Agradecemos ao Dr. Craig Roy e Dr. Dario Zamboni para fornecer um modelo para infecções protozoário. Agradecemos ao Dr. Jagdeep Obhrai, Dr. Georgiana Purdy, Dr. Fred Heffron e Todd Wisner para equipamentos e reagentes; Dr. Lulu Cambronne para revisão crítica do manuscrito. Citometria de fluxo foi realizada no Fluxo de instalação Citometria de Recursos OHSU compartilhado. Este trabalho foi financiado em parte por uma doação da Fundação de Pesquisa Médica de Oregon e uma concessão de NIH R21 AI088275 (EDC).

1. Preparação das Culturas Legionella pneumophila para infecções Estágio Escorvante <ol><li> Transformar toda L. estirpes pneumophila utilizados no ensaio com as pAM239 plasmídeo, que codificam uma isopropil β-D-1-tiogalactopiranósido (IPTG) induzível da proteína verde fluorescente (GFP) 28. Streak as estirpes bacterianas em ferro e de cisteína suplementado N-(2-Acetamido)-2-aminoetanossulfónico (ACES) tamponada carvão ágar com extracto de levedura (CYEA) contendo 6,25 ug / ml de cloranfenicol (CM) (para a manutenção do plasmídeo) e incubar durante 72 hr a 37 ° C. </li><li> Transferir um inoculo da estirpe bacteriana em ACES suplementados tamponada caldo de extracto de levedura (AYE) com 6,25 ug / ml de CM e IPTG 1 mM e cultivar até à fase estacionária (O / N) num agitador orbital a 37 ° C. </li><li> Confirmar a expressão da GFP em culturas in vitro, utilizando a microscopia de fluorescência. Transferem-se 10 ul de cultura sobre uma lâmina de vidro sob uma coberturadeslizamento e imagem através de uma objectiva 60X com excitação GFP / cubo de emissão (AMG EVOS fl). </li><li> Diluir uma alíquota de 100 ul de cada cultura in vitro utilizando a 1:10 estéril H 2 O. Faça um branco usando 100 ul AYE media com IPTG 1 mM e 6,25 ug / cm ml e água. Tome OD600 medições das diluições usando um espectrofotómetro (Bio-Rad inteligente Spec Plus). Calcula-se o volume necessário para infectar um bem a um MOI = 20 para cada cultura in vitro: V = [(ameba seeded) x MOI] / [OD 600 x (factor de diluição) x (constante)] = [(1 X 10 6 ) x (20)] / [OD 600 x (10) x (1 x 10 6)] = 2/OD 600. Concentração de bactérias é determinada como OD 600 = 1,0 = 1 x 10 9 CFU / ml. </li></ol> 2. Infecção Priming palco usando Acanthamoeba castellanii <ol><li> Manter e cultivar as amebas em meio ATCC PYG 712 em 175 cm 2 frascos de RT. </li><li> Substituir a mídia em cul amebasturas 24 h antes do início das culturas bacterianas de líquidos. No mesmo dia, líquidos culturas bacterianas são iniciados, recolha e contagem de células num microscópio de luz, utilizando um hemocitómetro. </li><li> Diluir as amebas com meio fresco para uma concentração final de 1 x 10 6 células / ml. Semente 12 poços de células placas de cultura com alíquotas de 1 ml da amebas utilizando um pipetador de repetição e incubar à temperatura ambiente O / N. </li><li> Após incubação, lava-se os poços das placas de 12 poços de cultura de células 3x com 1 ml de PBS estéril através de