Os autores gostariam de agradecer Resto Richard pelo dom generoso de anthrolysin O, Michael Caparon pelo dom generoso dos francos SLO plasmídeo e Jonathon de assistência técnica. Este trabalho foi financiado pelo NIH T32CA82084 (PAK), e R01AI072083 (RDS).

1. Purificação de Estreptolisina O (SLO) <ol><li> Inocular 20 mL de cultura durante a noite de células BL21 contendo ouro pBADgIII-SLOhis plasmídeo 9 em 0,5 L de caldo LB e adicionar 500 ul de 50 mg / ml de ampicilina. Agitar a cultura a 225 rpm a 37 ° C até DO600 = 0,6, geralmente ~ 1,5 horas. Centrifugar 1 ml (bactérias consideradas T 0) a 10.000 xg 5 min, dissolver o granulado em 140 ul de tampão de amostra SDS 1x / 1 OD e sonicar a DNA de cisalhamento para análise de pureza da proteína. </li><li> Induzir bactérias, com a adição de 5 ml de 20% de arabinose a cultura, agitar a 225 rpm à temperatura ambiente durante 3 hr. Colete 1 ml de cultura bacteriana, centrifugar e dissolver em 1x tampão de amostra de SDS como acima para T de tempo 3 para análise de pureza. </li><li> Recolher as bactérias remanescentes no frasco de 500 ml de centrífuga, girar 12000 xg durante 12 min a 4 ° C (8.500 rpm rotor Sorvall GS-3). Decantar o sobrenadante, armazenar pelete a -80 ° C durante a noite ou mais tempo, se necessário. </li><li> Ressuspender o frozen pellet em gelo em 10 ml de Lise / O tampão de lavagem (50 mM de NaH 2 PO 4, 300 mM NaCl, 10 mM de imidazole, pH 8,0) suplementado com 400 ul de 25% de Triton X-100, 100 uL de 100 mg / ml de lisozima, 50 200 ul mM de fluoreto de fenilmetilsulfonilo (PMSF). Transferir o sedimento ressuspenso para tubo de 50 ml de Ridge Oak. </li><li> Sonicar lisado 5 vezes durante 30 segundos em gelo, com intervalos de 30 seg. Spin-lisado sonicado em carvalho cume tubo a 39.000 xg durante 20 min a 4 ° C (18.000 rpm Sorvall SS-34 rotor). </li><li> Enquanto o lisado está girando, lave 1 ml de Ni-NTA agarose com 10 ml de Lise / tampão de lavagem e centrifugação a 1.200 rpm durante 5 min a 4 ° C. (Todas as lavagens adicionais / eluições utilizar a centrifugação sob estas condições). Aspirar lavar. </li><li> Adicionar sobrenadante bacteriano a partir do passo 1.5 a Ni-NTA agarose, agitar durante 2,5 horas a 4 ° C. Gire para recolher o sobrenadante. Combine 30 ul de sobrenadante com 10 tampão de amostra μ 4x SDS e salvar para análise de pureza. Lavar as pérolas 4 vezes em Lyse / tampão de lavagem e emce em Tris / sal tampão (50 mM Tris, 300 mM NaCl, pH 8,0). </li><li> Eluir 3 vezes por adição de 1 ml de tampão de eluição (250 mM de imidazole em Tris / tampão salino) e 5 ul de 1 M de ditiotreitol (DTT) a grânulos, incubação em gelo durante 10 min, centrifugação e recolhendo o sobrenadante. SLO é uma proteína muito sensível redox, e devem ser mantidos em gelo em todos os momentos. Uma vez reduzida, a proteína perderá rapidamente a sua actividade, se aquecido a 37 ° C, mesmo durante um curto período de tempo. </li><li> Lave 500 ul polimixina-conjugado de agarose em tampão de eluição, rotação a 10000 xg durante 1 min a 4 ° C e ressuspender em 500 ul. Para esgotar endotoxina, adicionar 200 ul de contas de cada eluição e agitar 4 ° C durante 30 min. Centrifugação a 10000 xg durante 1 min a 4 ° C, e guardar o sobrenadante. Combinar 30 ul de cada um de eluição com tampão de amostra de 10 ul 4x SDS para SDS-PAGE análise. </li><li> Testar a pureza de eluições por SDS-PAGE. Ferver as amostras guardadas para análise em gel a 95 ° C durante 5 min e 10 ul de carga sobre um gel a 10%. Mancha gel com Coomassie para visualizar proteína (Figura 2A). SLO é de 69 kDa. Executar um ensaio de Bradford para a determinação da concentração de proteína (rendimento típico é de 4 mg / ml para os primeiros dois eluições, mas pode variar de acordo com estirpe bacteriana e do plasmídeo utilizado). </li><li> Testar a actividade hemolítica de cada eluição (ver abaixo). Eluições piscina onde a atividade de concentração, pureza e hemolítica são satisfatórios. SLO alíquota em alíquotas de utilização única (ul tipicamente 5-10), em gelo seco congelar e armazenar a -80 ° C. </li></ol> 2. Ensaio hemolítico <ol><li> Lavar glóbulos vermelhos de ovelha (RBC) em RBC tampão de ensaio (0,3% de albumina de soro bovino (BSA), 2 mM de CaCl2, 10 mM de Hepes, pH 7,4), centrifugação a 1.200 rpm durante 5 min, e ressuspender em 2,5% em RBCs 10 ml de tampão de ensaio RBC. </li><li> Adicionar 10 ul de tampão de ensaio