Pesquisa em P. Cossart laboratório é apoiado pelo Instituto Pasteur, o Institut National de la Santé et de la Recherche Médicale, o Institut National de la Recherche Agronomique, ERC avançada Grant (233348), a Agence Nationale de la Recherche (Grant MIE- SignRupVac), a Fundação Louis-Jeantet ea Fondation Le Roch Les Mosqueteiros. AK é um beneficiário de uma bolsa de estudos do Pasteur-Paris International University Doutorado Programa / Institut Carnot Maladies Infecciosas. Agradecemos a Jason Mercer para otimizar o protocolo de transfecção celular.

1. Preparação de culturas celulares e bacterianos, ferramentas de transfecção e Anticorpos Primários <ol><li> Prepare uma placa de ágar fresco para isolar L. indivíduo monocytogenes colónias a partir de um estoque de glicerol bacteriana (50% glycerol/50% saturada de cultura durante a noite de bactérias líquido) mantida a -80 ° C. <ol><li> Usando uma cremalheira de alumínio (mantidos à temperatura de -80 ° C) para o transporte de um estoque de glicerol congelado de bactérias, bactérias estrias sobre uma infusão de cérebro e coração (BHI) em placas. </li><li> Incubar a placa a 37 ° C durante 48 horas (ou até que as colónias bacterianas individuais podem ser isoladas). </li><li> Manter esta placa a trabalhar depois a 4 ° C durante um período máximo de 1 mês (isto irá ser usado para semear culturas líquidas). </li><li> Em nosso protocolo, usamos a L. monocytogenes sorotipo 1/2a EGDe.PrfA * estirpe, mas como ilustrado na Figura 1, o L. monocytogenes sorotipo 4b P14.PrfA * tensão dá resultados semelhantes. </li></ol></li><li> EleAs células ATCC CCL2 La são cultivadas em Meio de Eagle Modificado por Dulbecco (DMEM) suplementado com 10% de soro fetal bovino (FBS) na ausência de antibióticos. </li><li> Piscinas independentes de mexidos (controlo) e os anti-Met siRNAs são carregados em 384 placas de cultura de células de microscopia pretas. <ol><li> Dilui-se 160 pmol de siRNA em 500 mL de água isenta de RNase e adicionam-se 5 ul desta solução por poço (concentração final: 1,6 pmol). </li><li> Manter esta placa à temperatura de -20 ° C durante um período máximo de 1 ano. </li></ol></li><li> Os anticorpos policlonais de coelho contra a proteína bacteriana inLC foram obtidos por imunização com uma proteína recombinante inLC-GST, conforme descrito anteriormente 25. </li></ol> 2. Reverter siRNA transfecção celular <ol><li> 72 horas antes da infecção trazer à temperatura ambiente, uma placa de 384 poços de cultura de células microscopia preto contendo 1,6 pmol siRNA em 5 mL de água livre de RNase em cada poço. </li><li> Centrifugar a placa durante 3 minutos a 300rcf à temperatura ambiente para reduzir o siRNA que poderia ter sido depositado nas paredes dos poços. </li><li> Prepare a temperatura ambiente DMEM suplementado com 0,4% de Lipofectamine RNAiMAX. </li><li> Adicionar 25 ul de meio de transfecção de DMEM / RNAiMAX a cada poço (não incubar o meio de transfecção mais de 20 minutos antes de adicionar o siRNA). </li><li> Mover a placa para trás e para misturar o siARN com a solução de transfecção, em seguida, manter a placa durante 1 hora à temperatura ambiente para permitir que os complexos de siRNA-lipofectamina a formar. </li><li> Lavar as células HeLa a partir de um balão de cultura de células confluentes ou sub-confluentes, uma vez com 10 ml de 37 ° C pré-aquecido PBS. </li><li> Separar as células HeLa pela adição de 1 ml 37 ° C pré-aquecido de tripsina para o frasco de cultura de células e incubar durante 3 a 5 min a 37 ° C. </li><li> Re-suspender as células em 10 ml de 37 ° C pré-aquecido DMEM suplementado com 16% de FBS. </li><li> Contagem de células e preparar uma suspensão de células de 12.000 células por ml em DMEM suplementado com16% de FBS. </li><li> Adicionar 50 ul de suspensão celular a cada poço da placa de 384 poços. </li><li> Mova a placa rapidamente para trás e para distribuir as células e deixar as células se acalmar por 10 