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

Denna studie stöddes av cancerprevention och Research Institute of Texas (CPRIT) Award RR150044, Welch stiftelsen forskning Grant C-1930, och av de nationella institut för hälsa K22 AI110552 tilldelas NVK. Finansiärerna hade ingen roll i studiedesign, datainsamling och analys, beslut om att offentliggöra eller beredning av manuskriptet.

Försiktighet: P. aeruginosa och E. faecalis är biosäkerhetsnivå 2 patogener, och korrekta säkerhetsåtgärder vidtas för att förhindra oavsiktlig infektion och för att minimera kontaminering av ytor. Alla media och material som kommer i kontakt med patogener måste vara steriliserade eller kasseras. Ytterligare riktlinjer finns tillgängliga från CDC publikationen biosäkerhet i Microbiological och biomedicinska laboratorier (BMBL), 5: e upplagan.
1. förberedelser och...

<ol>
	<li>Falagas, M. E., 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=Pandrug-resistant+Klebsiella+pneumoniae,+Pseudomonas+aeruginosa+and+Acinetobacter+baumannii+infections:+Characteristics+and+outcome+in+a+series+of+28+patients.">Pandrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii infections: Characteristics and outcome in a series of 28 patients.</a> International Journal of Antimicrobial Agents. 32 (5), 450-454 (2008).</li><li>Hsueh, P. 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=Pandrug-resistant+Acinetobacter+baumannii+causing+nosocomial+infections+in+a+university+hospital,+Taiwan.">Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan.</a> Emerging Infectious Diseases. 8 (8), 827-832 (2002).</li><li>Wang, C. Y., 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=Pandrug-resistant+Pseudomonas+aeruginosa+among+hospitalised+patients:+clinical+features,+risk-factors+and+outcomes.">Pandrug-resistant Pseudomonas aeruginosa among hospitalised patients: clinical features, risk-factors and outcomes.</a> Clinical Microbiology and Infection. 12 (1), 63-68 (2006).</li><li>Yao, Y., 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=Draft+genome+sequences+of+pandrug-resistant+Serratia+marcescens+clinical+isolates+harboring+blaNDM-1.">Draft genome sequences of pandrug-resistant Serratia marcescens clinical isolates harboring blaNDM-1.</a> Genome Announcements. 5 (3), (2017).</li><li>Utari, P. D., Quax, W. J. <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+reveals+novel+Pseudomonas+aeruginosa+virulence+mechanism.">Caenorhabditis elegans reveals novel Pseudomonas aeruginosa virulence mechanism.</a> Trends in Microbiology. 21 (7), 315-316 (2013).</li><li>Conery, A. L., Larkins-Ford, J., Ausubel, F. M., 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=High-throughput+screening+for+novel+anti-infectives+using+a+C.+elegans+pathogenesis+model.">High-throughput screening for novel anti-infectives using a C. elegans pathogenesis model.</a> Current Protocols in Chemical Biology. 6 (1), 25-37 (2014).</li><li>Kirienko, N. V., 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=Pseudomonas+aeruginosa+disrupts+Caenorhabditis+elegans+iron+homeostasis,+causing+a+hypoxic+response+and+death.">Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death.</a> Cell Host &amp; Microbe. 13 (4), 406-416 (2013).