This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1. Prepare-se com antecedência <ol><li> Quente fosfato estéril salina tamponada (PBS) a 37 ° C. </li><li> Modificação de Dulbecco quente de Meio de Eagle (DMEM) e Meio Essencial Mínimo (MEM) suplementado com 10% de soro fetal bovino (FBS), 100 UI / ml de penicilina, e 100 ug / ml de estreptomicina a 37 ° C. Nota: Estes representam crescimento normal de mídia (NGM) para as células RAW e HeLa, respectivamente. </li></ol> 2. Preparação de Células <ol><li> Crescer 264.7 RAW para 70-90% de confluência em NGM. <ol><li> Manter as células a 37 ° C numa atmosfera humidificada com 95% de ar / 5% de CO2. </li></ol></li><li> Cultivar células HeLa a 70-90% de confluência em NGM. <ol><li> Manter as células a 37 ° C numa atmosfera humidificada com 95% de ar / 5% de CO2. </li></ol></li><li> Antes da coleta, lave monocamada de células uma vez com PBS estéril. </li><li> Recolher as células pré-confluentes num tubo cónico estéril. <ol><li> Raspe suavemente as células RAWcom um policia de borracha em PBS, com pipetagem repetida para dispersar agregados de células. </li><li> Separar as células HeLa com solução de tripsina a 0,25% até que o exame visual mostra a dissociação a partir da superfície de cultura. Por exemplo, uso de 2 a 3 ml de um frasco T-75. Ajustar o volume da solução em conformidade para assegurar tripsina cobre toda a superfície de crescimento. <ol><li> Extingue-se reacção com dissociação NGM contendo 10% de FBS. </li><li> Use pelo menos duas vezes o volume de NGM a tripsina, com pipetagem repetida para dispersar agregações celulares. </li></ol></li></ol></li><li> Levemente peletizar as células por centrifugação a 900 xg durante 4 min. </li><li> Ressuspender em volume igual solução de tripsina / têmpera usando PBS estéril. </li><li> Contagem de células usando hemocitômetro ou contador de partículas. </li><li> Levemente peletizar as células por centrifugação a 900 xg durante 4 min. </li><li> Ressuspender em volume adequado de PBS para 5,5 x 10 6-6,0 x 10 6 células / ml. Nota: Por exemplo, um frasco de T-75 irá produzir aproximdamente 7,5 x 10 6 células 29. </li><li> Mantenha suspensão de células em gelo até eletroporação. </li></ol> 3. Preparação para electroporação <ol><li> Cuvetes Pré-chill eletroporação (0,4 centímetros de folga) em gelo. </li><li> Ligue aparelho de eletroporação e definir a tensão de 0,3 kV. NOTA: capacitância e resistência não eram configurações ajustáveis ​​em nosso electroporator (fixado em 10 iF e 600 Ω pelo fabricante). </li><li> Encha com placas de recuperação NGM e equilibrar em humidificada de 95% ar / 5% de CO2 a 37 ° C. </li></ol> 4. Eletroporação <ol><li> Colocar 400 mL da suspensão de células em cuvetes de pré-gelada e adiciona-se 20 ug de proteína seleccionada a cuvete (50 ug / ml). </li><li> Flick tina suavemente ~ 10 vezes para misturar sem danificar as células. Nota: A cuvete pode também ser invertida diversas vezes para misturar bem, mas não pipetar para cima e para baixo ou de vórtice, para evitar danificar as células. </li><li&gt; fora seco de tina com toalha de papel ou outro limpador de para evitar a formação de arco elétrico no