Somos gratos a Michael Feiss e Sippy Jean para a orientação sobre a criação de fagos e purificação. Agradecemos Michael Elowitz para fornecer o software de reconhecimento de célula, Schnitzcell. Trabalho no laboratório de Golding é suportado por subvenções dos Institutos Nacionais de Saúde (R01GM082837), a National Science Foundation (082,265, PFC: Centro de Física de Células Vivas), a Fundação Welch (Grant Q-1759) e Ciências Humanas da Fronteira Programa (RGY 70/2008).

<ol>
	<li>Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., Adhya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switches+in+bacteriophage+lambda+development.">Switches in bacteriophage lambda development.</a> Annu. Rev. Genet. 39, 409-429 (2005).</li><li>Ptashne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A genetic switch : phage lambda revisited. , (2004).</li><li>Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lambda II. , (1983).</li><li>Hershey, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Bacteriophage lambda. , (1971).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decision+making+at+a+subcellular+level+determines+the+outcome+of+bacteriophage+infection.">Decision making at a subcellular level determines the outcome of bacteriophage infection.</a> Cell. 141, 682-691 (2010).</li><li>Edgar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+infection+is+targeted+to+cellular+poles.">Bacteriophage infection is targeted to cellular poles.</a> Mol. Microbiol. , (2008).</li><li>Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in molecular biology. , (1994).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical handbook of biochemistry and molecular biology. , (1989).</li><li>Kaiser, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+internal+structure+of+bacteriophage+lambda.">On the internal structure of bacteriophage lambda.</a> J. Gen. Physiol. 49, 171-178 (1966).</li><li>Kobiler, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinetic+analysis+of+the+bacteriophage+lambda+genetic+network.">Quantitative kinetic analysis of the bacteriophage lambda genetic network.</a> Proc Natl Acad Sci. 102, 4470-4475 (2005).</li><li>Shaner, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Personal communication with M. Feiss. , .</li><li>Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adsorption+of+coliphage+lambda+to+its+host:+effect+of+variations+in+the+surface+density+of+receptor+and+in+phage-receptor+affinity.">The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity.</a> J. Mol. Biol. 103, 521-536 (1976).</li></ol>