</li><li>Kirienko, D. R., Revtovich, A. V., 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=A+high-content,+phenotypic+screen+identifies+fluorouridine+as+an+inhibitor+of+pyoverdine+biosynthesis+and+pseudomonas+aeruginosa+virulence.">A high-content, phenotypic screen identifies fluorouridine as an inhibitor of pyoverdine biosynthesis and pseudomonas aeruginosa virulence.</a> mSphere. 1 (4), (2016).</li><li>Manning, A. 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+high+content+microscopy+assay+to+determine+drug+activity+against+intracellular+Mycobacterium+tuberculosis.">A high content microscopy assay to determine drug activity against intracellular Mycobacterium tuberculosis.</a> Methods. 127, 3-11 (2017).</li><li>Marwaha, 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=N-acylated+derivatives+of+sulfamethoxazole+and+sulfafurazole+inhibit+intracellular+growth+of+Chlamydia+trachomatis.">N-acylated derivatives of sulfamethoxazole and sulfafurazole inhibit intracellular growth of Chlamydia trachomatis.</a> Antimicrobial Agents and Chemotherapy. 58 (5), 2968-2971 (2014).</li><li>Kota, K. P., 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=Integrating+high-content+imaging+and+chemical+genetics+to+probe+host+cellular+pathways+critical+for+Yersinia+pestis+infection.">Integrating high-content imaging and chemical genetics to probe host cellular pathways critical for Yersinia pestis infection.</a> PLoS One. 8 (1), e55167 (2013).</li><li>Arif, 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=Quantification+of+cell+infection+caused+by+Listeria+monocytogenes+invasion.">Quantification of cell infection caused by Listeria monocytogenes invasion.</a> Journal of Biotechnology. 154 (1), 76-83 (2011).</li><li>Jayamani, E., 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=Characterization+of+a+Francisella+tularensis-Caenorhabditis+elegans+pathosystem+for+the+evaluation+of+therapeutic+compounds.">Characterization of a Francisella tularensis-Caenorhabditis elegans pathosystem for the evaluation of therapeutic compounds.</a> Antimicrobial Agents and Chemotherapy. 61 (9), (2017).</li><li>Moy, T. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bZIP+transcription+factor+zip-2+mediates+an+early+response+to+Pseudomonas+aeruginosa+infection+in+Caenorhabditis+elegans.">bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (5), 2153-2158 (2010).</li><li>Tjahjono, E., 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=A+conserved+mitochondrial+surveillance+pathway+is+required+for+defense+against+Pseudomonas+aeruginosa.">A conserved mitochondrial surveillance pathway is required for defense against Pseudomonas aeruginosa.</a> PLoS Genetics. 13 (6), e1006876 (2017).</li><li>Moy, T. 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=Identification+of+novel+antimicrobials+using+a+live-animal+infection+model.">Identification of novel antimicrobials using a live-animal infection model.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (27), 10414-10419 (2006).</li><li>Henderson, S. T., Bonafe, M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-16+protects+the+nematode+Caenorhabditis+elegans+during+food+deprivation.">daf-16 protects the nematode Caenorhabditis elegans during food deprivation.</a> The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 61 (5), 444-460 (2006).</li><li>Beanan, M. J., Strome, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+germ-line+proliferation+mutation+in+C.+elegans.">Characterization of a germ-line proliferation mutation in C. elegans.</a> Development. 116 (3), 755-766 (1992).</li></ol>

