Ringraziamo il Dott. Diane Baronas-Lowell per la lettura critica del manoscritto. Questo lavoro è stato supportato da un FAU Charles E. Schmidt College of Science Seed Grant e una borsa di studio invecchiamento della Ellison Medical Foundation di KJ

1. Piastre XLD (xilosio lisina Desoxycholate) Agar XLD agar è un terreno di coltura selettivo per Salmonella, che appare come colonie nere su piastre di agar XLD. Tuttavia, se non ci sono problemi di contaminazione, un piatto regolare LB può essere sostituito. <ol><li> Pesare 5,5 g di agar XLD e risospendere in 5 ml di acqua deionizzata. </li><li> Mescolare accuratamente fino a quando tutti agar è bagnato. Aggiungere 95 ml di acqua deionizzata fino a quando tutti i grumi sono andati e il mezzo è completamente risospese. </li><li> Far bollire il mezzo per sciogliere completamente (non autoclave). </li><li> Raffreddare il terreno a temperatura ambiente a 50 ° C. </li><li> Versare 25 ml di agar-agar in ciascuno di 95 x 15 mm (diametro x altezza) targa (targhe sigillati con parafilm possono essere conservati a 4 ° C per un massimo di 1 mese). </li></ol> 2. Nematode Growth Medium (NGM) RNAi alimentazione Piatti Preparazione di C. piastre NGM elegans è stato descritto in precedenza 19. Qui una procedura viene brevemente descritta aggiungere ampicillina e l&#39;RNAi induttore chimico isopropil β-D-1-thiogalactopyranoside (IPTG) in media NGM per rendere le piastre di alimentazione RNAi. <ol><li> Sciogliere 3 g di NaCl e 2,5 g Bacto peptone in 1 L di acqua deionizzata. </li><li> Aggiungere 17 g di agar Bacto nei media. </li><li> Autoclavare media per 45 minuti e raffreddare il supporto a 50 ° C in un bagno d&#39;acqua. </li><li> Aggiungere le seguenti soluzioni: 1 ml di colesterolo (5 mg / ml in 95% etanolo), 1 ml di 1 M CaCl 2, 1 ml di 1 M MgSO4, e 25 ml di 1 M tampone fosfato di potassio (pH 6,0). Mescolare bene. </li><li> Aggiungere 1 ml di 1 M IPTG e 500 microlitri ampicillina (100 mg / ml in acqua sterile). </li><li> Mescolare bene la soluzione e versare in 60 x 15 mm (diametro x altezza) piastre di Petri con un apparecchio Pipettare e 25 ml pipetta sierologica seguenti procedure sterili. Riempire ogni piatto con 12 ml di agar. Le piastre possono essere conservati a 4 ° C per un massimo di 1 mese. </li></ol> Il gene essenziale autofagia bec-1 viene utilizzato come esempio per esaminare la funzione di un gene ospitante in difesa contro l&#39;infezione da Salmonella. Le procedure sperimentali sono illustrati nella figura 1 e nella tabella 1. Il protocollo per la preparazione RNAi animali trattati per l&#39;infezione segue, con il giorno di ogni fase sperimentale dato tra parentesi. <ol><li> Seminare bec-1 alimentazione RNAi e controllare vettore vuoto L4440 RNAi alimentazione batteri mettendo un fiocco di -80 ° C i batteri congelati in 2 ml di terreno LB integrato con 100 mg / ml ampicillina (1 ° giorno). Ripetere questa operazione una volta alla settimana durante l&#39;intero esperimento di avere batteri RNAi freschi. Conservare la cultura nel 4 ° C frigorifero quando non è utilizzato. </li><li> Seed 100 ml di coltura batterica durante la notte RNAi su lastre di RNAi. Preparare