Este trabalho foi financiado pelo Wellcome Trust (WT 0.795.880).

O protocolo a seguir irá descrever o estudo da invasão de células endoteliais por S. aureus, mas teoricamente pode ser usado para estudar invasão celular por qualquer bactéria cultivável. Estágios específicos para S. células endoteliais aureus e são indicados. 1. Preparação de bactérias <ol><li> Cultura S. cepas de Staphylococcus de 16/04 h (dependendo da fase de crescimento necessário) em 10 Cérebro-caldo Infusão ml (BHI) a 37 ° C no ar com agitação a 200 rpm. Estas condições de crescimento são específicos para S. aureus e pode precisar ser adaptado para outras bactérias. </li><li> Lavar as bactérias três vezes em meio Modified Dulbecco Águia (DMEM; Invitrogen) por rodadas alternadas de centrifugação a temperatura ambiente (5.000 x g, 10 min), a remoção do sobrenadante da cultura e ressuspensão do pellet bacteriano em um volume equivalente de DMEM. Medir a densidade óptica da suspensão resultante de bactérias que podem ser ajustados conforme a necessidade. Para S. aureus, nós preparamos uma suspensão na OD = 1 600, o que corresponde a ~ 10 9 ufc ml -1. </li></ol> 2. Cultura de células endoteliais <ol><li> Cultura da linhagem de células endoteliais EA.hy926 1 em DMEM suplementado com soro fetal bovino (FBS, 10%) e L-glutamina (2 mM) a 37 ° C em 5% CO 2. Alternativamente, agrupados células primárias de veias umbilicais humanas endoteliais (HUVECs) pode ser comprado de Lonza (Basel, Suíça) e cultivadas em meio basal endotelial suplementado com 2% de SFB, extrato de cérebro de bovinos (incluindo a heparina), fator de crescimento endotelial humana e hidrocortisona a 37 ° C em 5% de CO 2 de acordo com as instruções do fabricante (Lonza). Estas condições de crescimento são específicos para estas células e pode precisar ser adaptado para outros tipos de células host. </li><li> Cultivar células endoteliais em frascos de T75 para completar confluência, verificada por olho. </li><li> Prepare as placas de 24 poços para a inserção das lamelas. Pinças finas (chama esterilizado) são necessários para mover as lamelas, que têm uma superfície opaca e um brilhante. Lamínulas lugar nos poços da placa de 24 poços com a superfície opaca virada para cima para permitir a ligação celular. </li><li> Liberar as células do frasco T75 com 3 ml de tripsina-EDTA (0,25%) e adicionar a 10 ml do meio de cultura relevante. </li><li> Adicionar 500 mL de células em suspensão para placas de 24 poços contendo lamínulas de vidro Thermanox. Um frasco T75 confluentes de células desde células suficientes para duas placas de 24 poços, resultando em cerca de 5 × 10 5 células por poço (em 500 mL de médio). </li><li> Incubar as placas por 48 h, como descrito acima, e verificar 100% de células confluência por microscopia de luz invertida. </li><li> Dip-lavagem lamínulas em PBS e adicionar novas placas de 24 poços contendo 490 l contendo DMEM 10% FBS em cada poço. </li><li> Para examinar o papel de determinados processos metabólicos na invasão celular, os inibidores podem ser adicionados a culturas de células de 1 h antes da adição de bactérias e concentrações mantida durante o ensaio. Por exemplo, para determinar o papel do rearranjo de actina em S. aureus invasão das células endoteliais, 50 mM citocalasina D pode ser adicionada, ou para o papel de cavéolas, 5 mM metil-β-ciclodextrina pode ser adicionado. </li></ol> 3. Ensaio de invasão <ol><li> Adicionar 10 ml de bactérias lavada (resultando em cerca de 2 × 10 7 ufc ml -1 de S. aureus) a cada poço contendo uma lamela lavado com uma camada confluente de células endoteliais em 490 l contendo DMEM 10% FBS. </li><li> Incubar durante 