Gostaríamos de agradecer Elisabeth Guillemet, Christophe Buisson e Ludovic Bridoux para assistência técnica excelente. Temos uma grande dívida para Sylvie Salamitou e Fedhila Sinda para a configuração inicial do sistema.

1. A criação de insetos O ciclo completo de ovo a larva de último estádio dura cerca de 5 semanas a 25 ° C. Uma ou duas semanas adicionais são necessários para obter as borboletas adultas. <ol><li> Coloque pelo menos 100 pupas ou recém-fundidas adulto G. borboletas mellonella em 5 litros de gaiola de arame. Borboletas masculinos avaliar 10 a 15 mm. A mariposa adulta masculina é bege com luz fraca e marcas escuras. Medida borboletas fêmeas em torno de 20 mm. As fêmeas são mais escuras do que os homens com uma cor marrom / cinza. </li><li> Suspender dois maços de quatro camadas de papel na gaiola para postura de ovos. Após 2 dias, a fêmea adulta põe ovos no limite entre os papéis. </li><li> Duas vezes por semana, coloque os pacotes de ovo de papel em novas caixas de criação de plástico com grades para circulação de ar, contendo pólen e cera de abelha. Os ovos eclodem em cerca de 3 dias e pequenas larvas começam a se desenvolver através da alimentação em cera e pólen. Alternativamente, as larvas podem ser colocados em uma dieta artificial consisting de uma mistura de 500 g de mel líquido, 400 g de glicerina, 100 g de levedura de cerveja seca, 250 g de farinha de trigo, 200 g de leite em pó desnatado e 400 g polenta. Para proporcionar proporção adequada de alimento por larva, regularmente separar as larvas em caixas novas e reconstituir o alimento a cada dois dias. </li><li> As larvas são criados durante as seis fases de larva estádio. Cada estádio é caracterizado por o deslizamento da cápsula da cabeça de larvas. Quando as larvas atingem o último estágio antes pupação, eles param de alimentação e começar a construir um casulo de seda luz. </li><li> Em seus casulos protetores, as larvas vai descansar e se transformam em pupas. Worms cera permanecem na fase de pupa de uma a duas semanas para emergir em traças adultas. As mariposas adultas não beber nem comer. O acasalamento ocorre e ciclo dos vermes de cera &quot;a vida começa de novo. </li><li> Para padronizar o ensaio de patologia, tomar a larvas durante o último estágio larval. Durante esta fase, eles param de alimentação, mudar-se para a tampa da caixa de criação e começar a produzir seda. Este stage dura cerca de 5 dias. </li></ol> 2. Seleção de inseto para a Infecção <ol><li> Selecione larvas no último instar, que são 2-3 cm de comprimento e 180-250 mg de peso, 24 horas antes de infecções e colocá-los em uma caixa vazia para privá-los. </li><li> Remover o casulo de seda em torno da nascente larvas. </li></ol> 3. Preparação bacteriana <ol><li> Preparar suspensões bacterianas de Bacillus para se obter concentrações finais que variam de 10 4 unidades formadoras de colónias (ufc) / ml a 10 8 ufc / ml para injecção intra haemocoelic e de 3x10 6 ufc / ml para 1x10 8 ufc / ml para ingestão (a cfu obter um LD 50 depende das espécies de bactérias). No nosso caso, B. cereus é cultivada em meio Luria-Bertani broth (LB, 10 g / l de triptona, 5 g de extracto de levedura / l, 10 g / l NaCl) a 37 ° C, sob agitação, até à entrada em fase estacionária, que corresponde ao curvamento do crescimento curva. Medir o OD 600 nm (entre 1 e 2 para a B. cereus) e utilizá-la para determinar a concentração de bactérias, utilizando uma curva de titulação anteriormente feita. Colheita bactérias por centrifugação a 5000 xg durante 10 min à temperatura ambiente e ressuspender em tampão fosfato salino (PBS: 1M KH 2 PO 4, 1 M K 2 HPO 4, NaCl 5 M, pH 7,2). Cada larva receberá 10 ul de suspensão bacteriana na concentração desejada. </li><li> Em alternativa, a suspensão de esporos, podem ser preparados por infecção. Obter esporos por cultura das bactérias na esporulação meio HCT (5 g / L de triptona, 2 g / L de hidrolisado de caseína, 12,5 mM de K 2 HPO 4, 12,5 mM de MgSO4, 0,05 mM MnSO4, 1,2 mM de ZnSO 4, 1,2 mM Fe 2 (SO 4) 3, 0,5% de H 2 SO 4, 25 mM de CaCl 2) 20 a 30 ° C durante 3 dias. Esporos de colheita por centrifugação (10.000 xg, 15 min), e lavar duas vezes em água destilada estéril. Ressuspender o pel esporosdeixar em água destilada estéril e calor durante 15 min a 78 ° C para remover o restante bactérias vegetativas. Numerar a