Gostaríamos de agradecer Brandon Hassett e Carlos Ortiz por sua assistência técnica. Este trabalho foi financiado pela NSF subsídios IOB-0544428, 0951272-IOS e IOS-0925561 com o Dr. Michael Kolomiets, e pelo USDA Instituto Nacional de Alimentação e Agricultura (Nifa), Melhoramento de Plantas DRFA e Grant Educação # 2010-85117 -20539 para os drs. Seth Murray, Thomas Isakeit e Kolomiets Michael.

1. Bioensaio Kernel milho <ol><li> Duas semanas antes da cultura, os fungos patogénicos em Agar de Batata Dextrose (PDA) a 28 ° C. </li><li> Seleccione grãos com tamanho e forma semelhantes, de preferência achatada assim que colocam em nível com a parte inferior de frascos de bioensaio, e colocar em tubos de 50 ml Falcon. Kernels seleccionados devem ter sido produzidos simultaneamente em mesmo ambiente para garantir a idade de sementes semelhante e composição metabolito. </li><li> Tona esterilizar miolo por agitação tubos à temperatura ambiente durante 5 minutos com etanol a 70%, 1 minuto com água estéril, e 10 minutos com hipoclorito de sódio a 6%. Lavar, três vezes, durante 5 minutos de cada vez, com água estéril, agitando contínua. Kernels Secar em toalhas autoclavados. </li><li> Para facilitar a infecção de Aspergillus flavus, criar uma pequena ferida no embrião lado com uma agulha 18 G até à profundidade de 0,5 cm. No entanto, em nossa experiência maior ferida é necessário para a infecção Fusarium verticillioides. Therefore, utilizar uma lâmina de barbear para cortar no embrião lado-a uma profundidade de 0,5 mm. </li><li> Preparar suspensão do inóculo por raspagem placas de cultura em cerca de 5 ml de solução autoclavada 0,01% de Tween-20 para libertar os esporos e remover o micélio por filtração através de gravidade, pelo menos, 4 camadas de gaze esterilizada. Calcular a concentração de esporos com hemocitômetro e ajuste para 10 6 esporos / mL para A. flavus ou F. verticillioides. Se usando outras espécies de patógeno ou hospedeiro, é importante avaliar as concentrações de inoculo para a infecção apropriado. </li><li> Criar uma câmara de humidade num recipiente de plástico (29,2 cm X 18,7 cm X 8,3 cm) revestidas com cinco folhas de toalhas de papel e 100 ml de água estéril. Câmara não é a água ou ar apertado e água deve ser adicionado ao longo experimento para manter toalhas de papel úmido. </li><li> Coloque quatro núcleos em um frasco de 20 ml autoclavado cintilação de vidro e registrar sua massa. Inocular com 200 uL suspensão de esporos,boné e vortex para kernels uniformente com suspensão. Solte as tampas para permitir livremente a troca de ar e colocar em câmara de umidade. </li></ol> Nota: Para sementes ou outros fungos que os descritos nestes métodos, a quantidade de inoculo necessária pode ser diferente e pode ser derivada experimentalmente. <ol start="8"><li> Incubar grãos sob um fotoperíodo 12-h-light/12-h-dark a 28 ° C durante 7 dias ou até desejado. </li></ol> 2. Enumeração conídios <ol><li> Após o período de infecção, adicionar 5 ml de autoclavado 0,01% de Tween-20 para frascos de cintilação contendo grãos infectados e Vortex cuidadosamente durante 1 minuto. </li><li> Usando uma pipeta de ponta larga furo, imediatamente diluir pela transferência de duas separadas alíquotas de 200 ul de suspensão de esporos em tubos Eppendorf contendo 2,0 ml 1,8 ml de 0,01% de Tween-20 (ou diluir conforme necessário, dependendo da infecção). </li><li> Enumerar cada alíquota de 200 uL por duas vezes com um hemocitómetro 4e comparar os níveis de conídios entre os tratamentos. </li></ol> 3. Quantificação de aflatoxina <ol><li> Adicionar núcleos a partir de uma amostra de 50 ml misturador copo com 20 ml de metanol a 80% e 0,05 g de NaCl, a tampa e da mistura a alta velocidade durante 1 min. </li><li> Coloque um papel de filtro de pregas ao longo de um recipiente de coleta limpo e despeje o extrato em filtro. </li><li> Transferem-se 10 ml do filtrado para um recipiente limpo, adicionar 20 ml de H2O destilada, e misturar bem. </li><li> Amostra