uma pipeta de 10 ml serológicos e um auxiliar de pipeta manual. </li><li> Após a aspiração do PBS, adicionar 1 ml de meio de infecção (ATCC 712 media PYG menos glicose, peptona, extrato de levedura e) com IPTG 1 mM e 6,25 ug / ml cm a cada poço. Incubar as placas à temperatura ambiente durante 1 h. </li><li> Infect poços a um MOI = 20. Centrifugar a placa a 400 xg durante 5 min (Eppendorf 5810R) e flutuar num banho de água a 37 ° C durante 5 min. Transferir a placa para um de 37 ° C, 5% CO 2 incubadora durante 18 horas. </li><li> Confirmar as infecções usando imagens de células vivas em um microscópio de fluorescência antes de sediar a lise celular e colheita de bactérias. </li></ol> 3. Semeando células THP-1 para a Target Infecção estágio celular <ol><li> (Comece este processo 24 horas antes de definir-se líquidos culturas bacterianas). Cultivar células THP-1 a 75, em cm 2 frascos de cultura para quase-confluência em meio RPMI 1640 com 10% inactivado pelo calor de soro fetal bovino (FBS). </li><li> Contar as células em suspensão utilizando um hemocitómetro, diluir as células THP-1 em meio RPMI 1640 com FBS a 10% e 100 ng / ml de forbol-12-miristato-13-acetato (PMA) para uma concentração de 1 x 10 6 células / ml , e prato em alíquotas de 1 ml em placas de 12 poços de cultura de células. Incubar as placas durante 48 horas a 37 ° C, 5% CO 2. </li></ol> 4. Transformação de bactérias para a Target Infecção estágio de célula após a infecção Estágio Priming <ol><li> Aspirar a mídia da amebas preparado. </li><li> Lisam a ummoebae usando 500 ul de gelo frio, estéril de ultra-filtrado (UF) de H 2 O e incubar à temperatura ambiente durante 10 min. </li><li> O pool de lisados ​​de acordo com o tipo de estirpe e tomar uma medida nm E 512 para cada um dos lisados ​​agrupados utilizando um leitor de placas de fluorescência (Molecular Devices Spectramax Gêmeos EM). </li><li> Calcule um OD 600 de medição para os lisados ​​agrupados: calcOD 600 = 0,0008 (E 512 - fundo lisado) + 0,0019. A fórmula foi previamente determinada representando graficamente uma comparação directa de ambos o OD 600 e os E 512 nm medições de diluições de L. pneumophila GFP do tipo selvagem expressar de fase estacionária de cultura in vitro (Figura 1 *). Os lisados ​​de ameba não infectado, sujeitos às mesmas condições experimentais, a ameba infectada, são utilizadas como um espaço em branco, e desde um valor de correcção (por exemplo, fundo lisado), que foi incorporado na equação. O volume necessário tó infectar um bem a um MOI = 20 é calculada para cada pool de lisado: V = [(THP-1 sem sementes) MOI x] / [calcOD 600 x (constante)] = [(1 X 10 6) x (20)] / [calcOD 600 x (1 x 10 6)] = 20 / calcOD 600. </li><li> Lave as células THP-1 3x com PBS e adicionar 1 mL de RPMI fresco 1640 (10% de FBS) em cada poço. Incubar as células THP-1 durante 1 hora a 37 ° C, 5% CO 2. </li><li> Infectar as células THP-1 na MOI calculada usando os lisados ​​pool. Permitir que um