Este trabajo fue financiado por el Wellcome Trust subvención WT091939MA. Alain Filloux con el apoyo de la Royal Society.

1. Cepas bacterianas y Culturas <ol><li> Un ingeniero de Escherichia coli célula receptora (Prey, P) mediante la transformación (utilizando el estándar de CaCl2 tratamiento o electroporación) 15 E. coli DH5a células con un plásmido que permite la complementación de una-el gen lacZ (Tabla 1). Placa de las células transformadas en placas de agar Luria-Bertani (LBA, 1,5% de agar) que contenía 5-bromo-4-cloro-indolil-β-D-galactopiranósido (X-gal) a 40 mg / ml de concentración final y adecuada de antibióticos. Incubar a 37 ° C durante la noche y la mañana siguiente seleccionar los transformantes azules (véase § 1,3). </li><li> Cultivar las células de Pseudomonas aeruginosa que son donantes T6SS-activo (D +) o inactivo T6SS-(D-) (Tabla 1) durante la noche en LBA a 37 ° C. En el ejemplo presentado aquí se utilizó una P. RETS aeruginosa cepa que poseeun H1-T6SS constitutivamente activa 16 y un mutante isogénica con una deleción del grupo de genes H1-T6SS (Tabla 1). </li><li> El día siguiente, preparar un cultivo líquido durante la noche en un matraz de aireado por inoculación de un clon azul brillante entre la E. transformantes coli (P) de la placa descrita en § 1,1, en ​​5 ml de caldo de soja tríptico (TSB) suplementado con el antibiótico apropiado. Crecer con agitación a 37 ° C. Proceder igualmente con una única colonia de (D +) y de (D -) de la placa descrita en § 1,2. </li></ol> 2. Concurso de Ensayo <ol><li> Preparar placas de LBA para el experimento al día siguiente. Asegúrese de que estas placas se secan adecuadamente (ya sea cerca del quemador Bunsen o en el gabinete de flujo laminar). Preparar un &quot;Ensayo de entrada&quot; (A-Input) placa para las cepas (D +), (D -), y (P </strong&gt;). Preparar un &quot;Ensayo de salida&quot; (A-salida) placa para las cepas (D - + P) y (D + + P). Dividir y marcar las placas en consecuencia. </li><li> Después de una noche de crecimiento (véase el § 1,3), medir la densidad óptica (OD 600 nm) del cultivo bacteriano de entrada (D -, D + y P) y calcular el volumen necesario para obtener una densidad equivalente a 1 unidad celular OD 600 nm de cada cepa . Cada &quot;entrada&quot; de cultivo celular (D -, D + y P) inicialmente se recoge en un tubos estériles Eppendorf de 1,5 ml. </li><li> Centrifugar las muestras de bacterias a 13.000 rpm durante 1 min a temperatura ambiente y descartar el sobrenadante. </li><li> Resuspender los pellets de la D -, D + culturas, y P, en 100 l de TSB fresco por pipeteo suave y inocular 10 ml de cada correspondiente presión de un solo punto en el &quot;A-entrada&quot; placa (preparado en el § 2.1). </li><li> Inocular el &quot;A-output&quot; placa (preparado en el § 2.1). Más precisamente, mezclar suavemente 30 l de (D +) con 30 l (P) y 30 l de (D -) con 30 ml (P) separados en dos tubos Eppendorf (nota que los cultivos utilizados son los descritos en § 2,4) . Inocular 20 l de las mezclas (D + / P) y (D - / P) como puntos individuales en la &quot;A-salida&quot; placa. Deje secar las proximidades de un mechero de Bunsen y coloque la placa en una incubadora a 37 ° C durante un período adecuado de tiempo durante el cual la destrucción bacteriana está teniendo lugar. En el caso de P. aeruginosa, una eficiente destrucción bacteriana se observa después de 5 horas de incubación cuando se compara una T6SS activos con una cepa deficiente T6SS. </li></ol> 3. Observación