electroporator. </li><li> Amostra electroporate a 0,3 kV para 1,5-1,7 ms. Nota: Isso era típico para este estudo. </li><li> Imediatamente após a eletroporação filme tina suavemente ~ 10 vezes para misturar bem. </li></ol> 5. chapeamento de Células <ol><li> Tina loja com células eletroporados no gelo até que esteja pronto para colocar em placas de recuperação. </li><li> Para a maioria das análises a jusante, lave as células 1x com NGM pré-aquecido para remover a proteína estranha efectora. <ol><li> Remover as células para análise e suspender em 3-5 ml de NGM. </li><li> Peletizar as células por centrifugação a 900 xg durante 4 min. </li><li> Ressuspender em volume adequado de NGM para o tamanho da placa desejada (por exemplo 2 ml para 35 milímetros prato). </li></ol></li><li> Remover quantidade adequada de células para análise de downstream. <ol><li> Exemplo 1: placa em pratos com fundo de vidro para análise de microscopia. </li><li> Exemplo 2: placa into plástico de cultura de células para análise de proteínas, tais como purificação por afinidade. </li></ol></li><li> Permitir que as células em placas de recuperar humidificada equilibrada em 95% de ar / 5% de CO2 a 37 ° C durante pelo menos 4 horas. </li></ol> 6. Análise de Microscopia 6.1) Fixação / técnica de imunofluorescência <ol><li> Lave as células 1x com PBS estéril após o período de recuperação de 4 horas. </li><li> Fix células em 100% de metanol durante 2 minutos à temperatura ambiente. Use metanol suficiente para cobrir completamente as células (por exemplo, 2 ml para 35 milímetros prato). </li><li> Lavar 3x com PBS estéril. </li><li> Permeabilizar as células com 0,4% de Triton X-100 em PBS durante 15 min. Por exemplo, utilizar 1 ml para uma placa de diâmetro de 35 mm. <ol><li> Ajustar o comprimento de permeabilização e força de Triton X-100 de acordo com epitopo e localização da proteína alvo. Nota: Os melhores resultados terá de ser determinado empiricamente para cada alvo, no entanto, as condições acima mencionadas, deve ser adequada para a maioria cmetas ytosolic. </li></ol></li><li> Bloco com 5% Albumina de Soro Bovino (BSA) em PBS durante 1 h à TA. Por exemplo, utilizar 1 ml para uma placa de diâmetro de 35 mm. </li><li> Lavar 3x com PBS. </li><li> Incubar anticorpo primário em solução de ligação do anticorpo (0,1% de Triton X-100 e 1% de BSA em PBS) durante a noite a 4 &#160; ° C com agitação suave. Por exemplo, usar 0,5 ml para uma placa de diâmetro de 35 mm. <ol><li> Siga as recomendações do fabricante para a diluição do anticorpo. Nota: Por exemplo, o anticorpo marcador peptídico de ligação a estreptavidina (SBP-tag) foi utilizado a 1: 1000 de diluição, enquanto que o anticorpo PKN1 foi utilizado a 1: 200 de diluição. </li></ol></li><li> Lavar 4x com PBS. </li><li> Incubar com anticorpo secundário conjugado com fluorescência apropriado em solução de ligação do anticorpo durante 1 hora à temperatura ambiente, protegida da luz. <ol><li> Por exemplo, usar Alexa 488 ou Alexa 647, durante 1 h à temperatura ambiente. <ol><li> Siga as recomendações do fabricante para a formigadiluição ibody. (Por exemplo, cerca de 1: 1000 para este estudo). </li></ol></li><li> Adicionar outras manchas, conforme