Name: using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins
Uploaded: 2014-09-30T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

This work has been supported by Elizabeth E. Kennedy Research Award.

1 Preparación de la cepa productora <ol><li> Racha de una cepa neumocócica a ensayar para la producción de bacteriocinas en una sangre de oveja de soja tríptico placa de agar 5% desde -80 ° C congelador stocks. NOTA: La utilización de placas de sangre para el crecimiento inicial de la cepa a ensayar para la inhibición aumenta en gran medida la reproducibilidad del ensayo. Además, otras cepas no neumocócicas que producen bacteriocinas pueden ser utilizados aunque pueden necesitar ser modificados para un organismo específico condiciones de crecimiento. Hemos utilizado Streptococcus mitis y Lactococcus lactis en el ensayo de recubrimiento con éxito. </li><li> Incubar la placa durante la noche (O / N) a 37 ° C con 5% de CO 2. </li><li> Después de la incubación de la cepa productora, preparar la placa de agar de soja tríptico (TSA) pipeteando 3000 a 4000 unidades de catalasa sobre placas que contienen 25 ml de TSA y se extendió la solución de catalasa usando perlas de vidrio estériles. Deje que la placa se seque durante 10 minutos bajouna cabina de seguridad biológica. </li><li> Se recoge una cantidad visible de bacterias en una punta de la pipeta. Apuñalar a la punta de la pipeta en la placa de TSA seca. NOTA: Mas de una cepa productora puede ser apuñalado en una sola placa de TSA. Es importante espaciar las puñaladas de manera que halos resultantes no se solapan. <ol><li> Si es posible, incluir conocido productor de bacteriocina como un control positivo y la cepa para ser utilizado en la superposición como un control negativo. NOTA: Los controles positivos y negativos son han sido publicados con anterioridad y están disponibles bajo petición 10. </li></ol></li><li> Incubar la placa TSA contiene múltiples puñaladas a 37 ° C con CO2 al 5% durante 6 horas. </li></ol> 2. Hacer reservas de glicerina de la cepa Overlay <ol><li> Preparar stocks de glicerol (cultivos desarrollados a mediados de la fase exponencial y luego almacenados a -80 ° C después de la adición de glicerol) antes del día del ensayo de superposición. Inocular 5-10 colonias de str neumocócicaain a hacerse la prueba de sensibilidad o secreción de feromonas en medio Todd-Hewitt con extracto de levadura al 0,5% (THY). NOTA: Construcción de BLPC reporteros inducibles se ha descrito previamente 2 y cepas están disponibles bajo petición. </li><li> Incubar 5-10 colonias en tubos de vidrio con 5 ml de THY con tapas bien cerrados, en un baño de agua a 37 ° C sin agitación. </li><li> Incubar cultivos a 37 ° C hasta que llegan a un OD 620 de 0,3 a 0,5. NOTA: Esto tomará aproximadamente 4-5 horas, dependiendo de la cepa utilizada. <ol><li> Invierta suavemente el tubo un par de veces antes de la medición de la densidad óptica. </li></ol></li><li> Si el experimento no se puede realizar el mismo día o de la cepa se puede utilizar varias veces en el futuro, hacer stocks de glicerol y reanudar el experimento en una fecha posterior. Para las poblaciones de glicerol, glicerol añadir a los cultivos a una concentración final de 20%. Culturas alícuotas en tubos de microcentrífuga de 1,5 ml y almacenar a -80 ° C. NOTA: Las muestras pueden ser sreada durante meses a -80 ° C. Si no hacer stocks de glicerol, utilizar muestras directamente para el ensayo de superposición. </li></ol> 3. Overlay Ensayo <ol><li> Justo antes de la aplicación superponer, mezclar los componentes de superposición. En un tubo cónico de 15 ml, añadir 5 ml de THY, 3.000-4.000 U de catalasa, y 200 l de caldo de cultivo cepa superposición que se ha cultivado ese día o una acción de glicerol de la cultura que se descongela a temperatura ambiente. Si se utiliza la cepa reportero, añadir 50 l de X-gal (40 mg / ml) a la mezcla de superposición. Utilice un tubo cónico por placa. </li><li> Equilibrar mezcla de recubrimiento a temperatura ambiente antes de la adición de TSA fundido. </li><li> Retire la placa de TSA con la cepa apuñalado de la incubadora. Derretir sólido TSA en el microondas y mantener a 55 ° C baño de agua hasta que esté listo para usar. Añadir 3 ml de TSA fundido con 1,5% de agar se enfría a 55 ° C para superponer mezcla. <ol><li> Preparar la mezcla de superposición inmediatamente antes de pipeteo, la mezcla empezará a solidificar muyrápidamente. NOTA: Si la cubierta comienza a solidificarse antes de enchapado, los resultados serán imposibles de interpretar. </li></ol></li><li> Mezclar la mezcla de superposición pipeteando arriba y abajo varias veces con una pipeta de 5 ml con cuidado de no introducir burbujas en la mezcla. Lentamente pipetear mezcla de superposición directamente sobre la placa de TSA apuñalado. Mantenga la placa sobre la mesa de trabajo para pocos minutos permitiendo que el agar se endurezca. <ol><li> No gire la placa para distribuir la mezcla de superposición, este se tire el crecimiento de la cultura apuñalado en la superposición. </li></ol></li><li> Con cuidado, coloque la placa de TSA con plantilla a 37 ° C incubadora que contiene 5% de CO 2 O / N (aproximadamente 16 a 18 h). </li><li> Observar las placas después del crecimiento durante la noche. Las placas deben aparecer opaco de crecimiento de la cepa de superposición excepto en las zonas donde se ha producido la inhibición o la feromona de señalización. Estos aparecerán como zonas bien claros o azules alrededor de la cepa apuñalado, respectivamente. NOTA: Aunque measurelemen- del diámetro de la compensación de color azul o se pueden determinar para la evaluación cuantitativa, las diferencias en el diámetro también puede variar dependiendo de la cantidad de bacterias que se clavó en la placa. La superposición de ensayo es un ensayo cualitativo, cualquier evaluación cuantitativa debe ser interpretado con cautela. Cualquier compensación visible en comparación con el control negativo se considera como actividad. </li></ol>