minutos em temperatura ambiente. </li><li> Selar a placa com Parafilm e mantê-la durante 72 horas numa atmosfera húmida de 5% de CO 2 contendo atmosfera a 37 ° C. </li></ol> 3. Infecção Celular e Coloração <ol><li> O dia antes da infecção, tomar uma única colônia de L. monocytogenes da placa de agar BHI e ressuspender o em 5 mL de meio BHI líquido em um tubo de poliestireno de 15 ml. </li><li> Incubar durante a noite a 37 ° C num dispositivo Shacking para permitir o crescimento bacteriano. </li><li> No dia da infecção, lave 1 ml durante a noite de L. monocytogenes cultura por centrifugação 2 min a 10.600 rcf em uma centrífuga de mesa. </li><li> Descartar o sobrenadante (que contém a citotoxina segregada listeriolisina O) e re-suspender o sedimento em 1 ml de PBS (REPEAt a etapa de lavagem mais 3 vezes). </li><li> Ler a densidade óptica a 600 nm bacteriana e estimar o número de bactérias (OD = 1 é equivalente a 1E9 bactérias / ml). </li><li> Prepare o adequado L. monocytogenes diluição em DMEM suplementado com FBS a 1%: usando estirpes altamente invasivos como EGDe.PrfA *, sugere-se a utilização de bactérias 5E4 em 30 ul de meio por poço (a multiplicidade da infecção foi calculado como 25 por 2.000 células). </li><li> Remover o meio de cultura de células em cada cavidade (80 ul) e substituí-lo por adição de 30 ul do L. monocytogenes contendo meio. </li><li> Centrifugar a placa a 200 rcf durante 5 min à temperatura ambiente, para sincronizar o processo de infecção. </li><li> Incubar a placa durante 1 hora numa atmosfera húmida de 5% de CO 2 contendo atmosfera a 37 ° C sobre um bloco de alumínio pré-aquecido. </li><li> Remover o L. monocytogenes contendo meio de cada poço e adicionar 30 ul de DMEM pré-aquecido suplementado com 10% FBS e40 ug / ml de gentamicina para matar L. extracelular monocytogenes. </li><li> Incubar durante 4 horas em 5% de CO 2 contendo atmosfera a 37 ° C num bloco de metal pré-aquecido. </li><li> Prepara-se uma solução de PBS suplementado com 8% de formaldeído (esta solução deve ser preparada de modo a que os monómeros de formaldeído, em vez de os polímeros de paraformaldeído, são utilizados). </li><li> Sem descartar o meio de cultura celular, adicionar 30 ul de PBS suplementado com 8% de formaldeído (concentração final: 4%) e incubar durante 15 min à temperatura ambiente. </li><li> Retirar o fixador e lava-se as células três vezes com 80 ul de PBS por poço (manter as células num volume final de 80 ul de PBS por poço). </li><li> Prepara-se uma diluição de 1:250 de anticorpo de coelho anti-inLC soro em PBS suplementado com 0,2% de saponina. </li><li> Adicionar 10 ml da solução de anticorpo primário em cada poço após remoção do PBS e incubar durante 30 min à temperatura ambiente. </li><li> Descarte o primárioA solução de anticorpo e lavar quatro vezes com 40 ul de PBS por poço. </li><li> Dilui-se a Alexa Fluor 546 acoplado anticorpo secundário anti-coelho (1:250), a solução DAPI (1:1.500) e faloidina-Dy647 (1:150) em PBS suplementado com 0,2% de saponina. </li><li> Adicionar 10 ul desta solução de coloração secundária a cada poço e incubar durante 30 min à temperatura ambiente. </li><li> Descartar a solução de coloração secundário e lava-se quatro vezes com 40 ul de PBS por poço (deixar um volume final de 40 ul de cada poço e selar a placa). </li><li> A placa pode ser fotografada imediatamente ou pode ser armazenada a 4 ° C, protegida da luz (cobrir com folha de alumínio) para análise posterior. </li></ol> 4. Aquisição de Imagem e Análise <ol><li> Adquirir imagens em três canais diferentes (350 nm, 546 nm e 647 nm) utilizando uma objetiva de 10X montado em um microscópio automatizado (adquirir preferencialmente 9 imagens por bem). </li><li> O sinal inLC pode ser medido usando a imagemO software de análise como CellProfiler que permite a segmentação automatizada dos núcleos e corpos celulares utilizando o DAPI e a coloração faloidina, respectivamente. </li></ol>