15-90 minutos a 37 ° C em 5% CO 2. </li><li> Para medir o número total de bactérias associadas com as células (aderente e internalizadas), mergulhe-as lamelas lavar três vezes em PBS e adicione aos poços contendo 500 mL fresco Triton 0,5% X-100 em PBS. Para garantir as células totalmente lisar e liberar todas as bactérias internalizadas, pipetar o várias vezes, apontando a ponta da pipeta directamente na superfície da lamela. </li><li> Enumerar as bactérias por plaqueamento a suspensão líquida (ou diluições deste, sempre que necessário) sobre a superfície de placas de TSA. Desde TX-100 vai lisar muitas bactérias Gram-negativas, saponina pode ser usado em vez 2. </li><li> Para medir o número de bactérias internalizadas, remover o sobrenadante da cultura de cada bactéria unbound bem contendo e substituir com 500 mL DMEM/10% FBS suplementada com 200 mg ml -1 gentamicina. Rotineiramente usamos gentamicina, em vez de lysostaphin aqui como é mais barato e nos permite alternar entre experimentos usando diferentes tipos de bactérias (por exemplo, estafilococos e Lactococci) sem ter que mudar o protocolo experimental. </li><li> Incubar as placas a 37 ° C em 5% de CO 2 por 60 minutos para matar todas as bactérias extracelulares. </li><li> Lavar lamínulas 3 vezes em PBS,lisar e enumerar por plaqueamento em TSA, conforme descrito para o ensaio de adesão acima. </li><li> Em alguns casos, pode ser preferível visualmente contar o número de bactérias por microscopia de luz. Neste caso, e específicos para S. invasão celular aureus, use lysostaphin (10 mg ml -1), em vez de gentamicina para destruir fisicamente as bactérias extracelulares. </li><li> Incubar lamínulas por 20min a 37 ° C em CO 2 na solução lysostaphin, em seguida, lavar e consertar com Cytopath (Cellpath). </li><li> Flood as lamelas com cristal violeta (0,5%, w / v) para 5min. </li><li> Dip-enxágüe em água, ar seco e montagem em lâminas de vidro. O número de bactérias por mm 2 de células endoteliais confluentes podem ser quantificados utilizando microscopia de luz. </li></ol> 4. Resultados representativos: Expressão de proteínas de ligação a fibronectina (FnBPA e FnBPB) sobre a superfície da S. aureus confere a capacidade de invadir células endoteliais. Trabalho recente redefiniu o domínio de ligação de fibronectina FnBPA 3. Do tipo selvagem (WT) S. aureus 8.325,4 invade células endoteliais com alta eficiência, enquanto que uma cepa falta tanto FnBPA e B (Δ FNB) apresentaram níveis significativamente reduzida de internalização (Figura 1A). Complementação do mutante com um plasmídeo A FNB menos o domínio de ligação de fibronectina (pFnR0) não promover a invasão (Figura 1A). Por outro lado, a complementação do mutante com um plasmídeo FNB inteira Um gene (pFnBA4) restaurado invasão a níveis WT (Figura 1A). O papel do FnBPA na invasão de células endoteliais também pode ser demonstrada através dos expressão heteróloga Lactococcus lactis host. Expressão de plasmídeo codificado FnBPA em L. lactis (pRM9 9) não aumentar significativamente a adesão às células endoteliais em comparação com as bactérias não expressar FnBPA (CTL) (bares abertos, Figura 1B). Por outro lado, expressando-FnBPA L. lactis invadiram células endoteliais em níveis significativamente mais altos do que a cepa não expressar (bares fechados, Figura 1B). Experimentos foram realizados quatro vezes em duplicado e os média ± desvio padrão é apresentada. * Representam dados que são estatisticamente diferentes (p = &lt;0,05) da WT ou valores CTL. <img src="/files/ftp_upload/2693/2693fig1.jpg" alt="Figura 1" /> Figura 1. Invasão de EA. Hy926 células endoteliais por S. aureus (A) ou L. lactis (B).