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Estirpes bacterianas, fagos e plasmídeos: Strain LE392 é supF. Optou-se por suprimir a mutação SAM7 no genoma de fago (ver Tabela 1 para detalhes). Assim, lisogénios induzidas acabará por lise e liberar partículas de fagos, células infectadas como a vontade de que escolheram a via lítica. Células lisogénica são cultivadas a 30 ° C devido à presença do cl 857 sensível à temperatura alelo no genoma do fago. Após a indução de calor, gpD-EYFP e do tipo selvagem gpD são co-expressas a partir do genoma de λ LZ1 eo plasmídeo pPlate * D, respectivamente. Como resultado, a cápside do fago λ recém-criado LZ2 contém uma mistura de gpD-EYFP e proteínas gpd. Este fago mosaico é estruturalmente estável e suficientemente fluorescente para permitir a detecção de fagos individuais 5. pP RE - mCherry é um plasmídeo repórter utilizada para detectar escolha do pathwa lisogénicay. O promotor RE P é ativada pela CII durante o estabelecimento de lisogenia 1,11. pP RE - mCherry 5 foi derivado de pE-gfp 11 substituindo gfp com mCherry 12. Para mais detalhes veja o nosso trabalho anterior 5. Condição Parâmetros de crescimento: Durante a indução lisogénio (Seção 1), tremor leve a 180 rpm dá um rendimento bom vírus 13. O uso de glucose no meio de crescimento deve ser evitada, o metabolismo da glicose gera produtos metabólicos acídicos, e as partículas de lambda maduros são instáveis ​​a pH ácido 13. A adição de MgSO 4 destina-se a estabilizar a cápside do fago 3. Para fagos carregando tipo selvagem cI (em vez de cI 857), o lisogénio pode ser induzida usando o C-DNA prejudicial Mitomicina agente 3. No passo 1.3, a incubação a 37 ° C não deve exceder normalmente 90 minutos. É usef<html> ul para verificar a densidade celular por OD 600 a cada 30 min. Para uma boa lisado, OD 600 cai para cerca de 0,2 ou menos, e o restante OD 600 é um resultado de detritos de células. Incubação demasiado longo pode resultar num menor rendimento de fago uma vez que o fago pode começar a recentemente criada para injectar o DNA em fragmentos de células. Para se obter uma banda de fago visível (de pelo menos 1 x 10 11 partículas fágicas) nos passos 2.11 e 2.13, crescem, pelo menos, 500 ml de cultura na etapa 1.2. A adição de maltose a 0,2% para o meio de crescimento nas etapas 5.1 e 5.2 é destinado a induzir a expressão de LamB, o receptor para o fago lambda adsorção 3,14. A diluição de 1000 vezes, em vez de 100 vezes no passo 5.2 é destinado a reduzir o nível de fundo a partir do RE mCherry repórter plasmídeo pP - mCherry. No passo 5.5 para injecção de DNA de fago de disparo, a 35 ° C é escolhido para evitar a indução do alelo sensível à temperatura cI857. Purificação Phage: jove_content &quot;&gt; Os passos de purificação de fagos (Passos 2.1 a 2.11) pode ser substituído por outros protocolos de purificação 5, mas a ultracentrifugação de gradiente de equilíbrio final através de CsCl (Passos 2.12 e 2.13), é inevitável. rotores de caçamba móvel são necessários nos passos 2.10 e 2,12 para garantir afiadas bandas fago visíveis. Obtenção de um estoque fago puro pode facilmente levar até uma semana, por isso é necessário verificar o título de fagos ao longo do caminho para garantir que nada dê errado durante as etapas intermediárias. Manuseio Phage: Durante todos os procedimentos de purificação na Seção 2, é fundamental para lidar com fago lisado com cuidado para evitar corte caudas de fago de cabeças de fagos. Durante a infecção de células na Secção 5 (por exemplo, as etapas de 5,5 através de 5.7), é também crítica para evitar a ruptura das partículas de fago a partir da célula infectada. Note-se que, se o fago é cortado a partir da célula infectada, após injecção de seu DNA, o resultado é uma infecção &quot;escuro&quot;, ou seja, a defection resultado será observado na experiência, mas o fago infectando não. Para minimizar esses problemas, usamos uma ponteira larga sempre que manuseia fagos ou a mistura fago / célula. DAPI Teste: Manchando o estoque fago com DAPI (Seção 4) é um método rápido e eficiente para controlar a pureza do estoque de fagos. Ele também pode ser usado para testar a possível degradação de um estoque de fago existente ao longo do tempo. Para um estoque puro, a co-localização de sinais YFP e DAPI sob o microscópio de fluorescência deveria ser próximo de 100%. Nós normalmente observam que menos de 1% dos locais YFP não contêm DAPI (representando cãpsides sem o genoma viral), o que indica que estas partículas não êxito empacotar o DNA viral ou já tinham injectado o seu DNA em outro lugar. Menos de 1% dos locais DAPI não contêm YFP (correspondente a fagos não fluorescentes). Se este não for o caso, as etapas de 2,12 a 2,14 necessidade de ser repetido no order para purificar novamente. Com relação aos parâmetros de imagem, a configuração do microscópio no Passo 4.3 não é tão crítica como no capítulo 5, porque não a longo prazo imagem ao vivo de células é necessária aqui. No entanto, mantendo os ajustes