O Projeto descrito é suportado pelo Prêmio Número 5R01AI116895 do Instituto Nacional de Alergia e Doenças Infecciosas. O conteúdo é da exclusiva responsabilidade dos autores e não representam necessariamente as opiniões oficiais do Instituto Nacional de Alergia e Doenças Infecciosas ou o National Institutes of Health.

1. As amostras de Geração <ol><li> Calor ativar C. difficile esporos a 65 ° C durante 30 min e armazenar em gelo. Nota: C. esporos difficile pode ser produzida e purificada tal como descrito anteriormente 7,9,10. </li><li> Preparar a solução de germinação a X + 1 ml onde X é o número de amostras a serem tomadas durante o ensaio. Nota: A solução de germinação é específico para as espécies de bactérias esporos sendo estudado. Para ensaiar C. difficile a germinação de esporos, foi utilizada uma solução de Tris 10 mM (pH 7,5), NaCl 150 mM, glicina 100 mM e 10 mM de ácido taurocólico em água desionizada. </li><li> Antes de iniciar o ensaio, retirar uma amostra da solução de 1,0 ml sem germinação de esporos para servir como uma estrutura para a detecção fragmento córtex. </li><li> Adicionar esporos a um OD 600 de ~ 3.0 para a solução de germinação e vortex rapidamente. Nota: Para os nossos estudos de germinação em B. subtilis, foi utilizada a mesma OD 600 esporos como por C. difficile. Nota: Esta quantidade de esporos funcionou bem para monitoramento córtex liberação fragmento durante C. germinação de esporos difficile. A quantidade de esporos utilizada neste ensaio devem ser determinadas experimentalmente para cada bactéria ou estirpe. </li><li> Remover uma amostra de 1,2 ml, imediatamente após adição dos esporos e centrifugação à temperatura ambiente durante 1 min a 14.000 x g. Este é o ponto de tempo zero. </li><li> Transferir 1,0 ml do sobrenadante a partir da amostra centrifugada para um tubo fresco para a análise de fragmentos de córtex posterior. Nota: Se DPA necessitam de ser monitorizados, remover uma amostra de 100 ul em separado para a detecção de DPA na solução utilizando um ensaio baseado em fluorescência térbio 9,18,19. </li><li> Congelar a amostra recolhida a -80 ° C. </li><li> Repita os passos 1.4 e 1.5, em intervalos regulares, até que todos os pontos de tempo completo. Nota: Devido ao tempo necessário para a centrifugação e amostragem, momentos menos thum 2 min apart não fosse possível durante o nosso procedimento, para que os nossos intervalos de amostragem eram uma vez a cada 2 min para 14 min. </li><li> Como um controlo positivo para reduzir a detecção e quantificação de açúcar, incluem as seguintes quantidades de N-acetilglicosamina (nmol): 0, 12,5, 25, 50, 100, 250, 500, e 5000. Preparar estas amostras ao mesmo tempo como amostras de córtex e execute, juntamente com as amostras de córtex de modo que a