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 undersøgelse blev støttet af kræftforebyggelse og Research Institute of Texas (CPRIT) Award RR150044, Welch Foundation forskning tilskud C-1930, og af de nationale institutter for sundhed K22 AI110552 tildeles NVK. Finansieringskilderne spillede ingen rolle i undersøgelse design, dataindsamling og analyse, beslutningen om at offentliggøre eller forberedelse af manuskriptet.

Forsigtig: P. aeruginosa og E. faecalis er biosikkerhedsniveau 2 patogener, og korrekte sikkerhedsforanstaltninger skal træffes til at forhindre utilsigtet infektion og at minimere forurening af overflader. Alle medier og materialer, der kommer i kontakt med patogener skal være steriliseret og/eller kasseres. Yderligere retningslinjer er tilgængelige fra CDC publikation biosikkerhed i mikrobiologiske og biomedicinske laboratorier (BMBL), 5th edition.
1. forberedelse og vedli...

<ol>
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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. 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Denne analyse (eller lignende assays hvor andre patogener er stedet for P. aeruginosa eller E. faecalis) er nyttig for en række formål, herunder drug discovery. Det er også nyttigt for grundlæggende biologiske spørgsmål, såsom identificering af virulens faktorer, klarlæggelsen af vært forsvar, og fastlægge den lovgivningsmæssige maskiner involveret i vært-patogen interaktion.
P. aeruginosa flydende drab assay er robust, er der flere punkter hvor omhyggeli...

Identifikation og udvikling af effektiv, bredspektret antibiotika, nu næsten et århundrede siden, førte til en skelsættende øjeblik i offentlige sundhed hvor der var en udbredt tro det smitsomme sygdom ville være en svøbe af fortiden. Inden for et par korte årtier begyndte denne optimisme at aftage, som patogen efter patogen udviklet resistensmekanismer, der begrænsede disse engang mirakuløse behandlinger. For nogen tid syntes våbenkapløb mellem drug discovery indsats og patogener afbalanceret. Dog har misbrug af antimikrobielle stoffer for nylig kulminerede i fremkomsten af pan-resistente stammer af Klebsiella pneumoniae, Acinetobacter baumanii, Serratia marcescensog P. aeruginosa1, 2,3,4.
P. aeruginosa er en opportunistisk, gram negative, multi vært patogen, der er en alvorlig trussel mod patienter med alvorlige forbrændinger, dem, der er immunkompromitterede eller har cystisk fibrose. Det er også i stigende grad identificeret som en udløsende agens i svær nosokomielle infektioner, især på grund af dens igangværende erhvervelse af antimikrobiel resistens. Til at begynde at imødegå denne trussel, har vi brugt de veldokumenterede C. elegans-P. aeruginosa infektion system5. Vores lab har leveraged dette system til at udvikle en væskebaseret, høj overførselshastighed, high-indhold screening platform for at identificere nye forbindelser, der begrænser patogenet evne til at dræbe vært6. Intriguingly, synes disse forbindelser at tilhøre mindst tre generelle kategorier, herunder antimikrobielle stoffer7 og virulens hæmmere8. Andre high-indhold drug discovery assays i C. elegans er blevet rapporteret til Mycobacterium tuberculosum, Chlamydia trachomatis, Yersinia pestis, Listeria monocytogenes, Francisella tularensis, Staphylococcus aureus, Candida albicans, og Enterococcus faecalis, blandt andre9,10,11,12,13,14,15,16. Disse typer af assays har flere velkendt fordele, såsom begrænsning af falske positive hits, der kan være giftige for både vært og patogen, øget sandsynlighed for biotilgængelighed i forhold til en kemisk skærm, og evnen til at identificere hits ud over blot begrænse mikrobiel vækst, såsom anti-virulents, immun stimulerende molekyler eller forbindelser, der ellers vipbar balancen i vært-patogen interaktionen til fordel for førstnævnte. Derudover er forbindelser opdaget i disse skærme ofte effektiv i pattedyr værter.
Det er værd at bemærke, at mindst to andre assays17,18 rådighed til at udføre høj overførselshastighed skærme i C. elegans i væske. Hver af disse assays er imidlertid en ændring, der tillader den prototypiske tarm-kolonisering assay, kendt som sen-drab, der skal udføres i flydende, øge gennemløb og giver forbindelser til at være mere let screenet. Omhyggelig karakterisering har endegyldigt viste, at mekanismerne for bakteriel virulens er forskellige mellem disse assays og vores væske-baseret skærm7. Da begge typer af virulens er observeret i pattedyr systemer, er det vigtigt at overveje, hvilke virulens determinant er mest relevante for eksperimentatorens interesser forud for analysen udvalg.
Her viser vi en optimeret version af de væskebaseret C. elegans-P. aeruginosa assay. Vi rapporterer også tilpasning af vores væske-baseret analyse metode til at rumme de gram-positive bakteriel patogen Enterococcus faecalis. Ligesom P. aeruginosaidentificeres E. faecalis stadig som en alvorlig nosokomielle trussel med en voksende bevæbning af antimikrobiel resistens veje1. Selv om en tidligere metode for high throughput screening af E. faecalis findes14, kræver det preinfection med det patogen, der komplicerer proceduren og øger sandsynligheden for kontaminerende udstyr som COPAS FlowSort. Vores protokol eliminerer behovet for før inficeringen, forbedre sikkerhedsprofilen. Endelig, vi rapportere et middel, hvormed enten af disse assays kan kombineres med fodring RNAi, tillade bruger hen til ransage for værten faktorer, der spiller en rolle i oprettelsen af, eller resistens over for infektion.