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The authors have nothing to disclose.

Esta superposición de ensayo es una manera rápida de determinar la gama de actividades para los productores de bacteriocinas y para demostrar las interacciones competitivas que pueden ocurrir entre las cepas de neumococo. El ensayo de superposición se puede adaptar a utilizar para la detección de múltiples cepas para la producción de bacteriocina en una sola placa. Hemos analizado con éxito grandes colecciones de cepas con este ensayo mediante el uso de un replicador de 48 pines para inocular placas de SBA de la mitad de una placa de 96 pocillos. Después de incubación durante la noche, el crecimiento se transfiere a la placa de TSA utilizando el replicador y la perforación de la superficie de agar de la placa con los pasadores de la replicador. Otros péptidos antimicrobianos por bacterias secretadas que son reguladas de una manera dependiente de la densidad como lantibióticos también pueden ser examinadas usando la técnica de superposición 7,12,13. Esto es especialmente útil cuando es difícil la purificación de una bacteriocina o lantibiótico. Aunque los sobrenadantes se pueden ensayar para antimicrobianoactividad, expresión de las bacteriocinas BLP derivado en caldo es muy limitada o inexistente (datos no mostrados). El ensayo de superposición permite el crecimiento a una densidad más alta que no se logra cuando los organismos se cultivan en líquido. Crecimiento en un medio sólido también podría reflejar más estrechamente las condiciones de crecimiento in vivo de la nasofaringe. El ensayo de superposición también puede ser utilizado para examinar la capacidad de la cepa acuchillado para secretar la feromona de señalización específica bacteriocina. Esto requiere la construcción de una cepa reportero que expresa la proteína del receptor de feromonas y en el que un promotor sensible a feromona se coloca aguas arriba del gen lacZ. Valoración de la secreción de feromona por una cepa acuchillado es particularmente útil como una lectura de la actividad locus BLP si la cepa acuchillado no inhibe la cepa de superposición. La incapacidad para inhibir la cepa de superposición podría ser el resultado de un locus BLP no funcional (tal como en uncepa con una mutación transportador) o porque las bacteriocinas secretadas no son activos frente a la cepa en particular de superposición probado. La superposición reportero cepa proporciona información adicional que el locus en el productor tiene un transportador totalmente funcional sistema de dos componentes y BLPA lo que permite la regulación positiva y la secreción de la feromona. Esto implica que cualquier bacteriocinas codificados también son secretadas. Hay algunas limitaciones a este ensayo. El ensayo de superposición es un ensayo cualitativo, lo que las comparaciones entre la actividad inhibidora de la secreción de feromonas o de diferentes cepas tienen que hacerse cuidadosamente. De precios y condiciones de crecimiento se deben considerar cuando se comparan diferentes cepas debido a que el resultado de la prueba depende en gran medida la tasa de crecimiento de la superposición y cepas apuñalado. La adición de catalasa a la placa y la superposición es importante con el fin de eliminar los efectos inhibitorios de neumocócica H 2 O 2 de producción en lacrecimiento de la cepa de superposición, en particular cuando se usa organismo catalasa negativo en la superposición. Dada la diversidad conocida de contenido genómico en la población neumocócica, cualquier actividad inhibidora visto con este ensayo no puede atribuirse automáticamente a la locus BLP sin un análisis de deleción.