de microscopia mesmas como no ponto 5 é útil quando se deseja calibrar a intensidade de fluorescência de uma única partícula fágica. Se a placa de PBS-agarose não é muito limpa, ou muito DAPI corante é usado, alguns pontos DAPI correspondentes ao ADN do fago pode ser rodeada por um &quot;halo&quot;. Se for muito pouco corante DAPI é usado, o sinal do canal DAPI pode ser muito fraca. Sistema de microscópio: Para a imagem na seção 6, nós usamos um microscópio de epifluorescência comercial invertido (Eclipse TE2000-E, Nikon) com um objetivo 100x (Plano Fluo, abertura numérica 1,40, imersão em óleo) e padrão de conjuntos de filtros (Nikon). A fonte de luz de fluorescência é uma lâmpada de arco com o controlo da intensidade da luz. Os seguintes recursos são controlados por computador: x, y, z posição; campo brilhante e persianas de fluorescência, e opção de filtro de fluorescência. Um recurso de auto-foco é necessária. Caso contrário, o foco pode facilmente afastar-se durante o filme de lapso de tempo (normalmente 4 horas de duração). A capacidade de adquirir várias posições (x, y) em cada ponto de tempo é útil, uma vez que permite acompanhar a eventos de infecções múltiplas em paralelo. Nós normalmente adquirir 8 posições de palco em cada filme, seguindo até 100 ocorrências de infecção. A câmera que usamos é um resfriado 512x512 CCD com 16x16 pixel câmera mM com uma gama dinâmica de 16 bits (Cascade512, Fotometria). Aquisição é realizada usando software MetaMorph (Molecular Devices). O microscópio deve ser colocado numa sala com temperatura controlada, em alternativa, a platina do microscópio deve ser rodeada por uma câmara de temperatura controlada. Aquisição da Imagem: Para vivo de células de imagem, que é crítico para evitar a exposição desnecessária da amostra, o que poderia levar à descoloração e phototoxicity. Portanto, o melhor é primeiro caracterizar seu sistema para encontrar uma exposição de luz ideal que permite a detecção de fluorescência, enquanto não levando ao crescimento excessivo de células de branqueamento ou de inibição. Para obter uma boa imagem de fluorescência, jogar com a intensidade de luz emocionante, tempo de exposição e ganho de câmera. Nos Passos 6,2-6,3, o intervalo de quadro 10 min é escolhido com o fim de minimizar a exposição à luz. Em cada quadro, apenas uma imagem em foco único é necessário no contraste de fase (de reconhecimento de célula) e os canais de fluorescência (para determinar o destino da célula). No primeiro momento, no entanto, várias imagens z-posição através do canal de YFP são necessárias para capturar todos os fagos que infectam na superfície da célula. O tempo de exposição YFP no quadro inicial também pode precisar de ser maior do que o utilizado para o filme de lapso de tempo nos períodos de tempo posteriores. Análise de Imagem: Muito cuidadosamente contar partículas fágicas em torno da superfície da célula na etapa 7.1. Comomencionado acima, tomamos uma série de z-pilhas através YFP canal no Passo 6.2. No entanto, isso ainda pode deixar algumas partículas de fagos fluorescentes fora de foco, que desafia a contagem. O comprimento das células no espaço de tempo inicial é medida usando o software Metamorph. O comprimento da célula pode também ser medida por ImageJ ou outras ferramentas de software. Além disso, um sistema automatizado de casas construídas programa Matlab pode ser muito útil na obtenção de informações, tais como a mudança de fluorescência ao longo do tempo ao longo de linhagens celulares.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Clorofórmio </td><td> Fisher Scientific </td><td> C298-500 </td></tr><tr><td> NaCl </td><td> Fisher Scientific </td><td> S271-3 </td></tr><tr><td> DNase I </td><td> Sigma </td><td> D4527-10KU </td></tr><tr><td> RNase </td><td> Sigma </td><td> R4642-10mg </td></tr><tr><td> PEG8000 </td><td> Fisher Scientific </td><td> BP233-1 </td></tr><tr><td> Tampão SM </td><td> Teknova </td><td> S0249 </td></tr><tr><td> NZYM </td><td> Teknova </td><td> N2062 </td></tr><tr><td> CsCl </td><td> Sigma </td><td> C3011-250G </td></tr><tr><td> Seringa </td><td> Becton Dickinson </td><td> 309585 </td></tr><tr><td> Agulha </td><td> Becton Dickinson </td><td> 305176 </td></tr><tr><td> Cassete de diálise </td><td> Thermo Scientific </td><td> 66333 </td></tr><tr><td> Lâmina de microscópio </td><td> Corning </td><td> 2947-75x50 </td></tr><tr><td> Agarose </td><td> Fisher Scientific </td><td> BP160-100 </td></tr><tr><td> SW40Ti tubo ultra-claro </td><td> Beckman Coulter </td><td> 344060 </td></tr><tr><td> SW60Ti tubo ultra-claro </td><td> Beckman Coulter </td><td> 344062 </td></tr><tr><td> SW40Ti rotor </td><td> Beckman Coulter </td><td> 331302 </td></tr><tr><td> SW60Ti rotor </td><td> Beckman Coulter </td><td> 335649 </td></tr><tr><td> Refratômetro </td><td> Fisher Scientific </td><td> 13-947 </td></tr><tr><td> Microscópio de epifluorescência </td><td> Nikon </td><td> Eclipse TE2000-E </td></tr></tbody></table> Tabela 2. Reagentes e equipamentos.