conjunto de cavidades de não ficam infectadas, servindo como um controlo negativo para a análise de citometria de fluxo. Processamento da fase de escorvamento deve ser concluída em menos de 30 min. Os lisados ​​foram mantidos em gelo para limitar as alterações na expressão de genes de bactérias antes da infecção. </li><li> Centrifugar a placa, flutuar para aumentar a temperatura, e incubar como na fase de escorvamento. </li><li> Uma hora após a infecção, que remove o suporte por aspiração e lavar os poços 3x com PBS para remover as bactérias extracelulares. </li><li> Adicione 1 ml fresh RPMI 1640 (10% de FBS) aos poços e devolver a placa à incubadora. O tempo imediatamente após a troca de mídia serve como tempo zero (T 0). Incubar as células THP-1 de 14-16 horas a 37 ° C, 5% CO 2. </li></ol> 5. Análise Experimental 5,1 imagens de células vivas Imagem cavidades infectadas, utilizando um microscópio de fluorescência (AMG EVOS fl) em 10X ou 20X de ampliação (Figuras 1A-D). As imagens podem ser comparadas, quer qualitativamente ou quantitativamente para determinar os níveis de infecção em ambas a fase de escorvamento e de infecções alvo estágio de células. A citometria de fluxo 5,2 <ol><li> Os picos de emissão de infecções diferentes podem ser comparados através de citometria de fluxo (FACS Calibur BD). Tripsinizar as infectadas células THP-1 e gentilmente lavá-los a partir dos poços através da mistura em PBS com uma pipeta. </li><li> Reunir as células de tipo tensão em tubos Falcon de 15 ml e de pelotas a 1800 xg durante 2min na centrífuga de topo de mesa. </li><li> Suspender as pelotas em 1 ml de PBS e transferir para tubos de microcentrífuga de 1,5 ml. </li><li> Centrifugar as suspensões a 1.800 x g (Eppendorf 5424) e re-suspender as pelotas em 1 ml de PBS. Se as suspensões resultantes são altamente turva (OD 600 ≥ 1.0) eles devem ser diluídos em PBS adicional (1:3), para evitar o entupimento das linhas no citómetro de fluxo. </li><li> Use PBS estéril filtrada para equilíbrio do citómetro de fluxo e para as etapas de lavagem entre as injecções de amostra. Traça-se a dispersão para a frente / lateral de células alvo infectadas, antes da injecção de amostras-alvo da infecção das células. </li><li> Colete 20.000 eventos totais para cada condição utilizando 488 nm do laser de excitação e o canal FL1. </li><li> Portão populações de células pós-captura para excluir as células não infectadas e uma intensidade de fluorescência na trama de um histograma (FlowJo) (Figura 1E). </li></ol> 5,3 placas CFU <ol><li> Examinar a eficiência of infecção por CFU plaqueamento das células recolhidas por citometria de fluxo. Preparar diluições em série das amostras de ultra-filtrada H 2 O, 10 1, 10 -2, 10 -3 e. </li><li> Placa de 20 ul de cada diluição para 1/3 de uma placa CYEA com 6,25 ug / ml de CM. </li><li> Incubar as placas a 37 ° C durante 72 h. </li><li> Contar as colónias, usando um palito e contador de células. </li></ol>

