Este trabalho foi financiado pelo Wellcome Trust concessão WT091939MA. Alain Filloux é apoiado pela Royal Society.

1. As cepas bacterianas e Culturas <ol><li> Um engenheiro Escherichia coli célula receptora (Prey, P), transformando (usando padrão de CaCl 2 tratamento ou eletroporação) 15 E. coli DH5a células com um plasmídeo que permita a complementação do gene lacZ (Tabela 1). Placa das células transformadas em placas de ágar Luria-Bertani (LBA, agar 1,5%) contendo 5-bromo-4-cloro-indolil-β-D-galactopiranosido (X-gal) a 40 mg / ml de concentração final e antibiótico apropriado. Incubar a 37 ° C durante a noite e na manhã seguinte para selecionar transformantes azul (ver § 1.3). </li><li> Crescer as células de Pseudomonas aeruginosa de dadores que são T6SS-activo (D +) ou inactivos T6SS-(D-) (Tabela 1) em LBA durante a noite a 37 ° C. No exemplo aqui apresentado foi utilizado um P. Rets aeruginosa estirpe possuindoum H1-T6SS constitutivamente activo 16 e um mutante isogênica com uma deleção do aglomerado de genes de H1 T6SS-(Tabela 1). </li><li> No dia seguinte, preparar uma cultura durante a noite de líquido no frasco gaseificado através da inoculação de um clone azul brilhante entre a E. Os transformantes de E. coli (P) a partir da placa, conforme descrito em § 1.1, em 5 ml de caldo de soja tríptica (TSB) suplementada com o antibiótico apropriado. Crescer sob agitação a 37 ° C. Proceder igualmente com uma única colónia de (D +) e de (D -) a partir da placa, conforme descrito em § 1.2. </li></ol> 2. Ensaio de competição <ol><li> Prepare placas LBA para o experimento dia seguinte. Certifique-se estas placas são bem seco (ou perto do bico de Bunsen ou em câmara de fluxo laminar). Prepara-se uma placa de &quot;Ensaio de entrada&quot; (A-Input) para as amostras (D +), (D -), e (P </strong&gt;). Prepara-se uma &quot;Ensaio de saída&quot; (A saída) da placa para as estirpes (D - + P) e (D + + P). Dividir e rotular as placas em conformidade. </li><li> Após crescimento durante a noite (ver § 1.3), medir a densidade óptica (DO 600 nm) da cultura bacteriana de entrada (D -, D + e P) e calcular o volume necessário para obter uma densidade celular equivalente a 1 unidade de OD 600nm de cada estirpe . Cada &quot;entrada&quot; cultura de células (D -, D + e P) é inicialmente coletado em um recipiente estéril tubos de 1,5 ml Eppendorf. </li><li> Centrifugar as amostras bacterianas a 13.000 rpm durante 1 min à temperatura ambiente e desprezar o sobrenadante. </li><li> Voltar a suspender as pílulas da D -, as culturas de D +, P e, em 100 ul de TSB fresco por pipetagem suave e inocular 10 ml de cada correspondente estirpe como uma única mancha no &quot;A entrada de&quot; prato (preparado em § 2.1). </li><li> Inocular o &quot;A saída de&quot; placa (preparado no § 2.1). Mais precisamente, misturar suavemente ul de 30 (D +) com 30 ul de (P) e 30 ul de (D -) com 30 ml (P) em dois tubos Eppendorf separados (note que as culturas utilizadas são as descritas em § 2.4) . Inocular 20 ul da mistura (D + / P) e (D - / P) como pontos individuais na &quot;A-saída&quot; placa. Permitir que os pontos de secar de um queimador de Bunsen próxima e colocar a placa numa incubadora a 37 ° C, durante um período adequado de tempo durante o qual a morte bacteriana está a ter lugar. No caso de P. aeruginosa, uma matança eficaz bacteriana é observada após 5 horas de incubação quando se