tre bec-1 RNAi e tre di controllo vuota vettore piatti RNAi. Incubare le piastre a 37 ° C per una notte (Day 2). </li><li> Rimuovere le piastre di RNAi dal 37 ° C incubatore e permettere loro di raffreddare a temperatura sulla panchina stanza. Pick up ben nutriti L4 wild type ermafroditi N2 e trasferire loro di Bec-1 RNAi e controllo vettoriale piatti RNAi vuoti. Inserire tre vermi per piastra, su piastre in triplo. Lo stesso giorno, preparare piatti RNAi come descritto al punto 3.2 (3 ° giorno). </li><li> Incubare le piastre RNAi con i vermi in incubatore a 20 ° C per 36-40 hr e trasferire i vermi a piatti freschi RNAi corrispondenti preparati nella fase 3.3. Dopo i vermi vengono trasferiti, incubare le piastre nel 20 ° C incubatore per 64 ore (giorno 4). </li></ol> 4. Preparare Salmonella per infezione <ol><li> Streak Salmonella -80 ° C Si congelati su 1 piastra XLD agar ed incubare la piastra a 37 ° C durante la notte (Day 5 </strong&gt;). </li><li> Scegli una ben isolata Salmonella bianco colonia e inoculare in 2 ml di terreno LB a 37 ° C con agitazione tutta la notte (Giorno 6). </li><li> Seed 80 ml ​​Salmonella cultura durante la notte il 1 ° C. elegans 60 x 15 mm (diametro x altezza) piastra NGM agar e preparare 6 piatti in totale. Incubare le piastre a temperatura ambiente per 6 ore. La coltura batterica devono asciugare e formare un prato sulla piastra (7 ° giorno). </li></ol> 5. Worms Infect RNAi trattati con Salmonella <ol><li> Trasferimento bec-1 RNAi-trattati e controllo vettoriale RNAi trattati ermafroditi L4 N2 vuoti (progenie di vermi istituito nel passaggio 3) alle piastre Salmonella. Mettere 40 vermi per piastra su 3 piastre per ogni gruppo. Incubare le piastre a vite senza fine a 20 ° C per 48 ore (7 ° giorno). </li><li> Preparare una serie di lastre di RNAi freschi come descritto nei punti 3.1 e 3.2 (Day 7 e <strong&gt; Giorno 8). </li><li> Dopo 48 ore l&#39;infezione, trasferire i vermi Salmonella-infetti ai corrispondenti piastre RNAi preparati in fase 5.2 e incubare a 20 ° C (9 ° giorno). </li></ol> 6. Sopravvivenza Assay <ol><li> Punteggio la sopravvivenza di vermi quotidiano e trasferire i vermi di corrispondenti lastre di RNAi freschi durante il periodo di deposizione delle uova. Preparare una serie di lastre di RNAi fresco prima di ogni trasferimento verme come descritto nei punti 3.1 e 3.2. Toccare il corpo senza fine (testa, parte centrale e la coda) delicatamente con un filo di platino end-appiattita. Un worm è segnato come morta, se si osserva alcun movimento del corpo worm. </li><li> Punteggio la sopravvivenza di vermi giorno o ogni altro giorno, e trasferire i vermi alle corrispondenti piastre RNAi freschi due volte a settimana dopo i vermi fermano la deposizione delle uova. </li><li> Dopo che tutti i vermi muoiono, riunire i dati di sopravvivenza da lastre triplice copia come un set di dati. Inserire i dati di sopravvivenza di ogni gruppo nel caso di software statistico come GraphPad Prism per generare curve di sopravvivenza e di effettuare analisi di sopravvivenza di Kaplan-Meier. L&#39;intero esperimento viene ripetuto almeno una volta a confermare la conclusione. </li></ol>