curva padrão é relevante para os dados gerados pela germinação de esporos. </li><li> Depois de todas as amostras ponto de tempo são recolhidas, liofilizar as amostras. Nota: Nós liofilizado nossas amostras em tubos de 2 ml com tampa de rosca. </li></ol> 2. Medir Cortex Fragments <ol><li> Ressuspender as amostras liofilizadas em 120 mL de HCl 3 N suplementado com 1% de fenol e 0,5% β-mercaptoetanol. </li><li> Transferir as amostras em suspensão para 2 ml tubos com tampa de rosca. Nota: Para reprodutibilidade, assegurar toda a amostra liofilizada ï¿½ressuspenso e transferidos. </li> &lt;li&gt; Incubar as amostras num banho de água em recirculação, a 95 ° C durante 4 h. </li><li> Após a incubação, colocar as amostras em gelo até que eles são legais. </li><li> Neutraliza-se a amostras com 120 uL de NaOH 3 M. </li><li> Adicionar 80 ul de uma solução saturada de bicarbonato de sódio e 80 mL de uma solução de anidrido acético a 5% a cada amostra e vortex. </li><li> Incubam-se as amostras à temperatura ambiente durante 10 min. </li><li> Transferir as amostras de volta para o banho de água C 95 ° para exatamente 3 min. Nota: Esta etapa é hora vezes sensíveis e mais longas podem afetar o desenvolvimento do sinal a jusante. </li><li> Retirar amostras a partir do banho de água e arrefece-se em gelo. </li><li> Adicionar 400 uL de 6,54% K 2 B 4 O 7 · 4H 2 O para cada tubo e vortex. </li><li> Incubam-se as amostras num banho de água em recirculação, a 95 ° C durante exactamente 7 min. Nota: Esta etapa é hora vezes sensíveis e mais longas podem afetar o desenvolvimento do sinal a jusante. </li><li> Removeras amostras e colocar em gelo durante 5 min. </li></ol> 3. Cor Reagente <ol><li> Enquanto as amostras estão no gelo (Passo 2,12), preparar o reagente de cor. Dissolve-se 0,320 g de p -dimethylaminobenzaldehyde (DMAB; reagente de Ehrlich) em 1,9 ml de ácido acético glacial. Nota: O reagente é o tempo, temperatura e sensível à luz e não devem ser feitas com antecedência. </li><li> Após o DMAB se dissolva completamente, adicionar 100 mL de HCl 10 N e vortex. </li><li> Adicionar 5 ml de ácido acético glacial e agitar com vortex. </li></ol> 4. reacção colorimétrica <ol><li> Transferir 100 uL de cada amostra arrefecida córtex para um novo tubo de microcentrífuga de 1,5 ml. </li><li> Adicionar 700 ul da solução de cor recém-preparadas para cada amostra. </li><li> transferir imediatamente as amostras para um banho de água C 37 ° e incubar por exatamente 20 min. </li><li> Transferir 200 ul de cada amostra para uma placa de 96 poços clara. </li><li> Quantificar as amostras a 585 nm em um pratoleitor. As amostras devem começar em uma cor amarela e uma reacção positiva se deslocará para roxo. Quanto mais córtex presente, quanto mais profunda a cor roxa. </li></ol>

