Esta pesquisa foi suportada por uma concessão do Instituto Nacional de Saúde e da Universidade do Texas em Arlington criar fundos para MM.

1. As plantas que crescem e Bactérias Preparação <ol><li> Para iniciar este procedimento semear Arabidopsis em um v 01:01:01: v: v mistura de meio de cultura (Redi-earth plug and mix de mudas, Sun Gro), vermiculita fina, e perlite. </li><li> Crescer as plantas em câmara de crescimento (22 ° C umidade, 12 horas de luz de 100 mmol / mm 2 / s por dia e 65 ± 5%) e água, conforme necessário. As plantas estão prontas para uso em 4-6 semanas quando têm folhas jovens completamente expandidas antes do pendoamento. </li><li> Dois dias antes de iniciar o experimento preparar a cultura bactéria. Streak Pseudomonas syringae do estoque de glicerol em meio LB modificado (10 g / L triptona, 5 g / L de extrato de levedura, 5 g / L NaCl pH 7,0, e ágar 1,5%) e incubar durante 24 horas a 28 ° C. Use cultura fresco para preparar o inóculo e sempre começam placas de cultura de stocks glicerol como bactérias torna-se menos virulentas após o sub-cultura. </li><li> Use as bactérias que cresceram na placa para começar uma cultura de 10 mL de líquido de P. syringae em um erlenmeyer 50 mL. Incubar a cultura durante a noite a 28 ° C com agitação vigorosa até atingir densidade óptica ou OD a 600 nm entre 0,8 e 1. </li><li> Medir o OD a 600 e colher as células por centrifugação a 1360 xg por 10 minutos. Ressuspender as células em água destilada de modo que o OD final é 0,2. Este OD corresponde a 10 8 unidades formadoras de colônia (UFC) / mL. Com as bactérias pronto, vá para preparar as folhas e realizar o ensaio. </li></ol> 2. A coloração das folhas e incubação com bactérias <ol><li> , A fim de mancha as folhas primeiro preparar 20 iodeto de propídio mM ou solução PI na água. 10 mL de solução é suficiente para manchar 3 folhas pequenas de cada vez. </li><li> Recuperar as plantas da câmara de crescimento e com uma pinça retire três jovens, completamente expandidas folhas. Mergulhe as folhas inteiras em solução de PI por 5 minutos. Em seguida, retire as folhas e lavá-los rapidamente com água destilada. </li><li> Em seguida coloque uma folha com a superfície inferior virado para baixo sobre uma lâmina de microscópio. Corte a folha com uma lâmina afiada em quatro pedaços excluindo a veia meados de modo que a folha plana estabelece no slide. </li><li> Para incubar a bactéria com as folhas, adicionar 300 mL de suspensão bacteriana sob os pedaços de folhas no slide. Certifique-se que a superfície inferior da folha está em contato com o inóculo. </li><li> Incubar as amostras nas mesmas condições ambientais que foram usados ​​para crescer as plantas. No momento em que deixa de observar ao microscópio, transferir os pedaços de folhas para um novo slide com superfície inferior voltada para cima. Note-se que lamínula não é utilizado durante a montagem dos slides. A amostra foliar mesmo pode ser fotografada várias vezes durante a incubação e um curso de tempo pode ser configurado de acordo com os objetivos da pesquisa. </li></ol> 3. Análise da Medição Microscopia e Dados <ol><li> Aqui, um microscópio confocal de varredura a laser (LSM 510 Meta, Carl Zeiss Inc.) é usado para examinar as folhas. Observar a superfície inferior das folhas para detectar a fluorescência de PI (excitação 453 nm, a emissão de 543 e 620 nm). </li><li> Utilizando as amostras de folhas mesmo capturar imagens de estômatos ao longo do tempo. Guardar as imagens para medir a largura da abertura estomática. </li><li> Medir a largura de abertura estomática de pelo menos 60 estômatos para cada tratamento em cada momento usando o navegador de imagem LSM. </li><li> Calcular a média e erro padrão para a largura da abertura estomática. Significância estatística dos resultados pode ser calculada usando 2-tailed, Student pareado sábio t-teste. </li></ol> 4. Imagens representativas da Resposta estomática para incubação com bactérias <ol><li> Aqui está uma imagem típica microscópica (utilizando objetiva de 20x) de uma superfície foliar de Arabidopsis sob microscópio confocal de varredura a laser em vista de campo claro (Figura 1). </li><li> Manchas de iodeto de propídio da parede celular de células viáveis ​​aumentar a visibilidade de células-guarda, além de permitir a identificação de células intactas de coleta de dados (Figura 2). Uma série de aberturas de poro estomático pode ser visto nestas micrografias, a partir de estômatos completamente fechados para estômatos abertos. </li><li> Estômatos completamente fechados são identificados pela forma das células guarda (Figura 3). A largura de abertura é considerado como sendo 0 mM. </li><li> Para estômatos abertos (Figura 4) a largura de abertura mostrada é medido por meio de uma linha reta traçada através da maior área do poro estomático. </li><li> Estes são típicos os resultados experimentais deste ensaio estomática. Deixa Arabidopis foram incubadas no escuro com três diferentes cepas de Pseudomonas syringae. Apenas a bactéria Pst DC3000 foi capaz de abrir-escuro fechado estômatos, medido pela largura de abertura de estômatos selecionados aleatoriamente 60 por estirpe bacteriana (Figura 5). </li></ol&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="Figura 1" /> Figura 1. Micrografia da superfície de uma folha de Arabidopsis. Um campo de visão da superfície foliar sob microscópio confocal de varredura a laser (LSCM) usando o objetivo de 20x. Note-se que a abertura estomática não é tão evidente quando comparado com o ponto de vista fluorescentes auxiliado por coloração iodeto de propídio (Figura 2). <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="Figura 2" /> Figura 2. Micrografia da superfície de uma folha de Arabidopsis propidium iodeto de manchado. Um campo de visão da superfície foliar sob microscópio confocal de varredura a laser (LSCM) usando o objetivo de 20x. Nota uma gama de abertura do poro estomático. Setas amarelas são apontados para fechar completamente estômatos e setas verdes são apontados estômatos abertos. <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="Figura 3" /> Figura 3. Estômatos completamente perto identificados pela forma e da abertura entre as células guarda. A largura de abertura é considerado como sendo 0 mM. <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="Figura 4" /> Figura 4. Estômatos abertos mostrando a largura de abertura em mM. As medidas foram feitas usando o navegador LSCM baseado em uma linha reta traçada através da maior área do poro estomático. <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="Figura 5" /> Figura 5. Abertura estomática das folhas de Arabidopsis incubadas com três linhagens de Pseudomonas syringae pv tomate:.. DC3118, DB29, e DC3000 As plantas foram mantidas no escuro durante o tempo de experimentação. Os resultados são apresentados como a média (n = 50-70) ± erro padrão. Significância estatística foi detectada com bicaudal de Student t-test. O experimento foi repetido três vezes com resultados semelhantes.