Denna assay (eller liknande analyser där andra patogener ersätts för P. aeruginosa eller E. faecalis) är användbar för en mängd ändamål, inklusive läkemedelsutveckling. Det är också användbart för att hantera grundläggande biologiska frågor, såsom att fastställa virulensfaktorer, förtydligandet av värd försvar vägar, och att fastställa rättsliga maskineriet inblandad i värd-patogen interaktionen.
Även om P. aeruginosa flytande dödande analy...

Identifiering och utveckling av effektiva, brett spektrum antibiotika, nu nästan ett sekel sedan, ledde till en vattendelare ögonblick i folkhälsa där det fanns en utbredd tro att smittsam sjukdom skulle vara ett gissel av förflutnan. Inom några korta decennier började denna optimism avta, som patogen efter patogen framkallade resistensmekanismer som begränsade dessa en gång mirakulösa behandlingar. För en tid verkade kapprustningen mellan drug discovery ansträngningar och patogenerna balanserad. Missbruk av antimikrobiella medel har dock nyligen kulminerade i uppkomsten av pan-resistenta stammar av Klebsiella pneumoniae, Acinetobacter baumanii, Serratia marcescensoch P. aeruginosa1, 2,3,4.
P. aeruginosa är en opportunistisk, gram negativ, multi-värd patogen som är ett allvarligt hot mot patienter med svåra brännskador, dem som är immunsupprimerade, eller har cystisk fibros. Det är också alltmer identifierad som en orsakande agens i allvarliga nosokomiala infektioner, särskilt på grund av dess pågående förvärv av antimikrobiell resistens. Till att börja med att ta itu med detta hot har vi använt den väldokumenterade C. elegans-P. aeruginosa infektion system5. Vårt labb har leveraged detta system för att utveckla en vätskebaserade, hög kapacitet, hög-innehåll screening plattform för att identifiera nya föreningar som begränsar sjukdomsalstrande förmåga att döda den värd6. Dessa föreningar verkar spännande, tillhör minst tre allmänna kategorier, inklusive antimikrobiella medel7 och virulens hämmare8. Andra high-innehåll drug discovery analyser i C. elegans har rapporterats för Mycobacterium tuberculosum, Chlamydia trachomatis, Yersinia pestis, Listeria monocytogenes, Francisella tularensis, Staphylococcus aureus, Candida albicans, och Enterococcus faecalis, bland annat9,10,11,12,13,14,15,16. Dessa typer av analyser har flera välkända fördelar, till exempel begränsa falska positiva träffar som kan vara giftigt för både värd och patogen, ökad sannolikhet för biotillgänglighet jämfört med en kemisk skärm och förmågan att identifiera träffar utanför helt enkelt begränsa mikrobiell tillväxt, såsom anti-virulents, immun stimulerande molekyler eller föreningar som annars lutar balansen i värd-patogen interaktionen till förmån för den tidigare. De föreningar som upptäcktes i dessa skärmar är dessutom ofta effektiva i däggdjur värdar.
Det är värt att notera att det finns minst två andra analyser17,18 att utföra hög genomströmning skärmar i C. elegans i vätska. Dock var och en av dessa analyser är en ändring som tillåter prototypiska intestinal-colonization analysen, känd som långsam-dödande, ska utföras i flytande öka genomströmningen och låta föreningar elektronikkedjan lättare. Noggrann karakterisering har slutgiltigt visat att bakteriell virulens mekanismer är olika mellan dessa analyser och vår vätskebaserade skärmen7. Eftersom båda typerna av virulens observeras i däggdjur system, är det viktigt att överväga vilka virulens avgörande är mest relevanta för de experimenter's intressen före assay urval.
Här visar vi en optimerad version av de vätskebaserade C. elegans-P. aeruginosa assay. Vi rapporterar också anpassningen av vår vätskebaserade analys metod att rymma grampositiva bakterie patogenen Enterococcus faecalis. Som P. aeruginosaidentifieras E. faecalis alltmer som ett allvarliga nosokomiala hot med en växande rustningen av antimikrobiell resistens vägar1. Även om det finns14en föregående metod för high-throughput screening av E. faecalis , kräver det preinfection med patogener, vilket försvårar förfarandet och ökar risken för kontamination med utrustning som de COPAS FlowSort. Våra protokoll eliminerar behovet av före infektion, förbättra säkerhetsprofilen. Slutligen, Vi rapporterar ett medel genom vilket antingen av dessa analyser kan kombineras med utfodring RNAi, tillåt förbrukaren till söka för värd faktorer som spelar en roll i etableringen av, eller motstånd mot, 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

Antalet nya läkemedel som identifierats av traditionella, in vitro- skärmar har avtagit, minska framgången av detta tillvägagångssätt i sökandet efter nya vapen att bekämpa flera läkemedelsresistens. Detta har lett till slutsatsen att forskare inte bara behovet av att hitta nya läkemedel, men också behöver utveckla nya sätt att hitta dem. Bland de mest lovande kandidaten metoder är hel-organismen, in-vivo -analyser att använda hög genomströmning, fenotypiska utläsningar och värd som spänner från Caenorhabditis elegans till Danio rerio. Dessa värdar har flera starka fördelar, inklusive dramatiska minskningar falska positiva träffar, som föreningar som är giftiga för de mottagande och/eller biounavailable släpps normalt i den första skärmen, före kostsamma följa upp.
Här visar vi hur vår analys har använts för att förhöra värd variation i den väldokumenterade C. elegans—Pseudomonas aeruginosa flytande döda pathosystem. Vi visar också flera förlängningar av denna väl fungerade ut teknik. Exempelvis kan vi utföra hög genomströmning genetiska skärmar med hjälp av RNAi i 24 - eller 96-bra platta format till fråga värd faktorer i denna värd-patogen interaktion. Med denna analys, kan hela genomet skärmar fyllas i bara några månader, som dramatiskt förenklar uppgiften att identifiera läkemedelsmål, potentiellt utan behov av mödosamma biokemisk rening metoder.
Vi rapporterar också här en variant av vår metod som ersätter grampositiva bakterien Enterococcus faecalis för gramnegativ patogen P. aeruginosa. Mycket som är fallet för P. aeruginosa, är dödandet av E. faecalis tidsberoende. Till skillnad från tidigare C. elegans—E. faecalis analyser, vår analys för E. faecalis inte kräver preinfection, förbättra dess säkerhetsprofil och minska risken för kontamination med vätska-hanteringsutrustning. Analysen är mycket robust, visar ~ 95% dödstalen 96 h post infektion.

