Questo studio è stato sostenuto dal Research Institute del Texas (CPRIT) premio RR150044, Welch Foundation Research Grant C-1930 e la prevenzione di cancro e dell'istituti nazionali di salute K22 AI110552 assegnato a NVK. I finanziatori non avevano alcun ruolo nel disegno dello studio, raccolta dati e analisi, decisione di pubblicare o preparazione del manoscritto.

<ol>
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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>

Gli autori dichiarano di non avere conflitti di interesse a divulgare.

Questo saggio (o simili saggi dove altri agenti patogeni vengono sostituiti con p. aeruginosa o E. faecalis) sono utile per una varietà di scopi, tra cui la scoperta di nuovi farmaci. È anche utile per affrontare questioni biologiche fondamentali, come identificare i fattori di virulenza, la delucidazione dei meccanismi di difesa ospite e determinare il regolamentazione macchinari coinvolti nell'interazione ospite-patogeno.
Anche se il dosaggio di uccisione liquido p. a...

L'identificazione e lo sviluppo di efficaci antibiotici ad ampio spettro, ora quasi un secolo fa, ha portato a un momento di svolta nella sanità pubblica dove c'era una credenza diffusa che infettiva malattia sarebbe un flagello del passato. Entro pochi decenni, questo ottimismo iniziò a scemare, come agente patogeno dopo agente patogeno ha sviluppato meccanismi di resistenza che limitato questi trattamenti una volta miracolosi. Per qualche tempo, la corsa agli armamenti tra azioni di individuazione di droga e gli agenti patogeni sembrava equilibrata. Tuttavia, l'uso improprio degli antimicrobici è recentemente culminato nell'emergere di ceppi di Klebsiella pneumoniae, Acinetobacter baumanii, Serratia marcescense p. aeruginosa1, pan-farmaco resistenti 2,3,4.
P. aeruginosa è un patogeno di negativo, multi-host di grammo di opportunisti, che è una grave minaccia ai pazienti con gravi ustioni, coloro che sono immunocompromessi, o hanno la fibrosi cistica. È inoltre sempre più identificato come un agente eziologico nelle infezioni nosocomial severa, particolarmente dovuto la sua acquisizione in corso della resistenza antimicrobica. Per cominciare ad affrontare questa minaccia, abbiamo usato la ben documentata elegans del c.-p. aeruginosa infezione sistema5. Il nostro laboratorio ha sfruttato questo sistema per sviluppare una piattaforma di base liquida, ad alta velocità, ad alto contenuto di screening per identificare nuove molecole che limitano la capacità del patogeno di uccidere l' host6. Curiosamente, questi composti sembrano appartenere ad almeno tre categorie generali, compresi gli antimicrobici7 e virulenza inibitori8. Altri saggi di scoperta di droga ad alto contenuto in c. elegans sono stati segnalati per del micobatterio tuberculosum, Chlamydia trachomatis, pestis di Yersinia, Listeria monocytogenes, Francisella tularensis, Staphylococcus aureus, Candida albicans, e Enterococcus faecalis, tra gli altri9,10,11,12,13,14,15,16. Questi tipi di analisi presentano parecchi vantaggi ben riconosciuti, ad esempio limitando falsi positivi successi che possono essere tossici per sia l'host e l'agente patogeno, probabilità aumentata della biodisponibilità rispetto a uno schermo di chimico e la capacità di identificare successi di là semplicemente limitando la crescita microbica, come anti-virulents, stimolatore immunitario molecole o composti che altrimenti inclinare la bilancia dell'interazione ospite-patogeno in favore della ex. Inoltre, i composti scoperti in queste schermate sono spesso efficaci in mammiferi padroni di casa.
Vale la pena notare che almeno due altri saggi17,18 sono disponibili per realizzare schermi di alto-rendimento in c. elegans in liquido. Tuttavia, ciascuno di questi test è una modifica che permette il dosaggio di colonizzazione intestinale prototipo, conosciuto come lento-uccisione, da eseguirsi in liquido, aumentando la produttività e consentendo di composti più facilmente essere schermato. Un'attenta caratterizzazione ha dimostrato conclusivamente che i meccanismi di virulenza batterica sono diversi tra questi saggi e nostra base liquida schermo7. Poiché entrambi i tipi di virulenza sono osservati nei sistemi mammiferi, è importante considerare quali determinanti di virulenza sono più rilevante per gli interessi dello sperimentatore prima della selezione di test.
Qui dimostriamo una versione ottimizzata del liquido basato su analisi di c. elegans-P. aeruginosa . Segnaliamo anche l'adattamento del nostro metodo di analisi liquido-basata per ospitare l'agente patogeno batterico gram-positivo Enterococcus faecalis. Come p. aeruginosa, E. faecalis è sempre più identificato come una minaccia seria nosocomial con un armamento crescente di resistenza antimicrobica vie1. Anche se esiste un metodo precedente per high throughput screening di E. faecalis 14, richiede preinfection con l'agente patogeno, che complica la procedura e aumenta la probabilità di contaminazione di attrezzature come il FlowSort COPAS. Il nostro protocollo Elimina la necessità di pre-infezione, miglioramento del profilo di sicurezza. Infine, segnaliamo un mezzo mediante il quale uno di questi test possono essere combinato con alimentazione di RNAi, permettendo all'utente di cercare per fattori ospite che giocano un ruolo nella creazione di, o resistenza a, infezione.

