Este trabajo fue financiado por el Wellcome Trust (WT 0.795.880).

El protocolo siguiente se describe el estudio de la invasión de las células endoteliales por S. aureus, pero en teoría puede ser utilizada para estudiar la invasión celular por cualquier bacteria cultivables. Etapas específicas de S. Las células endoteliales aureus y se indican. 1. Preparación de las bacterias <ol><li> Cultura S. por cepas de Staphylococcus 04.16 h (en función de la fase de crecimiento es necesario) en 10 ml de infusión cerebro-corazón (BHI) caldo de cultivo a 37 ° C en el aire con agitación a 200 rpm. Estas condiciones de crecimiento son específicos para S. aureus y pueden necesitar ser adaptado para otras bacterias. </li><li> Lávese las bacterias en tres ocasiones en la Dulbecco modificado de Eagle (DMEM; Invitrogen) por rondas alternas de centrifugación a temperatura ambiente (5.000 x g, 10 min), la eliminación del sobrenadante del cultivo y la resuspensión del sedimento bacteriano en un volumen equivalente de DMEM. Medir la densidad óptica de la suspensión resultante de bacterias que se pueden ajustar según sea necesario. Para S. aureus, preparamos una suspensión a OD 600 = 1, que corresponde a ~ 10 9 ufc ml -1. </li></ol> 2. Cultivo de células endoteliales <ol><li> La cultura de la línea de células endoteliales EA.hy926 1 en DMEM suplementado con suero bovino fetal (FBS; 10%) y L-glutamina (2 mM) a 37 ° C en CO2 al 5%. Por otra parte, se agruparon las células humanas primarias endoteliales de vena umbilical (HUVEC) se pueden adquirir de Lonza (Basilea, Suiza) y se cultivaron en un medio endotelial basal suplementado con FBS al 2%, extracto de cerebro bovino (incluida la heparina), el factor humano de crecimiento del endotelio y la hidrocortisona a 37 ° C en 5% de CO 2 de acuerdo con las instrucciones del fabricante (Lonza). Estas condiciones de crecimiento son específicos para estas células y pueden necesitar ser adaptados para otros tipos de la célula huésped. </li><li> Se cultivan las células endoteliales en frascos T75 para completar la confluencia, verificado por los ojos. </li><li> Preparar las placas de 24 pozos para la inserción de los cubreobjetos. Pinzas finas (llama esterilización) se necesitan para mover el cubreobjetos, que tienen una superficie brillante y opaco uno. Cubreobjetos a cabo en los pozos de la placa de 24 pocillos con la superficie opaca hacia arriba para permitir la adhesión celular. </li><li> Liberar a las células del frasco T75 con 3 ml de tripsina-EDTA (0,25%) y añadir 10 ml del medio de cultivo correspondiente. </li><li> Añadir 500 l de células se resuspendió en 24 y placas que contienen cubreobjetos Thermanox vidrio. Un frasco T75 confluentes de células proporcionan suficientes células por dos placas de 24 pozos, lo que resulta en aproximadamente 5 × 10 5 células por pozo (en 500 l medio). </li><li> Incubar las placas durante 48 horas como se describe anteriormente, y verificar 100% de confluencia de células al microscopio de luz invertida. </li><li> Dip de lavado cubreobjetos en PBS y añadir nuevas placas de 24 pocillos que contiene 490 l MEDM con 10% de SFB en cada pozo. </li><li> Para examinar el papel de determinados procesos metabólicos en la invasión celular, los inhibidores se pueden añadir a las células cultivadas 1 h antes de la adición de las bacterias y las concentraciones de mantener durante el ensayo. Por ejemplo, para determinar el papel de la reorganización de la actina en S. aureus invasión de las células endoteliales, 50 M citocalasina D se pueden añadir, o para el papel de caveolas, 5 mM metil-β-ciclodextrina se puede añadir. </li></ol> 3. La invasión de ensayo <ol><li> Añadir 10 l de bacterias lavado (aproximadamente, 2 x 10 7 ufc ml -1 S. aureus) a cada pocillo con un cubreobjetos se lavaron con una capa confluente de células endoteliales en 490 l DMEM con 10% de SFB. </li><li> Incubar durante 15-90 