de filtro através de um filtro de microfibra 1,5 mM de vidro em um copo limpo. </li><li> Aplicar 1 ml de extracto filtrado para Afla coluna de teste e, utilizando ar comprimido, forçar o extracto filtrado através da coluna a uma taxa de 1-2 gotas por segundo até esvaziadas. </li><li> Lavar a coluna duas vezes com 1 ml de H2O destilada e passar através da coluna a 1-2 gota / segundo até que o ar passa. </li><li> Coluna eluir com 1 ml de metanol a 100% HPLC numa 1-2 gota / segundo taxa recolhido numa cuvete de vidro. </li><li> Adicionar1 ml de Afla Desenvolvedor de teste para eluato e misture bem. Cuvete lugar em fluorómetro calibrado e lida a 60 seg. </li></ol> Nota: Quando se utiliza o protocolo Aflatest FGIS, a medição a partir do fluorómetro é calculada com base um período inicial de 50 gramas de amostra extraída em 100 ml. Se se considera a diluição da amostra com a água, o 1 ml aplicado a uma coluna representa 0,166 gramas de amostra. Assim, ao modificar o protocolo, você precisa levar em conta as diferenças de tamanho da amostra e da solução de extração inicial. Por exemplo, 2 gramas de grãos são extraídos em 20 ml de metanol 80%. Este é de 0,1 gramas / ml. Quando 1 ml desta amostra é misturada com 2 ml de água, a proporção de amostra no líquido é agora 0,033 gramas / ml. Se esta amostra dá uma leitura fluorómetro de 100 ppb, a concentração real baseia-se na proporção de amostra para 0,166, isto é, ppb = (0,166 grama X 100 ppb) / 0,1 grama), ou 166 ppb. Para quantificação de aflatoxina alternativamétodos de cátions, ver referência 7. 4. Fumonisina B1 (FB1) Análise <ol><li> Quando fungo é cultivado em grãos de milho, extrair amostras durante a noite com 10 ml de acetonitrilo / água (50/50, v / v) à temperatura ambiente sem agitação. Subsequentemente, misturar o extracto (2 ml) com água desionizada (6 ml), e aplicar esta mistura (8 ml) directamente para a extracção de fase sólida C-18. </li><li> Antes de carregar amostra pré-condição, a coluna de extracção C-18 de fase sólida, por lavagem com 2 ml de acetonitrilo seguido por 2 ml de água. </li><li> Após o carregamento da amostra, a coluna é lavada com 2 ml de água seguido por 2 ml de acetonitrilo / água (15/85, v / v). Amostra para análise por HPLC (contendo FB1) é eluída com 2 ml de acetonitrilo / água (70/30, v / v). </li><li> Derivatizar FB1 com o-phthaldehyde (OPA) por transferência de 0,1 ml do eluato da coluna para um frasco contendo 0,1 ml de tampão de borato (0,05 M bórico acid/0.05 de sódio M de borato [50/50, v / v] pH, 8,5) e 0,1 OPA ml (0,1 mg / ml em acetonaitrile com 0,5% - mercaptoetanol). </li><li> Parar a reacção após 10 min por adição de 0,5 ml de acetonitrile/0.01 M de ácido bórico (40/60, v / v). </li><li> Analise FB1 numa HPLC Shimadzu LC-20AT sistema equipado com uma coluna analítica ODS Zorbax (4,6 mm 150) e um comprimento de onda variável Shimatzu detector RF-10Axl de fluorescência (excitação 335 nm / emissão 440 nm). </li><li> Um gradiente linear é usada (solvente A: acetonitrile/0.1 M fosfato de sódio (40/60), pH 3,3, solvente B: acetonitrilo / fosfato de sódio 0,1 M (60/40), pH 3,3) eo programa de gradiente é como se segue: 100% de A. a 100% de B em 10 min B, 100% durante 5 min </li><li> Analise FB1 padrões por HPLC, e medição da área de pico são utilizados para gerar a curva padrão. Subsequentemente, quantificar FB1 nível de uma amostra por comparação com as áreas dos picos FB1 curva padrão. </li></ol> 5. Ergosterol Análise <ol><li> Cultura do fungo em grãos de milho por 7-14 dias. </li><li> Após o período de incubação, adicionam-se 10 ml de clorofórmio: metanol (2:1, v / v) em cada frasco. Uma vez adicionado, agitar amostras bem e depois incubar frascos no escuro à temperatura ambiente durante 24 horas. </li><li> Após 24 hrs, centrifugar as amostras, e recolher o sobrenadante e filtrar através de membrana de nylon 0,45 um. </li><li> Injectar a amostra directamente para um sistema de HPLC Shimadzu LC-20AT equipado com um 4,6 U coluna ODS-C18 (200 A, 