compara um T6SS activas com uma estirpe T6SS defeituoso. </li></ol> 3. A observação qualitativa da tele Killing bacteriana <ol><li> Preparar as placas de LBA contendo 40 ug / ml de X-gal para a leitura do ensaio. As placas serão chamados de &quot;entrada de Leitura&quot; ou &quot;R-entrada&quot; para detectar bactérias isoladas ou &quot;saída de Leitura&quot; ou &quot;R-saída&quot; para detectar culturas mistas de bactérias e assim ler o desempenho de matar. Estas placas também devem ser bem seco. Divida o prato em quatro partes iguais em suas costas e anotar essas peças 0, 10 -1, 10 -2 e 10 -3, designando as diluições de cultura bacteriana de detectar as placas de ágar (ver § 3.3). A diluição permitirá uma avaliação semi-quantitativa da taxa de bactérias azul / branco dentro do mesmo local. </li><li> Recolher com uma agulha esterilizada os pontos individuais de bactérias do &quot;A entrada de&quot; placa (ver § 2.4) e &quot;A-saída&quot; placa (ver § 2.5) e ressuspender cada local em diferentes tubos de 1,5 ml Eppendorf contendo 1 ml de TSB. Coloque em um agitador Eppendorf bloco durante 30 minutos para voltar a suspender de forma eficiente obactérias. </li><li> Prepara-se uma série de 5 vezes 3 tubos de Eppendorf contendo 900 uL de TSB e prossiga para cada mancha ressuspenso a 10 diluições em série até 10 -3. Certifique-se de trocar a ponteira e vortex 5 segundos entre cada etapa de diluição. </li><li> Avance para a focagem de suas diluições bacterianas preparadas em § 3.3 em placas bem secas LBA contendo 40 ug / ml de X-gal, começando com o mais diluída à suspensão não diluída, tornando estes os &quot;R-&quot; placas de entrada para o &quot;A -entrada &quot;pontos e os&quot; R-saída &quot;placas para o&quot; A-saída &quot;pontos (Figura 2). Vortex brevemente cada tubo, antes de prosseguir para a mancha para manter uma suspensão homogénea bacteriana. Para a reprodutibilidade dos resultados, o ponto 20 ul, em triplicado, num quadrante (Figura 2). A secura relativa da sua placa é importante, nesta fase, uma vez que as manchas têm de permanecer separados individualmente na placa e não deslizam um contra o outro. A quantificação do ensaio de competição é também possível nesta fase do experimento. A seguir ao passo 3.3, espalhar 100 ul de uma diluição 10 -3 em placas LBA contendo 40 ug / ml de X-gal. Para a reprodutibilidade dos resultados, a uma cultura em placas em triplicado para cada ponto do &quot;A-saída&quot; placa. Colocar as placas de diluição de 37 ° C incubadora durante a noite (o que corresponde a 16 horas de incubação). Proceder à contagem das colônias azuis (as células P que permaneceram vivos). Os resultados típicos obtidos após 5 horas de competição são apresentados na Figura 3. </li><li> Deixar a placa aberta (sem tampa) dentro de uma zona estéril, para permitir a absorção do líquido em excesso, dentro de cada local. </li><li> Colocar a placa numa incubadora a 37 ° C durante a noite. Durante este período de incubação, a morte ocorre ainda na mistura (D + / P) a partir de ambas as estirpes estão ainda em contacto. </li><li> Tire uma fotoou digitalizar suas chapas para a análise de saída (Figura 2). Onde manchas permanecem em grande parte azul indica que E. coli não foi morto por P. aeruginosa. É esse o caso quando E. coli é misturado com um p-T6SS defeituoso estirpe aeruginosa (D-) (Figura 2, a placa na parte inferior direita). </li></ol>