necessário, por exemplo, 5 uM de gérmen de trigo agglutinnin (WGA) conjugada com Alexa 647 ou DAPI de acordo com a recomendação do fabricante, durante 1 hora à temperatura ambiente. </li></ol></li><li> Wash 5x com PBS e armazenar a 4 ° C, protegido da luz até que esteja pronto para a imagem. </li></ol> 6.2) microscopia confocal e Análise de Imagem <ol><li> Amostras de imagem em um microscópio confocal invertido utilizando uma objectiva de imersão em óleo de 63x. <ol><li> Imagem canal verde usando a linha de 488 nm de um laser de argônio, com emissão de largura de banda entre 492-542 nm. </li><li> Imagem canal vermelho usando um laser de diodo 633 nm, com emissão de largura de banda entre 640-718 nm. </li><li> Imagem do canal de azul usando um laser de diodo 405 nm, com emissão de largura de banda entre 407-453 nm. </li><li> Assegurar o canal múltiplo z pilhas cobrir a totalidade do volume celular. </li></ol></li><li> Processar imagens com o software de processamento de imagem apropriado. </li></ol> 7. Affinity Purificação <ol><li> Lave as células eletroporados duas vezes com 4 ° C PBS após o período de recuperação de 4 horas. </li><li> Lise com ~ 1,0 ml de tampão de lise (1% Triton X-100 com um cocktail inibidor de protease e inibidores de fosfatase em PBS) em gelo. Use inibidores de acordo com as instruções do fabricante. Nota: Por exemplo, ambos os inibidores da protease e da fosfatase utilizados neste estudo foram fornecidas a 100x, mas outras formulações deve funcionar igualmente bem. </li><li> Prontamente raspar células com polícia de borracha e recolher em tubos cônicos. </li><li> Lise por vórtice vigoroso e sonicação. Nota: Outros métodos de lise pode funcionar igualmente bem, mas a eficiência será necessário ser empiricamente determinada como a adequação dos requisitos do ensaio. <ol><li> Sonicar 3 x 30 segundos com agitação em vórtex intermitente. </li></ol></li><li> Centrifugar a 10.000 xg durante 10 min a 4 ° C para recolheros detritos celulares e agregados insolúveis; salvar o sobrenadante. </li><li> Combinar volumes iguais (~ 1,0 ml) de lisado de células electroporadas com 50 ul de suspensão de estreptavidina resina de agarose a 4 ° C durante a noite com uma rotação de extremidade sobre extremidade. Nota: Esta resina captura a proteína electroporadas (e complexos associados), através do seu péptido marcador de afinidade de ligação a estreptavidina. </li><li> Centrifugar a 2.500 xg por 2 min e elimine o sobrenadante. </li><li> Lavar duas vezes com 40 volumes de leito (~ 1 ml de PBS). </li><li> Adicionar 30 ul de tampão de carga 4x LDS (141 mM de base Tris, 2% de LDS, 10% glicerol, 0,51 mM de EDTA, 0,22 mM de L SERVA azul, vermelho de fenol 0,175 mM, pH 8,5), 20 ul dih 2 O, e 1 ul de 0,5 M de tris (2-carboxietil) fosfina (TCEP), permitindo um certo volume para permanecer em grânulos. </li><li> Aquece-se a 95 ° C durante 10 min e arrefecer em gelo. </li><li> Rotação&gt; 10.000 xg, a 4 ° C durante 5 min. </li><li> Recolher o sobrenadante. </li><li> Realizar um western blot utilizando anticorpos apropriados Nota: Elere, anti-PKN1 anticorpo primário é usado. </li></ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

