Nos gustaría dar las gracias a Brandon Hassett y Carlos Ortiz por su asistencia técnica. Este trabajo fue apoyado por la NSF subvenciones IOB-0544428, el IOS-0951272, 0925561 y IOS-con el Dr. Michael Kolomiets, y por el Instituto Nacional del USDA de Alimentos y Agricultura (NIFA), Mejoramiento Vegetal y AFRI Educación Grant # 2010-85117 -20539 a los Dres. Seth Murray, Isakeit Thomas y Michael Kolomiets.

1. Bioensayo grano de maíz <ol><li> Dos semanas antes, la cultura de los hongos patógenos en Agar Papa Dextrosa (PDA) a 28 ° C. </li><li> Seleccione granos con un tamaño y forma similar, preferentemente plana por lo que estaba a nivel con la parte inferior de los viales de bioensayo, y el lugar en 50 ml tubos Falcon. Granos seleccionados deben haber sido producidos simultáneamente en un mismo entorno para asegurarse de edad de la semilla o composición similar metabolito. </li><li> Surface esterilizar núcleos agitando los tubos a temperatura ambiente durante 5 minutos con 70% de etanol, 1 minuto con agua estéril, y 10 minutos con hipoclorito de sodio al 6%. Enjuague tres veces, durante 5 minutos cada vez, con agua estéril y agitando continua. Séquelos con toallas esterilizadas de granos. </li><li> Para facilitar la infección de Aspergillus flavus, crear una pequeña herida en el embrión del lado con una aguja de 18 G a la profundidad de 0,5 cm. Sin embargo, en nuestra experiencia una herida más grande es necesario para la infección por Fusarium verticillioides. Therefore, utilizar una hoja de afeitar para cortar en el embrión del lado a una profundidad de 0,5 mm. </li><li> Preparar suspensión del inóculo raspando placas cultivadas en aproximadamente 5 ml de autoclave 0,01% de Tween-20 solución para liberar las esporas y eliminar micelio por filtración a través de la gravedad por lo menos 4 capas de gasa autoclave. Calcular la concentración de esporas con hemocitómetro y ajustar a 10 6 esporas / ml para A. flavus o F. verticillioides. Si se utiliza otra especie de patógeno o huésped, es importante para evaluar las concentraciones de inóculo para la infección apropiado. </li><li> Crear una cámara de humedad en un recipiente de plástico (29,2 cm X 18,7 cm X 8,3 cm) forrada con cinco hojas de toallas de papel y 100 ml de agua estéril. Sala no es de agua o hermético y el agua se debe agregar todo experimento para mantener las toallas de papel húmedo. </li><li> Coloque los cuatro núcleos en un autoclave 20 ml vial de centelleo de vidrio y registrar su masa. Se inoculan con 200 l suspensión de esporas,la tapa, y agitar a los huesos de cubrir bien con la suspensión. Afloje las tapas para permitir el libre intercambio de aire y el lugar en la cámara de humedad. </li></ol> Nota: Para las semillas u hongos distintos de los descritos en estos métodos, la cantidad de inóculo requerida puede ser diferente y debe ser establecido de forma experimental. <ol start="8"><li> Incubar los granos bajo un fotoperíodo 12-h-light/12-h-dark a 28 ° C durante 7 días o hasta que se desee. </li></ol> 2. Enumeración conidios <ol><li> Después del período de infección, añadir 5 ml de autoclave 0,01% de Tween-20 a viales de centelleo que contienen granos infectados y mezclar bien durante 1 minuto. </li><li> Usando una punta de gran calibre pipeta, inmediatamente diluir mediante la transferencia de dos separadas 200 ml de alícuotas de la suspensión de esporas en 2,0 ml tubos Eppendorf conteniendo 1,8 ml de 0,01% de Tween-20 (o diluir como sea necesario dependiendo de la infección). </li><li> Enumerar cada una alícuota de 200 l dos veces con un hemocitómetro 4y comparar los niveles de conidios entre los tratamientos. </li></ol> 3. La cuantificación de aflatoxinas <ol><li> Añadir núcleos a partir de 1 muestra a 50 ml de taza mezcladora con 20 ml de 80% de metanol y 0,05 g de NaCl, la cubierta y la mezcla a alta velocidad durante 1 minuto. </li><li> Colocar un filtro plegado sobre un recipiente de recogida limpia y se vierte en extracto de filtro. </li><li> Transferir 10 ml de filtrado a un recipiente limpio y añadir 20 ml de H 2 O destilada y mezclar bien. </li><li> Filtro de la muestra a través de un filtro de microfibra de vidrio de 1,5 m en una taza limpia. </li><li> Aplicar 1 ml de