Damos las gracias a la Dra. Diane Baronas-Lowell para la lectura crítica del manuscrito. Este trabajo fue apoyado por un Charles E. Schmidt Colegio FAU del Fondo Semilla de Ciencia y una Beca de Envejecimiento de la Fundación Médica Ellison a KJ

1. Placas XLD (xilosa lisina desoxicolato) Agar Agar XLD es un medio de crecimiento selectivo para Salmonella, que aparece como colonias negras en placas de agar XLD. Sin embargo, si no hay problemas de contaminación, una placa regular de LB puede ser sustituido. <ol><li> Pesar 5,5 g de agar XLD y resuspender en 5 ml de agua desionizada. </li><li> Mezclar bien hasta que todo el agar está mojado. Añadir 95 ml de agua desionizada hasta que todos queden grumos y el medio se volvieron a suspender por completo. </li><li> Hervir el medio para disolver por completo (no en autoclave). </li><li> Enfriar el medio a temperatura ambiente a 50 ° C. </li><li> Verter 25 ml de agar de cada 95 x 15 mm (diámetro x altura) de la placa (placas selladas con Parafilm pueden almacenarse a 4 ° C durante un máximo de 1 mes). </li></ol> 2. Nematodo Medio de Crecimiento (NGM) de alimentación RNAi placas Preparación de C. placas elegans NGM se ha descrito anteriormente 19. Aquí un procedimiento se describe brevemente para añadir el antibiótico ampicilina y el ARNi inductor químico isopropil β-D-1-tiogalactopiranósido (IPTG) en los medios de comunicación NGM para hacer las placas de alimentación de RNAi. <ol><li> Disolver 3 g de NaCl y 2,5 g de Bacto peptona en 1 L de agua desionizada. </li><li> Añadir 17 g de agar Bacto en los medios de comunicación. </li><li> Autoclave los medios de comunicación durante 45 minutos y enfriar el medio a 50 ° C en un baño de agua. </li><li> Añadir las siguientes soluciones: 1 ml de colesterol (5 mg / ml en etanol al 95%), 1 ml de 1 M de CaCl2, 1 ml de 1 M MgSO4, y 25 ml de 1 M de tampón de fosfato de potasio (pH 6,0). Mezclar bien. </li><li> Añadir 1 ml de 1 M de IPTG y 500 l de ampicilina (100 mg / ml en agua estéril). </li><li> Mezcle bien la solución y verter en placas de Petri de 60 x 15 mm (diámetro x altura) utilizando una pipeta de Ayuda y 25 ml de pipeta serológica siguientes procedimientos estériles. Llene cada plato con 12 ml de agar. Las placas se pueden almacenar a 4 ° C durante hasta 1 mes. </li></ol> El gen esencial autofagia BEC-1 se utiliza como un ejemplo para examinar la función de un gen de acogida en la defensa contra la infección por Salmonella. Los procedimientos experimentales se ilustran en la Figura 1 y la Tabla 1. El protocolo para la preparación de los animales tratados con ARNi para la infección sigue, con el día de cada paso experimentales dados en paréntesis. <ol><li> Inocular bec-1 RNAi alimentación y el control de vector vacío L4440 alimentación RNAi bacterias mediante la colocación de un copo de -80 ° C las bacterias congeladas en 2 ml de medio LB suplementado con 100 mg / ml de ampicilina (Día 1). Repita este paso una vez a la semana durante todo el experimento de tener bacterias RNAi frescas. Guarde la cultura en el 4 ° C refrigerador cuando no se utiliza. </li><li> Seed 100 ml de cultivo bacteriano durante la noche en placas de RNAi RNAi. Prepare de tres bec-1 RNAi y el control de tres v vacíaector placas de RNAi. Incubar