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Efectores secretadas a partir de bactérias patogénicas evoluíram a funcionar dentro do ambiente da célula hospedeira e, assim, é útil para estudá-los in situ dentro do hospedeiro. Introdução de efetores específicos de interesse em células hospedeiras permite a interação patógeno-hospedeiro relevantes a serem estudados de forma isolada, sem a interferência de outras proteínas bacterianas. O objectivo foi o de explorar a electroporação como um meio para introduzir proteínas efectoras bacteriana...

Muitas bactérias Gram-negativas empregam sistemas de secreção especializados para injetar proteínas relacionadas à virulência (referidas como efetores) diretamente nas células hospedeiras 1-5. Estes efectores têm uma ampla gama de funções biológicas, incluindo: supressão da imunidade do hospedeiro, alterações do citoesqueleto, a modificação do tráfico intracelular e de sinalização, alterações da transcrição, e as alterações de proteassoma hospedeiro 6-9. As funções de alguns efectores são conhecidos, no entanto, os alvos do hospedeiro e de acção bioquímica (s) de muitos outros permanece a ser determinada. Ao se comparar o tipo selvagem e infecções bacterianas recombinantes é uma aproximação válida para estudar os mecanismos de virulência efector intracelular, é muitas vezes vantajoso para introduzir um indivíduo efectora na célula hospedeira. Assim, os métodos simples para a introdução e caracterização de proteínas efectoras bacterianas no contexto de células hospedeiras é altamente desejável. Simplificar o wi análise experimentalth um único efetor é crítica como outros efetores podem ter funções opostas ou redundantes. Para realizar esta simplificação, os pesquisadores já introduziu macromoléculas em células por muitos métodos diferentes, incluindo a transdução viral 10, microinjeção 11, raspar o carregamento 12,13, fusão celular com microinjeção induzido quimicamente 14, proteína proprietária &quot;transfecção&quot; reagentes 15, precipitações de fosfato de cálcio 16, 17-20 e electroporação. As moléculas introduzidas variam de ácidos nucleicos, incluindo as espécies de ADN, ARN, e proteínas de ARNi a, corantes de células-impermeável, e anticorpos para alvos intracelulares 21,22. Alguns métodos têm limitações, incluindo o tipo de macromoléculas que podem ser introduzidas, e em particular análises a jusante pode ser limitada devido à elevada toxicidade celular, prejudiciais mecanismos de acção, a baixa eficácia, ou eficiência de introdução. A transfecção, uma oftemétodo de n-utilizado para a expressão de genes bacterianos em células de mamífero, também sofre da limitação de que alguns tipos de células hospedeiras relevantes, tais como macrófagos e células primárias, são particularmente resistentes às transfecção. Além disso, é difícil controlar os níveis de proteína bacteriana produzida após a introdução de ADN estranho. Muito trabalho estabeleceu eletroporação de ácidos nucleicos em ambas as células bacterianas e de mamíferos como uma técnica de laboratório comum; no entanto, há investigação em curso sobre os melhores métodos para a entrega de proteínas nas células sob condições fisiológicas. Relatórios sobre a transfecção de proteína são promissores, mas requerem reagentes e otimização caros. O desejo de introduzir efectores bacterianas potencialmente tóxicos em uma grande variedade de alvos celulares com um custo mínimo nos levou a considerar electroporação como um método para o estudo destas proteínas in vivo. Electroporação é uma proteína de 23-25 ​​method para introduzir as proteínas em células vivas através electropermeabilization, também conhecidos como electro-transfecção ou electro-injeção 26. Esta técnica utiliza-alta intensidade impulsos eléctricos para criar poros em membranas celulares. Estes poros reversíveis permitir macromoléculas que são normalmente excluídos do espaço intracelular para entrar na célula. Após a remoção do campo eléctrico externo, a membrana pode selar novamente, permitindo que a célula de reter moléculas que passaram pelos poros 27,28. Um electroporador padrão foi utilizada neste estudo para introduzir consistentemente efectores bacterianas em ambas as células tipo macrófagos do rato e as células epiteliais humanas. O método é rápido, eficiente e barato, sem redução apreciável na viabilidade celular. As proteínas podem ser introduzidos visualizado por microscopia de imunofluorescência ou utilizadas para ensaios funcionais. Isto foi demonstrado utilizando a proteína fluorescente verde (GFP) como um padrão não-tóxicos, bem comoduas proteínas efectoras Salmonella, SspH1 e GTGE. Propomos electroporação proteína como uma ferramenta adicional para o repertório para o estudo de proteínas de virulência da bactéria e das suas funções em células hospedeiras eucarióticas.