extracto se filtró a la columna de prueba Afla y, utilizando aire comprimido, forzar el extracto se filtró a través de la columna a una velocidad de 1-2 gotas por segundo hasta vaciados. </li><li> Lavar la columna dos veces con 1 ml de H 2 O destilada y pasar a través de la columna a 1.2 gotas / segundo hasta que el aire llega a través de. </li><li> La columna se eluye con 1 ml de metanol al 100% grado HPLC en una caída de 2.1 / segunda tasa recogida en una cubeta de vidrio. </li><li> Añadir1 ml de desarrollador de pruebas Afla de eluido y mezclar bien. Coloque la cubeta en fluorómetro calibrado y leído en 60 seg. </li></ol> Nota: Cuando se utiliza el protocolo de Aflatest FGIS, la medición de la fluorómetro se calcula en base a un período inicial de 50 gramos de muestra extraída en 100 ml. Si se tiene en cuenta la dilución de la muestra con agua, 1 ml de la aplica a una columna representa 0,166 gramos de muestra. Por lo tanto, al modificar el protocolo, es necesario tener en cuenta las diferencias en el tamaño de la muestra y la solución de extracción inicial. Por ejemplo, 2 gramos de granos se extraen en 20 ml de metanol 80%. Esto es de 0,1 gramos por ml. Cuando 1 ml de esta muestra se mezcló con 2 ml de agua, la proporción de la muestra en el líquido es ahora 0,033 gramos por ml. Si esta muestra da una lectura fluorómetro de 100 ppb, la concentración real se basa en la proporción de la muestra a 0,166, es decir, ppb = (0,166 gramos X 100 ppb) / gramo 0,1), o ppb 166. Para las aflatoxinas alternativa cuantificaciónmétodos de cationes, véase la referencia 7. 4. Fumonisina B1 (FB1) Análisis <ol><li> Cuando hongo se cultiva en granos de maíz, extraer muestras durante la noche con 10 ml de acetonitrilo / agua (50/50, v / v) a temperatura ambiente sin agitación. Posteriormente, mezclar el extracto (2 ml) con agua desionizada (6 ml), y aplicar esta mezcla (8 ml) directamente a la extracción en fase sólida C-18. </li><li> Antes de cargar la muestra, la columna condición de extracción en fase sólida C-18 de un enjuague con 2 ml de acetonitrilo seguido por 2 ml de agua. </li><li> Después de cargar la muestra, la columna se lavó con 2 ml de agua seguido por 2 ml de acetonitrilo / agua (15/85, v / v). Muestra para análisis por HPLC (que contenía FB1) se eluyó con 2 ml de acetonitrilo / agua (70/30, v / v). </li><li> Derivatizar FB1 con o-ftaldehído (OPA) mediante la transferencia de 0,1 ml de eluato de la columna a un vial que contiene 0,1 ml de tampón borato (0,05 M bórico acid/0.05 M de borato de sodio [50/50, v / v], pH 8,5) y 0,1 OPA ml (0,1 mg / ml en acetonaitrile con 0,5% - mercaptoetanol). </li><li> Se detiene la reacción después de 10 minutos mediante la adición de 0,5 ml de acetonitrile/0.01 M de ácido bórico (40/60, v / v). </li><li> Analizar FB1 en un HPLC Shimadzu LC-20AT sistema equipado con una columna analítica Zorbax ODS (4,6 150 mm) y una longitud de onda variable Shimatzu RF-10Axl detector de fluorescencia (excitación 335 nm / emisión 440 nm). </li><li> Un gradiente lineal se utiliza (disolvente A: acetonitrile/0.1 M de fosfato sódico (40/60), pH 3,3; disolvente B: acetonitrilo / fosfato sódico 0,1 M (60/40), pH 3,3) y el programa de gradiente es como sigue: 100% de A. a 100% de B en 10 min, B 100% durante 5 min </li><li> Analizar FB1 estándares por HPLC, y las mediciones de área de los picos se utilizan para generar la curva estándar. Posteriormente, cuantificar FB1 nivel en una muestra mediante la comparación de áreas de los picos con FB1 curva estándar. </li></ol> 5. Análisis de ergosterol <ol><li> Cultura del hongo en los granos de maíz durante 7-14 días. </li><li> Después de que el período de incubación, agregar 10 ml de cloroformo: methanol (2:1, v / v) en cada vial. Una vez añadido, agitar bien y luego las muestras se incuban los frascos en la oscuridad a temperatura ambiente durante 24 horas. </li><li> Después de 24 horas, centrifugar las muestras, y recoger el sobrenadante y filtrar a través de una membrana de nylon 0,45 um. </li><li> Inyectar la muestra directamente en un HPLC Shimadzu LC-20AT sistema equipado con un 4,6 U SAO columna C18 (200 A y 250 ± 4,6 mm) y un Shimadzu SPD-20A detector UV / VIS configurado para monitorear a 