<ol>
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<html> O ensaio descrevemos baseia-se no ensaio de proteção a gentamicina, que tem sido amplamente utilizada para estudar a invasão das células do hospedeiro por bactérias. Observações da incapacidade dos antibióticos gentamicina e outros para matar as bactérias intracelulares foram utilizados nos primeiros estudos sobre a invasão das células do hospedeiro por bactérias 4-8. O uso de 24, 48 ou mesmo placas de 96 poços permite a geração de grandes quantidades de quantitativos, dados reprodutíveis em um curto período de tempo e de custo relativamente baixo. O ensaio também é atraente, porque não requer equipamento especializado, ele pode ser adaptado para trabalhar com várias bactérias e células, e pode ser usado para estudar o papel dos processos celulares tanto bacterianas e de acolhimento na invasão 9. De fato, desde os primeiros experimentos, o ensaio de proteção gentamicina tem sido amplamente empregada para medir a invasão da célula hospedeira por uma gama de bactérias diferentes, ou mesmo combinações de bactérias 10,11. Embora os princípios fundamentais são os mesmos, muitas variações sutis no método tem sido relatado. Neste artigo descrevemos a nossa versão e indicou onde as modificações podem ser necessários para outras bactérias ou células do hospedeiro. Ao projetar um experimento para medir a invasão da célula hospedeira usando essa abordagem, há uma série de pontos importantes a considerar: Sensibilidade bacteriana à gentamicina. Isto pode parecer óbvio, mas é importante garantir que a bactéria em estudo é suscetível a gentamicina, na concentração, temperatura e ao longo da duração do tempo a ser utilizado. No caso de insensibilidade, pode ser possível a utilização de outros antibióticos 5. Uma abordagem alternativa pode ser a utilização de enzimas líticas (lysostaphin, mutanolysin, lisozima) 12. Incubação e inóculo. Ao executar este teste pela primeira vez é importante para estabelecer o período de incubação ideal e inóculo bacteriano a ser utilizado. Portanto, experimentos iniciais deve examinar tanto a adesão e invasão ao longo do tempo (5 min para até 6 h). Também é importante avaliar como a invasão é afetado pela multiplicidade de infecção (MOI, o número de bactérias por célula). Em geral, é melhor usar o menor número possível de bactérias como isso irá reduzir os danos às células em cultura. Importantes diferenças entre cepas podem ser perdidas se os períodos de incubação prolongado ou inóculos grandes são usadas 9,13. Danos celulares. É importante lavar as bactérias antes da utilização. No entanto, os danos celulares não se restringe à produção de toxinas. Invasão bacteriana, indução de apoptose e interrupção das funções normais celulares podem causar danos celulares. Isso pode levar a penetração de gentamicina na célula, matando as bactérias internalizadas e dando a impressão de que a invasão não ocorreu 14. Condições de incubação. A temperatura ea composição do meio de cultura pode ter um impacto significativo sobre a invasão. Por exemplo, a invasão de células HeLa por Streptococcus pyogenes é dependente da presença de fibronectina solúvel, que é normalmente presentes no soro 15. Outra consideração é o crescimento bacteriano no meio de cultura durante o curso do experimento. Cultura e quantificação de bactérias intracelulares. Embora TX-100 não pode matar as bactérias intracelulares, pode inibir o seu crescimento. Como tal, é importante verificar a replicação bacteriana em meios sólidos na presença do detergente. Onde afetados, pode ser possível a utilização de menores concentrações do detergente ou para usar uma alternativa, como saponina. Um benefício do ensaio de proteção gentamicina sobre a coloração violeta cristal e ensaio de microscopia é que é menos trabalhoso, e permite a detecção e diferenciação de sub-populações de bactérias internalizadas. Por exemplo, S. aureus pode produzir tanto um tipo de colônia normal (NCT) ou a variante pequena colônia (SCV) 16, que não seriam distinguíveis por microscopia de células bacterianas individuais. Um benefício da coloração violeta cristal e ensaio de microscopia sobre o ensaio de proteção gentamicina é a aglutinação de células podem ser contabilizadas e que as bactérias intracelulares que digitar um &#39;viável, mas não cultivável &quot;estado será detectado 17. Examinar o papel dos processos da célula hospedeira na invasão bacteriana. Muitos estudos têm empregado inibidores da função da célula hospedeira tais como citocalasina D, que interfere com rearranjo de actina. Tais estudos também podem incluir anticorpos que alvejam receptores de superfície celular e knockdown siRNA de genes específicos 18. É de vital importância para assegurar que estes tratamentos não afetam a viabilidade ou a fixação de células em cultura ou ter efeitos tóxicos sobre bactérias. Futuras direções: &gt; ONTEÚDO &quot;Porque este ensaio é normalmente realizado em multi-bem placas de cultura de células, é teoricamente possível adaptá-lo a uma operação de rastreio de alto rendimento, que pode envolver o exame de bibliotecas de potenciais inibidores da função das células, anti-infecciosos ou antibióticos projetado para atacar bactérias intracelulares. Alternativamente, essa abordagem pode ser usada para a tela mutante bibliotecas para identificar genes envolvidos na adesão, invasão e sobrevivência intracelular. Em alguns casos, pode ser desejável incluir forças de cisalhamento (fluxo) no sistema modelo, por exemplo, quando a modelagem do endotélio, para mais perto imitar o no ambiente vivo. avanços atuais na concepção e fabrico de células de fluxo e sistemas de cultura de células microfluídicos oferecem a possibilidade de empregar o ensaio de proteção gentamicina na patógeno-hospedeiro modelos que incorporam as forças de cisalhamento. Em resumo, o protocolo aqui descrito é baseado em abordagens similares utilizados por muitos anos. É amplamente aplicável a muitos tipos de células cultivadas e espécies de bactérias e pode gerar grandes quantidades de dados rapidamente ea um custo relativamente pequeno.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31885-023</td>
<td>&nbsp;</td>
<tr>
<td>Brain heart infusion</td>
<td>&nbsp;</td>
<td>BioChemika</td>
<td>53286</td>
<td>&nbsp;</td>
<tr>
<td>Foetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>HUVECs</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-2519</td>
<td>&nbsp;</td>
<tr>
<td>Endothelial basal medium</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3121</td>
<td>&nbsp;</td>
<tr>
<td>Bovine brain extract (including heparin)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092</td>
<td>&nbsp;</td>
<tr>
<td>Human endothelial growth factor</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4017C</td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4035C</td>
<td>&nbsp;</td>
<tr>
<td>GA-1000</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092C</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Lysostaphin</td>
<td>&nbsp;</td>
<td>AMBI</td>
<td>LSPN-50</td>
<td>&nbsp;</td>
<tr>
<td>Thermanox coverslips</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>TKT-210-330P</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Cytopath</td>
<td>&nbsp;</td>
<td>TCS Biosciences</td>
<td>HC8595</td>
<td>&nbsp;</td>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3886</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4049</td>
<td>&nbsp;</td>
<tr>
<td>T75 flasks</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>430641</td>
<td>&nbsp;</td>
<tr>
<td>Cytochalasin D</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2618</td>
<td>&nbsp;</td>
<tr>
<td>Methyl-B-cyclodextrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>332615</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

