øke gjennomstrømming og gir forbindelser til screenes lettere. Forsiktig karakterisering har definitivt vist at mekanismer for bakteriell virulens er forskjellige mellom disse analyser og våre vannbasert skjermen7. Siden begge typer virulens er observert i pattedyr systemer, er det viktig å vurdere hvilke virulens determinant er mest relevante for den eksperimentator interesser før analysen valget.
Her viser vi en optimalisert versjon av den vannbasert C. elegans-P. aeruginosa analysen. Vi rapporterer også tilpasningen av vår vannbasert analysen metode å imøtekomme gram-positive bakteriell patogen Enterococcus faecalis. Som P. aeruginosa, er E. faecalis stadig identifisert som en alvorlig nosocomial trussel med en voksende armament resistensutvikling veier1. Selv om en tidligere metode for høy gjennomstrømming screening av E. faecalis finnes14, krever det preinfection med patogen, som kompliserer prosedyren og øker sannsynligheten for forurensende utstyr som COPAS FlowSort. Våre protokollen eliminerer behovet for pre-smitte, forbedre sikkerhet profilen. Til slutt, vi rapporterer et middel som en av disse analyser kan kombineres med mating RNAi, tillater brukeren å søke for vert faktorer som spiller en rolle i etableringen av, eller resistens mot infeksjon.

<table>
	<tbody>
		<tr>
			<td>COPAS FP BioSorter</td>
			<td>Union Biometrica</td>
			<td></td>
			<td>Large object flow cytometer/worm sorter</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EL406 Washer Dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron Pro</td>
			<td>Infors HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 Deep-Well RB Block</td>
			<td>Thermo Fisher Scientific</td>
			<td>CS15124</td>
			<td></td>
		</tr>
		<tr>
			<td>384-Well plate</td>
			<td>Greiner Bio-One</td>
			<td>MPG-781091</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Media (NGM)</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) plates</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) media</td>
			<td></td>
			<td></td>
			<td>Amount per liter:&#160; 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>USBiological Life Sciences</td>
			<td>L1520</td>
			<td></td>
		</tr>
		<tr>
			<td>Brian Heart Infusion broth (BHI)</td>
			<td>Research Products International Corp</td>
			<td>50-488-526</td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Bleach Solution</td>
			<td></td>
			<td></td>
			<td>Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water</td>
		</tr>
		<tr>
			<td>S Basal</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 5.85 grams NaCl, 6 grams KH2PO4, 1 gram K2HPO4, and 1 Liter of milli-Q water</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>USBiological Life Sciences</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>USBiological Life Sciences</td>
			<td>S5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>USBiological Life Sciences</td>
			<td>P3300</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>USBiological Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffer</td>
			<td></td>
			<td></td>
			<td>amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Acros Organics</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>USBiological Life Sciences</td>
			<td>P5100</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Sodium Hypochlorite Solution</td>
			<td>BICCA</td>
			<td>7495.5-32</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution</td>
			<td>Fisher Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-easy</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Nucleic Acid Stain</td>
			<td>Fisher Scientific</td>
			<td>S11368</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P. aeruginosa (PA14)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. faecalis(OG1RF)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli superfood (OP50)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAi expressing bacteria (HT115)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput, high-content, liquid-based c. elegans pathosystem

Antall nye stoffer identifisert av tradisjonelle i vitro skjermer har svekket, redusere suksessen til denne tilnærmingen i letingen etter nye våpen mot flere resistens. Dette har ført til konklusjonen at forskerne ikke trenger bare å finne nye stoffer, men må også utvikle nye måter å finne dem. Blant de mest lovende kandidaten metoder er hele-organisme, i vivo søk at bruk høy gjennomstrømning og fenotypiske readouts og vert som spenner fra Caenorhabditis elegans til Danio rerio. Disse vertene har flere kraftige fordeler, inkludert dramatiske reduksjoner i falske positive treff, som forbindelser som er giftige for vert og/eller biounavailable utelates vanligvis første skjermen før kostbare følge opp.
Her viser vi hvordan våre analysen er brukt til å forhøre vert variasjon i den godt dokumentert C. elegans,Pseudomonas aeruginosa flytende drap pathosystem. Vi viser også flere utvidelser av denne godt jobbet ut teknikken. For eksempel kan vi utføre høy gjennomstrømming genetisk skjermer bruker RNAi i 24 - eller 96-brønnen plate formater spørringen vert faktorer i denne verten-patogen samhandlingen. Bruker denne analysen, kan hele genomet skjermer gjennomføres på bare noen få måneder, som kan dramatisk forenkler oppgaven med å identifisere narkotika mål, potensielt uten behov for arbeidskrevende biokjemiske rensing tilnærminger.
Vi rapporterer også her en variant av vår metode som erstatter gram-positive bakterien Enterococcus faecalis for gram-negative patogen P. aeruginosa. Mye som er tilfellet for P. aeruginosa, er drepe av E. faecalis tidsavhengige. I motsetning til tidligere C. elegans-E. faecalis analyser, analysen vår E. faecalis ikke krever preinfection, forbedre sin sikkerhet profil og redusere sjansene for forurensende væske-håndtering av utstyr. Analysen er svært robust, viser ~ 95% dødelighet 96 h innlegget infeksjon.

Viktige parametere for analysen ytelse
En riktig forståelse av biologi underliggende denne analysen er nødvendig for feilsøking og optimalisering analysen. Derfor, vi viser først til flere viktige papirer Klargjørende mekanismer av patogenesen av P. aeruginosa-mediert drap i flytende7,20. Forutsatt at fremgangsmåten ovenfor følges (se <strong cla...

Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Her beskriver vi en protokoll som er tilpasningsdyktige, hele vert, høyt innhold screeningsverktøy som kan brukes til å studere vert-patogen interaksjoner og brukes for medisiner.

En høy gjennomstrømning, høyt innhold og vannbasert C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Research

JoVE Journal

Immunology and Infection

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Tilrettelegging Drug Discovery: En automatisert høyt innhold Betennelse Assay i sebrafisk

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Automatisert Separering av<em&gt; C. elegans</em&gt; Trinnløs kolonisert av en bakteriell Patogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

En mikroskopisk fenotypisk analyse for kvantifisering av intracellulær mykobakterier tilpasset høygjennomstrømning / screening av høyt innhold

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Høy gjennomstrømming analysen til sin fenotype<em&gt; Salmonella ente</em&gt; Typhimurium Association, Invasion, og Replication i makrofager

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Høy gjennomstrømming Kvantitativ Real-time RT-PCR-analyse for fastsetting Expression Profiler av type I og III Interferon Subtyper

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Etablering av en High-throughput Oppsett for screening små molekyler som modulerer c-di-GMP signalering i<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Høy throughput Påvisning av respiratoriske patogener i dyr eksemplarer av nanoskala PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Cell-free biokjemiske Fluorometric enzymatisk analysen for høy gjennomstrømming-måling av Lipid Peroxidation in høy tetthet Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automatisert atferdsdata analyse av store C. elegans bestander bruke et bredt felt-of-view sporing Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

En høy gjennomstrømming Shigella-spesifikke bakteriedrepende analysen

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...