following cell-fate in e. coli after infection by phage lambda

Compreendendo o sistema bacteriófago (fago) lambda e a bactéria E. coli serviu durante muito tempo como um paradigma para a célula destino, 1,2 determinação. Seguindo a infecção simultânea da célula de um número de fagos, uma de duas vias é escolhido: lítico (virulento) ou lisogênico (latentes) 3,4. Recentemente, desenvolveu um método para etiquetar fluorescentemente fagos individuais, e foram capazes de estudar a decisão de pós-infecção, em tempo real, ao microscópio, a nível de fagos individuais e as células 5. Aqui, descrevemos o procedimento completo para a realização dos experimentos de infecção descritas em nossos trabalhos anteriores 5. Isto inclui a criação de fagos fluorescentes, a infecção das células, sob o microscópio de imagem e análise de dados. O fago fluorescente é um &quot;híbrido&quot;, co-expressando as versões de tipo selvagem e YFP-fusão da proteína da cápside gpD. Um ligado fágico bruto é primeiro obtido por indução de um lisogénio de o (gpD-EYFP EnhYellow Fluorescent Protein ANCED fago), portadora de um plasmídeo que expressa tipo selvagem gpD. Uma série de passos de purificação são então realizadas, seguido por DAPI-rotulagem e de imagem sob o microscópio. Isto é feito a fim de verificar a uniformidade, a eficiência de empacotamento do ADN, o sinal de fluorescência e a estabilidade estrutural do stock de fagos. A adsorção inicial de fagos às bactérias é realizada em gelo, seguido por uma incubação breve, a 35 ° C para provocar a injecção do ADN virai 6. O fago / bactérias mistura é, então, transferido para a superfície de uma laje fina agar nutriente, coberta com uma lamela e visualizados sob um microscópio de epifluorescência. O processo de pós-infecção é seguida durante 4 h, a 10 minutos de intervalo. Posições de múltiplos estágios são monitorados tais que ~ 100 infecções celulares podem ser rastreados em um único experimento. Em cada ponto de posição e de tempo, as imagens são adquiridas no contraste de fase e canais de vermelho e verde fluorescente. A imagem de contraste de fase é usado posteriormente para automatizado celreconhecimento l enquanto os canais fluorescentes são usados ​​para caracterizar o resultado de infecção: produção de novos fagos fluorescentes (verde) seguido de lise das células, ou a expressão de factores de lisogenia (vermelho), seguido de crescimento das células é reiniciada e divisão. Os adquiridos lapso de tempo os filmes são processadas utilizando uma combinação de métodos manuais e automatizados. Dados os resultados da análise para a identificação de parâmetros de infecção para cada evento de infecção (por exemplo, número e posições de infectar fagos), bem como resultado da infecção (lise / lisogenia). Os parâmetros adicionais podem ser extraídas se desejado.

Watch this Scientific Journal Video about Following Cell-fate in E. coli After Infection by Phage Lambda at JoVE.com

Following Cell-fate in E. coli After Infection by Phage Lambda

Este artigo descreve o procedimento para a preparação de uma versão marcada com fluorescência do bacteriófago lambda, infecção de<em&gt; E. coli</em&gt; Bactérias, após o resultado da infecção sob o microscópio, e análise dos resultados de infecção.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

following-cell-fate-in-e-coli-after-infection-by-phage-lambda

Research

JoVE Journal

Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

23.2K visualizações.  University of Illinois at Urbana-Champaign. Este artigo descreve o procedimento para a preparação de uma versão marcada com fluorescência do bacteriófago lambda, infecção de E. coli Bactérias, após o resultado da infecção sob o microscópio, e análise dos resultados de infecção.

Vídeo: Following Cell-fate in E. coli After Infection by Phage Lambda

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Monitoramento células imunes Tráfico Rods Horas Prion fluorescente após a infecção intraperitoneal

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Imunologia e Infecção

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de meio de escarro Artificial para testar a eficácia antibiótica contra<em&gt; Pseudomonas aeruginosa</em&gt; Em condições mais relevantes para o Pulmão Fibrose Cística

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extração de proteínas e 2-D métodos Eletroforese em Gel de<em&gt; Burkholderia</em&gt; Espécies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Selecção de alta Scalable Rendimento de bibliotecas de anticorpos em fagos sintética exibida-

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Modelo não-invasiva da neuropatogênicos<em&gt; Escherichia coli</em&gt; Infecção no Rato Neonatal

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Phenomics fago: Abordagens fisiológicas para Caracterizar Novel Proteínas Virais

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Transformação de levedura probiótica e sua recuperação a partir gastrointestinais imunitário tecidos após a sonda oral em camundongos

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Patógeno de origem alimentar rastreio usando Nanosensor Magneto-fluorescente: Rápida detecção de Escherichia Coli O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Deteção de colonização Enterohemorrhagic Escherichia Coli murino Host pelo sistema não-invasivo na Vivo bioluminescência

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Criação de um denso Transposon inserção biblioteca usando bacteriana conjugação em entérobactérienne cepas tais como Escherichia Coli ou Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...