<ol>
	<li>Filloux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterial+type+VI+secretion+machine:+yet+another+player+for+protein+transport+across+membranes.">The bacterial type VI secretion machine: yet another player for protein transport across membranes.</a> Microbiology. 154 (6), 1570 (2008).</li><li>Cascales, E., Cambillau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+of+type+VI+secretion+systems.">Structural biology of type VI secretion systems.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 367 (1592), 1102 (2012).</li><li>Leiman, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+T4+tail+and+tail+fibers.">Morphogenesis of the T4 tail and tail fibers.</a> Virol. J. 7, 355 (2010).</li><li>Ballister, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+self-assembly+of+tailorable+nanotubes+from+a+simple+protein+building+block.">In vitro self-assembly of tailorable nanotubes from a simple protein building block.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (10), 3733 (2008).</li><li>Leiman, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+VI+secretion+apparatus+and+phage+tail-associated+protein+complexes+share+a+common+evolutionary+origin.">Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (11), 4154 (2009).</li><li>Pukatzki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+VI+secretion+system+translocates+a+phage+tail+spike-like+protein+into+target+cells+where+it+cross-links+actin.">Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (39), 15508 (2007).</li><li>Ma, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+a+Vibrio+cholerae+type+VI+secretion+effector+requires+bacterial+endocytosis+by+host+cells.">Translocation of a Vibrio cholerae type VI secretion effector requires bacterial endocytosis by host cells.</a> Cell Host Microbe. 5 (3), 234 (2009).</li><li>Hood, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+type+VI+secretion+system+of+Pseudomonas+aeruginosa+targets+a+toxin+to+bacteria.">A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria.</a> Cell Host Microbe. 7 (1), 25 (2010).</li><li>Russell, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+VI+secretion+delivers+bacteriolytic+effectors+to+target+cells.">Type VI secretion delivers bacteriolytic effectors to target cells.</a> Nature. 475 (7356), 343 (2011).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+type+VI+secretion+effector+recognition+by+a+cognate+immunity+protein.">Structural basis for type VI secretion effector recognition by a cognate immunity protein.</a> PLoS Pathog. 8 (4), e1002613 (2012).</li><li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+anti-amoebae+and+anti-bacterial+activities+of+the+type+VI+secretion+system+in+Vibrio+cholerae.">Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae.</a> PLoS One. 6 (8), (2011).</li><li>Schwarz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burkholderia+type+VI+secretion+systems+have+distinct+roles+in+eukaryotic+and+bacterial+cell+interactions.">Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.</a> PLoS Pathog. 6 (8), e23876 (2010).</li><li>Murdoch, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+opportunistic+pathogen+Serratia+marcescens+utilizes+type+VI+secretion+to+target+bacterial+competitors.">The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors.</a> J. Bacteriol. 193 (21), 6057 (2011).</li><li>Boyer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+bacterial+type+VI+secretion+system+by+a+genome+wide+in+silico+analysis:+what+can+be+learned+from+available+microbial+genomic+resources?.">Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?.</a> BMC Genomics. 10, 104 (2009).</li><li>Maniatis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A Laboratory Manual. , (1982).</li><li>Mougous, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+virulence+locus+of+Pseudomonas+aeruginosa+encodes+a+protein+secretion+apparatus.">A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.</a> Science. 312 (5779), 1526 (2006).</li><li>Records, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+VI+secretion+system:+a+multipurpose+delivery+system+with+a+phage-like+machinery.">The type VI secretion system: a multipurpose delivery system with a phage-like machinery.</a> Mol. Plant Microbe Interact. 24 (7), 751 (2011).</li><li>Hachani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+VI+secretion+system+in+Pseudomonas+aeruginosa:+secretion+and+multimerization+of+VgrG+proteins.">Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins.</a> J. Biol. Chem. 286 (14), 12317 (2011).</li></ol>