cualitativa de tél Killing bacteriana <ol><li> Preparar placas de LBA que contenían 40 ug / ml de X-gal para la lectura del ensayo. Las placas se llamará &quot;Lectura de entrada&quot; o &quot;R-entrada&quot; para detectar bacterias aisladas o &quot;Salida de Lectura&quot; o &quot;R-salida&quot; para detectar cultivos mixtos de bacterias y por lo tanto leer el desempeño matando. Estas placas también deben ser bien seca. Divida el plato en cuatro partes iguales en la espalda y anotar estas partes 0, 10 -1, 10 -2 y -3 10, la designación de las diluciones del cultivo bacteriano de detectar en las placas de agar (véase § 3.3). La dilución permitirá una evaluación semi-cuantitativa de la relación azul / blanco de las bacterias en el mismo lugar. </li><li> Recoger con un asa estéril las manchas bacterianas individuales de la &quot;A input-&quot; la placa (ver § 2.4) y &quot;A-output&quot; placa (ver § 2.5) y resuspender cada lugar en distintos tubos Eppendorf de 1,5 ml que contienen 1 ml de TSB. Coloque en un bloque de Eppendorf agitador durante 30 minutos para volver a suspender de manera eficiente elbacterias. </li><li> Preparar una serie de 5 veces 3 tubos Eppendorf que contiene 900 l de TSB y seguir por cada punto resuspendido a diez veces diluciones seriadas hasta 10 -3. Asegúrese de cambiar las puntas de pipeta y vórtice 5 segundos entre cada paso de dilución. </li><li> Proceder a la localización de sus diluciones bacterianas preparadas en el § 3,3 en bien secos placas de LBA que contenían 40 ug / ml de X-gal por empezando por la más diluida a la suspensión diluida, por lo que estos la &quot;entrada de R-&quot; placas para el &quot;A de insumos y los &quot;puntos de salida&quot; R &quot;para las&quot; placas de salida A-&quot;puntos (Figura 2). Vórtice brevemente cada tubo antes de proceder a la localización para mantener una suspensión bacteriana homogénea. Para la reproducibilidad de los resultados, punto 20 l en triplicado dentro de un cuadrante (Figura 2). La sequedad relativa de la placa es importante en esta etapa ya que las manchas deben permanecer separados individualmente en el plato y no se deslizan el uno hacia el otro. Una cuantificación del ensayo de competición también es posible en esta fase del experimento. Tras la etapa de 3,3, se extendió 100 l de la dilución 10 -3 en placas de LBA que contenían 40 ug / ml de X-gal. Para la reproducibilidad de los resultados, proceder a un recubrimiento por triplicado para cada punto de la &quot;A-output&quot; plato. Colocar las placas de dilución en una incubadora ° C durante la noche (correspondiente a 16 horas de incubación) 37. Proceder al recuento de las colonias azules (las células que quedaban vivos P). Los resultados típicos obtenidos después de 5 horas de competencia se muestran en la Figura 3. </li><li> Dejar la placa abierta (sin tapa) dentro de una zona estéril para permitir la absorción del líquido en exceso dentro de cada mancha. </li><li> Colocar la placa en una incubadora a 37 ° C durante la noche. Durante este tiempo de incubación, la matanza todavía se producen en la mezcla (D + / P) puesto que ambas cepas están todavía en contacto. </li><li> Tome una fotografíao escanear sus placas para el análisis de la producción (Figura 2). Cuando las manchas permanecen en gran parte azul indica que E. coli no ha sido asesinado por P. aeruginosa. Este es el caso cuando E. coli se mezcla con un p-T6SS defectuoso Aeruginosa cepa (D-) (figura 2, la placa en la parte inferior derecha). </li></ol>