RBC / poço em placas de 96 poços de fundo em V de gelo. Série diluir cada eluição em duplicata. Gamas de diluição típicos são de 1:1.000 1:512,000. Incluem 3 poços sem toxina e 3 com 10 poçosul de Triton X-2% 100 como poços mínimo e máximo, respectivamente. </li><li> Cobrir a placa e incubar a 37 ° C 30 min. Centrifugar a 1200 rpm durante 5 min. Transferir 70 uL de sobrenadante para uma placa de fundo plano de 96 poços. Leia A 405. Uma unidade é a diluição da toxina necessária para a lise de 50% dos glóbulos vermelhos. Atividade toxina é uma unidade * diluição factor/0.01 ml. </li></ol> 3. Ensaio de célula Lytic <ol><li> Células de colheita, a contagem, spin. Ressuspender a 2x10 6 / ml em tampão de R / B (RPMI com CaCl2 2 mM, BSA 0,5%) e 20 ug / ml de iodeto de propídio. Adicionar 100 ul de células de uma placa de 96 poços de fundo em V. </li><li> Seriadamente diluído SLO para 2x a concentração final em tampão de R / B, adicionar 100 ul de toxina ou 100 ul de tampão P / B às células. Incubar 5 min a 37 ° C. Gama típica de concentrações finais de 2,000 U / ml para 31,25 U / mL. </li><li> Executar células em citômetro de fluxo e recolher dados com filtros de ficoeritrina (PE). Uma mudança de um log-cel representa transitoriamente permeabilizadasls, enquanto um desvio de log 3 indica as células mortas 4. Calcula-se a lise específica de células, subtraindo a percentagem de células mortas no controlo (% PI elevado ctl) a partir da (exp% PI elevado) experimental, como se segue: a lise específica = (% exp PI elevado -% PI elevado ctl) / (100 -% PI elevado ctl) * 100 </li></ol> 4. Micropipeta Entrega de Toxina de células em placas de cultura <ol><li> Um dia antes da experiência, placa 2x10 5 macrófagos em um colágeno revestido prato milímetros com fundo de vidro-35. </li><li> Ligue microscópio e microinjetor. Permitir tempo fase aquecida a aquecer até 37 ° C. A escolha do microscópio geralmente depende do que está localmente disponível. O microscópio de fase invertida precisa de uma, uma fase capaz de aquecer os pratos a 37 ° C, os cubos de excitação / emissão de filtro apropriadas para o corante escolhido, e o espaço de ligar fisicamente o microinjector. Uma lente Bertrandé útil para a micro-injecção, mas não exigida. O computador dirigir o microscópio precisa de memória suficiente para coletar e armazenar dados. </li><li> Células do rótulo para 30 min com corante, a 37 ° C. Dependendo do ensaio, etiquetagem pode ser feita com 5 uL Fura2 AM em 1 ml de PBS ou 2 ul de calceína AM em 1 ml de meio completo. Para Fura2, excitação / emissão é recolhida para 340/510 nm e 380/510 nm e a razão de 340/380 determina os sinais de fluxo de cálcio. Para calceína, excitação / emissão é 495/515 nm, enquanto etídio homodímero é 525/620 nm e APC é 650/660 nm. Outros rótulos podem ser escolhidos também. </li><li> Lavar as células com PBS e colocar em 1 ml de RPMI suplementado com 2 mM de CaCl 2. Montar em microscópio. </li><li> Diluir a toxina e o dextrano em água e centrifugar a 20000 xg durante 10 min 4 ° C. Tipicamente, uma SLO ul e 4 uL de 10 mg / ml de dextrano-555 é diluído em 6 ul de água. Dextrano ou outra molécula de fase fluida fluorescente é usado para verificar que o femto-ponta não estiver obstruído, e injects, como desejado. </li><li> Carregar femto ponta da parte traseira com 0,07 ul de toxina diluída utilizando uma microloader. </li><li> Carregar femto-ponta na microinjetor. Ajustar o ângulo do injector de modo a que a ponta se sentar sobre o centro das células, com espaço para se mover em todas as direções. Limpar z-limite. Configurações de injeção deve ser para injetar 0,5 segundos a 120 psi com pressão de 20 psi de volta. Diminuir ponta até que entra no meio. </li><li> Usando a lente Bertrand, centro da ponta e seguem a ponta, uma vez que é reduzida mais perto das células. Depois de perder o foco, voltar a ótica normais. A sombra da agulha deve ser aparente no campo. Concentre-se as células acima e abaixe a agulha até que esteja em foco. Concentre-se novamente as células e cuidadosamente trazer a agulha adjacente a uma célula. Definir o z-limite para a injeção. Iniciar imaging, mover a ponta para a posição desejada, injectar a libertar a toxina no ponto de tempo desejado. Levante a agulha, passar para uma nova região de células e injectar. Mover agulha para a posição inicial para evitar indesejadavazamento toxina da agulha. </li></ol>