las placas a 37 ° C durante la noche (Día 2). </li><li> Retire las placas de RNAi de la 37 ° C incubadora y deje que se enfríen a temperatura ambiente en el banquillo. Recoge bien alimentados L4 tipo salvaje hermafroditas N2 y transferirlos a Bec-1 RNAi y controlan placas RNAi vector vacío. Coloque tres gusanos por placa, en placas por triplicado. El mismo día, se preparan platos de RNAi como se describe en el paso 3.2 (día 3). </li><li> Incubar las placas de RNAi con gusanos en la incubadora de 20 ° C para 36 a 40 hy de transferencia de los gusanos a las placas de RNAi correspondientes frescos preparados en el paso 3.3. Después de gusanos se transfieren, incubar las placas en el 20 ° C incubadora durante 64 horas (día 4). </li></ol> 4. Preparar para la infección por Salmonella <ol><li> Salmonella racha de -80 ° C madre congelada en 1 placa de agar XLD e incubar la placa a 37 ° C durante la noche (el día 5 </stRong&gt;). </li><li> Elige un bien aislada de Salmonella negro colonia e inocular en 2 ml de medio LB a 37 ° C con agitación durante la noche (Día 6). </li><li> Seed 80 ml ​​Salmonella cultivo de una noche en 1 C. elegans 60 x 15 mm (diámetro x altura) placa de agar NGM y preparar 6 placas en total. Las placas se incuban a temperatura ambiente durante 6 horas. El cultivo bacteriano debe secarse y formar un césped en la placa (día 7). </li></ol> 5. Worms tratados con RNAi Infect con Salmonella <ol><li> Traslado bec-1 RNAi tratados y control de vectores hermafroditas L4 N2 vacías RNAi tratados (la progenie de gusanos creados en el paso 3) para placas de Salmonella. Coloque 40 gusanos por placa en 3 placas para cada grupo. Incubar las placas de gusanos a 20 ° C durante 48 horas (día 7). </li><li> Prepare un juego de placas de RNAi frescas tal como se describe en los pasos 3.1 y 3.2 (Día 7 y <strong&gt; día 8). </li><li> Después de 48 horas de infección, transferir gusanos infectados por Salmonella en las correspondientes placas de RNAi preparados en la etapa 5.2 y se incuba a 20 ° C (día 9). </li></ol> 6. Ensayo de supervivencia <ol><li> Puntuación de la supervivencia de los gusanos diario y transferir gusanos correspondientes placas de RNAi frescas durante la época de puesta de huevos. Prepare un conjunto de placas de RNAi frescas antes de cada transferencia de gusano como se describe en los pasos 3.1 y 3.2. Toque el cuerpo del gusano (cabeza, parte media y cola) suavemente con un alambre de platino final aplanado. Un gusano se califica como muerto si no se observa el movimiento del cuerpo del gusano. </li><li> Puntuación de la supervivencia de los gusanos al día o cada dos días, y la transferencia de los gusanos de las placas correspondientes RNAi frescas dos veces a la semana después de que los gusanos dejan de poner huevos. </li><li> Después de todos los gusanos mueren, agrupar los datos de supervivencia de placas por triplicado como un conjunto de datos. Introduzca los datos de supervivencia de cada grupo en el software estadístico apropiado como GraphPad Prismo para generar curvas de supervivencia y para llevar a cabo el análisis de supervivencia de Kaplan-Meier. Todo el experimento se repite al menos una vez para confirmar la conclusión. </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>