minutos a 37 ° C en 5% de CO 2. </li><li> Para medir el número total de bacterias asociadas con las células (adherente e internalizado), baño de lavado de los cubreobjetos tres veces en PBS y se añade a los pozos nuevos con 500 l 0,5% de Triton X-100 en PBS. Para asegurarse de que las células totalmente lisis y la liberación de todas las bacterias internalizadas, pipeta varias veces, señalando que la punta de la pipeta directamente en la superficie del cubreobjetos. </li><li> Enumerar las bacterias en placas de la suspensión líquida (o diluciones de esta en caso necesario) sobre la superficie de las placas de TSA. Desde TX-100 se lisar muchas bacterias Gram-negativas, la saponina se puede utilizar en lugar de dos. </li><li> Para medir el número de bacterias internalizadas, separar el sobrenadante de cultivo de cada bacteria no consolidados y que contiene y reemplazar con 500 l de FBS DMEM/10% suplementado con 200 mg ml -1 gentamicina. Nosotros usamos la gentamicina en lugar de lysostaphin aquí, ya que es más barato y nos permite cambiar entre los experimentos con diferentes tipos de bacterias (por ejemplo, los estafilococos y Lactococci) sin tener que cambiar el protocolo experimental. </li><li> Incubar las placas a 37 ° C en 5% CO 2 durante 60 minutos para matar todas las bacterias extracelulares. </li><li> Lave cubreobjetos 3 veces en PBS,lisis y enumerar en placas TSA a la descrita para el ensayo de adhesión anteriormente. </li><li> En algunos casos puede ser preferible contar visualmente el número de bacterias mediante microscopía de luz. En este caso, y específico para S. aureus invasión celular, el uso lysostaphin (10 mg ml -1) en lugar de gentamicina para destruir físicamente las bacterias extracelulares. </li><li> Incubar cubreobjetos de 20 minutos a 37 ° C en CO 2 en la solución de lysostaphin, luego enjuague y fijar con Cytopath (Cellpath). </li><li> Inundar el cubreobjetos con cristal violeta (0,5% w / v) durante 5 minutos. </li><li> Dip-enjuague con agua, aire seco y montaje en portaobjetos de vidrio. El número de bacterias por mm 2 de las células endoteliales confluentes se puede cuantificar mediante microscopía de luz. </li></ol> 4. Los resultados representativos: Expresión de proteínas de unión a la fibronectina (FnBPA y FnBPB) en la superficie de S. aureus confiere la capacidad de invadir las células endoteliales. Un trabajo reciente ha redefinido el dominio de unión a fibronectina de FnBPA 3. De tipo salvaje (WT) S. aureus 8.325,4 invade las células endoteliales con una alta eficiencia, mientras que una cepa que carecen de ambos FnBPA y B (Δ FNB) mostraron niveles significativamente reducidos de internalización (Figura 1). Complementación del mutante con un plásmido que codifica una FNB menos el dominio de unión a fibronectina (pFnR0) no promover la invasión (Figura 1). Por el contrario, la complementación de los mutantes con un plásmido que codifica la FNB todo un gen (pFnBA4) restauró la invasión de los niveles de WT (Figura 1). El papel de FnBPA en la invasión de las células endoteliales también se puede demostrar utilizando la expresión heteróloga Lactococcus lactis anfitrión. Expresión de plásmidos codifican FnBPA en L. lactis (pRM9 9) no mejoran significativamente la adhesión a células endoteliales en comparación con las bacterias que expresan no FnBPA (CTL) (bares abiertos, Figura 1B). Por el contrario, FnBPA expresan L. lactis invadido las células endoteliales a niveles significativamente más altos que la cepa no expresa (bares cerrados, Figura 1B). Los experimentos se llevaron a cabo cuatro veces por duplicado y la media ± desviación estándar se presenta. * Representan los datos que son estadísticamente significativa (p = &lt;0,05) de los valores de WT o CTL. <img src="/files/ftp_upload/2693/2693fig1.jpg" alt="Figura 1" /> Figura 1. La invasión de EA. Hy926 las células endoteliales de S. aureus (A) o L. lactis (B).