suspensão através do plaqueamento de diluições em série em placas de agar LB. Cada larva receberá 10 ul de suspensão de esporos para a concentração desejada. </li></ol> 4. Cry Preparação Toxina <ol><li> Preparar a toxina do B. Cry1C thuringiensis 407 transformado com o plasmídeo pHTF31C 21 transportando o gene que codifica Cry1C. Cultura da estirpe em meio HCT 100 ml a 30 ° C durante 72 horas com antibiótico (eritromicina 10mg/ml) para permitir a esporulação total, a produção de toxina de cristal e da libertação no sobrenadante da cultura. </li><li> Purifica-se cristais a partir do sobrenadante em um gradiente de sacarose de 72% -79%. Primeiro adicione 17 ml de sacarose de 79% em um tubo de 40 ml. Cuidadosamente camada de 17 ml de 72% de sacarose em cima de sacarose a 79%. Finalmente, a camada de 5-6 ml da cultura concentrada 10X esporulado e fechar o tubo. Centrifugar o tubo durante 14 horas a 20,000 xg, 4 ° C, para separar os cristais a partir dos esporos. Recolher os cristais na interface do gradiente. Ressuspender o sedimento em 25 ml de cristais de água fria estéril para lavá-la. Centrifugar a 8.000 xg durante 20 min. Este passo é repetido três vezes para remover todos sacarose aderindo aos cristais. Ressuspender os cristais em 5 ml de água estéril e observar ao microscópio para avaliação da pureza. </li><li> Avaliar a concentração de proteína toxina pelo clássico coloração Bradford em solução de cristal presolubilized em 50 mM NaOH. Cristais de toxina podem ser armazenadas a -20 ° C até à sua utilização. </li></ol> 5. Injector Preparação <ol><li> Para controlar o volume de injecção de precisão, utilizar uma bomba de seringa automatizada (por exemplo KD Scientific KDS 100) com uma velocidade de instalação para 1 ^ l / s (Figura 1A). </li><li> Encher uma seringa hipodérmica uma com 300 ml de água ou ul da solução bacteriana (uma seringa por condição) para a infecção de larvas de 20-25. Remover cuidadosamente as bolhas por tapping a seringa, em seguida, juntar uma agulha 0,45 x 12 mm para a seringa. </li><li> Colocar a seringa no injector. </li><li> Realizar uma injecção de 10 ul em branco em um tubo vazio para controlar o volume injectado. </li></ol> 6. Injeção Intrahaemocoelic inseto <ol><li> Coloque o inseto manualmente entre o polegar eo indicador. Em seguida, inserir a agulha fixada no injector na pele larva (cutícula) (Figura 1C). </li><li> Mantenha o inseto no local, enquanto injetar a solução de 10 ul bacteriana (bactérias ou esporos vegetativo). </li><li> Remova cuidadosamente o inseto da agulha. </li><li> Coloque o insecto infectado em um prato de Petri pequenas (5 cm de diâmetro), 5 larvas por placa. </li><li> Infectar pelo menos 20 larvas para cada condição experimental. </li><li> Colocar as placas a 37 ° C numa incubadora durante 48 horas. </li></ol> 7. Alimentação Força inseto (Ingestão) <ol><li> Num tubo Eppendorf 2 ml, misturar 0,2 ug / &amp; mu, l de toxina Cry1C quer com suspensão de esporos ou células vegetativas para obter concentrações finais que variam de 4 a 3x10 1x10 7 por 10 ul. </li><li> Encher uma seringa de 1 ml com uma 30G, 25 mm, com agulha hipodérmica de 300 ul da solução de toxina bacteriana, Cry1C na infecção de larvas de 20-25. Remover as bolhas a partir da seringa. </li><li> Coloque o insecto manualmente, de modo que a sua boca entra na agulha fixa no injector (Figura 1D), e da força de alimentá-la com 10 ul da solução bacteriana usando a bomba de seringa automatizada. </li><li> Infectar pelo menos 20 larvas para cada condição experimental. </li><li> Coloque o inseto infectado em um pequeno 5 centímetros de Petri (5 larvas por prato). Colocar as placas a 37 ° C numa incubadora durante 48 horas. </li></ol> 8. Mortalidade dos insetos gravação <ol><li> Depois de experiências de alimentação forçada ou injecção, manter as larvas na placa de Petri, sem comida, a 25 ° C ou 37 ° C. Verifique larval mortality regularmente ao longo de um período de 48 h. Larvas mortas são inertes e, geralmente, ficam pretas (Figura 1B). </li><li> Os dados de mortalidade pode ser analisado utilizando o programa de log-probit 14. Este programa testa a linearidade das curvas de mortalidade de dose e fornece doses letais (LD 50). Alternativamente, outros programas, tais como o Prism (Graph Pad), análise de sobrevivência ou dados de mortalidade pode ser usado. </li></ol>