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The authors have nothing to disclose.

Após estimulação, os esporos submetidos ao processo de germinação perder as suas propriedades de resistência, sem a necessidade de síntese macromolecular. Quando a germinação de esporos é acionado, o núcleo de esporos libera DPA em troca de água 2. Devido ao elevado teor de DPA do esporo dormente, o núcleo é de esporos sob intensa pressão osmótica e o peptidoglicano especializado, córtex, ajuda a impedir que o núcleo de inchaço, agindo, presumivelmente, como uma barreira para a expansão 2. O método acima descrito utiliza reagente de Ehrlich (uma solução amarela) para detectar a presença de açúcares redutores como uma mudança mensurável para uma tonalidade púrpura. Durante a hidrólise córtex, NAG e NAM açúcares são libertados (dependendo do grau de hidrolisar enzimas, os açúcares podem ser libertados como dissacáridos ou polissacáridos). A hidrólise das ligações glicosídicas, por HCl (Passo 2.1) gera monossacáridos com extremidades redutoras livres 7. após neutralizção do ácido com uma quantidade equivalente de hidróxido de sódio (Passo 2.5), os álcoois em torno da NAG e monossacarídeos NAM são acilado com anidrido acético (Passo 2.6) 13. Subsequentemente, o anidrido acético que não reagiu é convertido em ácido acético por aquecimento da amostra (Passo 2.8). Como hipótese por Morgan e Elson 20, o aldeído terminal de converte-se espontaneamente para a forma enol. Sob condições alcalinas (passo 2.10), esta forma de enol pode reagir com o acetil adjacente para gerar um derivado de oxazol, que reage com o reagente de Ehrlich (Passo 4.3) para gerar uma cor púrpura 15,20. Este método descrito acima produz resultados consistentes e fiáveis ​​para a detecção de fragmentos de córtex de germinação de esporos, tanto B. subtilis e C. difficile 9,16,17. O ensaio requer uma atenção especial para garantir a reação vezes, temperaturas e procedimentos são realizados com precisão. Sem a devida atençãoaos detalhes, é fácil para as condições da reacção a falhar ou não reproduzir entre experiências 13. No entanto, a inclusão de um padrão NAG SIDA na aplicação do ensaio entre ambientes laboratoriais. A norma de controlo positivo NAG incluídos em cada controlos experimentais para qualquer variabilidade inter-experimental (por exemplo, pequenas variações de tempos de reação ou a eficácia do reagente de cor). Reações usando apenas condições tampão e reações com esporos incapazes de hidrolisar córtex ambos produziram nenhuma mudança na cor. O problema mais comum com este método é a variabilidade inter-experimental ou completa falta de sinal. Para controlar a variabilidade, as preparações de esporos devem ser livres de quaisquer detritos célula vegetativa. Este detritos celulares irá conter fragmentos de peptidoglicano (NAG e NAM) e resultará em um sinal de alta fundo para os esporos germinam. Para garantir uma preparação de esporos pura (&gt; 99,9% esporos), referem-se à literature de métodos para preparar as suspensões de esporos puros no organismo a ser estudado 7,9,21-23. Se o ensaio não está rendendo qualquer sinal detectável, observou-se que a etapa de liofilização pode introduzir variabilidade. Devido à força com que o vácuo é liberado, observou-se que o pó de amostra seca pode ser soprado para fora do tubo de amostra. Para reduzir / evitar que isso ocorra, use uma agulha de seringa pequena para picar furos nas tampas de tubos de amostra. A pequena área de os furos introduzidos irá reduzir a probabilidade de a amostra liofilizada (agora na forma de pó) seja perturbada. Finalmente, como observado nos métodos acima, os vários passos de incubação são muito sensíveis ao tempo. Certifique-se de que todas as etapas cronometradas são monitorados com cuidado. Uma consideração importante para este ensaio é que os fragmentos de córtex deve ser libertado a partir da germinação de esporos, de modo a ser detectado. Se o córtex é hidrolisado em grandes fragmentos, estes fragmentos podem ser demasiado grande para ser released através do revestimento de esporos. Embora uma consideração importante ao solucionar um experimento que não deu um sinal, recomendamos confirmar que as condições usado para germinar a maioria dos esporos na solução e as outras opções de resolução de problemas listados aqui antes de se considerar o tamanho do fragmento córtex como um problema potencial no o ensaio. Nem todos os tampões de germinação / condições são adequadas para este ensaio. Uma vez que este ensaio detecta a presença de açúcares redutores libertados durante o processo de germinação 13, qualquer tampão de germinação que contém açúcares redutores ou qualquer germinativo que é, em si, um açúcar redutor (por exemplo, glicose) irá resultar num sinal forte neste ensaio. Se este for o caso, a cromatografia líquida de alta pressão (HPLC) pode ser usado para detectar a presença de fragmentos de córtex 24-26. No entanto, porque nem todos os laboratórios estão equipados com tais instrumentos, o método aqui descrito pode ser utilizado pela maiorialaboratórios e oferece resultados simples e facilmente quantificáveis. Germinação de esporos de bactérias tem sido amplamente estudado em Bacillus e algumas espécies de Clostridium 2,8. O processo geralmente é conservada, no entanto, existem diferenças. Importante, nem todos os organismos germinar usando mecanismos de germinação de esporos, como observado no modelo de esporos formadores de 9,27. Porque a germinação está sendo estudado em mais diversos organismos, o ensaio descrito acima pode ser aplicada a estes organismos para detectar fragmentos de córtex durante a germinação dos esporos. Ao aplicar este método para estes organismos recém-estudados, os pesquisadores podem determinar o papel proteínas diferentes germinação jogar na hidrólise córtex.