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No tenemos nada que revelar.

El método presentado en este artículo permite la observación visual de T6SS actividad bactericida mediada / bacteriostasis. El ensayo se realiza en la superficie de una placa de agar. Se ha demostrado previamente que T6SS dependiente de ensayo de destrucción realiza con cultivo mixto líquido bacteriano no es eficiente, probablemente debido a la falta de contacto constante entre las dos bacterias 8. El T6SS se cree que funcionan con un mecanismo similar al utilizado por los bacteriófagos para inyectar ADN en células diana 17. En la cultura líquida, la estructura tubular de la T6SS pueden romper con más facilidad, entre otras-bacterial contacto se pueden perder, y las toxinas no se entregan de manera eficiente. En términos de tiempos de incubación, las 5 horas iniciales de contacto que describimos entre la cepa donante y la presa son suficientes para observar la destrucción bacteriana entre P. aeruginosa y E. coli, como se ilustra en la figura 3 </strong&gt;. Sin embargo, es conveniente ajustar el tiempo de incubación mediante la realización de una cinética con el fin de optimizar las condiciones experimentales. Dado que este método es una técnica basada en el color, los resultados de salida puede verse comprometida por la pigmentación de la cepa donante. Por ejemplo, en el caso de P. aeruginosa, algunas cepas producen altos niveles de pigmentos coloreados tales como piocianina y pyoverdine, que pueden interferir con la lectura del ensayo, haciendo que la distinción de la presa relativamente difícil. Otros cromógenos galactosidasa β-sustratos, tales como el magenta-gal o el rojo-gal, se puede utilizar en lugar de la X-gal (Tabla 1). El ensayo de competición se puede hacer uso de otros genes indicadores para la lectura. Por ejemplo, ensayo similar también se ha realizado mediante el uso de proteína verde fluorescente marcada con presas 12. Nuestro ensayo, mientras que no cuantitativa, da una buena indicaciónde la actividad T6SS ya que se basa en la supervivencia o la muerte de una presa reportero. Esta técnica presenta la ventaja de ser fácil y conveniente para evaluar la actividad bactericida / bacteriostasis de T6SSs de cualquier especie bacteriana. Hasta ahora, la actividad de la T6SS se ha demostrado contra bacterias Gram-negativas y no hay ningún ejemplo claro de T6SS sensibles a bacterias Gram-positivas se ha informado todavía 12. También es obvio que la incompatibilidad en la cultura de las diferentes especies bacterianas a prueba (por ejemplo la temperatura de crecimiento, la oxigenación, los medios de comunicación específicos) es para ser considerada. Nuestro ensayo también se puede utilizar para evaluar cuál de los componentes T6SS son absolutamente esenciales ya que incluso trazas de una toxina secretada podría ser suficiente para matar a la presa. Incluso débil actividad de los T6SS entonces podría claramente ser detectado por nuestro ensayo en comparación con el procedimiento estándar de ensayo T6SS dependiente de la secreción utilizando blot sobrenadante de cultivo y occidentalanálisis. Sin embargo, una adecuada unidad formadora de colonias (CFU) recuento todavía se requiere para la cuantificación exacta de esta actividad T6SS.

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> P. aeruginosa PAKΔ TER (D +) (T6SS activos) </td><td> Laboratorio de tensión </td><td></td><td> Descrito en la Referencia 16 </td></tr><tr><td> P. aeruginosa PAKΔ RETS Δ H1-T6SS (D -) (T6SS inactivos) </td><td> Este estudio </td><td></td><td> El clúster H1-T6SS (que comprende los genes PA0070 a PA0095) ha sido borrado por intercambio alélico siguiendo el procedimiento descrito en la Referencia 18. El fragmento mutador fue generado con el siguiente conjunto de cebadores: Los cebadores fragmento Subir: 5&#39;-ATGGTCAACGACATGGAGCTGGAG-3 &#39;, y 5&#39;CGAGGCCGATCAGGCCTTCAGAACTGA-3 &#39;. Los primers de fragmentos de Down: 5&#39;-TCAGGCCTTCAGAACTGAAGCGGCGCA-3 &#39;, <pre> 5&#39;-GGTGGCGTTCAACAGTTCCATGTC-3 &#39; </pre></td></tr><tr><td> E. coli DH5a </td><td> Invitrogen </td><td> 18258-012 </td><td> F-φ80 lac ZΔM15 Δ (lac ZYA-arg F) U169 rec A1 A1 final hsd R17 (r k -, m k +) pho A sup E44-thi λ -1 gyr A96 rel A1 </td></tr><tr><td> pBluescript II SK (+) </td><td> Agilent </td><td> 212205 </td><td> Este vector expresa el péptido α de β-galactosidasa utilizado para α-complementación. </td></tr><tr><td> X-gal </td><td> Invitrogen </td><td> 15520-018 </td><td> Utilizar en 40 ug / ml </td></tr><tr><td> Agar Luria Bertani </td><td> Merck-químicos </td><td> 1.10283.0500 </td><td></td></tr><tr><td> TSB (caseína de soja caldo) </td><td> Oxoid </td><td> CM109 </td><td></td&gt; </tr><tr><td> Vortex agitador Genius 3 </td><td> IKA </td><td> 3340000 </td><td></td></tr><tr><td> Escáner </td><td> Epson </td><td> V700 </td><td></td></tr><tr><td> Espectrofotómetro </td><td> WPA Biowave </td><td> CO8000 celular densímetro </td><td></td></tr><tr><td> Magenta-gal </td><td> Bioworld </td><td> 30350001-1 (715241) </td><td></td></tr><tr><td> Red-gal </td><td> Research Organics </td><td> 1364c </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 1. Strain, material de plásmido, y el reactivo usado. </td></tr></tbody></table>