<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

Antallet af nye stoffer identificeret af traditionelle, in vitro- skærme er aftaget, reducere succes af denne tilgang i søgen efter nye våben til at bekæmpe flere medicinresistens. Dette har ført til den konklusion, at forskere ikke kun behovet for at finde nye lægemidler, men også nødt til at udvikle nye måder at finde dem. Blandt de mest lovende kandidat metoder er hele-organisme, i vivo -undersøgelser, at bruge høj overførselshastighed, fænotypisk readouts og vært der spænder fra Caenorhabditis elegans til Danio rerio. Disse værter har flere stærke fordele, herunder dramatiske nedskæringer i falske positive hits, som forbindelser, der er giftige for værten og/eller biounavailable er typisk faldt i begyndelsesskærmen før dyre følge op.
Her viser vi, hvordan vores assay er blevet brugt til at afhøre vært variation i de veldokumenterede C. elegans—Pseudomonas aeruginosa flydende drab pathosystem. Vi viser også flere udvidelser af denne gennemarbejdet ud teknik. For eksempel, er vi i stand til at udføre høj overførselshastighed genetiske skærme ved hjælp af RNAi i 24 - eller 96-brønd plade formater til forespørgsel vært faktorer i denne vært-patogen interaktion. Ved hjælp af denne analyse, kan samlede genom skærme være afsluttet i kun et par måneder, der drastisk kan forenkle opgaven med at identificere stoffet mål, potentielt uden brug af besværlige biokemiske rensning tilgange.
Vi rapporterer også her en variation af vores metode, der erstatter de gram-positive bakterie Enterococcus faecalis for gram-negative patogenet P. aeruginosa. Meget som det er tilfældet for P. aeruginosa, er drab af E. faecalis tidsafhængig. I modsætning til tidligere C. elegans—E. faecalis assays, vores assay for E. faecalis ikke kræver preinfection, forbedre dens sikkerhedsprofil og reducere chancerne for kontaminerende væske-håndtering udstyr. Analysen er meget robust, viser ~ 95% dødelighed 96 timer efter infektion.

Vigtige parametre for analysen ydeevne
En korrekt forståelse af den biologi, der ligger til grund for denne analyse er nødvendig for fejlfinding og optimering af analysen. I forbindelse henviser vi først til flere vigtige papirer belyse mekanismerne i patogenesen af P. aeruginosa-medieret drab i flydende7,20. Forudsat at trinene ovenfor følges (Se <...

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 protokol, der er en fleksibel, hele vært, high-indhold screeningsværktøj, der kan udnyttes til at studere vært-patogen interaktioner og bruges for drug discovery.

En høj overførselshastighed, High-indhold, væskebaseret 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 ...

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Immunology and Infection

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

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

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