Viktiga parametrar för analysens prestanda
En riktig förståelse av biologin bakom denna analys krävs för felsökning och optimering av analysen. Därför hänvisar vi först till flera viktiga papper klarlägga mekanismerna i patogenesen av P. aeruginosa-medierad döda i flytande7,20. Förutsatt att de steg som beskrivs ovan följs (se <strong clas...

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

Här beskriver vi ett protokoll som är en anpassningsbar, hela värd, hög-innehåll screeningverktyg som kan utnyttjas för att studera värd-patogen interaktioner och användas för läkemedelsutveckling.

En hög genomströmning, hög-innehåll, vätskebaserade 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 ...

This method can answer questions in host pathogen interaction and drug discovery, such as the the importance of various virulence factors and the ability of small molecules to ameliorate with the genesis. The main advantage of this technique is that it takes place in a liquid format with small volumes. To begin, while working inside a BSL 2 biosafety cabinet, streak P.aeruginosa from a frozen stock onto an LB Agar plate.Incubate the plate at 37 degrees C for 16 to 24 hours, then store the plate at four degrees Celsius for up to one week. Two days prior to setting up assay plates, use a single colony from the plate to inoculate three to five milliliters of sterile LB broth. Incubate the culture at 37 degrees Celsius for 12 to 16 hours.After preparing the slow kill or SK media according to the text protocol, with 350 milliliters of p. Aeruginosa from the fresh overnight LB culture, seed each 10 centimeter SK plate. Use a sterile bacterial spreader to evenly spread the bacteria, and allow the plates to dry in the biosafety cabinet.Then, incubate the plates at 37 degrees Celsius for 24 hours. To test multiple RNAi bacterial strains in parallel in a single well of a 24 well deep well plate, inoculate a single colony from each clone of previously prepared RNAi containing bacteria into four milliliters of carbenicillin supplemented LB.Place the plates into a shaking incubator optimized for multi-well plates at 37 degrees Celsius and 950 rpm for 16 hours, then collect the bacteria by centrifugation at 2, 000 g for 5 minutes. Decant the supernatant by inverting the plate and vigorously shaking it.Using 100 microliters of S Basal resuspend the RNAi bacteria. Pipette the resuspended bacteria into an appropriate number of wells of a multi-well NGM plate supplemented with IPTG and carbenicillin and allow the plate to dry. After preparing L1 worms according to the text protocol, prepare plates for basic experiments by pipetting approximately 5, 000 worms per 10 centimeter plate, seeded with RNAi or OP50 superfood.To set up for an RNAi screen, pipette approximately 300 worms per well in a 24 well plate seeded with RNAi. If the strain being used is temperature-sterile, incubate the worms at 15 degrees Celsius for approximately 16 hours and then transfer them to 25 degrees Celsius for 44 hours to complete development and prevent embryogenesis. To carry out a liquid killing assay use a cell scraper to remove the P.aeruginosa from an SK plateand resuspend the bacteria in approximately 5 milliliters of S.Basal.Using a spectrophotometer meaure the OD 600 of the bacterial suspension. Prepare 24 milliliters of diluted stock of P.Aeruginosa in S.Basal at an OD 600 equal to 09, then add 21 milliliters of liquid SK media, and, using a multichannel pipette, transfer 45 microliters of bacteria and media to each well of a 384 well plate. Wash the worms from their source into a 50 milliliter conical tube, and allow them to settle under gravitational force.Aspirate the supernatant to 5 milliliters, then use a total of 50 milliliters of S.Basal to resuspend the worms, and repeat the wash twice more. Using a worm sorter, sort approximately 22 worms into each well of a 384 well plate, according to the text protocol, then use a gas-permeable film to seal the plate and incubate it at 25 degrees Celsius for 24 to 48 hours. At the desired time, use a microplate washer and S.Basal to wash the 384 well plate a total of 5 times.One of the easiest mistakes to make is to aspirate the 384 well plate before worms have completely settled. It takes practice to accurately see whether the worms have settled completely. After the final wash, aspirate the supernatant down to 20 microliters.Then, add 50 microliters of 98 micromolar nucleic acid stain per well, for a final concentration of 0.7 micromolar. Incubate the plate at room temperature for 12 to 16 hours. After the desired incubation period, use the microplate washer