<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

Il numero di nuovi farmaci identificati da tradizionale, schermi in vitro è scemato, riducendo il successo di questo approccio nella ricerca di nuove armi combattere la Resistenza multifarmaco. Questo ha portato alla conclusione che i ricercatori non solo la necessità di trovare nuovi farmaci, ma anche bisogno di sviluppare nuovi modi di trovare loro. Tra i candidati più promettenti metodi sono intero-organismo, in vivo dosaggi che uso alto-rendimento, fenotipica letture e ospita che vanno da Caenorhabditis elegans di Danio rerio. Questi host presentano parecchi vantaggi potenti, tra cui le riduzioni drammatiche in falsi positivi colpisce, come composti che sono tossici per l'ospite e/o biounavailable vengono in genere eliminati nella schermata iniziale, prima di seguire costosi fino.
Qui vi mostriamo come nostra analisi sono stato utilizzato per interrogare variazione host la ben documentata di c. elegans— patosistema come contributo liquido uccisione diPseudomonas aeruginosa . Inoltre dimostriamo diverse estensioni di questa tecnica ben lavorata fuori. Per esempio, siamo in grado di svolgere high throughput genetiche schermate mediante RNAi in 24 o 96 pozzetti formati piastra di fattori dell'ospite query in questa interazione ospite-patogeno. Usando questa analisi, schermi intero genoma possono essere completati in pochi mesi, che possono di semplificare notevolmente il compito di individuare bersagli farmacologici, potenzialmente senza la necessità di approcci biochimici laboriosa purificazione.
Inoltre segnaliamo qui una variazione del nostro metodo che sostituisce il batterio gram-positivo Enterococcus faecalis per l'agente patogeno gram-negativo p. aeruginosa. Molto come è il caso per p. aeruginosa, uccisione di E. faecalis è dipendente dal tempo. A differenza del precedente c. elegans— saggi diE. faecalis , nostra analisi per E. faecalis non richiede preinfection, migliorando il suo profilo di sicurezza e riducendo le possibilità di contaminazione liquido-per la movimentazione. Il dosaggio è estremamente robusto, mostrando ~ 95% tassi di mortalità 96h post infezione.

Parametri importanti per le prestazioni
Una corretta comprensione della biologia alla base di questa analisi è necessaria per risolvere i problemi e ottimizzare il dosaggio. A tal fine, ci riferiamo in primo luogo a parecchie carte chiave delucidamento dei meccanismi di patogenesi di p. aeruginosa-mediata uccidendo in liquido7,20. Condizione che i pass...

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

Qui descriviamo un protocollo che è un host adattabile, intero, strumento di screening ad alto contenuto che possa essere utilizzata per studiare le interazioni ospite-patogeno ed essere utilizzato per la scoperta di nuovi farmaci.

Un ad alta velocità, ad alto contenuto, liquido a base di c. elegans patosistema come contributo

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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Research

JoVE Journal

Immunology and Infection

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

14.4K Views.  Rice University. Qui descriviamo un protocollo che è un host adattabile, intero, strumento di screening ad alto contenuto che possa essere utilizzata per studiare le interazioni ospite-patogeno ed essere utilizzato per la scoperta di nuovi farmaci.

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

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

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 of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

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

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Separazione automatica di<em&gt; C. elegans</em&gt; Variabilmente Colonizzato da un batterio patogeno

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 ...

Ricerca

Immunologia e infezioni

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

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Un test fenotipico microscopico per la quantificazione dei micobatteri intracellulari adattati per lo screening ad alta produttività / alto contenuto

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

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

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

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

High-throughput Assay per Fenotipo<em&gt; Salmonella enterica</em&gt; Typhimurium Associazione, Invasion, e replica nei macrofagi

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

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 locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

High-throughput Quantitative Real-time RT-PCR test per la determinazione di espressione Profili di tipo I e III interferone sottotipi

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

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

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

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Creazione di una configurazione high-throughput screening per piccole molecole che modulano c-di-GMP Segnalazione di<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 Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

High-throughput di Gene Tagging in<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale 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 combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Detection High-throughput respiratorie patogeni nei campioni animali da Nanoscale 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

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Analisi enzimatica fluorometrica cell-free biochimico per la misura di High-throughput di perossidazione lipidica in lipoproteina ad alta densità

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

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

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

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Analisi del comportamento di grande c. elegans popolazioni utilizzando un ampio campo di vista Monitoraggio piattaforma automatizzata

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

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un High-throughput Shigella-specifica analisi battericida

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