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Os autores declaram que não têm interesses conflitantes financeiros.

A expressão do gene bacteriano é rigorosamente controlado por meio de uma combinação de progressão do ciclo de vida e resposta a sinais do micro-ambiente circundante. Patógenos vacuolares, tais como L. pneumophila responder a uma grande variedade de hospedeiros de células derivadas de pistas quando compartimentado em um fagossoma. Como resultado colectivo de exaustão de nutrientes na célula hospedeira, a bactéria compensa por expressar factores necessários para a difusão bem sucedida para uma célul...

Numerosos agentes patogénicos bacterianos adaptaram estratégias generalizadas de explorar as células hospedeiras para a sua sobrevivência e replicação num compartimento intracelular. Em muitos casos, os mecanismos patogénicos são semelhantes entre protozoários e metazoários células. No entanto, estes dois microambientes são muito diferentes e podem resultar em expressão diferencial de factores de virulência 1-4. A doença dos legionários bactéria Legionella pneumophila é onipresente associada com ambientes de água doce em todo o mundo 5. Importante, L. pneumophila cultivado em células de protozoários antes da infecção dos monócitos humanos ganhar uma vantagem patogénicas, o que sugere que os perfis de expressão gênica global da bactéria que sai de uma célula de protozoários são diferentes do que a do organismo cultivado in vitro em 6-8. Na natureza, a água doce amebas fornecer nutrientes limita ricos para amplificação rápida de uma bactéria invasora. Aquisição humana de L. pneumophila é o mais frequentemente atribuída a inalação de gotículas de água contaminada que contém a bactéria. É provável que essas gotículas porto protozoários associados a células de bactérias, células de protozoários, onde são mais resistentes a práticas convencionais de tratamento de água 9,10. Infecção de macrófagos alveolares do pulmão procede de uma maneira praticamente idêntica à do ciclo de vida da bactéria intracelular em células hospedeiras de protozoários 11-13. A fim de sobreviver e de se replicar em células eucariotas, L. pneumophila utiliza um tipo especializado sistema de secreção IVb denominado Dot / Icm para entregar cerca de 300 &#39;&quot;proteínas efectoras para o citosol da célula hospedeira 14-16. Estas proteínas efectoras funcionar colectivamente subverter processos celulares, a fim de gerar um compartimento de replicação para a bactéria permissiva 17,18. Deleções em qualquer um dos 26 genes que compreendem o resultado transportador Dot / Icm de estirpes defeituosas de mult intracelulariplication 19-23. Historicamente, a deleção de genes individuais que codificam efetores raramente resultou em estirpes atenuadas para o crescimento intracelular. Esse fenômeno tem sido atribuída a várias hipóteses, incluindo função redundante e cópias parálogas de efetores. Alguns factores de virulência são apenas expressas no contexto do hospedeiro associada a células de crescimento intracelular 24. Nós racionalizado que se um efector específico foi expresso apenas no contexto da infecção por protozoários, em seguida, a contribuição do efector não pode ser comparada com uma estirpe de tipo selvagem, quando ambos foram cultivadas in vitro. L. pneumophila transições de um replicativa a uma fase transmissivo enquanto entra na fase estacionária da cultura 25. O fenótipo de comutação fase representa o esgotamento de nutrientes encontrados durante crescimento intracelular e é exemplificada através de montagem de flagelos para a motilidade 26. Porque L. pneumophila é mais invasive e virulenta quando colhido a partir de células de protozoários, buscou-se desenvolver um ensaio que mais fielmente representado o estado patogênico da bactéria quando encontrou macrófagos de acolhimento. Para este fim, foi desenvolvido um ensaio de escorvamento versátil protozoário que pode acomodar qualquer hospedeiro adequado para ambos os primeiros (cell priming) e segunda (infecções de células alvo) de palco. O processo de infecção é tratável através da utilização de bactérias que expressam de forma estável a proteína fluorescente verde (GFP). O modelo de infecção pelo protozoário Acanthamoeba castellanii segue uma metodologia