<ol>
	<li>Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, 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=.">.</a> C. elegans II. , (1997).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genetics+of+Caenorhabditis+elegans.">The Genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Aballay, A., Ausubel, F. M. <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+as+a+host+for+the+study+of+host-pathogen+interactions.">Caenorhabditis elegans as a host for the study of host-pathogen interactions.</a> Curr Opin Microbiol. 5, 97-101 (2002).</li><li>Kurz, C. L., Ewbank, J. 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:+an+emerging+genetic+model+for+the+study+of+innate+immunity.">Caenorhabditis elegans: an emerging genetic model for the study of innate immunity.</a> Nat Rev Genet. 4, 380-390 (2003).</li><li>Mylonakis, E., Aballay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worms+and+flies+as+genetically+tractable+animal+models+to+study+host-pathogen+interactions.">Worms and flies as genetically tractable animal models to study host-pathogen interactions.</a> Infection and Immunity. 73, 3833-3841 (2005).</li><li>Ford, M. W., 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+descriptive+study+of+human+Salmonella+serotype+typhimurium+infections+reported+in+Ontario+from+1990+to+1997.">A descriptive study of human Salmonella serotype typhimurium infections reported in Ontario from 1990 to 1997.</a> Can J Infect Dis. 14, 267-273 (2003).</li><li>Voetsch, A. C., 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=FoodNet+estimate+of+the+burden+of+illness+caused+by+nontyphoidal+Salmonella+infections+in+the+United+States.">FoodNet estimate of the burden of illness caused by nontyphoidal Salmonella infections in the United States.</a> Clin Infect Dis. 38 Suppl 3, (2004).</li><li>Aballay, A., Yorgey, P., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+proliferates+and+establishes+a+persistent+infection+in+the+intestine+of+Caenorhabditis+elegans.">Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans.</a> Curr Biol. 10, 1539-1542 (2000).</li><li>Alegado, R. A., Tan, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antimicrobial+peptides+contributes+to+persistence+of+Salmonella+typhimurium+in+the+C.+elegans+intestine.">Resistance to antimicrobial peptides contributes to persistence of Salmonella typhimurium in the C. elegans intestine.</a> Cell Microbiol. 10, 1259-1273 (2008).</li><li>Jia, K., 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=Autophagy+genes+protect+against+Salmonella+typhimurium+infection+and+mediate+insulin+signaling-regulated+pathogen+resistance.">Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 14564-14569 (2009).</li><li>Tenor, J. L., McCormick, B. A., Ausubel, F. M., Aballay, A. <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-based+screen+identifies+Salmonella+virulence+factors+required+for+conserved+host-pathogen+interactions.">Caenorhabditis elegans-based screen identifies Salmonella virulence factors required for conserved host-pathogen interactions.</a> Curr Biol. 14, 1018-1024 (2004).</li><li>Levine, B., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+by+self-digestion:+molecular+mechanisms+and+biological+functions+of+autophagy.">Development by self-digestion: molecular mechanisms and biological functions of autophagy.</a> Developmental Cell. 6, 463-477 (2004).</li><li>Birmingham, C. L., Smith, A. C., Bakowski, M. A., Yoshimori, T., Brumell, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+controls+Salmonella+infection+in+response+to+damage+to+the+Salmonella-containing+vacuole.">Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole.</a> J Biol Chem. 281, 11374-11383 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+Sequencing+Consortium.+Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">The C. elegans Sequencing Consortium. Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282, 2012-2018 (1998).</li><li>Fire, 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=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome Biol. 2, 1-10 (2001).</li><li>Liang, J., Xiong, S., Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNA-mediated+interference+feeding+strategy+to+screen+for+genes+involved+in+body+size+regulation+in+the+nematode+C+elegans.">Using RNA-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode C elegans.</a> J. Vis. Exp. (72), (2013).</li><li>Fraser, A. G., 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=Functional+genomic+analysis+of+C.+elegans+chromosome+I+by+systematic+RNA+interference.">Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.</a> Nature. 408, 325-330 (2000).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: the online review of C elegans biology. , 1-11 (2006).</li><li>Aballay, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+mediated+by+ced-3+and+ced-4+protects+Caenorhabditis+elegans+from+Salmonella+typhimurium-mediated+killing.">Programmed cell death mediated by ced-3 and ced-4 protects Caenorhabditis elegans from Salmonella typhimurium-mediated killing.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 2735-2739 (2001).</li><li>Melendez, 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=Autophagy+genes+are+essential+for+dauer+development+and+lifespan+extension+in+C.+elegans.">Autophagy genes are essential for dauer development and lifespan extension in C. elegans.</a> Science. 301, 1387-1391 (2003).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