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Não temos nada a divulgar.

Vários parâmetros são fundamentais para o sucesso do nosso protocolo de detecção inLC, incluindo o uso de linhas de células saudáveis ​​que apresentam um número suficiente de citoplasma para permitir uma detecção inequívoca do sinal inLC. No ensaio que apresentamos neste artigo, propomos o uso de células HeLa CCL2, que são particularmente adequados para o nosso ensaio, devido à extensão de seu espaço citosólico; outros clones HeLa como as células HeLa Kyoto exibir um citoplasma menor, mas pode ser ...

A correction was made to <a href="http://www.jove.com/video/51043">Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes</a>. Three authors were omitted from the article at the time of publication. The acknowledgments section was also updated. The author list has been updated from:
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020
to
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Jason Mercer4, Mario Emmenlauer5, Christoph Dehio5, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute,2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Z&#252;rich 5Focal Area Infection Biology, Biozentrum, University of Basel
The acknowledgments where update from:
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We thank Jason Mercer for optimizing the cellular transfection protocol.
to
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We acknowledge support by grant 51RT 0_126008 for the Research and Technology Development (RTD) project InfectX in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology (to C.D.).

A correction to author list has been made for the article Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes. Three authors were omitted from the article at the time of publication.

Erratum: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

A bactéria Gram positiva Listeria monocytogenes é um agente patogénico de origem alimentar que invade células hospedeiras, interrompe a sua internalização vacúolo e replica-se no citoplasma das células hospedeiras 1. L. monocytogenes facilidade de manipulação no âmbito de laboratório (crescimento rápido, uma baixa toxicidade para os indivíduos saudáveis) associada à persistência de traços de virulência da bactéria observadas em modelos celulares e animais (actividade hemolítica, leucocitose) permitiu o seu uso inicial em 1960 como um modelo para a maior o estudo do parasitismo intracelular e para o estabelecimento dos fundamentos teóricos da imunidade celular contra a infecção 2. No final de 1980 e início de 1990, a dissecação do ciclo intracelular bacteriana 3, bem como a caracterização molecular da virulência bacteriana mais importante fatores 4-7 favoreceu o uso de L. monocytogenes como ferramenta molecular chave para a manipulação e cravoy de funções da célula hospedeira. A presença de avirulent (L. innocua) e (L. monocytogenes) espécies virulentas do gênero Listeria pavimentou o caminho para estudos genômicos comparativos 8 que, juntamente com a recente criação da L. completo monocytogenes transcriptoma 9, aumentamos nossa compreensão da evolução de L. Listeria como um patogénico humano e como um sistema modelo para estudos de infecção de 10. L. monocytogenes induz sua internalização em células hospedeiras após a interação das proteínas da superfície bacteriana inla e inlB com seus receptores da célula hospedeira E-caderina e MET, respectivamente 11-12. Estudos de base candidata inicial levou à identificação de uma ligação cateninas-actina α / β como um componente significativo da via inla-13 e invasão da fosfoinositida 3-quinase (PI 3-K) como um efector crucial do inlB- dependente casca invasãode 14-15. Ensaios de proteômica e baseados em funcional, posteriormente, permitiu a identificação de novos elementos do citoesqueleto 16 e lipídios segundos mensageiros 17 necessários para a invasão da célula hospedeira. Estudos de transcrição 18 e proteômica baseada em espectrometria de massa quantitativos 19 lançaram recentemente uma nova luz sobre a ativação da sinalização de acolhimento cascatas ea repressão da resposta imune durante L. monocytogenes infecção. Biologia de sistemas abordagens baseadas na inativação de grandes conjuntos de genes (kinomes, genomas completos) por pequeno RNA de interferência (siRNA) silenciamento abriram recentemente novos caminhos para a análise do acolhimento global de cascatas de sinalização no contexto de funções celulares específicas, incluindo a fagocitose e patógeno internalização 20. Telas siRNA Genome-wide foram previamente realizados para investigar cascatas celulares necessários para a infecção de L. monocytogenes em fagocitária Drosophila </em&gt; 21-22 células S2, mas este tipo de análise não foi realizada em células não fagocíticas, que representam alvos importantes para a infecção in vivo. Nós otimizamos um protocolo para a detecção microscópica dos estágios finais da infecção por L. monocytogenes, que é adequada para estudos de alta produtividade siRNA de entrada de bactérias no interior das células epiteliais. Nosso ensaio se aproveita de uma L. altamente invasivo monocytogenes cepa que apresenta uma mutação pontual na PrfA, o principal regulador da transcrição de L. monocytogenes virulência 6: esta mutação (chamado PrfA *) torna PrfA constitutivamente activa 23 e conduz a um aumento da expressão das proteínas de invasão inla inlB e, portanto, favorecer a entrada de bactérias em células não fagocíticas outro modo mal infectados. A nossa leitura de infecção baseia-se na detecção da acumulação citosólica do inLC proteína bacteriana segregada: esta molécula éum efector pleiotrópico que é expressa preferencialmente pelas intra-citoplasmático L. monocytogenes 9 e que não só participa na célula-a-célula bacteriana espalhar 24, mas que também modula as respostas imunitárias do hospedeiro 25. A marcação fluorescente de secreção inLC por bactérias intracelulares não só permite distinguir claramente infectada a partir de células não-infectadas, mas também representa uma leitura de ponto final que pode ser usada para dissecar posteriormente infecção em suas diferentes etapas: entrada, escape vacuolar, citosólica bacteriana proliferação e disseminação célula-a-célula. Este protocolo baseado em microscopia pode ser acoplado a telas de siRNA, por conseguinte, para estudar vias celulares envolvidas na infecção de células hospedeiras por L. monocytogenes.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td>For liquid BHI preparation</td>
		 </tr>
		 <tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>Supplement to liquid BHI for BHI agar plates </td>
		 </tr>
		 <tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51830-500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamicin</td>
			<td>Sigma </td>
			<td>G1397-10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Formaldehyde (16%)</td>
			<td>EMS</td>
			<td>15710</td>
			<td>Prepare fresh before each experiment</td>
		 </tr>
		 <tr>
			<td>Anti-Rabbit Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A-11035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D-1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phalloidin Dy647</td>
			<td>Dyomics</td>
			<td>647-33</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Scramble</td>
			<td>Dharmacon</td>
			<td>D-001810-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Met</td>
			<td>Dharmacon</td>
			<td>L-003156-00-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 384-well microscopy cell culture plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AxioObserver Z1 microscope</td>
			<td>Zeiss</td>
			<td>431007 9901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Neo</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Metamorph analysis software</td>
			<td>Molecular Devices</td>
			<td>4000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CellProfiler analysis software</td>
			<td>Broad Institute</td>
			<td>Public software available at <a href="http://www.cellprofiler.org/" target="_blank">http://www.cellprofiler.org/</a></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes

Patógenos intracelulares bacterianas podem ser concebidas como ferramentas moleculares para dissecar as cascatas de sinalização celular, devido à sua capacidade de manipular requintadamente e subverter as funções das células que são necessários para a infecção dos tecidos-alvo do hospedeiro. Entre estes agentes patogénicos bacterianos, Listeria monocytogenes é um microrganismo gram-positivo que tem sido usada como um paradigma para parasitismo intracelular na caracterização das respostas imunes celulares, e que tem desempenhado as funções instrumentais na descoberta de vias moleculares que controlam citoesqueleto e membrana dinâmica de tráfico. Neste artigo, os autores descrevem um ensaio microscópico robusto para a detecção de níveis de infecção celular final de L. monocytogenes baseado na marcação fluorescente do inLC, uma proteína bacteriana segregada que se acumula no citoplasma de células infectadas, o ensaio pode ser acoplado a high-throughput telas pequenas automatizados ARN interferentes, de modo a characterize vias de sinalização celular envolvidas no cima ou para baixo-regulação da infecção.

Marcação fluorescente de citoplasmática inLC fornece uma leitura robusta para a infecção de células por L. monocytogenes, como ilustrado na Figura 1: a célula central na micrografia é altamente infectados pela estirpe P14.PrfA * 23, uma vez que pode ser observada na imagem de contraste de fase (setas, Figura 1A) e confirma-se por o sinal de DAPI onde bactérias individuais podem ser claramente distinguidos (Figura 1B). A coloração inLC (sob...

Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

A Listeria monocytogenes é uma bactéria patogénica gram positiva frequentemente utilizado como um importante modelo para o estudo de parasitismo intracelular. Imagiologia tarde L. monocytogenes fases de infecção dentro do contexto de telas de ARN pequenos interferentes permite o estudo global de vias celulares necessários para a infecção bacteriana de células hospedeiras alvo.

Imagiologia inLC Secreção para investigar infecção celular pelo patógeno bacteriano<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

imaging-inlc-secretion-to-investigate-cellular-infection-bacterial

Research

JoVE Journal

Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

9.8K visualizações.  Pasteur Institute.  A Listeria monocytogenes é uma bactéria patogénica gram positiva frequentemente utilizado como um importante modelo para o estudo de parasitismo intracelular. Imagiologia tarde L. monocytogenes fases de infecção dentro do contexto de telas de ARN pequenos interferentes permite o estudo global de vias celulares necessários para a infecção bacteriana de células hospedeiras alvo. 

Vídeo: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Imunologia e Infecção

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 ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Medir a carga bacteriana e resposta imune em camundongos infectados com Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

O inseto<em&gt; Galleria mellonella</em&gt; Como um modelo de infecção poderosa para investigar Patogênese Bacteriana

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Transmissão Oral de<em&gt; Listeria monocytogenes</em&gt; Em camundongos através de ingestão de alimentos contaminados

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

A separação automática de<em&gt; C. elegans</em&gt; Variably Colonizada por um patógeno bacteriano

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

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 ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

A purificação a partir de ARN intracelularmente Grown<em&gt; Listeria monocytogenes</em&gt; Em macrófagos

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Infecção experimental com<em&gt; Listeria monocytogenes</em&gt; Como um modelo para estudar o anfitrião Respostas interferão-y

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.