250 ± 4,6 mm) e um Shimadzu UV SPD-20A / detector VIS definido para monitorar a 282 nm. </li><li> Use metanol (100%) a um caudal de 1,5 mL / min como a fase móvel. Determinar a quantificação de ergosterol em amostras por comparação das áreas de pico de amostras com uma curva padrão gerada a partir de HPLC ergosterol. </li></ol> 6. Os resultados representativos Após a inoculação e incubação numa câmara de humidade durante dois a três dias, o crescimento de fungos deve começar a aparecer sobre os núcleos. Sete dias após o tratamento, o crescimento vegetativo das plantas tratadas devem ser claramente visíveis, whilcontrolos electrónicos simulados deve ser não infectados (Figura 1B, no topo). Os períodos de incubação mais longo são favoráveis ​​a mais abundante crescimento vegetativo (Figura 1B, inferior). Sob as condições aqui descritas (Figura 2G), A. flavus de tipo selvagem NRRL 3357 indicadas valores máximos de colonização, a acumulação de aflatoxina, e produção de conídios de 8, 6, e 8 dias, respectivamente (Figura 2, CA). No entanto, quando a contaminação com micotoxinas e conidiação foram comparados por unidade de ergosterol, os maiores níveis foram observados 4 e 6 dias, respectivamente (Figura 2, DE). Figura 2F resume estes fungos biomassa-dependentes valor máximo-observações. Curiosamente, entre 4-6 dias após a inoculação, os grãos sofrem degradação de ácido nucleico, como visto pelo RNA total a partir de amostras sobre o tempo de curso-(Figura 2H, parte inferior). Vários estudos, incluindo a nossa, têm exaed F. verticilliodies infecção em grãos de milho 2,8,9,10,11,12 e utilizado com sucesso 7-13 dias como tempo-pontos para as observações. Utilizando os métodos aqui descritos, fumonisina níveis variam de 3,500-8,000 ng / g gama núcleo 4,13 e os níveis de ergosterol de 5.000-10.000 ng / g núcleo 14,13. Figura 3 mostra picos representativos para fumonisina (superior) e ergosterol (inferior ) de HPLC cromatogramas. Medição do ergosterol também pode ser realizada através de espectros de absorvância 15. <img alt="A Figura 1" src="/files/ftp_upload/3727/3727fig1.jpg" /> Figura 1. Fluxograma para a quantificação da biomassa fúngica, esporogênese, e produção de micotoxinas e os resultados representativos para o crescimento vegetativo de bioensaios de núcleo. A) Fluxograma delineando o método descrito para a quantificação de parâmetros biológicos fundamentais utilizados para avaliar patogénese do fungo sobre grãos de milho. B) Representanteresultados para bioensaios de kernel. Top, A. flavus (NRRL3357) crescimento vegetativo em grãos de milho no fundo B73 genética. As sementes foram inoculadas com 200 ul de 10 6 esporos / mL e fotografias foram tiradas 7 dias após a inoculação. Inferior, F. verticillioides inoculados em grãos B73 fundo de 13 dias após a infecção. Grãos foram inoculados com 200 ul de 10 6 esporos / mL. <img alt="A Figura 2" src="/files/ftp_upload/3727/3727fig2.jpg" /> Figura 2. Aspergillus flavus bioensaio kernel do tempo do curso. B73 núcleos foram inoculados com 200 ul de 10 6 esporos / ml de suspensão de Aspergillus flavus conídios e incubadas durante 2-8 dias (G). Todos os valores foram determinados a partir da média de peso seco de 4 grãos (n = 3-4; média ± SE). A) A colonização (com base no ergosterol), (B) aflatoxina, e (C) conidiação foram quantificados utilizando métodos descritos acima. E) A aflatoxina e (F) conidiação são exibidascomo uma função da biomassa fúngica como medido através h ergosterol. F) os valores máximos de ergosterol, a acumulação de aflatoxina, e conidiação como uma função da biomassa fúngica - valores máximos para cada quantidade observada foi definido como 100%. (H) 1 ug por pista de RNA total de tempo-curso. <img alt="A Figura 3" src="/files/ftp_upload/3727/3727fig3.jpg" /> Figura 3. Representativos Cromatografia Líquida de Alta Eficiência (HPLC) cromatogramas de fumonisina B1 (topo) e ergosterol (inferior) isolado a partir de grãos infectados com Fusarium verticillioides (M3125).