to wash the plates a minimum of three times.For a data acquisition use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence. Add one milliliter of S Basal per well of a 24 well plate containing 300 worms per well. Gently agitate the worms by shaking the plate, then transfer worms to an empty, sterile 24 deep well plate.Allow the worms to settle gravitationally, about five minutes. Aspirate the supernatant, leaving approximately one milliliter per well, Then add 7 milliliters of S Basal to each well. Repeat the wash two more times, and then after the final wash aspirate all but approximately 400 microliters of supernatant.After pipetting the bacteria into 384 well plates to avoid starvation, using the resampler function of the worm sorter sort 22 worms into each well of a 384 well plate. Finally, following sorting, add small molecules or other experiment specific materials. As illustrated in this graph, when the steps outlined in this video are followed, a time dependent killing of C.elegans will be observed only in the presence of P.aeruginosa.In contrast, in the absence of key bacterial nutritional supplements, little to no killing will be observed. As shown here, the two step incubation of P.aeruginosa at 37 degrees Celsius and then 25 degrees Celsius, which is critical for conventional slow killing assays, and was originally implemented in liquid killing, is dispensable in this assay. The protocol tolerates a wide range of initial bacterial concentrations, from as low as an OD 600 equal to 0025, and still exhibits time and concentration dependent killing, although the timing does shift.In addition, as few as four wells is frequently sufficient to obtain statistically significant data. An example of the utility of processing is shown here when the signal-to-noise ratio is high, as in this example, analysis is simple and discriminating between positive and negative conditions is trivial. In these cases, even weak hits can be readily identified.Once mastered, this technique can be performed in about 60 to 75 minutes of hands on time per 384 well plate, not including sorting. While attempting this procedure, it's critical to remember to add all the components to your media or virulence will be compromised. This assay simplifies the application of whole organism phenotype based screening to drug discovery in host-pathogen research.After watching this video, you should have a good understanding of how to carry out liquid-based C elegans pathogenesis assays. This procedure can be modified by substituting with other pathogens, such as E.Faecalis for P.Aeruginosa, facilitating the search for treatments for other bacterial infections. Don't forget that working with infectious bacteria like P.Aeruginosa or E.Faecalis can be dangerous.Appropriate precautions, such as proper training, good personal protective equipment, and proper technique, should always be taken while performing this procedure.

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

Underlätta Drug Discovery: En automatiserad hög halt Inflammation analys i zebrafisk

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

Automatiserad Separation av<em&gt; C. elegans</em&gt; Steglös koloniseras 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 analys för kvantifiering av intracellulär mykobakteri anpassad för screening med högt genomströmning/högt innehåll

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

Hög kapacitet analys till Phenotype<em&gt; Salmonella enterica</em&gt; Typhimurium Association, Invasion och replikering 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

Hög-throughput kvantitativ realtids-RT-PCR-analys för bestämning av uttryck Profiler av typ I och 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

Inrättandet av en hög kapacitet Setup för screening av små molekyler som modulerar 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. ...

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

Cell-free biokemiska Fluorometric enzymatisk analys för High-throughput mätning av lipidperoxidation i hög Density Lipoprotein

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

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

En hög genomströmning plattform för Screening av Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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

Automatiserad beteendevetenskaplig analys av stor C. elegans populationer med ett brett fält-of-view spårning plattform

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 hög genomströmning Shigella-specifika bakteriedödande analysmetod

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