Los autores desean agradecer a los miembros anteriores del laboratorio Goodrich-Blair: Samantha Orchard, Cowles Kimberly, Erin Herbert-Tran, Greg Richards, Menard Megan y Parque Youngjin por sus contribuciones al desarrollo de este protocolo. Este trabajo fue financiado por la National Science Foundation de subvención IOS-0950873 y los Institutos Nacionales de Salud NRSA compañerismo FAI084441Z.

1. Esterilización de Insectos huevo y Cría <ol><li> Preparar dieta por primera autoclave 15 g de agar proporcionado en la 900-1,000 ml H 2 O. Inmediatamente después de la esterilización en autoclave, se mezclan con 166 g de germen de trigo dieta y mezcla bien en un mezclador de laboratorio. Vierta en un plato (o platos) para enfriar y transferir dieta para papel de aluminio, envolver herméticamente, y se almacena a 4 ° C. </li><li> A su llegada, esterilizar M. Sexta huevos con 250 ml de solución de lejía al 0,6% durante 2-3 min en un soporte de filtro de vidrio y aparatos termo con un papel de filtro de 90 mm, revolviendo de vez en cuando. </li><li> Encienda el vacío para drenar la solución de lejía y lavado de los huevos de 3-4 veces con 250 ml de agua estéril destilada H 2 O por lavado. </li><li> Traslado huevos a papel nuevo de 90 mm filtro colocado en una placa de Petri tapa y deje que se sequen hasta que ya no estamos juntos (unos 20-30 minutos). </li><li> Transferir los huevos a la parte inferior de los recipientes de plástico con dieta de insectos, colocando aproximadamente 40 huevos en cada envase(Figura 1A). Los huevos se separan de la dieta para evitar que la humedad inducida por la contaminación fúngica. Mantenga los huevos a 26 ° C con una luz de 16 horas: 8 hr fotoperíodo oscuro. Los huevos se aclarará a un color amarillo-blanco antes de la eclosión. </li><li> Cuando la incubación es completa, transferir cuidadosamente aproximadamente 25 larvas cada uno de los nuevos contenedores con la dieta en la parte inferior (Figura 1B). Es importante recoger cada larva por el cuerno largo y negro con unas pinzas para evitar dañar a ellos durante sus primeras etapas de desarrollo frágiles. Incubar durante 2 días como anteriormente. Si bien la muerte larval es inusual en esta etapa, algunos muertos o, más probablemente, larvas de desarrollo atrofiado puede ser observado. Tales insectos deben excluirse del estudio y se sacrificaron por congelación. </li><li> Para evitar comportamientos caníbales, larvas transfiera a recipientes individuales con pequeños trozos de alimentos (Figura 1 C) y se incuba como antes. Supervisar el desarrollo de insectos, heces sustitución de alimentos y limpieza out de recipientes cada día hasta que se someten a la tercera muda larval (normalmente 6-9 días después de la eclosión). Este es el 4 º instar etapa larval, que se caracteriza por la aparición de negros en forma de gancho crochets en cada prominencia Proleg y aumento de rayas a lo largo del cuerpo del insecto (Figura 1D). Las larvas pueden variar considerablemente en tamaño, pero normalmente pesan 0.15 a 0.4 g al entrar en la 4 ª etapa instar. Las larvas de tamaño similar debe ser elegido para la inyección. </li></ol> 2. Preparación de bacterias para Inyección <ol><li> Crecer la cepa bacteriana (s) a ensayar de acuerdo con procedimientos estándar para la fase de crecimiento que se desea probar. </li><li> Pellet 500 l de cada cepa bacteriana para ser probado por hilado durante 2 min en una microcentrífuga a 13.000 rpm a temperatura ambiente. </li><li> Resuspender las células en 1 ml de 1x solución salina tamponada con fosfato estéril (PBS) y el sedimento de nuevo como anteriormente. </li><li> Resuspender las células en 0,5 ml de PBS 1x estéril y meaAsegúrese de que la densidad óptica (DO) de la suspensión. Diluir las suspensiones para el mismo diámetro exterior, si es necesario. Medios estériles de crecimiento también se puede utilizar para la resuspensión celular y la dilución en lugar de PBS 1x, si se desea. </li></ol> 3. Inyección de 4 º estadio las larvas <ol><li> Preparar serie 10-veces diluciones de la cepa bacteriana primero en 6 pocillos de una placa de microtitulación de 96 pocillos, en PBS 1x estéril, usando una punta de pipeta nueva para cada dilución (Figura 2A). El volumen final de la suspensión celular en cada pocillo debe exceder el volumen necesario para la inyección (10 l por insectos), además de 20 l para la placa de inóculo (ver los pasos 3,2 y 3,7). </li><li> Punto 10 l cada uno de 5 diluciones (10 -2 a través de 10 -6) a lo largo de la parte superior de una placa de Petri que contiene un medio de crecimiento apropiado. Inclinar la placa de modo que los puntos se extendió al centro (Figura 2B). </li><li> Retire dieta de insectos y excrementos e insectos lugar en recipientes con hielo paraaproximadamente 5 min. </li><li> Esterilizar la aguja de la jeringa mediante enjuague 3 veces cada una en 2 tubos de 70-100% de etanol, seguidos por un tubo estéril de O H 2 (Figura 2C). El volumen de líquido debe ser suficiente para sumergir la aguja. </li><li> Cuantificar las suspensiones de células diluidas con un microscopio utilizando un hemocitómetro. Dependiendo del inóculo deseado, dibujar 10 l de la microtitulación correspondientes pocillos (Figura 2D). </li><li> Torunda la superficie de insecto con 95% de etanol, y se inyecta la suspensión de 10 ml de células en el insecto en un ángulo (de menos de 45 °) detrás de la una de las patas abdominales abdominales. Tener cuidado de no perforar el intestino e inyectar el contenido justo debajo de la capa epidérmica (2E Figura). Si bien una cierta pérdida de hemolinfa (claro, verde-azul líquido) es normal, las partículas y el líquido amarillo que emerge de la zona de inyección son indicativos de punción intestino. Si esto ocurre, sacrificar el insecto y obtener una larva de nuevo para volvercolocarlo. </li><li> Repita el paso 3.6 para cada insecto restante en el grupo de la primera inyección. Aguja de la esterilización no es necesaria entre cada insecto inyecta con la misma dilución y la cepa de bacterias a menos que se produzca contaminación. Alternativamente, un dispensador de repetición se puede utilizar para una mayor consistencia en el volumen de inyección entre las muestras. </li><li> Después de inyectar cada insecto con la primera cepa de bacteria, repita el paso 3,2, esta vez manchado suspensiones de células en el medio de la placa y dejar que el flujo hacia la parte inferior (Figura 2F). Incubar las placas a temperatura adecuada y contar las colonias de enumerar el inóculo. Este paso chapado segunda se utiliza para verificar que las muestras no estaban contaminadas durante el proceso de inyección, ya que esto daría lugar a números de colonias dispares entre los chapados primero y segundo. </li><li> Repetir los pasos a través de 3,1 3,8 para cada cepa de bacteria en el experimento. Finalmente, se inyectan 3-5 insectos con PBS 1x estéril utilizando un sterile aguja como un grupo de control negativo. Incubar los insectos a 26 ° C con una luz de 16 horas: 8 hr fotoperíodo oscuro. </li><li> Supervisar la supervivencia del insecto con el tiempo después de la inyección. La muerte del insecto se caracteriza por una falta de movimiento voluntario a la estimulación. Los insectos suelen presentar pérdida del apetito, diarrea (excremento aguado, de color amarillo), y / o pérdida de agua (&quot;sudor&quot;) durante la infección antes de la muerte; estas características puede ser digno de mención también. </li></ol>