Los autores declaran que no tienen intereses financieros en competencia.

C. elegans es un simple organismo modelo genético que come bacterias como fuente de nutrientes. Por lo tanto, es fácil de sustituir su alimentaria bacteriana normal con un patógeno intestinal para investigar las interacciones entre C. elegans y el patógeno elegido. En la presente memoria se describe un protocolo para combinar la infección por Salmonella y C. elegans alimentación RNAi tratamiento para examinar el papel de los genes del huésped en la defensa contra la infección ...

La vida libre de nematodos Caenorhabditis elegans suelo es un simple y manejable de forma genética organismo modelo utilizado para estudiar muchas cuestiones biológicas. C. elegans existe predominantemente como hermafroditas autógamas. Los varones son generados espontáneamente por la no disyunción del cromosoma X durante 1,2 gametogénesis. En la presencia de abundante comida, C. elegans desarrollar continuamente a través de cuatro estadios larvarios a adulto. La temperatura también influye en C. desarrollo elegans; desarrollo más rápido se observó a temperaturas más altas. En el laboratorio, C. elegans se cultiva a una temperatura estándar de 20 ° C en placas de agar con cabeza de serie bacteria Escherichia coli (cepa OP50) como alimento 1,2. En la última década, C. elegans se ha convertido en un organismo invertebrado para estudiar las interacciones huésped-patógeno 3-5. En la naturaleza, C. elegans come bacterias como su nutrien1,2 t fuente. Su comida normal de laboratorio bacteriana, OP50, puede ser fácilmente sustituido con otros patógenos para examinar las interacciones entre C. elegans y cualquier patógeno elegido. En estas condiciones, el intestino es el sitio primario de la infección. De hecho, se ha demostrado una amplia gama de patógenos bacterianos para infectar letalmente C. elegans 3-5. La bacteria Salmonella gramnegativos es un patógeno gastrointestinal que causa la enfermedad transmitida por los alimentos humanos en todo el mundo 6,7. C. elegans es un buen modelo de acogida de Salmonella typhimurium ya que esta bacteria se replica y exhibe las infecciones intestinales persistentes 8-10. C. elegans ha sido utilizado para identificar tanto novedoso y anteriormente conocido virulencia de Salmonella factores 11. Curiosamente, la C. sistema inmune elegans limita con éxito la replicación de Salmonella. Se ha informado anteriormente de que no habilition de genes autofagia hace que el aumento de la replicación de Salmonella en C. elegans, lo que resulta en la muerte temprana de los gusanos infectados 10. Macroautofagia (aquí referido como la autofagia) es un proceso dinámico que implica la reorganización de las membranas subcelulares para secuestrar citoplasma y orgánulos para la entrega a los lisosomas para degradación 12. La autofagia ha sido reportado para limitar la replicación de Salmonella en C. elegans y en los mamíferos 10,13. El C. elegans genoma fue el primer genoma eucariota multicelular secuenciado; que es sensible a RNAi tratamiento 14-16. Por otra parte, el ARNi puede ser administrado eficazmente sometiendo gusanos de ingerir las bacterias que contienen el ARN de doble cadena del gen diana, conocido como alimentación RNAi 16,17. Genoma bibliotecas de alimentación RNAi enteras han sido generados para la selección de ARNi de todo el genoma 16,18. En la presente memoria, una infección de Salmonella Proprotocolo es, junto con alimentación RNAi para permitir pruebas de C. genes elegans de interés por su capacidad de proteger contra la infección por Salmonella.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">BP9723-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XLD agar</td>
			<td>EMD Chemicals</td>
			<td>1.05287.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Fisher</td>
			<td>DF0140-01-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Fisher</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Fisher</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Fisher</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td>Gold Biotechnology</td>
			<td>12481C50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Fisher&#160;</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 95 x 15mm</td>
			<td>Fisher</td>
			<td>FB0875714G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish 60 x 15mm&#160;</td>
			<td>Fisher</td>
			<td>08-757-13A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Serological pipet</td>
			<td>Fisher</td>
			<td>13-668-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Express Pipet-Aid</td>
			<td>Fisher</td>
			<td>13-675-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ6000 shaking incubator&#160;</td>
			<td>Thermo Scientific</td>
			<td>SHKE6000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Percival</td>
			<td>I-36DL</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