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	<li>Edgell, C. J., McDonald, C. C., Graham, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc. Natl. Acad. Sci. U. S. A. 80, 3734-3737 (1983).</li><li>Makino, S., van Putten, J. P., Meyer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+variation+of+the+opacity+outer+membrane+protein+controls+invasion+by+Neisseria+gonorrhoeae+into+human+epithelial+cells.">Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.</a> EMBO J. 10, 1307-1315 (1991).</li><li>Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Gurusiddappa, S., Kim, J. H., Pilka, E. S., Briggs, J. A., Gough, T. S., Höök, M., Campbell, I. D., Potts, 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=Pathogenic+bacteria+attach+to+human+fibronectin+through+a+tandem+beta-zipper.">Pathogenic bacteria attach to human fibronectin through a tandem beta-zipper.</a> Nature. 423, 177-181 (2003).</li><li>Magoffin, R. L., Spink, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protection+of+intracellular+Brucella+against+streptomycin+alone+and+in+combination+with+other+antibiotics.">The protection of intracellular Brucella against streptomycin alone and in combination with other antibiotics.</a> J. Lab. Clin. Med. 37, 924-930 (1951).</li><li>Mandell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+intraleukocytic+bacteria.">Interaction of intraleukocytic bacteria.</a> Antimicrob. Agents Chemother. 52, 1673-1679 (1973).</li><li>Vaudaux, P., Waldvogel, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+antibacterial+activity+in+the+presence+of+human+polymorphonuclear+leukocytes.">Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes.</a> Antimicrob. Agents Chemother. 16, 743-749 (1979).</li><li>De Melo, M. A., Pechere, J. 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<html> El ensayo se describe se basa en el ensayo de protección de la gentamicina, que se ha utilizado ampliamente para estudiar la invasión de las células huésped por las bacterias. Las observaciones de la incapacidad de los antibióticos gentamicina y otros para matar las bacterias intracelulares se utilizaron en los primeros estudios sobre la invasión de las células huésped por 4-8 bacterias. El uso de 24, 48 o incluso 96 y placas permite la generación de grandes cantidades de datos cuantitativos y reproducibles en un corto período de tiempo ya un costo relativamente bajo. El ensayo también es atractivo porque no requiere de equipos especializados, que pueden ser adaptados para trabajar con varias bacterias y células, y puede ser utilizado para estudiar el papel de los procesos celulares bacterianas y de acogida en la invasión 9. De hecho, desde los primeros experimentos, el ensayo de protección de la gentamicina se ha empleado ampliamente para medir la invasión de la célula huésped por una serie de diferentes bacterias o incluso combinaciones de bacterias 10,11. Si bien los principios básicos son las mismas variaciones, muchas sutiles en el método se han reportado. En este artículo hemos descrito nuestra versión e indicó que las modificaciones que sean necesarias para otras bacterias o las células huésped. Al diseñar un experimento para medir la invasión de la célula huésped utilizando este enfoque, hay una serie de puntos importantes a considerar: Sensibilidad bacteriana a la gentamicina. Esto puede parecer obvio, pero es importante asegurarse de que la bacteria se está estudiando es susceptible a la gentamicina a la concentración, la temperatura y el tiempo de duración que se utilizará. En el caso de falta de sensibilidad, puede ser posible usar otros antibióticos 5. Un enfoque alternativo puede ser el uso de enzimas líticas (lysostaphin, mutanolisina, lisozima) 12. La incubación y el inóculo. Al realizar esta prueba por primera vez es importante para establecer los tiempos de incubación óptimo y inóculo bacteriano que se utilizará. Por lo tanto, los experimentos iniciales debe examinar tanto la adherencia y la invasión a través del tiempo (5 min a un máximo de 6 h). También es importante para evaluar la invasión se ve afectada por la multiplicidad de infección (MOI, el número de bacterias por célula). En general, lo mejor es utilizar el menor número de bacterias es posible ya que esto reducirá el daño a las células cultivadas. Importantes diferencias entre las cepas se puede perder si durante largos períodos de incubación o inóculos grandes se utilizan 9,13. El daño celular. Es importante lavarse las bacterias antes de su uso. Sin embargo, el daño celular no se limita a la producción de toxinas. La invasión bacteriana, la inducción de la apoptosis y la alteración de las funciones celulares pueden causar daño celular. Esto puede llevar a la penetración de la gentamicina en la célula, matando a las bacterias internalizadas y dando la impresión de que la invasión no se ha producido 14. Las condiciones de incubación. La temperatura y la composición del medio de cultivo puede tener un impacto significativo