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As técnicas descritas aqui permitem a análise das respostas de células imunes para as toxinas bacterianas. A etapa mais crítica é o manuseamento e a dosagem da toxina. A actividade da toxina pode ser extremamente variável, mesmo entre diferentes alíquotas da mesma preparação, devido à sua fragilidade. Isto requer tanto o teste de cada aliquota de toxina contra uma linha de células de referência, ou RBCs ou utilizando gradientes de toxina. Gradientes de toxina, tal como entregue por micropipeta, permitir que ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Ni-NTA agarose </td><td> Qiagen </td><td> 30210 </td><td></td></tr><tr><td> polimixina-agarose </td><td> Sigma </td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba dessalinizar rotação col </td><td> Pescador </td><td> PI-89891 </td><td></td></tr><tr><td> hemácias de ovinos </td><td> Pescador </td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> ver ref 9 </td></tr><tr><td> Cy5 corante monoreactive </td><td> GE Healthcare </td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies </td><td> F1221 </td><td></td></tr><tr><td> Calceína AM / Ethidium homodímero </td><td> Life Technologies </td><td> L3224 </td><td></td></tr><tr><td> Anti-CD11c-APC </td><td> BD Biosciences </td><td> 550261 </td><td></td></tr><tr><td> colágeno revestido prato fundo de vidro </td><td> Mattek </td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td> femto-ponta II </td><td> Pescador </td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td> Pescador </td><td> E5242956003 </td><td></td></tr><tr><td> dextrano Alexa 555 </td><td> Life Technologies </td><td> D34679 </td><td></td></tr><tr><td> InjectMan NI 2 </td><td> Eppendorf </td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf </td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Lista Tabela 1. Ea fonte de reagentes específicos e Equipent necessário. Equipamentos específicos e reagentes utilizados neste protocolo, juntamente com a empresa e número de catálogo estão listados. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Tampão </td><td> Composição </td><td> Passo Usado </td></tr><tr><td> Lyse / Wash </td><td> 50 mM de NaH 2 PO 4 300 mM de NaCl Imidazole 10 mM, pH 8,0 </td><td> 1,4 </td></tr><tr><td> Tris / sal </td><td> 50 mM Tris, pH 8,0 300 mM de NaCl </td><td> 1,7 </td></tr><tr><td> Tampão de eluição </td><td> 50 mM Tris, pH 8,0 300 mM de NaCl Imidazole 250 mM </td><td> 1,8 </td></tr><tr><td> O tampão de ensaio RBC </td><td> 0,3% de BSA 2 mM de CaCl 2 HEPES 10 mM, pH 7,4 </td><td> 2,1 </td></tr><tr><td> tampão R / B </td><td> Meio de cultura RPMI célula 2 mM de CaCl 2 BSA a 0,5% </td><td&gt; 3,1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> Lista Tabela 2. Dos buffers usados ​​neste protocolo. Os tampões utilizados, a sua composição e a primeira etapa em que são usados ​​no protocolo estão listadas. </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Toxinas bacterianas ligar ao colesterol em membranas, formando poros que permitem o vazamento do conteúdo celular e influxo de materiais a partir do ambiente externo. A célula pode recuperar-se este insulto, o que requer processos de reparação activos de membrana, ou morrem, dependendo da quantidade de exposição a uma toxina e tipo de célula 1. Além disso, estas toxinas induzir fortes respostas inflamatórias em hospedeiros infectados através da activação de células imunitárias, incluindo macrófagos, que produzem uma variedade de citocinas pró-inflamatórias 2. Muitas bactérias Gram-positivas produzem toxinas colesterol vinculativos que foram mostrados para contribuir para a sua virulência através de mecanismos largamente descaracterizados. Alterações morfológicas na membrana plasmática das células expostas a estas toxinas incluem a sua sequestração em saliências colesterol enriquecidas de superfície, que podem ser derramadas para o espaço extracelular, sugerindo uma defesa celular intrínsecomecanismo de 3,4. Este processo ocorre em todas as células na ausência de actividade metabólica, e pode ser visualizada usando EM após 4 a fixação química. Em células do sistema imunológico, tais como os macrófagos, que medeiam a inflamação em resposta a exposição a uma toxina, vesículas de membrana induzidas são sugeridos para conter citocinas da família IL-1, e pode ser responsável tanto para o derramamento de toxinas e disseminação destas citoquinas pró-inflamatórias 5,6,7. A ligação entre a IL-1β libertação e um tipo específico de morte celular, denominado pyroptosis tem sido sugerido, como ambos os processos são dependentes da caspase-1 8. Para resolver as complexidades desta resposta de macrófagos, que inclui a ligação da toxina, o derramamento de vesículas de membrana, a libertação de citoquinas, e da morte de células potencialmente, desenvolvemos técnicas de etiquetagem e de métodos de microscopia de fluorescência que permitem a visualização em tempo real da toxina de células-interacções, incluindo medidas de disfunção e morte (Figura 1). Usarde imagens de células vivas é necessária devido às limitações das outras técnicas. Abordagens bioquímicas não pode resolver os efeitos que ocorrem em células individuais, ao passo que a citometria de fluxo não oferece alta resolução, a visualização em tempo real das células individuais. Os métodos descritos aqui podem ser aplicados a análise cinética de respostas induzidas por estímulos que envolvem complexos alterações fenotípicas em células.