Não temos nada a revelar.

O método apresentado neste artigo permite uma observação visual de T6SS mediada atividade bactericida / bacteriostase. O ensaio é efectuado sobre a superfície de uma placa de agar. Foi previamente demonstrado que T6SS dependente de ensaio realizado com matança cultura bacteriana mista líquido não é eficaz, provavelmente devido à falta de contacto constante entre as duas bactérias 8. O T6SS é acreditado para operar com um mecanismo semelhante ao utilizado por bacteriófagos para injectar o DNA em células-alvo 17. Na cultura líquido, a estrutura tubular do T6SS podem quebrar com mais facilidade, inter-bacteriana contato pode ser perdida, e as toxinas não são eficientemente entregue. Em termos de tempos de incubação, as 5 primeiras horas de contacto que descrevem entre a estirpe doadora e a presa são suficientes para observar a morte bacteriana entre P. aeruginosa e E. coli, tal como ilustrado na Figura 3 </strong&gt;. No entanto, é aconselhável ajustar o tempo de incubação através da realização de uma cinética de modo a optimizar as condições experimentais. Uma vez que este método é uma técnica baseada em cor, os resultados de saída pode ser comprometida pela pigmentação da estirpe doadora. Por exemplo, no caso do P. aeruginosa, algumas linhagens produzem níveis elevados de pigmentos de cor, tais como piocianina e pyoverdine, o que pode interferir com a leitura de ensaio, fazendo com que a distinção da presa relativamente difícil. Outros cromogénicos β-galactosidase substratos, tais como o magenta gal-gal ou o vermelho-, podem ser usados ​​em vez de X-gal (Tabela 1). O ensaio de competição pode fazer uso de outros genes repórter para a leitura. Por exemplo, o ensaio semelhante foi também realizada usando a proteína verde fluorescente marcado com presas 12. O nosso ensaio, ao passo que não quantitativas, dá uma boa indicaçãoda actividade T6SS uma vez que é baseada na sobrevivência ou a morte de uma presa repórter. Esta técnica apresenta a vantagem de ser fácil e conveniente para avaliar a actividade bactericida / bacteriostase de T6SSs de quaisquer espécies de bactérias. Até agora, a actividade do T6SS foi demonstrado contra bactérias Gram-negativas e nenhum exemplo claro de T6SS sensíveis bactérias Gram-positivas foi relatado ainda 12. É também óbvio que a incompatibilidade na cultura das diferentes espécies bacterianas para testar (por exemplo, temperatura de crescimento, oxigenação, meios específicos), deve ser considerado. O nosso ensaio pode também ser utilizado para avaliar quais dos componentes T6SS são absolutamente essenciais uma vez que mesmo vestígios de uma toxina secretada pode ser suficiente para matar a presa. Mesmo fraca actividade dos T6SS poderia então ser claramente detectado pelo nosso ensaio, em comparação com o padrão de secreção procedimento de teste dependente da T6SS blot utilizando sobrenadante de cultura e ocidentalanálise. No entanto, uma adequada colónia formadora de unidade de contagem (UFC) é ainda necessário para a quantificação exacta desta actividade T6SS.

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> P. aeruginosa PAKΔ RETS (D +) (T6SS ativos) </td><td> Tensão laboratório </td><td></td><td> Descrito na referência 16 </td></tr><tr><td> P. aeruginosa PAKΔ Rets Δ H1-T6SS (D -) (T6SS inativos) </td><td> Este estudo </td><td></td><td> O cluster H1 T6SS-(englobando os genes para PA0070 PA0095) foi eliminado por troca alélica seguindo o procedimento descrito na referência 18. O fragmento mutante foi gerado com o seguinte conjunto de iniciadores: Os primers dos fragmentos acima: 5&#39;-ATGGTCAACGACATGGAGCTGGAG-3 &#39;, e 5&#39;CGAGGCCGATCAGGCCTTCAGAACTGA-3 &#39;. Os primers de Down fragmento: 5&#39;-TCAGGCCTTCAGAACTGAAGCGGCGCA-3 &#39;, <pre> 5&#39;-GGTGGCGTTCAACAGTTCCATGTC-3 &#39; </pre></td></tr><tr><td> E. coli DH5a </td><td> Invitrogen </td><td> 18258-012 </td><td> F-φ80 lac ZΔM15 Δ (lac ZYA-arg F) U169 rec final A1 A1 HSD R17 (r k -, m k +) pho um sup E44 λ-thi -1 Gir A96 rel A1 </td></tr><tr><td> pBluescript II SK (+) </td><td> Agilent </td><td> 212205 </td><td> Este vector expressa o peptídeo α de β-galactosidase usado para α-complementação. </td></tr><tr><td> X-gal </td><td> Invitrogen </td><td> 15520-018 </td><td> Utilize a 40 ug / ml </td></tr><tr><td> Luria Bertani agar </td><td> Merck-químicos </td><td> 1.10283.0500 </td><td></td></tr><tr><td> TSB (caldo de caseína de soja) </td><td> Oxoid </td><td> CM109 </td><td></td&gt; </tr><tr><td> Vortex Genius agitador 3 </td><td> IKA </td><td> 3340000 </td><td></td></tr><tr><td> Scanner </td><td> Epson </td><td> V700 </td><td></td></tr><tr><td> Espectrofotômetro </td><td> WPA Biowave </td><td> CO8000 medidor de densidade celular </td><td></td></tr><tr><td> Magenta-gal </td><td> Bioworld </td><td> 30350001-1 (715241) </td><td></td></tr><tr><td> Red-gal </td><td> Research Organics </td><td> 1364c </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Strain, material, plasmídeo e do reagente utilizado. </td></tr></tbody></table>