282 nm. </li><li> El uso de metanol (100%) a un caudal de 1,5 ml / min como fase móvil. Determinar la cuantificación de ergosterol en las muestras mediante la comparación de áreas de los picos de las muestras a una curva estándar generada a partir de grado HPLC ergosterol. </li></ol> 6. Los resultados representativos Después de la inoculación y la incubación en una cámara de humedad durante dos a tres días, el crecimiento de hongos debe comenzar a aparecer en los granos. Siete días después del tratamiento, el crecimiento vegetativo en las plantas tratadas deben ser claramente visibles, rodeado pocontroles electrónicos falsos que no está infectada (Figura 1B, arriba). Los períodos de incubación más largo son propicias para el crecimiento vegetativo más abundante (Figura 1B, abajo). En las condiciones descritas en este documento (Figura 2G), A. flavus de tipo salvaje NRRL 3357 se muestran los valores máximos de la colonización, la acumulación de aflatoxina, y la producción de conidios a las 8, 6 y 8 días, respectivamente (Figura 2, CA). Sin embargo, cuando la contaminación por micotoxinas y la formación de conidios fueron comparados por unidad de ergosterol, los mayores niveles se observaron a las 4 y 6 días, respectivamente (Figura 2, DE). Figura 2F resumen de estos hongos que dependen de la biomasa de valor máximo de las observaciones. Curiosamente, entre los 4-6 días después de la inoculación, los granos sufren una degradación de ácidos nucleicos, como se ve por el ARN total de muestras en el tiempo de duración (Figura 2H, abajo). Varios estudios, incluyendo la nuestra, tienen exaed F. verticilliodies infección en granos de maíz y utilizado con éxito 2,8,9,10,11,12 7-13 días como los puntos de tiempo para las observaciones. Utilizando los métodos descritos en este documento, las fumonisinas niveles van de 3,500-8,000 ng / g de gama kernel 4,13 y los niveles de ergosterol de 5.000-10.000 ng / g del núcleo 14,13. La Figura 3 muestra los picos representativos de las fumonisinas (arriba) y el ergosterol (parte inferior ) de cromatogramas de HPLC. Medición de ergosterol también puede llevarse a cabo a través de espectros de absorbancia 15. <img alt="Figura 1" src="/files/ftp_upload/3727/3727fig1.jpg" /> Figura 1. Diagrama de flujo para la cuantificación de la biomasa de hongos, esporogenesis, y la producción de micotoxinas y los resultados representativos para el crecimiento vegetativo de los bioensayos del kernel. A) Diagrama de flujo esbozar el método descrito para la cuantificación de los parámetros biológicos fundamentales utilizados para evaluar la patogénesis de hongos en los granos de maíz. B) Representanteresultados de los bioensayos del kernel. Arriba, A. flavus (NRRL3357) el crecimiento vegetativo de los granos de maíz en el fondo B73 genética. Las semillas se inocularon con 200 l de 10 6 esporas / ml y se tomaron fotografías 7 días post inoculación. En pocas palabras, F. verticillioides inocularon granos en el fondo de la infección por B73 13 días después. Los granos se inocularon con 200 l de 10 6 esporas / ml. <img alt="Figura 2" src="/files/ftp_upload/3727/3727fig2.jpg" /> Figura 2. Aspergillus flavus bioensayo sistema de tiempo-por supuesto. B73 granos fueron inoculados con 200 l de 10 6 esporas / ml Aspergillus flavus suspensión de conidios y se incubaron durante 2-8 días (G). Todos los valores se determinaron a partir de la media de peso en seco de 4 núcleos (n = 3-4, con una media ± DE). A) La colonización (basado en ergosterol), aflatoxinas (B), y conidiación (C) se cuantificaron utilizando métodos descritos anteriormente. E) La aflatoxina y la formación de conidios (F) se muestrancomo una función de la biomasa fúngica, medido a través de ergosterol h. F) Los valores máximos de ergosterol, la acumulación de aflatoxina, y conidios en función de la biomasa de hongos: los valores máximos para cada cantidad se observó a 100%. (H) 1 g por carril de RNA total de tiempo-curso. <img alt="Figura 3" src="/files/ftp_upload/3727/3727fig3.jpg" /> Figura 3. Alto Representante cromatografía líquida (HPLC) cromatogramas de las fumonisinas B1 (arriba) y el ergosterol (abajo) aisladas a partir de granos infectados con Fusarium verticillioides (M3125).