<ol>
	<li>Brooks, D. M., Hernandez-Guzman, G., Kloek, A. P., Alarcon-Chaidez, F., Sreedharan, A., Rangaswamy, V., Penaloza-Vazquez, A., Bender, C. L., Kunkel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+well-defined+series+of+coronatine+biosynthetic+mutants+of+Pseudomonas+syringae+pv.+tomato+DC3000.">Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.</a> Mol Plant-Microbe Interact. 17, 162-174 (2004).</li><li>Katagiri, F., Thilmony, R., He, S. Y., Somerville, C. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> The Arabidopsis Book. , (2002).</li><li>Ma, S. W., Morris, V. L., Cuppels, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+DNA+region+required+for+production+of+the+phytotoxin+coronatine+by+Pseudomonas+syringae+pv.+tomato.">Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato.</a> Mol Plant-Microbe Interact. 4, 69-74 (1991).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126, 969-980 (2006).</li></ol>

Não há conflitos de interesse declarados.

Nós apresentamos um procedimento para a frente para medir a abertura estomática em tecido foliar intacta permitindo uma fácil avaliação da resposta estomática a diferentes tratamentos. Embora tenhamos apresentado resultados utilizando o sistema modelo Arabidopsis / Pseudomonas syringae, o ensaio da folha intacta estomática pode ser potencialmente realizada com qualquer combinação de plantas bactéria. O protocolo pode ser facilmente modificado para atender às necessidades de crescimento de outras plantas e patógenos bacterianos. O princípio geral e procedimento permanece o mesmo. Além disso, este método pode ser benéfica para pesquisadores que desejam estudar saída funcional de células-guarda não só para viver os micróbios, mas também a outros estímulos e agentes químicos em condições que mantenham o ambiente natural da folha. Folha inteira pode ser difícil de imagem devido a sua espessura e topografia irregular. Este problema pode ser aliviado com a remoção da veia meados da folha de modo que estabelece plano no slide e microscopia confocal. No entanto, outros tipos de microscópio de fluorescência pode ser usado para obter imagens de alta resolução da superfície da folha.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Propidium iodide</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P4864</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1421-1</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0127-01-7</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Bio Express</td>
<td>J637-2500G</td>
<td>&nbsp;</td>
<tr>
<td>Plant growth chamber</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Shaker incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Laser scanning confocal microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assessing stomatal response to live bacterial cells using whole leaf imaging