Endospores são metabolicamente formas latentes de bactérias que permitem que as bactérias para persistir em ambientes desfavoráveis. Em muitas bactérias formadoras de esporos, a formação de esporos é induzida pela privação de nutrientes, mas este processo pode ser controlada por alterações de pH, exposição ao oxigénio ou outros stresses 1. Embora em sua forma de esporos metabolicamente dormente, as bactérias resistir à radiação UV, a dessecação, temperaturas mais elevadas e que congela 2. A maior parte do conhecimento sobre o processo de esporulação vem de estudos no organismo modelo, Bacillus subtilis. O processo de esporulação começa com a replicação do ADN e a formação de um filamento de 3,4 axial. Um septo assimétrica, em seguida, divide a célula em dois compartimentos de tamanho desigual. O compartimento maior, a célula mãe, engolfa o compartimento mais pequeno, o forespore. Tanto a célula mãe e forespore coordenar a expressão do gene para amadurecer o esporo e a célula mãe, eventualmente, lisa, liberando o Dormant de esporos para o ambiente circundante um. A estrutura e a composição de esporos é conservada entre várias espécies bacterianas. O núcleo de esporos tem um teor de água inferior a célula vegetativa e é rica em ácido dipicolínico (DPA) 1,2. Durante a formação de esporos, DPA é bombeado para dentro do núcleo de esporos em troca de água. Em torno do núcleo é uma membrana de esporos interior, onde, na maioria das bactérias formadoras de esporos, os receptores que reconhecem as pequenas moléculas que estimulam a germinação (germinants) estão localizados a 2. Localizado no exterior membrana interna do esporo é uma camada de peptidoglicano da parede celular. Uma camada de peptidoglicano especializado (córtex) rodeia o peptidoglicano da parede celular e é composta de muitos dos mesmos componentes como peptidoglicano da parede celular [alternando N-acetilglucosamina (NAG) e ácido N-acetilmurâmico (NAM)]. No entanto, aproximadamente 50% dos resíduos de NAM no córtex foram convertidos para murâmico-δ-lactama 5,6 </s-se&gt;. Durante a germinação de esporos, estes resíduos murâmico-δ-lactâmicos são reconhecidos pelos esporos do córtex enzimas líticas (SCLEs), permitindo assim que o córtex para ser degradada (um processo essencial para completar a germinação) mas não a parede celular. Rodeando o córtex é uma membrana exterior e várias camadas de proteínas de revestimento 2. Controlando o processo de germinação é criticamente importante para as bactérias formadoras de esporos. A germinação é iniciada quando os receptores germinativa interagir com seus respectivos germinants 2. Em muitas bactérias formadoras de esporos, estes são germinants amino ácidos, açúcares, nucleótidos, iões, ou combinações destes dois. C. difficile germinação de esporos é iniciada por meio de combinações de certos ácidos biliares, [por exemplo, ácido taurocólico (TA)] e aminoácidos (por exemplo, glicina) 7-10. Embora existam diferenças entre o C. via de germinação difficile e as vias estudadas em outra forma de esporobactérias, tais como B. subtilis, comum a todos é a exigência absoluta para degradar o córtex esporos para permitir que uma célula vegetativa a crescer a partir do esporos germinados 2,8. Córtex degradação pode ser conseguida pela SCLEs CwlJ / SleB (como encontrado em B. subtilis) ou SLEC (como encontrado em muitos Clostridia). hidrólise córtex reduz a restrição na parede da célula e esporo. Isto permite uma re-hidratação completa do núcleo, um passo necessário para a reactivação muitas das proteínas necessárias para o metabolismo celular 2. Quando esporos germinam, as mudanças de esporos a partir de um estado dormente de fase brilhante para um estado de fase-escuro e este processo pode ser medido por uma alteração na densidade óptica (DO) a 600 nm 11. Um relatório anterior sugere que grande parte dessa mudança de OD é devido à liberação da DPA do esporo 12. Durante a nossa recente estudo, buscou-se comparar o tempo de C. difficile germinação de esporos e acompanhou olançamento do DPA e córtex fragmentos 9. Para este estudo foi fundamental para monitorar o lançamento de fragmentos de córtex, quando começaram a ser lançado pela germinação de esporos. O ensaio colorimétrico aqui utilizado baseou-se num método para a detecção de açúcares com extremidades redutoras desenvolvido Ghuysen et ai. 13. Porque os outros têm descrito protocolos para a detecção de açúcares redutores 14 ou modificou os protocolos 15, a literatura sobre este assunto pode ser confuso. Aqui, nós detalhe um método passo-a-passo para a detecção colorimétrica de açúcares redutores libertados de germinação C. esporos difficile. Embora este estudo utiliza C. esporos difficile, os açúcares redutores libertados durante a germinação de esporos a partir de outras bactérias formadoras de esporos são capazes de serem detectadas com este protocolo 9,16,17.