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			<td>25-050-Cl</td>
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			<td>165-2088</td>
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			<td>Flammable, Toxic</td>
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			<td>Ziess</td>
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			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
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			<td>Any</td>
			<td>N/A</td>
			<td></td>
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			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
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			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
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			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
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			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
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			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
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electroporation of functional bacterial effectors into mammalian cells

O estudo das interações proteína no contexto de células vivas podem gerar informações importantes sobre a localização, dinâmica e parceiros interagem. Esta informação é particularmente valiosa no contexto das interacções hospedeiro-agente patogénico. Muitas proteínas de agentes patogénicos funcionar dentro das células hospedeiras na forma de uma variedade tais como, permitindo a evasão do sistema imunitário do hospedeiro e a sobrevivência no ambiente intracelular. Para estudar essas interações célula-hospedeiro de patógenos em proteínas, diversas abordagens são comumente usados, incluindo: infecção in vivo com uma estirpe que expressa uma proteína marcada ou mutante, ou a introdução de genes de patógenos via transfecção ou transdução. Cada uma destas abordagens tem vantagens e desvantagens. Buscamos um meio para introduzir directamente proteínas exógenas nas células. A electroporação é geralmente utilizado para introduzir ácidos nucleicos em células, mas foi mais raramente aplicado a proteínas, embora a base biofísica é exactamente o mesmo.Um electroporador padrão foi utilizada para introduzir efectores bacterianas de afinidade marcado com em células de mamífero. Macrófagos epiteliais e mouse Humanos foram cultivados pelos métodos tradicionais, individual, e colocados em 0,4 centímetros cuvetes gap eletroporação com uma proteína exógena bacteriana patógeno de interesse (por exemplo, Salmonella Typhimurium GTGE). Após a eletroporação (0,3 kV) e um curto período de recuperação (4 horas), proteína intracelular foi verificada por fluorescently rotulando a proteína através da sua marca de afinidade e examinar a distribuição espacial e temporal por microscopia confocal. A proteína também electroporação mostrou ser funcional no interior da célula e capaz de tráfico subcelular correcta e interacção proteína-proteína. Enquanto as proteínas exógenas tendia a acumular-se sobre a superfície das células, as amostras electroporadas teve grandes aumentos na concentração intracelular de efector em relação à incubação sozinho. O protocolo é simples e rápido o suficiente para ser feito em apmoda arallel, permitindo a caracterização de alta taxa de transferência de proteínas de patógenos em células hospedeiras incluindo segmentação e função das proteínas de virulência subcelular.

Como um teste de conceito, a proteína fluorescente verde purificada foi introduzida com sucesso em células de mamíferos utilizando eletroporação. GFP, um valor aproximado de 27 kD, uma proteína de peso molecular é geralmente introduzido nas células de mamífero (normalmente expressos a partir de ADN de plasmídeo), como uma ferramenta de biologia molecular, sem toxicidade celular significativa. As células HeLa foram incubadas (Figura 1A) ou electroporação (Figura 1B) com 25 u...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

A electroporação de efectores bacterianos funcionais em células de mamíferos

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K visualizações.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

Vídeo: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Invasão das células humanas por um agente patogénico

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Imunologia e Infecção

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Ensaio in vitro da adesão bacteriana em mamíferos Células Epiteliais

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolamento de células do sistema imunológico funcional cardíaca

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imagens em tempo real de células mielóides em Dynamics<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores girando Disk microscopia confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Detecção de glicogênio no sangue periférico células mononucleares com Periodic Acid Schiff Coloração

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Bacteriana Artificial Chromosomes: Uma Ferramenta genómica funcional para o Estudo da Positivo-fio vírus RNA

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Implementação de um Sistema de Infecção baseado em Inserir Permeável Membrana para estudar os efeitos de secretada bacterianas toxinas em células hospedeiras de mamíferos

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Desvendando a função de uma bacteriana Effector de um patógeno não-cultiváveis ​​meio de uma levedura Screen Two-hybrid

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Análise de imunofluorescência da formação de grânulos de estresse após o desafio bacteriano de células de mamífero

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...