a visual assay to monitor t6ss-mediated bacterial competition

Sistemas de secreción de tipo VI (T6SSs) son nanomáquinas moleculares que permiten bacterias Gram-negativas para transportar e inyectar proteínas en una amplia variedad de células diana 1,2. El T6SS está integrado por 13 componentes principales y muestra similitudes estructurales con el tubo de cola de bacteriófagos 3. El fago se utiliza un tubo y un dispositivo de punción para penetrar la envoltura celular de las bacterias diana e inyectar ADN. Se propone que la T6SS es un dispositivo de bacteriófago invertida crear una ruta específica en la envoltura celular bacteriana para conducir efectores y toxinas a la superficie. El proceso podría continuarse y el dispositivo T6SS podría perforar otras células con la que la bacteria está en contacto, con lo que la inyección de los efectores en estos objetivos. El tubo de cola y partes de punción del dispositivo de las T6SS se hacen con Hcp y proteínas VgrG, respectivamente, 4,5. La versatilidad de la T6SS se ha demostrado a través de sos estudios con diversos patógenos bacterianos. Los T6SS Vibrio cholerae puede remodelar el citoesqueleto de las células huésped eucariotas mediante la inyección de un &quot;evolucionado&quot; VgrG llevando una actina C-terminal de dominio de reticulación 6,7. Otro ejemplo notable se documentó recientemente usando Pseudomonas aeruginosa, que es capaz de atacar y matar a las bacterias de una manera T6SS-dependiente, por lo tanto, promover el establecimiento de bacterias en nichos microbianos específicos y el entorno competitivo 8,9,10. En este último caso, tres T6SS proteínas secretadas, a saber Tse1, Tse2 y TSE3 han sido identificados como las toxinas inyectadas en las bacterias diana (Figura 1). La célula donante está protegida de los efectos perjudiciales de estos efectores a través de un mecanismo de anti-toxina, mediada por las proteínas de la inmunidad Tsi1, Tsi2 y Tsi3 8,9,10. Esta actividad antimicrobiana se puede controlar cuando T6SS que dominan las bacterias son co-cultivadas en sOlid superficies en competencia con otras especies bacterianas o con T6SS-inactivos bacterias de la misma especie 8,11,12,13. Los datos disponibles hincapié en un método numérico para el ensayo de competición bacteriana, incluyendo el conteo de CFU mucho tiempo y que depende en gran medida de los fabricantes de antibióticos. En el caso de cepas resistentes a antibióticos tales como P. aeruginosa, estos métodos pueden ser inapropiado. Además, con la identificación de unos 200 loci diferentes T6SS en más de 100 genomas de bacterias 14, una herramienta de cribado conveniente es altamente deseable. Hemos desarrollado un ensayo que es fácil de utilizar y necesita material de laboratorio estándar y los reactivos. El método ofrece una técnica rápida y cualitativa para monitorizar la actividad bactericida dependiente de T6SS / bacteriostasis mediante el uso de una cepa reportero como una presa (en este caso Escherichia coli DH5a) permitiendo una complementación del gen lacZ. En general, este método es grapHIC y permite la identificación rápida de T6SS fenotipos relacionados sobre placas de agar. Este protocolo experimental se puede adaptar a otras cepas o especies bacterianas, teniendo en cuenta las condiciones específicas, tales como medios de crecimiento, la temperatura o el tiempo de contacto.