amplamente utilizado no campo 27. Para o passo de activação, L. estirpes pneumophila são cultivados in vitro até à fase estacionária em meios líquidos para produzir rotulados &#39;transmissivos &quot;bactérias (Figura 1A). As bactérias são depois utilizadas para infectar monocamadas de A. castellanii durante 18 horas para atingir uma fase tardia do ciclo de vida intracelular. Grandes vacúolos contêmbactérias ção pode ser visualizada nesta altura utilizando a microscopia de fluorescência (Figura 1A). As células são então lisadas por protozoários e bactérias recuperadas a partir do lisado são medidos para a emissão a 512 nm utilizando um leitor de placas de fluorescência. A fluorescência é proporcional à densidade óptica para calcular multiplicidade de infecção (MOI) durante a infecção das células alvo (figura 1, * Curva de correlação). Após invasão (T 0) e 18 horas pós-invasão (T 18), as células alvo são quantificadas por fluorescência, que representa as bactérias intracelulares. A fluorescência pode ser monitorizada por microscopia e citometria de fluxo, e a contagem de viáveis ​​pode ser medido através de plaqueamento de colónias. O ensaio de priming é sempre acompanhada por infecções de tipo selvagem L. pneumophila e de uma estirpe deficiente em Dot / Icm sistema de secreção de tipo IV (Δ DOTA) (Figura 1A). Este importante fornece controles internos para comparações diretas entre um tipo selvagemnd quaisquer estirpes isogénicas mutantes utilizados no processo de infecção. A inclusão da estirpe avirulenta DOTA Δ durante a fase de escorvamento estabelece um limiar para a observação de fenótipos de crescimento atenuado associados isogénicas estirpes mutantes que são cultivadas in vitro.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>chloramphenicol</td>
 <td>Fisher Scientific</td>
 <td>BP904-100</td>
 <td>antibiotic</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Fisher Scientific</td>
 <td>BP1755-10</td>
 <td></td>
 </tr>
 <tr>
 <td>ACES</td>
 <td>Sigma</td>
 <td>A9758-1KG</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>ATCC medium: 712 PYG</td>
 <td>ATCC</td>
 <td></td>
 <td>growth media for protozoans</td>
 </tr>
 <tr>
 <td>1X PBS</td>
 <td>Fisher Scientific</td>
 <td>SH30256FS</td>
 <td>phosphate buffered saline</td>
 </tr>
 <tr>
 <td>activated charcoal</td>
 <td>Fisher Scientific</td>
 <td>C272-212</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>yeast extract</td>
 <td>Fisher Scientific</td>
 <td>BP1422-500</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>peptone</td>
 <td>BD Diagnostics</td>
 <td>211677</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>Fisher Scientific</td>
 <td>BP1423-2</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>L-cysteine, 99%+</td>
 <td>Acros organics</td>
 <td>173601000</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td>Ferric nitrate nonahydrate</td>
 <td>Fisher Scientific</td>
 <td>I110-100</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>EVOS fl</td>
 <td>AMG</td>
 <td>EVOS fl</td>
 <td>fluorescence microscope</td>
 </tr>
 <tr>
 <td>Smart Spec Plus</td>
 <td>Bio-Rad</td>
 <td>170-2525</td>
 <td>spectrophotometer</td>
 </tr>
 <tr>
 <td>5810 R centrifuge</td>
 <td>Eppendorf</td>
 <td>22627023</td>
 <td>bench top centrifuge</td>
 </tr>
 <tr>
 <td>Repeater plus</td>
 <td>Eppendorf</td>
 <td>22230201</td>
 <td>repeating pipette</td>
 </tr>
 <tr>
 <td>SpectraMax Gemini EM</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>microplate reader</td>
 </tr>
 <tr>
 <td>Softmax Pro 5.3</td>
 <td>Molecular Devices</td>
 <td>0200-310</td>
 <td>microplate reader software</td>
 </tr>
 <tr>
 <td>5424 microfuge</td>
 <td>Eppendorf</td>
 <td>22620401</td>
 <td>table top microcentrifuge</td>
 </tr>
 <tr>
 <td>Fast-release pipette pump II</td>
 <td>Scienceware</td>
 <td>379111010</td>
 <td>pipette aid</td>
 </tr>
 <tr>
 <td>FACS Calibur</td>
 <td>BD Bioscience</td>
 <td></td>
 <td>flow cytometer</td>
 </tr>
 <tr>
 <td>FlowJo 7.6.1</td>
 <td>FlowJo</td>
 <td>license</td>
 <td>flow cytometery software</td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>polypropylene conical tube</td>
 </tr>
 <tr>
 <td>1.6 ml microfuge tube</td>
 <td>Neptune</td>
 <td>3745.X</td>
 <td>microcentrifuge tubes</td>
 </tr>
 </tbody>
</table>