En la última década, C. elegans se ha convertido en un organismo invertebrado para estudiar las interacciones entre los hosts y patógenos, incluyendo la defensa del huésped contra bacterias gram-negativas bacteria Salmonella typhimurium. Salmonella establece una infección persistente en el intestino de C. elegans y resulta en la muerte temprana de los animales infectados. Un número de mecanismos de la inmunidad se ha identificado en C. elegans para defenderse de las infecciones por Salmonella. La autofagia, una vía de degradación lisosomal conservadas evolutivamente, se ha demostrado que limitar la replicación de Salmonella en C. elegans y en los mamíferos. Aquí, un protocolo se describe para infectar C. elegans con Salmonella typhimurium, en la que los gusanos están expuestos a la Salmonella por un tiempo limitado, similar a la infección por Salmonella en los seres humanos. infección por Salmonella acorta significativamente la vida útil de C. elegans &lt;/ Em&gt;. Usando el gen de la autofagia esencial BEC-1 como un ejemplo, se combinaron este método de infección con C. elegans alimentación RNAi enfoque y mostró este protocolo puede ser utilizado para examinar la función de C. genes del huésped elegans en la defensa contra la infección por Salmonella. Desde C. elegans todo el genoma RNAi bibliotecas están disponibles, este protocolo permite a la pantalla exhaustivamente para C. genes elegans que protegen contra la Salmonella y otros patógenos intestinales utilizando RNAi bibliotecas de todo el genoma.

A 20 ° C, la esperanza de vida media de tipo salvaje gusanos N2 es de 17 días (Figura 2A y Tabla 2). Infección por Salmonella disminuye significativamente la esperanza de vida media de los gusanos N2 a 10,5 días (p = 0,0002, prueba de log-rank) (Figura 2A ). Si una C elegans gen desempeña un papel importante en la defensa contra la infección por Salmonella, se prevé que su inhibición impartirá la susceptib...

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 se ha convertido en un nuevo modelo genético para estudiar las interacciones huésped-patógeno. Aquí se describe un protocolo para infectar C. elegans con Salmonella typhimurium, junto con la técnica de RNAi interferencia de doble hebra para examinar el papel de los genes del huésped en la defensa contra la infección por Salmonella.

Un protocolo para infectar<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 ...

a-protocol-to-infect-caenorhabditis-elegans-with-salmonella

Research

JoVE Journal

Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K vistas.  Florida Atlantic University.  C. elegans se ha convertido en un nuevo modelo genético para estudiar las interacciones huésped-patógeno. Aquí se describe un protocolo para infectar C. elegans con Salmonella typhimurium, junto con la técnica de RNAi interferencia de doble hebra para examinar el papel de los genes del huésped en la defensa contra la infección por Salmonella. 

Video: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Una PCR para la identificación de Allelotyping Salmonella enterica Serotipos Enteritidis, Hadar, Heidelberg, y Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Investigación

Inmunología e Infección

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.

Ensayo de alto rendimiento para Fenotipo<em&gt; Salmonella enterica</em&gt; Asociación Typhimurium, Invasión y replicación en macrófagos

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Un protocolo fácil de generar proteínas Site-Specifically acetiladas en Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

A High-throughput Platform for the Screening of 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 spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

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

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una plataforma de alto rendimiento para la detección de 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 ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

Evaluación de la virulencia y patogenia de la infección por Aeromonas en un modelo elegans de Caenorhabditis

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Aplicación de Perturbaciones Consistentes Similares al Masaje en Los terneros de ratón y Monitoreo de los Cambios de Presión Intramuscular Resultantes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

Usando organoides intestinales derivados de células madre pluripotentes inducidos por humanos para estudiar y modificar la protección de células epiteliales contra Salmonella y otros patógenos

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

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.

Infección crónica por salmonela fibrosis intestinal inducida

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

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

NF-&amp;#954;B-dependent Luciferase Activation and Quantification of Gene Expression in &lt;em&gt;Salmonella&lt;/em&gt; Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

Activación y cuantificación de la expresión génica dependiente de NF--B en células de cultivo de tejido infectado por Salmonella

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...