<ol>
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<p class="jove_content"&gt; Não tenho nada para revelar.</p

<p class="jove_content"&gt; Os métodos descritos aqui foram testados extensivamente e provou ser robusto na geração de resultados quantificáveis ​​para a colonização de fungos, esporogênese, e produção de micotoxinas. Além disso, estes métodos devem ser aplicável a sementes de outras espécies de plantas que são susceptíveis de contaminação com fungos micotoxigênico (amendoim, por exemplo, trigo, algodão, pistácios, etc.) Para competentes análises planta-patógeno de interação, é imperativo que as sementes se manter vivo. Um local da ferida pequena no lado do embrião do núcleo facilita a infecção enquanto se mantém a viabilidade das sementes.</p&gt;<p class="jove_content"&gt; Ensaios de kernel efectivamente administrada têm sido mostrados para os resultados paralelas a partir do campo. Em um estudo anterior<sup&gt; 4</sup&gt;, Bioensaios do kernel mostrou que<em&gt; Lox3-4 mutante</em&gt; Linhas foram mais suscetíveis à contaminação por aflatoxinas do tipo selvagem plantas. Esse resultado foi diretamente em linha com os resultados vistos em quatro experimentos de campo independentes<sup&gt; 4</sup&gt;. Isto sugere que os bioensaios de kernel podem ser ferramentas eficazes para determinar e / ou prever o resultado da planta-patógeno interações em larga escala configurações.</p

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Catalogo </td></tr><tr><td> Agar Batata Dextrose </td><td> Fisher Cientificos </td><td> S71659A </td></tr><tr><td> Tween-20 </td><td> Fisher Cientificos </td><td> BP337-100 </td></tr><tr><td> Recipiente plástico de incubação </td><td> Sterilite </td><td> 1713LAB06 </td></tr><tr><td> Liqüidificador </td><td> Vicam </td><td> 20200 </td></tr><tr><td> 24 centímetros filtro de pregas Papers </td><td> Vicam </td><td> 31240 </td></tr><tr><td> 1,5 mM microfibra de vidro </td><td> Vicam </td><td> 31955 </td></tr><tr><td> Afla coluna Teste </td><td> Vicam </td><td> G1024 </td></tr><tr><td> Afrla Desenvolvedor Teste </td><td> Vicam </td><td> 32010 </td></tr><tr><td> Metanol </td><td> Vicam </td><td> 35016 </td></tr><tr><td> Acetonitrila </td><td> Fisher Cientificos </td><td> AC14952-0025 </td></tr><tr><td> Etanol </td><td> Fisher Scientifc </td><td> AC39769-0025 </td></tr><tr><td> Coluna C-18 de extracção em fase sólida (SPE Prep setembro Coluna C18) </td><td> Fisher Cientificos </td><td> 60108-304 </td></tr><tr><td> O-ftalaldeído (OPA) </td><td> Sigma Chemical Co </td><td> 79.760-5g </td></tr><tr><td> Ácido bórico </td><td> Fisher Cientificos </td><td> BP168-500 </td></tr><tr><td> Borato de sódio </td><td> Fisher Cientificos </td><td> RDCS0330500 </td></tr><tr><td> Mercaptoetanol </td><td> Fisher Cientificos </td><td> 45-000-231 </td></tr><tr><td> Shimadzu HPLC LC-20AT (Pump) </td><td> Shimadzu Scientific Instruments, Inc. </td><td> LC-20AT </td></tr><tr><td> Coluna Zorbax ODS (4.6x150mm) </td><td> Agilent Technologies </td><td> 443905-902 </td></tr><tr><td> Shimatzu detector RF-10Axl fluorescência </td><td> Shimadzu Scientific Instruments, Inc. </td><td> RF-10AXL </td></tr><tr><td> Fosfato de sódio </td><td> Fisher Cientificos </td><td> AC38987-0010 </td></tr><tr><td> FB1 padrões </td><td> Sigma Chemical Co. </td><td> F1147-1mg </td></tr><tr><td> Clorofórmio </td><td> VWR </td><td> MK444410 </td></tr><tr><td> 13 mm com filtro de seringa de 0,45 membrana de nylon UM (HPLC) </td><td> Ciências da Vida Pall </td><td> 4426 </td></tr><tr><td> Ergosterol </td><td> Sigma-Aldrich </td><td> 45480-50G-F </td></tr><tr><td> Cintilação frascos </td><td> VWR </td><td> 66021-602 </td></tr><tr><td> Cloreto de Sódio </td><td> Vicam </td><td> G1124 </td></tr></tbody></table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