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O uso de insetos e principalmente o estágio larval, como modelos de infecção por vários patógenos, está se tornando freqüente. Um modelo de escolha para alguns aspectos é Drosophila (o modelo mosca), utilizado como adultos e 1,2 estágio larval. O inseto lepidóptero G. mellonella também tem sido principalmente utilizado para o ensaio de virulência bacteriana por injecção. A vantagem de tolerar temperaturas mais elevadas (acima de 37 ° C) do que a Drosophila (máximo 25...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Cera e pólen </td><td> La Ruche Roanaise </td><td> 303000 </td><td> Qualquer produtor de mel </td></tr><tr><td> Bomba de seringa automatizada </td><td> KD Scientific </td><td> KDS 100 </td><td></td></tr><tr><td> Seringa de 1 ml </td><td> Terumo </td><td> BS 01T </td><td></td></tr><tr><td> Agulha 0,45 x 12 mm </td><td> Terumo </td><td> NN 2613R </td><td></td></tr><tr><td> Petri 5 centímetros </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> 30G agulha, hipodérmica 25 milímetros </td><td> Burkard Mfg Co. Ltd. </td><td> PDE0005 </td><td></td></tr></tbody></table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

O estudo da virulência bacteriana requer frequentemente um modelo animal adequado. Modelos de mamíferos de infecção são caros e pode levantar questões éticas. O uso de insetos como modelos de infecção proporciona uma alternativa valiosa. Comparado com outros hospedeiros não vertebrados modelo tais como nemátodos, insectos têm um sistema relativamente avançado de defesas antimicrobianas e são por isso mais susceptíveis de produzir a informação relevante para o processo de infecção de mamíferos. Como mamíferos, insetos possuem um sistema complexo imune inata 1. As células na hemolinfa são capazes de encapsular ou fagocitando invasores microbianos, e as respostas humorais incluem a produção induzida de lisozima e pequenos péptidos antibacterianos 2,3. Além disso, as analogias são encontrados entre as células epiteliais do intestino médio de insectos e larvas de células intestinais de sistemas digestivo de mamíferos. Por fim, vários componentes básicos essenciais para o processo de infecção bacteriana, como a adesão celular, a resistência àpéptidos antimicrobianos, a degradação do tecido e de adaptação ao stress oxidativo são susceptíveis de ser importante, em ambos os insectos e mamíferos 1. Assim, os insectos são ferramentas polivalentes para a identificação e caracterização de factores de virulência microbianas envolvidas em infecções de mamíferos. As larvas de traça da cera maior Galleria mellonella foram mostrados para fornecer uma visão útil para a patogénese de uma grande variedade de infecções microbianas, incluindo fungos de mamíferos (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) e os agentes patogénicos bacterianos, tais como Staphylococcus aureus, Proteus vulgaris , Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes ou Enterococcus faecalis 4-7. Independentemente das espécies bacterianas, os resultados obtidos com Galleria larvas infectadas por injecção directa através da cutícula consistentemente correlacionados com aqueles de simiLar estudos de mamíferos: estirpes de bactérias que são atenuadas em modelos de mamíferos demonstram menor virulência em Galleria, e as tensões que causam graves infecções humanas são também altamente virulenta no modelo Galleria 8-11. A infecção oral de Galleria é muito menos utilizada e adicional de compostos, como as toxinas específicas, são necessárias para atingir a mortalidade. Larvas de G. mellonella apresentam várias vantagens técnicas: são relativamente grandes (larvas de último estádio antes pupação são cerca de 2 cm de comprimento e de peso mg 250), permitindo assim a injecção