Los resultados típicos se muestran en la Figura 1 con las cepas y reactivos descritos en la Tabla 1. Las placas que se muestran en esta figura se escanea después de una incubación durante la noche. La &quot;entrada&quot; Lectura-placas muestran un patrón de dilución en serie para las cepas utilizadas en este ensayo. Como era de esperar, la E. coli presa puntos (P) que sobreexpresan el gen lacZ aparecen azules en medios suplementados con X-gal, mientras que el P. donante aeruginosa cepas (D +, T6SS activo) y (D -, T6SS inactivo) sigue siendo blanco. Los &quot;resultados&quot; Lectura-placas en que se haya la mezcla entre la presa y una cepa T6SS activo (D + / P) ¿Ha encontrado demostrar la desaparición de la presa azul indicando así que ha sido asesinado. Esto demuestra la capacidad del donante para competir directamente con la presa. La persistencia del color azul en la (D - / P) Placa demuestra la incapacidad de un donante T6SS inactivo para matar a la presa azul. <img src="/files/ftp_upload/50103/50103fig1.jpg" alt="Figura 1"/> Figura 1. Asesinato de E. coli por T6SS-competente P. aeruginosa. P. aeruginosa inyecta toxinas en la E. coli células diana de una manera dependiente de T6SS (mostrada por la flecha blanca). Dos toxinas Tse1 y TSE3 (naranja y círculos rojos) se inyectan en la E. coli periplasma y degradar el peptidoglicano 9. La toxina Tse2 (círculo amarillo) se inyecta en la E. coli citoplasma y tiene una actividad bacteriostática 8,10. La acción combinada de las toxinas mata a las células diana (relámpago y el cráneo). La supervivencia de las células diana puede ser detectada mediante la supervisión de la actividad de la β-galactosidasa producida (véase también la Figura 2). P. uneruginosa está protegido contra la actividad de las toxinas por las proteínas de la inmunidad Tsi1, Tsi2 y Tsi3 (naranja, cuadrados amarillo y rojo, respectivamente) 8,9,10. <img src="/files/ftp_upload/50103/50103fig2.jpg" alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/50103/50103fig2highres.jpg"/> Figura 2. Ensayo en placa de agar para supervisar T6SS dependiente de muerte bacteriana En esta figura se muestran en la parte superior de la &quot;entrada&quot; Lectura-planchas compuestas de las diluciones en serie de la D -., D +, y células de entrada P. Las celdas de entrada P son de color azul debido a la α-complementación del gen lacZ y por lo tanto produce β-galactosidasa que escinde X-gal. En la parte inferior se muestra la &quot;salida&quot; Lectura-cajas placas de diluciones en serie de la mezcla bacteriana entre un activo (D + / P) </strong&gt;, o una forma inactiva (D - / P), T6SS donante P. aeruginosa cepa con la E. coli presa. <a href="https://www.jove.com/files/ftp_upload/50103/50103fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img src="/files/ftp_upload/50103/50103fig3.jpg" alt="Figura 3"/> Figura 3. La cuantificación de la muerte de E. coli por P. aeruginosa después de 5 horas de incubación. El gráfico presenta el recuento de CFU de E. coli se describe en el paso 3,4. Los resultados aquí presentados muestran una diferencia de 3 veces entre el T6SS + y las cepas T6SS-, lo que sugiere que la mayoría de la matanza se lleva a cabo durante las 5 horas iniciales de contacto.

Watch this Scientific Journal Video about A Visual Assay to Monitor T6SS-mediated Bacterial Competition at JoVE.com

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Se describe un ensayo cualitativo para supervisar la competencia bacteriana mediada por el<em&gt; Pseudomonas aeruginosa</em&gt; Tipo VI secreción del sistema (T6SS). El ensayo se basa en la supervivencia / muerte de células de Escherichia coli que llevan un objetivo<em&gt; LacZ</em&gt;-Reportero. Esta técnica es ajustable para evaluar la actividad bactericida / bacteriostasis de T6SS que dominan los microorganismos.

Un ensayo visual para monitorear T6SS mediada por la competencia bacteriana

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

a-visual-assay-to-monitor-t6ss-mediated-bacterial-competition

Research

JoVE Journal

Immunology and Infection

15.5K vistas.  Imperial College London. Se describe un ensayo cualitativo para supervisar la competencia bacteriana mediada por el Pseudomonas aeruginosa Tipo VI secreción del sistema (T6SS). El ensayo se basa en la supervivencia / muerte de células de Escherichia coli que llevan un objetivo LacZ-Reportero. Esta técnica es ajustable para evaluar la actividad bactericida / bacteriostasis de T6SS que dominan los microorganismos.

Video: A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Luciferasa utilizando a las infecciones bacterianas de la imagen en ratones

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Investigación

Inmunología e Infección

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 ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Uso de la superposición de ensayo para medir cualitativamente la producción bacteriana y sensibilidad hacia neumocócica bacteriocinas

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Crecimiento por parejas Ensayo de competición para determinar la aptitud de replicación de virus de inmunodeficiencia Humanos

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Aplicación de Ensayo inmunoensayo de largo plazo culta Interferón-γ ligado a enzimas para la evaluación efectoras y de memoria T respuestas celulares en el ganado

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

La detección de la corteza Fragmentos Durante la germinación de esporas bacterianas

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Evaluación visual y microscópica de mutantes desarrollo de Streptomyces

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

A High-throughput Shigella-specific Bactericidal Assay

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

A High-throughput Shigella-specific Bactericidal Assay

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

Un alto rendimiento Shigella-ensayo bactericida específico

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

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonofagocítico asesinato ensayo para evaluar las respuestas inmunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...