Streptococcus pneumoniae, un colonizador común de la comunidad polimicrobiana de la nasofaringe, es capaz de competir con otros neumococos y especies estrechamente relacionadas con la producción de péptidos antimicrobianos producidos por bacterias (bacteriocinas). Cada cepa de neumococo totalmente secuenciado-examinado hasta la fecha contiene una versión de la l ike locus p eptide b acteriocin-, BLP. La producción de bacteriocina por el neumococo ha demostrado ser importante en el resultado de tanto in vitro como in vivo en la competición 1-4. Dinámica competitiva están influenciados por la expresión de diversas bacteriocinas junto con proteínas de la inmunidad afines en respuesta a una feromona específica de péptido. Inducción del locus BLP está controlado por un sistema de detección de quórum en la que una feromona péptido, BLPC, se une a la quinasa sensor, BlpH, e inicia una cascada de señalización que resulta en lala transcripción del locus 5,6 BLP. Hay cuatro variaciones alélicas de BLPC que se unen cada uno a su BlpH afines 6. Para la célula para sobrevivir a los efectos de su propia bacteriocina, los genes que codifican proteínas de la inmunidad afines se transcriben en el mismo operón como cada uno de los genes de bacteriocina. Killing se produce si una cepa competidor no es capaz de regular positivamente el locus BLP en respuesta a la feromona exógeno o, si la cepa carece del gen de inmunidad específica necesaria para la protección. El complejo transportador, BlpAB se requiere para la secreción y el procesamiento de la feromona de péptidos y los péptidos de bacteriocina 2,4. Recientemente, se ha demostrado que una proporción significativa de cepas neumocócicas son &quot;tramposos&quot; que significa que tienen una mutación en el gen conservado BLPA que las hace incapaces de secretar feromonas y bacteriocinas 1,2. Estas cepas son capaces de responder a BLPC exógeno 2 secretada por relinchocepas aburridas que permitan la producción de proteínas de la inmunidad. Aunque, preparaciones crudas de bacteriocinas a partir de sobrenadantes se pueden preparar a partir de cepas de productores usando métodos pasos bioquímicos para lograr la pureza, este enfoque no es útil para el cribado de grandes colecciones de cepas y si el nivel de expresión de bacteriocina es baja en caldos de cultivo crecido 2,7- 9. La superposición de ensayo se utiliza como una forma rápida para investigar la dinámica competitiva entre las cepas que puedan existir en vitro. Para ello, una cepa neumocócica es pre-inocularon en una placa de agar y se dejaron crecer hasta alcanzar concentraciones bacterianas locales suficientes para la inducción del locus BLP. Una segunda cepa se inocula luego en una solución de agar blando fundido que se aplica entonces sobre la cepa de pre inoculado para la prueba de sensibilidad. Para evaluar la actividad del locus BLP (independiente de la actividad inhibitoria), las cepas pueden ser superpuestos con una serie de juree reportero cepas BLPC descritos anteriormente. Se construyeron estas cepas para contener uno de los tres alelos blpH diferentes que responden a las tres feromonas BLPC más comunes secretadas por el neumococo. Las cepas indicadoras tienen un gen lacZ integrado que está bajo el control de un promotor dependiente de BlpH y llevar a una deleción en el gen BLPC que impide la auto-estimulación 10,11.