<ol>
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<p class="jove_content"&gt; No tengo nada que revelar.</p

<p class="jove_content"&gt; Los métodos descritos aquí se ha probado extensamente y ha demostrado ser robusta en la generación de resultados cuantificables para la colonización por hongos, esporogenesis, y la producción de micotoxinas. Por otra parte, estos métodos deben ser aplicables a las semillas de otras especies de plantas que son susceptibles a la contaminación con hongos micotoxigénicos (por ejemplo, los cacahuetes, trigo, algodón, pistachos, etc.) Para competentes los análisis de interacción planta-patógeno, es imperativo q...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Catálogo # </td></tr><tr><td> Agar Patata Dextrosa </td><td> Fisher científico se </td><td> S71659A </td></tr><tr><td> Tween-20 al </td><td> Fisher científico se </td><td> BP337-100 </td></tr><tr><td> Recipiente de incubación de plástico </td><td> Sterilite </td><td> 1713LAB06 </td></tr><tr><td> Licuadora </td><td> Vicam </td><td> 20200 </td></tr><tr><td> 24 cm filtro plegado documentos </td><td> Vicam </td><td> 31240 </td></tr><tr><td> 1,5 m de microfibra de vidrio </td><td> Vicam </td><td> 31955 </td></tr><tr><td> Afla columna de prueba </td><td> Vicam </td><td> G1024 </td></tr><tr><td> Afrla prueba para desarrolladores </td><td> Vicam </td><td> 32010 </td></tr><tr><td> El metanol </td><td> Vicam </td><td> 35016 </td></tr><tr><td> Acetonitrilo </td><td> Fisher científico se </td><td> AC14952-0025 </td></tr><tr><td> Etanol </td><td> Fisher Scientifc </td><td> AC39769-0025 </td></tr><tr><td> C-18 de fase sólida columna de extracción (Prep septiembre Columna SPE C18) </td><td> Fisher científico se </td><td> 60108-304 </td></tr><tr><td> O-ftalaldehído (OPA) </td><td> Sigma Chemical Co </td><td> 79760-5g </td></tr><tr><td> El ácido bórico </td><td> Fisher científico se </td><td> BP168-500 </td></tr><tr><td> Borato de sodio </td><td> Fisher científico se </td><td> RDCS0330500 </td></tr><tr><td> Mercaptoetanol </td><td> Fisher científico se </td><td> 45-000-231 </td></tr><tr><td> HPLC Shimadzu LC-20AT (bomba) </td><td> Shimadzu Scientific Instruments, Inc. </td><td> LC-20AT </td></tr><tr><td> Zorbax ODS columna (4.6x150mm) </td><td> Agilent Technologies </td><td> 443905-902 </td></tr><tr><td> Shimatzu RF-10Axl detector de fluorescencia </td><td> Shimadzu Scientific Instruments, Inc. </td><td> RF-10AXL </td></tr><tr><td> Fosfato sódico </td><td> Fisher científico se </td><td> AC38987-0010 </td></tr><tr><td> FB1 normas </td><td> Sigma Chemical Co. </td><td> F1147-1mg </td></tr><tr><td> Cloroformo </td><td> VWR </td><td> MK444410 </td></tr><tr><td> 13 mm de filtro jeringa con membrana de nylon 0,45 um (HPLC) </td><td> Pall Life Science </td><td> 4426 </td></tr><tr><td> Ergosterol </td><td> Sigma-Aldrich </td><td> 45480-50G-F </td></tr><tr><td> Centelleo viales </td><td> VWR </td><td> 66021-602 </td></tr><tr><td> Cloruro de Sodio </td><td> Vicam </td><td> G1124 </td></tr></tbody></table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