C. elegans è un organismo semplice modello genetico che mangia i batteri come fonte di nutrienti. Così, è facile da sostituire la alimentare batterica normale con un patogeno intestinale per indagare le interazioni tra C. elegans e l&#39;agente patogeno scelto. Qui un protocollo è descritto combinare Salmonella infezione e C. elegans RNAi alimentazione trattamento per esaminare il ruolo dei geni ospitanti in difesa contro le infezioni da Salmonella. Protocolli di preceden...

Il nematode del suolo a vita libera Caenorhabditis elegans è un semplice e geneticamente trattabili organismo modello utilizzato per studiare molte questioni biologiche. C. elegans esiste prevalentemente come ermafroditi auto-fertilizzazione. I maschi sono spontaneamente generati da non disgiunzione del cromosoma X durante la gametogenesi 1,2. In presenza di cibo abbondante, C. elegans sviluppare continuamente attraverso quattro stadi larvali all&#39;adulto. La temperatura influisce anche C. sviluppo elegans; sviluppo più rapido si osserva a temperature più elevate. In laboratorio, C. elegans è coltivato ad una temperatura standard di 20 ° C su piastre di agar con seminato batterio Escherichia coli (ceppo OP50) come alimento 1,2. Nell&#39;ultimo decennio, C. elegans è emerso come un organismo invertebrato per studiare le interazioni ospite-patogeno 3-5. In natura, C. elegans mangia i batteri come nutrient 1,2 fonte. La sua normale cibo laboratorio batterica, OP50, può essere facilmente sostituito con altri patogeni per esaminare le interazioni tra C. elegans e qualsiasi agente patogeno scelta. In queste condizioni, l&#39;intestino è il sito primario dell&#39;infezione. Infatti, una vasta gamma di patogeni batterici ha dimostrato di infettare lethally C. elegans 3-5. Il batterio Salmonella gram-negativa è un agente patogeno gastrointestinale che causa malattie di origine alimentare umana in tutto il mondo 6,7. C. elegans è un buon modello di accoglienza per la Salmonella typhimurium come questo batterio si replica e presenta infezioni intestinali persistenti 8-10. C. elegans è stato usato per identificare sia nuovo e precedentemente noto virulenza di Salmonella fattori 11. È interessante notare che il C. sistema immunitario elegans limita con successo la replica Salmonella. E &#39;stato riportato in precedenza che inibition di geni autofagia rende maggiore la replica di Salmonella in C. elegans, con conseguente morte precoce di vermi infetti 10. Macroautofagia (di seguito denominato autofagia) è un processo dinamico che coinvolge il riassetto delle membrane subcellulari di sequestrare citoplasma e organelli per la consegna agli lisosomi per la degradazione 12. Autofagia è stato segnalato per limitare la replica Salmonella in C. elegans e nei mammiferi 10,13. La C. elegans genoma è stato il primo genoma degli eucarioti multicellulari sequenziato; è sensibile alle RNAi trattamento 14-16. Inoltre, RNAi può essere somministrato efficacemente sottoponendo vermi ingerire batteri contenenti l&#39;RNA a doppio filamento del gene bersaglio, noto come RNAi alimentazione 16,17. Genoma biblioteche alimentazione RNAi interi sono stati generati per il genoma di screening RNAi 16,18. Qui, una Salmonella infezione proprotocollo è accoppiato con RNAi alimentazione per consentire la sperimentazione C. elegans geni di interesse per la loro capacità di proteggere contro l&#39;infezione da Salmonella.

<table><tbody><tr><td>LB Broth</td><td>Fisher</td><td>BP9723-500</td><td></td></tr><tr><td>XLD agar</td><td>EMD Chemicals</td><td>1.05287.0500</td><td></td></tr><tr><td>Bacto Agar</td><td>Fisher</td><td>DF0140-01-0</td><td></td></tr><tr><td>Peptone</td><td>Fisher</td><td>BP1420-500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher</td><td>S671-500</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher</td><td>C69-500</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher</td><td>M65-500</td><td></td></tr><tr><td>IPTG</td><td>Gold Biotechnology</td><td>12481C50</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8667-25G</td><td></td></tr><tr><td>Ampicillin</td><td>Fisher</td><td>BP1760-25</td><td></td></tr><tr><td>&lt;em&gt;Salmonella typhimurium&lt;/em&gt;</td><td>ATCC</td><td>ATCC14028</td><td></td></tr><tr><td>Petri Dish 95 x 15 mm</td><td>Fisher</td><td>FB0875714G</td><td></td></tr><tr><td>Petri Dish 60 x 15 mm&amp;nbsp;</td><td>Fisher</td><td>08-757-13A</td><td></td></tr><tr><td>Falcon Serological pipet</td><td>Fisher</td><td>13-668-2</td><td></td></tr><tr><td>Falcon Express Pipet-Aid</td><td>Fisher</td><td>13-675-42</td><td></td></tr><tr><td>MaxQ6000 shaking incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>SHKE6000-7</td><td></td></tr><tr><td>Incubator</td><td>Percival</td><td>I-36DL</td><td></td></tr></tbody></table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