<table><tbody><tr><td>2.0 ml screw cap tube</td><td>USA scientific</td><td>1420-3700</td><td></td></tr><tr><td>p1000 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>p200 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>p20 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>100 - 1,250 &amp;#956;l pipet tips</td><td>VWR</td><td>89079-486</td><td></td></tr><tr><td>1 - 200 &amp;#956;ll pipet tips</td><td>VWR</td><td>89079-458</td><td></td></tr><tr><td>N-acetyl-D-glucosamine</td><td>Sigma</td><td>A3286-25G</td><td></td></tr><tr><td>Hydrochloric Acid - 10 N</td><td>BDH-Aristar</td><td>BDH3032-3.8LP</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Alfa Aesar</td><td>L04295</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>BDH</td><td>BDH0280-500G</td><td></td></tr><tr><td>Potassium Tetraborate tetrahydrate</td><td>Alfa Aesar</td><td>39435</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>BDH</td><td>BDH8019-500G</td><td></td></tr><tr><td>4-(Dimethylamino)benzaldehyde</td><td>Sigma</td><td>156477-100G</td><td></td></tr><tr><td>Saturated Phenol</td><td>Fisher</td><td>BP1750-400</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Aldrich</td><td>M6250-100ML</td><td></td></tr><tr><td>SpectraMax M3</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Acetic Acid, Glacial</td><td>BDH</td><td>BDH3098-3.8LP</td><td></td></tr><tr><td>Heated, Circulating Water Bath</td><td>VWR Scientific</td><td>Model 1136</td><td></td></tr><tr><td>Microtest 96</td><td>Falcon</td><td>353072</td><td>96 well clear tissue culture plates</td></tr><tr><td>Culture tubes</td><td>VWR</td><td>89000-506</td><td></td></tr><tr><td>Lyophilizer</td><td></td><td></td><td></td></tr><tr><td>Heat Blocks</td><td></td><td></td><td></td></tr><tr><td>Vortex Machine</td><td></td><td></td><td></td></tr></tbody></table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