Estômatos são aberturas naturais na epiderme planta responsável pela troca de gases entre o interior da planta e ambiente. Eles são formados por um par de células-guarda, que são capazes de fechar o poro estomático em resposta a uma série de fatores externos, incluindo a intensidade da luz, a concentração de dióxido de carbono e umidade relativa (UR). O poro estomático é também a principal via para a entrada do patógeno em folhas, um passo crucial para o desenvolvimento da doença. Estudos recentes têm revelado que o fechamento do poro é eficiente na minimização de desenvolvimento de doenças bacterianas em plantas de Arabidopsis, uma parte integrante da imunidade inata de plantas. Anteriormente, nós usamos as cascas da epiderme para avaliar a resposta estomática ao vivo bactérias (Melotto et al 2006).; No entanto manter condições ambientais favoráveis ​​para ambas as cascas de plantas da epiderme e células bacterianas tem sido um desafio. Epiderme da folha pode ser mantido vivo e saudável, com MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6,15) para experimentos eletrofisiológicos das células guarda. No entanto, essa reserva não é apropriado para a obtenção de suspensão bacteriana. Por outro lado, as células bacterianas podem ser mantidos vivos em água que não é adequada para manter as cascas da epiderme por longo período de tempo. Quando uma casca flutua sobre a água da epiderme, as células na casca que estão expostos ao ar seco dentro de 4 horas limitando o tempo para realizar o experimento. Um método ideal para avaliar o efeito de um estímulo especial sobre células-guarda deve apresentar o mínimo de interferência para a fisiologia dos estômatos e ao meio ambiente natural da planta, tanto quanto possível. Nós, portanto, desenvolveu um novo método para avaliar a resposta estomática ao vivo em que as bactérias ferindo folha e manipulação é bastante minimizado com o objetivo de fornecer um ensaio facilmente reproduzível e confiável estomática. O protocolo se baseia na coloração da folha intacta com iodeto de propídio (PI), a incubação de coloração das folhas com suspensão bacteriana, e observação das folhas sob microscópio confocal de varredura a laser. Finalmente, esse método permite a observação da amostra da folha mesmo viver durante longos períodos de tempo usando condições que simulam as condições naturais em que as plantas são atacadas por patógenos.

Watch this Scientific Journal Video about Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging at JoVE.com

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Nós desenvolvemos um protocolo simples e reprodutível para acessar a resposta estomática ao bactérias vivas. Este método minimiza ferimento e manipulação da folha, em comparação com o uso de peelings epidérmicos relatado anteriormente.

Avaliar a resposta estomática ao vivo células bacterianas usando imagens Folha Inteira

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

assessing-stomatal-response-to-live-bacterial-cells-using-whole-leaf

Research

JoVE Journal

Immunology and Infection

18.3K visualizações.  University of Texas at Arlington. Nós desenvolvemos um protocolo simples e reprodutível para acessar a resposta estomática ao bactérias vivas. Este método minimiza ferimento e manipulação da folha, em comparação com o uso de peelings epidérmicos relatado anteriormente.

Vídeo: Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Using Luciferase to Image Bacterial Infections in Mice

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

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

Usando a luciferase Infecções bacterianas Imagem em camundongos

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

Imunologia e Infecção

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imagens de células vivas de Bacillus subtilis E Streptococcus pneumoniae Usando Microscopia lapso de tempo Automated

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualização das respostas toxina bacteriana induzida por microscopia de fluorescência de células vivas

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Imagem bacteriana Bioluminescent<em&gt; Em Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualizando interações DNA-proteína em células bacterianas ao vivo usando fotoativado única molécula de Rastreamento

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

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.

Avaliação da invasão bacteriana de células cardíacas na cultura e colonização cardíaca em camundongos infectados usando listeria monocytogenes

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

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

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

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

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

Geração e Multi-fenotípica rastreio de alto teor de<em&gt; Coxiella burnetii</em&gt; mutantes por transposões

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

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Bacteriana Folha Infiltração Ensaio para Belas Caracterização das respostas de defesa da planta usando o Arabidopsis thaliana-Pseudomonas syringae Patossistema

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Medindo respostas fisiológicas de<em&gt; Drosophila</em&gt; Neurônios sensoriais para Lipid feromonas Usando Imagem de cálcio ao vivo

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Imagens ao vivo de antifúngicos Atividade por neutrófilos humanos primários e monócitos em resposta à<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...