a visual assay to monitor t6ss-mediated bacterial competition

Sistemas de secreção de tipo VI (T6SSs) são nanomáquinas moleculares que permitem bactérias Gram-negativas e para o transporte de proteínas para injectar uma ampla variedade de células de 1,2-alvo. O T6SS é composta por 13 componentes do núcleo e exibe semelhanças estruturais com a cauda de bacteriófagos tubo 3. O fago usa um tubo e um dispositivo de puncionar para penetrar o envelope celular das bactérias alvo e injectar o DNA. Propõe-se que o T6SS é um dispositivo de bacteriófago invertido criação de um caminho específico no envelope da célula bacteriana para dirigir efectores e toxinas para a superfície. O processo pode ser levado mais longe e o dispositivo pode perfurar T6SS outras células com as quais a bactéria estiver em contacto, assim injectar os efectores para estes alvos. O tubo de cauda e partes do dispositivo de punção dos T6SS são feitos com Hcp e proteínas VgrG, respectivamente 4,5. A versatilidade do T6SS tem sido demonstrada através datudies usando vários patógenos bacterianos. Os T6SS Vibrio cholerae pode remodelar o citoesqueleto das células hospedeiras eucarióticas injectando uma &quot;evoluiu&quot; VgrG carregando uma actina C-terminal de ligação cruzada de domínio 6,7. Outro exemplo notável foi recentemente documentado usando Pseudomonas aeruginosa, que é capaz de atacar e eliminar as bactérias de uma maneira dependente da T6SS, promovendo assim o estabelecimento de bactérias em nichos microbianos específicos e ambiente competitivo 8,9,10. Neste último caso, três T6SS-secretadas proteínas, ou seja, Tse1, Tse2 Tse3 e têm sido identificados como as toxinas injectados na bactéria alvo (figura 1). A célula do dador é protegido contra o efeito prejudicial destes efectores por meio de um mecanismo de anti-toxina, mediados pelas proteínas Tsi1, Tsi2 e Tsi3 imunidade 8,9,10. Esta actividade anti-microbiana pode ser monitorizado quando T6SS-proficientes bactérias são co-cultivados em sOlid superfícies em competição com outras espécies bacterianas ou com T6SS-inactivos bactérias da mesma espécie 8,11,12,13. Os dados disponíveis enfatizou uma abordagem numérica para o ensaio de competição de bactérias, incluindo demorada contagem CFU que depende em grande parte responsáveis ​​antibióticas. No caso de estirpes resistentes aos antibióticos, como P. aeruginosa, estes métodos podem ser inadequadas. Além disso, com a identificação de cerca de 200 diferentes loci T6SS em mais de 100 genomas bacterianos 14, uma ferramenta de rastreio conveniente é altamente desejável. Foi desenvolvido um ensaio que é fácil de utilizar e requer materiais e reagentes de laboratório padrão. O método oferece uma técnica rápida e qualitativa para monitorizar a actividade bactericida dependente do T6SS / bacteriostase usando uma estirpe repórter como presa (neste caso, a Escherichia coli DH5a), permitindo uma complementação do gene lacZ. No geral, este método é graphic e permite a rápida identificação de fenótipos relacionados T6SS em placas de agar. Este protocolo experimental pode ser adaptado para outras estirpes ou espécies bacterianas, tomando em consideração as condições específicas, tais como meios de crescimento, a temperatura ou o tempo de contacto.