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No hay conflictos de interés declarado.

La inyección directa de M. larvas sexta con bacterias entomopatógenos, como se describe aquí, sirve como un medio sencillo y eficaz para analizar la virulencia bacteriana. El método es también muy adaptable para adaptarse a diferentes sujetos experimentales y / o condiciones. Las bacterias se pueden preparar de diversas maneras antes de la inyección. En el caso de X. nematophila, las células de tipo salvaje cultivadas en rica en nutrientes Luria-Bertani (LB) a mitad de la fase log son t...

<table border="1"><tbody><tr><td> Reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 90 mm papel de filtro </td><td> Whatman </td><td> 1001 090 </td><td></td></tr><tr><td> Filtro de vidrio titular </td><td> Millipore </td><td> XX1004700 </td><td></td></tr><tr><td> Manduca sexta huevos </td><td> Carolina Biological Supply </td><td> 143880 </td><td></td></tr><tr><td> Dieta Gypsy Moth agar + </td><td> MP Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5,5 oz envases de plástico y tapas </td><td> Solo Cup Company </td><td> URC55-0090-0090 PI4 </td><td></td></tr><tr><td> 1 oz envases de plástico y tapas </td><td> Dart Container Corporation </td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1x PBS </td><td></td&gt; <td></td><td> 137 mM NaCl, 2,7 mM KCl, 8 mM Na 2 HPO 4, 1,46 mM KH 2 PO 4, pH 7,4 </td></tr><tr><td> Jeringa </td><td> Hamilton </td><td> 80208 </td><td> Calibre 30, 0,375 &quot;de largo, estilo, punto 2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