en la invasión. Por ejemplo, la invasión de las células HeLa por Streptococcus pyogenes depende de la presencia de fibronectina soluble, la cual está normalmente presente en el suero 15. Otra consideración es el crecimiento de bacterias en el medio de cultivo durante el transcurso del experimento. Cultura y cuantificación de bacterias intracelulares. A pesar de TX-100 no puede matar a las bacterias intracelulares, que pueden inhibir su crecimiento. Como tal, es importante comprobar la replicación bacteriana en el medio sólido en presencia del detergente. Cuando existan, puede ser posible utilizar concentraciones más bajas del detergente o usar una alternativa como la saponina. Una de las ventajas de la protección de ensayo gentamicina sobre el cristal violeta y el ensayo de microscopía es que es menos mano de obra, y permite la detección y diferenciación de las sub-poblaciones de bacterias internalizadas. Por ejemplo, S. aureus puede producir un tipo de colonia normal (NCT), o la variante pequeña colonia (SCV) 16, que no se distinguiría por microscopía de las células bacterianas individuales. Una de las ventajas de la tinción de cristal violeta y el ensayo de microscopía en el ensayo de protección de la gentamicina es aglutinación de células pueden ser tenidos en cuenta y que las bacterias intracelulares que introducir una &quot;viables pero no cultivables&quot; voluntad estatal se detectaron 17. Examinar el papel de los procesos de la célula huésped en la invasión bacteriana. Muchos estudios han utilizado inhibidores de la función de la célula huésped, como la citocalasina D, que interfiere con la reorganización de actina. Estos estudios también pueden incluir anticuerpos que se dirigen a los receptores de la superficie celular y caída siRNA de genes específicos 18. Es de vital importancia para garantizar que estos tratamientos no afectan a la viabilidad o la unión de las células cultivadas o tener efectos tóxicos sobre las bacterias. Orientación futura: CONTENIDO &quot;&gt; Debido a que este ensayo se realiza típicamente en múltiples placas de cultivo celular, es teóricamente posible adaptarlo a una operación de selección de alto rendimiento, lo que podría implicar el examen de las bibliotecas de los posibles inhibidores de la función celular, antiinfecciosos o antibióticos diseñados para atacar las bacterias intracelulares. Por otra parte, este enfoque podría utilizarse para detectar bibliotecas mutantes para identificar genes implicados en la adhesión, la invasión o la supervivencia intracelular. En algunos casos puede ser deseable incluir las fuerzas de corte (de flujo) en el sistema modelo, por ejemplo, cuando se modela el endotelio, para imitar más de cerca el ambiente en vivo. Los avances actuales en el diseño y la fabricación de celdas de flujo y sistemas de cultivo celular de microfluidos ofrecer la posibilidad de emplear el ensayo de protección de la gentamicina en huésped-patógeno modelos que incorporan las fuerzas de cizalla. En resumen, el protocolo descrito aquí se basa en métodos similares usados ​​durante muchos años. Que es ampliamente aplicable a muchos tipos de células cultivadas y especies de bacterias y puede generar grandes cantidades de datos de forma rápida ya un costo relativamente bajo.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31885-023</td>
<td>&nbsp;</td>
<tr>
<td>Brain heart infusion</td>
<td>&nbsp;</td>
<td>BioChemika</td>
<td>53286</td>
<td>&nbsp;</td>
<tr>
<td>Foetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>HUVECs</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-2519</td>
<td>&nbsp;</td>
<tr>
<td>Endothelial basal medium</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3121</td>
<td>&nbsp;</td>
<tr>
<td>Bovine brain extract (including heparin)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092</td>
<td>&nbsp;</td>
<tr>
<td>Human endothelial growth factor</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4017C</td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4035C</td>
<td>&nbsp;</td>
<tr>
<td>GA-1000</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092C</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Lysostaphin</td>
<td>&nbsp;</td>
<td>AMBI</td>
<td>LSPN-50</td>
<td>&nbsp;</td>
<tr>
<td>Thermanox coverslips</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>TKT-210-330P</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Cytopath</td>
<td>&nbsp;</td>
<td>TCS Biosciences</td>
<td>HC8595</td>
<td>&nbsp;</td>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3886</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4049</td>
<td>&nbsp;</td>
<tr>
<td>T75 flasks</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>430641</td>
<td>&nbsp;</td>
<tr>
<td>Cytochalasin D</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2618</td>
<td>&nbsp;</td>
<tr>
<td>Methyl-B-cyclodextrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>332615</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