tractable mammalian cell infections with protozoan-primed bacteria

Muitos agentes patogénicos bacterianos intracelulares, protozoários usar água doce como um reservatório natural para a proliferação no ambiente. Legionella pneumophila, o agente causador do legionário pneumonia, ganha uma vantagem sobre patogénico em bactérias cultivadas in vitro quando a primeira colheita a partir de células de protozoários antes da infecção de macrófagos de mamíferos. Isto sugere que os factores de virulência importantes não podem ser adequadamente expressas in vitro. Nós desenvolvemos um sistema tratável para o priming L. pneumophila através do seu hospedeiro natural protozoário Acanthamoeba castellanii antes da infecção de células de mamíferos. A contribuição de qualquer factor de virulência podem ser examinados, comparando o crescimento intracelular de uma estirpe mutante de tipo selvagem de bactérias depois de priming protozoário. Expressando GFP-L. de tipo selvagem e mutante estirpes pneumophila são utilizadas para infectar monocamadas de protozoários num passo de escorvamento e deixada atingir a fase tardia de crescimento intracelular. Bactérias fluorescentes são então colhidas a partir destas células infectadas e normalizados por espectrofotometria para gerar números comparáveis ​​de bactérias para uma subsequente infecção em macrófagos de mamíferos. Para quantificação, as bactérias vivas são monitorizados após a infecção através de microscopia de fluorescência, citometria de fluxo, e por plaqueamento de colónias. Esta técnica destaca e conta com a contribuição de anfitrião a expressão do gene de células-dependente, imitando o ambiente que seria encontrado em uma rota de aquisição natural. Esta abordagem pode ser modificado para acomodar qualquer bactéria que utiliza um hospedeiro intermediário como um meio para a obtenção de uma vantagem patogénico.

Um resultado típico para o processo de infecção toda está delineado na Figura 1. Viver micrografias de células de fluorescência mostrando monocamadas de A.castellanii infectadas com o tipo selvagem L. pneumophila durante a fase de escorvamento é mostrado na Figura 1A. Uma medida de sucesso da etapa de iniciação levaria a uma população de cerca de 90% das células hospedeiras que contêm grandes vacúolos preenchidos com bactérias marcadas com GFP neste MOI....

Watch this Scientific Journal Video about Tractable Mammalian Cell Infections with Protozoan-primed Bacteria at JoVE.com

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Esta técnica proporciona um método para colher, normalizar e quantificar o crescimento intracelular das bactérias, que são pré-cultivadas em células hospedeiras naturais protozoários antes da infecção de células de mamíferos. Este método pode ser modificado para acomodar uma grande variedade de células hospedeiras para a fase de escorvamento, bem como tipos de células-alvo.

Tratáveis ​​mamíferos Infecções celulares com Protozoário-condicionadas Bactérias

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

tractable-mammalian-cell-infections-with-protozoan-primed-bacteria

Research

JoVE Journal

Immunology and Infection

10.4K visualizações.  Oregon Health & Science University. Esta técnica proporciona um método para colher, normalizar e quantificar o crescimento intracelular das bactérias, que são pré-cultivadas em células hospedeiras naturais protozoários antes da infecção de células de mamíferos. Este método pode ser modificado para acomodar uma grande variedade de células hospedeiras para a fase de escorvamento, bem como tipos de células-alvo.

Vídeo: Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Using Luciferase to Image Bacterial Infections in Mice

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

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

Usando a luciferase Infecções bacterianas Imagem em camundongos

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

Imunologia e Infecção

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Geração de células T específicas Multivirus para Prevenir / tratar infecções virais após transplante de células-tronco hematopoéticas alogênico

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D Multimodalidade de imagens de<em&gt; Citrobacter rodentium</em&gt; Infecções em Ratos

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescência métodos de microscopia para determinar a viabilidade de bactérias em associação com células de mamíferos

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

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 ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis como um organismo modelo para a Avaliação da Interação de bactérias anaeróbias com células do hospedeiro

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Usando modelos de Zebrafish de infecções por vírus gripe humana A para Screen antivirais e Caracterizar imune do hospedeiro respostas de células

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análise de Linfócitos extravasamento Usando um<em&gt; In Vitro</em&gt; Modelo do Sangue-Cérebro humano Barreira

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

O Modelo WinCF - Um Microcosmo barato e tratável de um muco Plugged Bronquíolo para estudar a Microbiologia do Pulmão Infecções

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Imagem latente confocal de RNA Double-Stranded e receptores de reconhecimento padrão na infecção pelo vírus de RNA de sentido negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Tratáveis ​​mamíferos Infecções celulares com Protozoário-condicionadas Bactérias

Tratáveis mamíferos Infecções celulares com Protozoário-condicionadas Bactérias