<table><tbody><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>Fisher</td><td>B21261X</td><td>For growth from freezer cultures</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C100-500mg</td><td>4,741 Units per plate</td></tr><tr><td>Todd Hewitt Broth + Yeast Extract (THY)</td><td>Fisher</td><td>DF0492-17-6&amp;nbsp;</td><td>30 g of THB, 5 g yeast extract, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>Trypticase Soy Broth + Agar plates (TSA)</td><td>Fisher</td><td>B11768&amp;nbsp;</td><td>30 g of TS, 15 g agar, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside (X-gal)</td><td>Sigma</td><td>B4252-50MG</td><td>Dissolve in dimethylformamide</td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Spectrophometer</td><td></td><td></td><td>Measures the OD&lt;sub&gt;620&lt;/sub&gt; of cultures</td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO2</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td></td><td></td><td></td></tr></tbody></table>

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.

Hay dos posibles resultados en el ensayo de recubrimiento. Debe ser posible ver el crecimiento de la cepa acuchillado después de la incubación durante la noche. En la Figura 1A, la cepa es sensible a la superposición de las bacteriocinas que se producen. Un espacio libre se puede visualizar que rodea la cepa apuñalado. Además, remolino de la cepa acuchillado en la mezcla de superposición es posible y se puede ver en la Figura 1A. En la Figura 1B, la cepa de superposición es inmune a las bacteriocinas siendo secretadas como se indica por la ausencia de un halo que rodea la puñalada. También es posible que la cepa acuchillado no está secretando bacteriocinas. Para el ensayo de señalización, dos resultados diferentes son posibles. La cepa acuchillado es capaz de secretar feromonas que interactúa con el histidina quinasa de la cepa de superposición, como se ve a través de la descomposición de X-gal como se muestra en la Figura 2A. Si la feromona no activa la transcripción delBLP locus en la cepa reportero, o si la cepa acuchillado no secreta feromona, no se producen descomposición de X-gal como se muestra en la Figura 2B. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51876/51876fig1highres.jpg" width="500" /> Figura 1. Aspecto de la inhibición y la inmunidad mediada por BLP en un ensayo de superposición. (A) Cepa P133, un productor de bacteriocina, se añadieron a una placa de TSA y se dejó crecer durante 6 h. P250 Strain, una cepa de bacteriocinas negativo, se inoculó en la superposición. Las placas fueron fotografiadas después de O / N de incubación. Un espacio libre se indica por la flecha. P133 (B) La cepa se inoculó en una placa de TSA y se incubaron durante 6 hr. Strain 130, que contiene una mutación de desplazamiento de marco en BLPA, se inoculó en la superposición. Después de O / N de la incubación, las placas fueron fotografiados. Figura 2. Pheromone mediada activación de locus BLP en ensayo de señalización. (A) P133 Strain, un tipo BLPC R6 secretor, se inoculó en una placa de TSA y se incubaron durante 6 horas. P981 Strain, un reportero R6 tipo BLPC con una deleción en BLPC, se inoculó en la superposición. Las fotografías fueron tomadas después de O / N de incubación. (B) P133, se añadieron a una placa de TSA. Después de la incubación durante 6 horas, P845, un reportero tipo P164 BLPC con una deleción en BLPC, se inoculó en la superposición. Las fotografías fueron tomadas después de O / N crecimiento.

Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

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

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 ...

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Immunology and Infection

13.4K vistas.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  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. 

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

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

Un ensayo para cuantificar la unión de proteínas y ARN en bacterias

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Investigación

Genética

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

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 ...

Inmunología e Infección

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Sola célula Medidas de Ruptura Vacuolar causadas por patógenos intracelulares

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Vigilar los cambios en la polaridad de la membrana, membrana Integridad, e intracelular de iones concentraciones en<em&gt; Streptococcus pneumoniae</em&gt; Uso de tintes fluorescentes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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 ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Materiales biomiméticos para caracterizar las interacciones bacteria-huésped

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Los ensayos cualitativos y cuantitativos para la detección y caracterización de proteínas de antimicrobianos

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

El uso de la técnica de superposición Soft-agar para la detección de compuestos inhibidores en bacterias producidas

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

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 ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Crecimiento diferida Ensayo de inhibición para cuantificar el efecto de antimicrobianos de bacterias derivadas de la Competencia

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

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

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