O apodrecimento de grãos por semente de fungos que infectam representa um dos maiores desafios econômicos à produção mundial de cereais, para não mencionar os riscos graves para a saúde humana e animal. Entre a produção de cereais, o milho é sem dúvida a cultura mais afetada, devido ao patógeno induzidas por perdas na integridade de grãos e sementes de contaminação por micotoxinas. As duas micotoxinas mais prevalentes e problemático para os produtores de milho e alimentos e processadores de alimentos são aflatoxina e fumonisina, produzida por Aspergillus flavus e Fusarium verticillioides, respectivamente. Recentes estudos moleculares em planta-patógeno interações têm demonstrado promessa em compreender os mecanismos específicos associados com a resposta das plantas à infecção por fungos e contaminação por micotoxinas 1,2,3,4,5,6. Porque muitos laboratórios são usando ensaios de kernel para estudar organismos patogénicos de plantas interacções, não há uma necessidade de um método padronizado para quantificar diferentes parâmetros biológicos, de modoresultados de diferentes laboratórios podem ser cross-interpretado. Para um meio robusto e reprodutível para análises quantitativas de sementes, temos desenvolvido ensaios em laboratório e os métodos do kernel posteriores a quantificar o crescimento de fungos, biomassa e contaminação com micotoxinas. Quatro grãos de milho esterilizados são inoculadas em frascos de vidro com a suspensão fúngica (10 6) e incubadas durante um período predeterminado. Frascos de amostra são então seleccionados para a enumeração de conídios por análise de biomassa hemocitómetro, ergosterol baseada por cromatografia líquida de alta eficiência (HPLC), a quantificação aflatoxina utilizando um método AflaTest fluorómetro, e quantificação de fumonisina por HPLC.

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

A devastação das culturas de cereais por fungos que infectam sementes de suscitou numerosos esforços de pesquisa para entender melhor planta-patógeno interações. Para estudar sementes de fungos interações em um ambiente de laboratório, nós desenvolvemos um método robusto para a quantificação de reprodução dos fungos, biomassa e contaminação por micotoxinas utilizando bioensaios de kernel.

Quantificação da colonização fúngica, esporogênese, e produção de micotoxinas Utilizando Bioensaios Kernel

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.0K visualizações.  Texas A&M University. A devastação das culturas de cereais por fungos que infectam sementes de suscitou numerosos esforços de pesquisa para entender melhor planta-patógeno interações. Para estudar sementes de fungos interações em um ambiente de laboratório, nós desenvolvemos um método robusto para a quantificação de reprodução dos fungos, biomassa e contaminação por micotoxinas utilizando bioensaios de kernel.

Vídeo: Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Colonização de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Imunologia e Infecção

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-célula Microscopia vídeo de Fagocitose patógeno fúngico

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterização de Respostas Inflamatórias Durante Colonização Intranasal com Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolamento, purificação e Rotulagem de neutrófilos da medula óssea do rato de Estudos funcionais e Experimentos de transferência adotiva

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Investigando os efeitos dos probióticos sobre pneumocócica Colonização Usando um<em&gt; In Vitro</em&gt; Adesão Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Avaliar a atividade anti-fúngica isolada de macrófagos alveolares por microscopia confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

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 ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

Histológica quantificação para determinar o pulmão carga fúngica em aspergilose Experimental

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...