Os resultados típicos são mostrados na Figura 1 com as estirpes e os reagentes descritos na Tabela 1. As placas mostradas nesta figura foram verificados após uma incubação de um dia para o outro. Os &quot;Leitura-Input&quot; placas mostram um padrão de diluição em série para as estirpes utilizadas neste ensaio. Como esperado, a E. coli rapina pontos (P) com superexpressão do gene lacZ azul aparecem no meio suplementado com X-gal, enquanto que a P. doador aeruginosa (D +, T6SS ativa) e (D -, inativos T6SS) permanecem branco. Os &quot;Leitura-saída&quot; placas em que a mistura entre a presa e uma estirpe T6SS ativa (D + / P) foi flagrada mostram o desaparecimento do azul presa, assim, indicando que foi morto. Isto demonstra a capacidade do doador para outcompete a presa. A persistência da cor azul na (D - / P placa) demonstra a incapacidade de um doador T6SS inativo para matar a presa azul. <img src="/files/ftp_upload/50103/50103fig1.jpg" alt="Figura 1"/> Figura 1. Matança de E. coli por T6SS-proficiente P. aeruginosa. P. aeruginosa injeta toxinas no E. coli célula alvo de uma forma dependente da T6SS (indicado pela seta branca). Duas toxinas Tse1 e Tse3 (laranja e círculos vermelhos) são injetados na E. periplasma coli e degradar o peptidoglicano 9. A toxina Tse2 (círculo amarelo) é injetado no E. coli citoplasma e tem uma atividade bacteriostática 8,10. A ação combinada das toxinas mata as células-alvo (relâmpago e crânio). A sobrevivência de células alvo pode ser detectada por monitorização da actividade de a-galactosidase produzida β (ver também a Figura 2). P. umeruginosa é protegida contra a actividade das toxinas pelas proteínas de imunidade Tsi1, Tsi2 e Tsi3 (laranja, quadrados amarelos e vermelhos, respectivamente) 8,9,10. <img src="/files/ftp_upload/50103/50103fig2.jpg" alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/50103/50103fig2highres.jpg"/> Figura 2. Ensaio de placa de agar para monitorar T6SS dependente de morte bacteriana Nesta figura são mostradas na parte superior do &quot;input&quot; Leitura placas constituídas por diluições em série do D -., D + e células de entrada P. As células de entrada P são azuis devido ao α-complementação do gene lacZ e assim produzido β-galactosidase que cliva X-gal. Na parte mais inferior é mostrada a &quot;Leitura-Output&quot; placas constituídas por uma série de diluições da mistura de bactérias entre um activo (D + / P) </strong&gt;, ou um inactivo (D - / P), T6SS doador P. aeruginosa com a estirpe de E. coli presa. <a href="https://www.jove.com/files/ftp_upload/50103/50103fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img src="/files/ftp_upload/50103/50103fig3.jpg" alt="Figura 3"/> Figura 3. Quantificação da morte de E. coli por P. aeruginosa, após 5 horas de incubação. O gráfico apresenta a contagem de CFU de E. coli descrito no passo 3.4. Os resultados aqui apresentados mostram uma diferença de 3 vezes entre o T6SS + e as T6SS-estirpes, sugerindo que a maior parte da morte tem lugar durante as 5 primeiras horas de contacto.

Watch this Scientific Journal Video about A Visual Assay to Monitor T6SS-mediated Bacterial Competition at JoVE.com

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Descreve-se um ensaio qualitativo para monitorar competição bacteriana mediada pela<em&gt; Pseudomonas aeruginosa</em&gt; Tipo de sistema de secreção VI (T6SS). O ensaio baseia-se na sobrevivência / morte de células de Escherichia coli que transportam um alvo<em&gt; LacZ</em&gt; Repórter. Esta técnica é ajustável para avaliar a actividade bactericida / bacteriostase de T6SS-proficientes microorganismos.

Um Ensaio visual para o Monitor T6SS mediada Competição bacteriana

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

a-visual-assay-to-monitor-t6ss-mediated-bacterial-competition

Research

JoVE Journal

Immunology and Infection

15.5K visualizações.  Imperial College London. Descreve-se um ensaio qualitativo para monitorar competição bacteriana mediada pela Pseudomonas aeruginosa Tipo de sistema de secreção VI (T6SS). O ensaio baseia-se na sobrevivência / morte de células de Escherichia coli que transportam um alvo LacZ Repórter. Esta técnica é ajustável para avaliar a actividade bactericida / bacteriostase de T6SS-proficientes microorganismos.

Vídeo: A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Using Luciferase to Image Bacterial Infections in Mice

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

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

Usando a luciferase Infecções bacterianas Imagem em camundongos

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

Imunologia e Infecção

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

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

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

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

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

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

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Usando a sobreposição de ensaio para medir qualitativamente produção bacteriana e sensibilidade para pneumocócica Bacteriocinas

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Crescimento Pairwise competição ensaio para determinar a replicação de Fitness de Imunodeficiência vírus humanos

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Aplicação de longo prazo cultivadas Interferon-γ Enzyme-linked Immunospot Ensaio para a Avaliação e efetoras de memória T respostas de células em gado

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Geração e Multi-fenotípica rastreio de alto teor de<em&gt; Coxiella burnetii</em&gt; mutantes por transposões

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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.

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 ...

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 ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Um do elevado-throughput Shigella-ensaio bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Ensaio de matar Opsonophagocytic para avaliar as respostas imunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...