invasion of human cells by a bacterial pathogen

Aquí vamos a describir la forma en que el estudio de la invasión de las células endoteliales humanas por Staphylococcus aureus patógeno bacteriano. El protocolo general se puede aplicar al estudio de la invasión de las células de prácticamente cualquier bacteria cultivables. Las etapas en las que los aspectos específicos de la invasión se puede estudiar, como por ejemplo el papel de la reorganización de actina o caveolas, se destacó. Las células hospedadoras se cultivan en frascos y cuando esté listo para su uso se siembran en placas de 24 pocillos que contienen cubreobjetos Thermanox. Utilizando cubreobjetos permite su posterior eliminación de las células de los pozos para reducir la interferencia de las proteínas del suero deposita en las paredes de los pozos (a la que se conceden por S. aureus). Las bacterias se cultivan a la densidad requerida y se lavan para eliminar cualquier proteínas secretadas (por ejemplo, toxinas). Cubreobjetos con capas confluentes de células endoteliales son transferidos a nuevas placas de 24 pocillos que contienen medio de cultivo fresco antes de la adición de bacterias. Las bacterias y las células se incuban juntos por la cantidad de tiempo requerido en el 5% de CO 2 a 37 ° C. Para S. aureus suele ser entre 15-90 minutos. Cubreobjetos Thermanox se eliminan de cada pozo y la inmersión se lavan en PBS para eliminar las bacterias sin ataduras. Si el total de bacterias asociadas (adherente e internalizado) se cuantifica, cubreobjetos se colocan en un lugar bien fresco que contiene 0,5% de Triton X-100 en PBS. Pipeteo suave lleva a completar la lisis celular y las bacterias son enumerados por la dilución de serie y placas en agar. Si el número de bacterias que han invadido las células que se necesita, cubreobjetos se añaden a los pocillos con 500 l medio de cultivo de tejidos suplementado con gentamicina y la continuación de incubación de 1 h, lo que va a matar todas las bacterias externas. Cubreobjetos puede ser lavado, las células lisadas y las bacterias enumeradas en placas en agar como se describió anteriormente. Si el experimento requiere la visualización directa, cubreobjetos se fijaron y se tiñeron para microscopía óptica, fluorescencia o confocal o preparados para microscopía electrónica.

Watch this Scientific Journal Video about Invasion of Human Cells by a Bacterial Pathogen at JoVE.com

Invasion of Human Cells by a Bacterial Pathogen

Un protocolo general para el estudio de la invasión de las células huésped por un patógeno bacteriano, centrándose en<em&gt; Staphylococcus aureus</em&gt; Y las células endoteliales humanas.

La invasión de las células humanas por un patógeno bacteriano

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

invasion-of-human-cells-by-a-bacterial-pathogen

Research

JoVE Journal

Immunology and Infection

34.6K vistas.  University of Bath. Un protocolo general para el estudio de la invasión de las células huésped por un patógeno bacteriano, centrándose en Staphylococcus aureus Y las células endoteliales humanas.

Video: Invasion of Human Cells by a Bacterial Pathogen

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

La aplicación de un ratón ligado Ensayo placas de Peyer asa intestinal para evaluar la absorción por las células bacterianas M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Investigación

Inmunología e Infección

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Un día de flujo de trabajo Método de detección de patógenos bacterianos y pruebas de resistencia a los antimicrobianos de cultivos de sangre

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Entérica invasión bacteriana de las células epiteliales intestinales<em&gt; In Vitro</em&gt; Se ha mejorado considerablemente gracias a un modelo de cámara de difusión vertical

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC secreción de investigar la infección celular por el patógeno bacteriano<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

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.

La separación automatizada de<em&gt; C. elegans</em&gt; Variablemente colonizada por un patógeno bacteriano

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

La evaluación de la innata de detección de VIH-1 CD4 infectados<sup&gt; +</sup&gt; Células T por las células dendríticas plasmocitoides Usando un<em&gt; Ex vivo</em&gt; Co-cultura del sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Desenredar la función de un efector bacteriana de una planta de patógenos no cultivables Uso de una levadura de dos híbridos pantalla

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.