Manduca sexta, comúnmente conocido como el gusano del tabaco, se considera una plaga agrícola importante, se alimentan de plantas solanáceas, incluyendo tabaco y tomate. La susceptibilidad de M. larvas sexta a una variedad de especies bacterianas entomopatógenos 1-5, así como la gran cantidad de información disponible sobre el sistema inmunológico del insecto 6-8, y la secuencia del genoma pendiente 9 lo convierten en un organismo modelo bueno para el uso en el estudio de la interacción huésped-microbio durante la patogénesis. Además, M. sexta larvas son relativamente grandes y fáciles de manipular y mantener en el laboratorio en relación a otras especies de insectos susceptibles. Su gran tamaño también facilita eficiente tejido / hemolinfa de extracción para el análisis de la respuesta del huésped a la infección. El método aquí presentado describe la inyección directa de bacterias en el hemocele (cavidad sangre) de M. sexta larvas. Este enfoquese puede utilizar para analizar y comparar las características de virulencia de diversas especies bacterianas, cepas o mutantes simplemente monitorizar el tiempo a la muerte del insecto después de la inyección. Este método fue desarrollado para estudiar la patogenicidad de Xenorhabdus y Photorhabdus especies, que suelen asociar con vectores de nematodos como un medio para ganar la entrada en el insecto. Nematodos entomopatógenos típicamente infectan larvas vía digestiva natural o aberturas respiratorias, y liberan su contenido de bacterias simbióticas en la hemolinfa del insecto (sangre) poco después 10. El método de inyección aquí descrito evita la necesidad de un vector de nematodos, así desacoplar los efectos de las bacterias y nematodos en el insecto. Este método permite la enumeración precisa de material infeccioso (células o proteínas) en el inóculo, que no es posible con otros métodos existentes para el análisis de entomopathogenesis, incluyendo formación de muescas 11 y los ensayos de toxicidad por vía oral 12 <em&gt;. Además, los ensayos de toxicidad por vía oral frente a la virulencia de las toxinas secretadas introducidos en el sistema digestivo de las larvas, mientras que el método de inyección directa se refiere a la virulencia de los inóculos de células enteras. La utilidad del método de inyección directa como se describe aquí es analizar la patogénesis bacteriana mediante la supervisión de mortalidad de los insectos. Sin embargo, este método puede ser fácilmente ampliado para su uso en el estudio de los efectos de la infección en el M. sexta sistema inmune. El insecto responde a la infección a través de ambas respuestas humoral y celular. La respuesta humoral se incluye el reconocimiento de bacterias asociadas con los patrones y la subsiguiente producción de diversos péptidos antimicrobianos 7, la expresión de genes que codifican estos péptidos pueden ser monitoreados posterior a la infección directa a través de la extracción de RNA y PCR cuantitativa 13. La respuesta celular a la infección implica la nodulación, encapsulación, y la fagocitosis de agentes infecciosos por hemocitos 6 </sup&gt;. Para analizar estas respuestas, insectos inyectadas pueden ser diseccionados y se visualizaron por microscopía 13, 14.

Un ejemplo representativo de un ensayo de mortalidad de los insectos se representa en la Figura 3. En este experimento, los insectos fueron inyectados con aproximadamente 50 unidades formadoras de colonias (UFC) de tipo salvaje (ATCC19061) o un mutante de la cepa atenuada (lrp 13) de Xenorhabdus nematophila crecer hasta la fase logarítmica media (n = 6 insectos por cepa). Los insectos fueron observados durante aproximadamente 72 horas, y registró el porcentaje de insectos ...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

El método descrito aquí utiliza la inyección directa de bacterias entomopatógenos en el hemocele de<em&gt; Manduca sexta</em&gt; Larvas de insectos.<em&gt; M. sexta</em&gt; Es un insecto disponibles comercialmente y bien estudiado. Por lo tanto, este método representa un método simple para analizar bacterianas interacción huésped-desde la perspectiva de uno o ambos socios.

Crianza y la inyección de<em&gt; Manduca sexta</em&gt; Las larvas para evaluar la virulencia bacteriana

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K vistas.  University of Wisconsin-Madison. El método descrito aquí utiliza la inyección directa de bacterias entomopatógenos en el hemocele de Manduca sexta Larvas de insectos. M. sexta Es un insecto disponibles comercialmente y bien estudiado. Por lo tanto, este método representa un método simple para analizar bacterianas interacción huésped-desde la perspectiva de uno o ambos socios.

Video: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

La saliva, la glándula salival, y la Colección hemolinfa de las garrapatas Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Investigación

Inmunología e Infección

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

La aplicación de un sistema de expresión inducible para el Estudio de la interferencia de los factores de virulencia bacteriana con Señalización Intracelular

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

La cucaracha que silba como un modelo alternativo de animales no mamíferos para investigar la eficacia de la droga, virulencia y patogenia de Madagascar

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

Identificación de marcadores de virulencia de Mycobacterium abscessus para la replicación intracelular de fagocitos

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

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

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

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

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

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

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

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modelo de ratón para evaluar respuesta inmune innata a la infección por Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

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 ...