La descomposición de granos por semilla-hongos que infectan plantea uno de los mayores desafíos económicos a la producción de cereales en todo el mundo, por no hablar de los riesgos graves para la salud humana y animal. Entre la producción de cereales, el maíz es sin duda el cultivo más afectado, debido a patógenos inducida por pérdidas en la integridad del grano y la contaminación de las semillas de micotoxinas. Los dos micotoxinas más comunes y problemáticas de los productores de maíz y de alimentos y procesadores de alimentos son las aflatoxinas y las fumonisinas, producida por Aspergillus flavus y Fusarium verticillioides, respectivamente. Recientes estudios moleculares de las interacciones planta-patógeno han demostrado promesa en la comprensión de los mecanismos específicos asociados con la respuesta de las plantas a la infección por hongos y la contaminación por micotoxinas 1,2,3,4,5,6. Debido a que muchos laboratorios están utilizando ensayos del núcleo para estudiar patógenos de plantas interacciones, existe una necesidad de un método normalizado para la cuantificación de diferentes parámetros biológicos, asíresultados de distintos laboratorios se puede interrogar a interpretar. Para un método eficaz y reproducible para el análisis cuantitativo de las semillas, se han desarrollado ensayos de laboratorio en el núcleo y los métodos siguientes para cuantificar el crecimiento de hongos, la biomasa y la contaminación por micotoxinas. Cuatro esterilizados granos de maíz se inoculan en viales de vidrio con una suspensión fúngica (10 6) y se incubaron durante un período predeterminado. Viales de muestras se seleccionan entonces para la enumeración de los conidios por análisis de biomasa hemocitómetro, ergosterol basada por cromatografía líquida de alta resolución (HPLC), la cuantificación de aflatoxinas utilizando un método AflaTest fluorómetro, y cuantificación fumonisina por HPLC.

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

La devastación de los cultivos de cereales de semillas por los hongos que infectan ha llevado a los numerosos esfuerzos de investigación para comprender mejor las interacciones planta-patógeno. Para estudiar las interacciones entre las semillas de hongos en un entorno de laboratorio, hemos desarrollado un método robusto para la cuantificación de reproducción de los hongos, la biomasa y la contaminación por micotoxinas mediante bioensayos del kernel.

La cuantificación de la colonización por hongos, esporogenesis y producción de micotoxinas mediante bioensayos del núcleo

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

Research

JoVE Journal

Immunology and Infection

18.0K vistas.  Texas A&M University. La devastación de los cultivos de cereales de semillas por los hongos que infectan ha llevado a los numerosos esfuerzos de investigación para comprender mejor las interacciones planta-patógeno. Para estudiar las interacciones entre las semillas de hongos en un entorno de laboratorio, hemos desarrollado un método robusto para la cuantificación de reproducción de los hongos, la biomasa y la contaminación por micotoxinas mediante bioensayos del kernel.

Video: Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

La colonización de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Investigación

Inmunología e Infección

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Vídeo en vivo de células Microscopía de fagocitosis hongo patógeno

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterización De Las Respuestas Inflamatorias Durante La Colonización Intranasal Con Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Aislamiento, Purificación y Etiquetado de neutrófilos de médula ósea de ratón de Estudios funcionales y experimentos de transferencia adoptivos

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

La investigación de los efectos de los probióticos en antineumocócica Colonización El uso de un<em&gt; In Vitro</em&gt; Ensayo de adherencia

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

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 ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonización vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

Cuantificación histológica para determinar la carga micótica pulmonar en aspergilosis Experimental

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Imagen en tiempo real y cuantificación del desarrollo de biopelículas fúngicas mediante un sistema recirculación de flujo bifásico

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...