Nell&#39;ultimo decennio, C. elegans è emerso come un organismo invertebrato per studiare le interazioni tra i padroni di casa e agenti patogeni, tra cui la difesa ospite contro i gram-negativi batterio Salmonella typhimurium. Salmonella stabilisce infezione persistente nell&#39;intestino di C. elegans e si traduce in morte precoce di animali infetti. Un certo numero di meccanismi immunitari sono stati identificati in C. elegans per difendersi contro le infezioni da Salmonella. Autofagia, un evolutivamente conservata degradazione lisosomiale percorso, ha dimostrato di limitare la replica Salmonella in C. elegans e nei mammiferi. Qui, un protocollo è descritto per infettare C. elegans con Salmonella typhimurium, in cui i vermi sono esposti a Salmonella per un tempo limitato, simile alle infezioni da Salmonella nell&#39;uomo. Salmonella infezione riduce notevolmente la durata di C. elegans &lt;/ Em&gt;. Utilizzando il gene autofagia essenziale bec-1 come esempio, abbiamo combinato questo metodo infezione da C. elegans RNAi alimentazione approccio e ha mostrato questo protocollo può essere utilizzato per esaminare la funzione di C. geni ospitanti elegans in difesa contro le infezioni da Salmonella. Poiché C. elegans intero genoma librerie RNAi sono disponibili, questo protocollo permette di schermare completamente per la C. elegans geni che proteggono contro la salmonella e altri patogeni intestinali utilizzando le librerie RNAi genome-wide.

A 20 ° C, la durata mediana di wild type vermi N2 è 17 giorni (Figura 2a e Tabella 2). Infezione da Salmonella diminuisce significativamente la durata mediana di vermi N2 a 10,5 giorni (p = 0,0002, log-rank test) (Figura 2A ). Se una C. gene elegans svolge un ruolo importante nella difesa contro le infezioni da Salmonella, si prevede che la sua inibizione impartirà suscettibilità alle infezioni Sal...

Watch this Scientific Journal Video about A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium at JoVE.com

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans è emerso come un nuovo modello genetico per studiare le interazioni ospite-patogeno. Qui si descrive un protocollo per infettare C. elegans con Salmonella typhimurium accoppiato con la tecnica RNAi doppio filamento interferenze per esaminare il ruolo dei geni ospitanti in difesa contro le infezioni da Salmonella.

Un protocollo Infettare<em&gt; Caenorhabditis elegans</em&gt; Con<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

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Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K Views.  Florida Atlantic University.  C. elegans è emerso come un nuovo modello genetico per studiare le interazioni ospite-patogeno. Qui si descrive un protocollo per infettare C. elegans con Salmonella typhimurium accoppiato con la tecnica RNAi doppio filamento interferenze per esaminare il ruolo dei geni ospitanti in difesa contro le infezioni da Salmonella. 

Video: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

Il Nematode Caenorhabditis Elegans - un modello Versatile In Vivo per studiare le interazioni ospite-microbo

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Ricerca

Genetica

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 ...

Immunologia e infezioni

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 ...

Dosaggio ad alto rendimento per misurare l'assunzione di cibo e la durata della vita di Caenorhabditis elegans

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Autore in primo piano: Esplorare la relazione tra lipotossicità e HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Quantificazione dell'assunzione di cibo in Caenorhabditis elegans misurando la clearance batterica

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Comportamento

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.

Utilizzando il protozoo Paramecium caudatum come veicolo di infezioni di origine alimentare nelle larve di Zebrafish

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Infezione da Salmonella cronica Fibrosi intestinale indotta

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

Studio dell'immunità ereditaria in un modello di infezione da microsporidi di Caenorhabditis elegans

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Caratterizzazione della peritonite settica indotta da Salmonella Typhimurium nei topi

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

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Il Nematode Caenorhabditis Elegans - un modello Versatile In Vivo per studiare le interazioni ospite-microbo

Separazione automatica di

High-throughput Assay per Fenotipo

Quantificazione dell'assunzione di cibo in Caenorhabditis elegans misurando la clearance batterica

Quantificazione dell'assunzione di cibo in Caenorhabditis elegans misurando la clearance batterica

Quantificazione dell'assunzione di cibo in Caenorhabditis elegans misurando la clearance batterica

Utilizzando il protozoo Paramecium caudatum come veicolo di infezioni di origine alimentare nelle larve di Zebrafish

Infezione da Salmonella cronica Fibrosi intestinale indotta

Studio dell'immunità ereditaria in un modello di infezione da microsporidi di Caenorhabditis elegans

Caratterizzazione della peritonite settica indotta da Salmonella Typhimurium nei topi