invasion of human cells by a bacterial pathogen

Aqui vamos descrever a forma como nós estudamos a invasão de células endoteliais humanas por Staphylococcus aureus bacteriana patogênica. O protocolo geral pode ser aplicada ao estudo da invasão celular por praticamente qualquer bactéria cultivável. Os estágios em que aspectos específicos de invasão pode ser estudado, como o papel do rearranjo de actina ou cavéolas, será destacado. Células hospedeiras são cultivadas em frascos e quando estiver pronto para o uso estão semeados em placas de 24 poços contendo lamínulas Thermanox. Usando lamínulas permite posterior remoção das células dos poços para reduzir a interferência de proteínas do soro depositado para os lados dos poços (a que S. aureus seria anexar). As bactérias são cultivadas com a densidade necessária e lavada para remover qualquer proteínas secretadas (toxinas, por exemplo). Lamínulas com camadas confluentes de células endoteliais são transferidos para nova placas de 24 poços contendo meio de cultura fresco antes da adição de bactérias. Bactérias e células são então incubadas juntas para a quantidade necessária de vez em 5% CO 2 a 37 ° C. Para S. aureus este é tipicamente entre 15-90 minutos. Lamínulas Thermanox são removidos de cada bem e dip-lavados em PBS para remover bactérias solto. Se o total bactérias associadas (aderente e internalizadas) devem ser quantificados, lamínulas são então colocados em um poço fresco contendo 0,5% Triton X-100 em PBS. Pipetagem suave leva à completa lise das células e bactérias são enumerados por diluição seriada e plaqueamento em ágar. Se o número de bactérias que invadiram as células é necessário, lamínulas são adicionados aos poços contendo 500 mL meio de cultura de tecidos suplementado com gentamicina e incubação continuou por 1 h, que vai matar todas as bactérias externas. Lamínulas pode então ser lavada, as células lisadas e bactérias enumeradas por plaqueamento em ágar, conforme descrito acima. Se o experimento requer visualização direta, lamelas podem ser fixadas e coradas para microscopia de luz, fluorescência confocal ou ou preparados para microscopia eletrônica.

Watch this Scientific Journal Video about Invasion of Human Cells by a Bacterial Pathogen at JoVE.com

Invasion of Human Cells by a Bacterial Pathogen

Um protocolo geral para o estudo da invasão das células do hospedeiro por um agente patogénico, com foco na<em&gt; Staphylococcus aureus</em&gt; E células endoteliais humanas.

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 ...

invasion-of-human-cells-by-a-bacterial-pathogen

Research

JoVE Journal

Immunology and Infection

34.6K visualizações.  University of Bath. Um protocolo geral para o estudo da invasão das células do hospedeiro por um agente patogénico, com foco na Staphylococcus aureus E células endoteliais humanas.

Vídeo: Invasion of Human Cells by a Bacterial Pathogen

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Aplicação de um rato ligado patch Peyer Assay alça intestinal para avaliar absorção pelas células bacterianas M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Imunologia e Infecção

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Um dia-Esquema de fluxo de trabalho para detecção de patógenos de bactérias e de resistência antimicrobiana de hemoculturas

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Entérico invasão bacteriana de células epiteliais intestinais<em&gt; In Vitro</em&gt; É dramaticamente aumentada usando um Vertical Modelo câmara de difusão

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

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

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

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 ...

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 the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

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 ...

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

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

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

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

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

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

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

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