de doses definidas de bactérias, podem ser criados em várias temperaturas (20 ° estudos C a 30 ° C) e a infecção pode ser realizada entre 15 ° C e acima de 37 ° C 12,13, permitindo experiências que simulam um ambiente de mamíferos. Além disso, a criação de insetos é fácil e relativamente barato. Infecção das larvas permite monitorar a virulência bacteriana por vários meios, including cálculo de LD 50 14, a medida de sobrevivência bacteriana 15,16 e exame do processo de infecção 17. Aqui, descrevemos a criação dos insetos, abrangendo todas as fases de vida de G. mellonella. Nós fornecemos um protocolo detalhado de infecção por duas vias de inoculação: haemocoelic oral e intra. O modelo bacteriano utilizado no presente protocolo é uma Bacillus cereus, de agentes patogénicos Gram-positivos implicada na gastrointestinal, bem como em outros locais ou sistémicas graves infecções oportunistas 18,19.

Injecção intra haemocoelic de bactérias em G. mellonella foi provado muito útil para a identificação de factores de virulência muitas tratam de danos nos tecidos e a resistência a factores imunes inatas de vários agentes patogénicos humanos. Como exemplo, a figura 2A representa a mortalidade de insectos após a injecção de várias doses de B. cereus (bactérias de tipo selvagem e estirpes mutantes) 22. Figura 2B representa a sobrevivência após ...

Watch this Scientific Journal Video about The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis at JoVE.com

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

Oral e intra infecção haemocolic de larvas de traça da cera<em&gt; Galleria mellonella</em&gt; É descrito. Este insecto pode ser utilizada para estudar factores de virulência de entomopatogênico bem como mamíferos bactérias oportunistas. Criação dos insetos, os métodos de infecção e exemplos de<em&gt; In vivo</em&gt; Análise são descritos.

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

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

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JoVE Journal

Immunology and Infection

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

31.6K visualizações.  INRA, Micalis UMR1319, France. Oral e intra infecção haemocolic de larvas de traça da cera Galleria mellonella É descrito. Este insecto pode ser utilizada para estudar factores de virulência de entomopatogênico bem como mamíferos bactérias oportunistas. Criação dos insetos, os métodos de infecção e exemplos de In vivo Análise são descritos.

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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

In vitro a formação de biofilme em um slide Câmara de 8 poços

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Imunologia e Infecção

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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Um Ensaio visual para o Monitor T6SS mediada Competição bacteriana

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Utilização de<em&gt; Galleria mellonella</em&gt; Como um organismo modelo para estudar<em&gt; Legionella pneumophila</em&gt; Infecção

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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 ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

Peixe-zebra como um modelo para avaliar o potencial teratogênico de nitrito

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

O Madagascar silvando barata como um modelo de animais não-mamíferos alternativo para investigar a eficácia da droga, patogenia e virulência

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Um modelo de cultura primária de células bolsas serosas Ex Vivo frango para estudar a patogênese de doenças infecciosas bolsas serosas vírus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...