A Tabela 1 mostra os resultados típicos usando padrões de NAG. Os dados são utilizados para gerar uma curva padrão. A Tabela 2 mostra os resultados típicos de um ensaio de germinação de germinação em tampão suplementado com 100 mM de glicina e TA 10 mM (condições de promoção da germinação) ou de glicina 100 mM apenas (condições não-germinação). Na ausência de TA, C. difficile esporos germinam e não há pouca alteração na presença de fragmentos do córtex na solução. No entanto, na presença de ambos TA e glicina, C. difficile esporos germinam e libertar os fragmentos do córtex para dentro da solução. Isto é observado como um aumento na OD a 585 ao longo do tempo das amostras que reagiram. A quantidade total de fragmentos de córtex (nmol) liberados durante este ensaio foi calculada utilizando uma curva padrão semelhante ao da Figura 1B. Neste ensaio, a maior parte do córtex é hidrolisado (ou solta) por 15 minutos pós-exposição germinativo, sugerindo que a maior parte dos esporos germinaram neste período de tempo (Figura 2; de acordo com uma publicação anterior 7). Depois de desenvolver as amostras no reagente de cor, a maioria das amostras podem não mostrar a cor púrpura a olho nu. A Figura 1 mostra uma amostra representativa produzida a partir de N-acetilglucosamina. Muitas das amostras não mostram uma cor púrpura visível. No entanto, os 250 nmol, 500 nmol e 5.000 nmol amostras aparecer visualmente diferente do controlo negativo (0 nmol). Quando medida a OD 585 nm, o sinal gerado melhor se adapta uma regressão linear (R2 = 0,999). <img alt="figura 1" src="/files/ftp_upload/54146/54146fig1.jpg" /> Figura 1:. Detecção N-acetilglucosamina amostras (A) N-acetilglucosamina (0 nmol, 12,5 nmol,25 nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, 5,000 nmol) foram liofilizadas e feito reagir de acordo com o procedimento descrito. (B) As amostras foram adicionadas a uma placa de 96 cavidades e a quantidade de material reagido detectado a 585 nm. O sinal foi usado para gerar uma curva padrão. <img alt="Figura 2" src="/files/ftp_upload/54146/54146fig2.jpg" /> Figura 2:. Representação gráfica dos resultados de germinação O córtex nmol calculado na Tabela 2 foi representada graficamente em função do tempo. (●) mM de glicina + TA mM 100 10; (■) glicina 100 mM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54146/54146fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> NAG Concentração (nmol) </td><td> OD 585 </td></tr><tr><td> 0 </td><td> 0,047 </td></tr><tr><td> 12,5</td><td> 0,054 </td></tr><tr><td> 25 </td><td> 0,066 </td></tr><tr><td> 50 </td><td> 0,084 </td></tr><tr><td> 100 </td><td> 0,128 </td></tr><tr><td> 250 </td><td> 0,285 </td></tr><tr><td> 500 </td><td> 0,556 </td></tr><tr><td> 5.000 </td><td> 4.0 </td></tr><tr><td> = em branco </td><td> 0,046 </td></tr></tbody></table> Quadro 1: Padrão de NAG. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="4"> Clostridium difficile germinação de esporos </td></tr><tr><td></td><td colspan="4"> Solução de Germinação (Tris 10 mM (pH 7,5), NaCl 150 mM) </td></tr><tr><td></td><td colspan="2"> 100 mM de glicina a 10 mM + TA </td><td colspan="2"> 100 mM de glicina </td></tr><tr><td> Tempo (minutos) </td><td> OD 585 </td><td>Calculado (nmol) </td><td> OD 585 </td><td> Calculado (nmol) </td></tr><tr><td> 0 </td><td> 0,046 </td><td> 0.00 </td><td> 0,047 </td><td> 0.00 </td></tr><tr><td> 2 </td><td> 0,048 </td><td> 2.54 </td><td> 0,048 </td><td> 1,27 </td></tr><tr><td> 4 </td><td> 0,05 </td><td> 5,07 </td><td> 0,049 </td><td> 2.54 </td></tr><tr><td> 6 </td><td> 0,055 </td><td> 11,41 </td><td> 0,05 </td><td> 3.80 </td></tr><tr><td> 8 </td><td> 0,059 </td><td> 16,49 </td><td> 0,05 </td><td> 3.80 </td></tr><tr><td> 10 </td><td> 0,06 </td><td> 17,76 </td><td> 0,051 </td><td> 5,07 </td></tr><tr><td> 12 </td><td> 0,061 </td><td> 19.02 </td><td> 0,05 </td><td> 3.80 </td></tr><tr><td> 14 </td><td> 0,063 </td><td> 21,56 </td><td> 0,051 </td><td> 5,07 </td></tr></tbody></table> Tabela 2: Exemplo de Spore Germinatiem resultados.

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Detecção de Cortex Fragmentos Durante bacteriana germinação de esporos

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

9.3K visualizações.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Vídeo: Detecting Cortex Fragments During Bacterial Spore Germination

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

O isolamento físico do Endospores de amostras ambientais por alvejado Lise de células vegetativas

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

Ambiente

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Detecção de bactérias utilizando DNAzymes fluorogénicas

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Imunologia e Infecção

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

Aprimoramento da Enumeração de Esporos de Bacillus subtilis e Análise de Marcação em Citometria de Fluxo

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

Bioquímica

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Visualização do Germinosomes e a membrana interna em esporos de Bacillus subtilis

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Bioengenharia

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Avaliação visual e microscópica de Streptomyces mutantes do desenvolvimento

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

Desenvolvimento Sexual e descarga de ascósporos em Fusarium graminearum</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Biologia

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

Adsorção de esporos, como um sistema de exibição Nonrecombinant por enzimas e antígenos

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

Usando Coculture detectar quimicamente mediada interespécies Interações

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigando os prejudiciais efeitos de baixa pressão Plasma esterilização na sobrevivência de esporos de Bacillus subtilis usando Live celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Investigando a germinação de Teliospore usando a análise de Microrespiration e Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...