Tipicamente 10 7 -10 8 U / ml SLO pode ser obtida com uma concentração de proteína de 4 mg / ml. A quantidade de toxina necessária para a lise das células varia com o tipo de célula, mas é geralmente 125-500 U / ml SLO (Figura 2B). Tipos de células, como os macrófagos podem ser mais resistentes (4000 U / ml), embora outras linhas de células (especialmente T) são mais sensíveis. Estas sensibilidades corresponder com SLO comercialmente disponível. A actividade da toxina ...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Os métodos para a purificação do colesterol de ligação da toxina a partir de estreptolisina O recombinante<em&gt; E. coli</em&gt; E visualização de toxina se ligar a células eucarióticas vivo são descritos. Entrega localizada de toxina induz a mudanças rápidas e complexas em células-alvo, revelando novos aspectos da biologia toxina.

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 ...

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JoVE Journal

Immunology and Infection

11.7K visualizações.  University of Pittsburgh School of Medicine. Os métodos para a purificação do colesterol de ligação da toxina a partir de estreptolisina O recombinante E. coli E visualização de toxina se ligar a células eucarióticas vivo são descritos. Entrega localizada de toxina induz a mudanças rápidas e complexas em células-alvo, revelando novos aspectos da biologia toxina.

Vídeo: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizando transferência de células-célula de HIV usando Clones fluorescente de HIV e Microscopia Confocal ao vivo

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Imunologia e Infecção

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

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 ...

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 ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Análise de uma única célula de Bacillus subtilis Os biofilmes utilizando microscopia de fluorescência e citometria de fluxo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

Live-célula Microscopia vídeo de Fagocitose patógeno fúngico

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

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

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

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

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

Aplicação de longo prazo cultivadas Interferon-γ Enzyme-linked Immunospot Ensaio para a Avaliação e efetoras de memória T respostas de células em gado

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

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

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

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

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

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 ...

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 ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantificação de Efferocytosis por microscopia de fluorescência de célula única

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.