Les auteurs tiennent à remercier les anciens membres du laboratoire de Goodrich-Blair: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard, et Youngjin parc pour leurs contributions à l&#39;élaboration de ce protocole. Ce travail a été financé par la National Science Foundation subvention IOS-0950873 et le National Institutes of Health NRSA FAI084441Z bourse.

1. Stérilisation des oeufs d&#39;insectes et d&#39;élevage <ol><li> Préparer l&#39;alimentation d&#39;abord autoclave 15 g de gélose fournie dans 900-1000 ml H O. 2 Immédiatement après passage à l&#39;autoclave, le mélange avec 166 g de nourriture de germe de blé et bien mélanger dans un mélangeur de laboratoire. Verser dans un plat (ou des plats) pour refroidir, puis transfert à l&#39;alimentation du papier d&#39;aluminium, enveloppez hermétiquement et conserver à 4 ° C. </li><li> À l&#39;arrivée, stériliser M. oeufs sexta avec 250 ml d&#39;eau de Javel 0,6% pendant 2-3 min dans un porte-filtre en verre et appareils fiole à vide avec un papier filtre de 90 mm, en remuant de temps en temps. </li><li> Mettez à vide pour purger l&#39;eau de Javel et laver les oeufs 3-4 fois avec 250 ml stérile distillée H 2 O par lavage. </li><li> Transfert des œufs frais à papier de 90 mm filtre placé dans une boîte de Pétri couvercle et laissez-les sécher jusqu&#39;à ce qu&#39;ils ne collent ensemble (environ 20-30 min). </li><li> Transférer les oeufs sur le fond des récipients en plastique avec un régime alimentaire des insectes, placer environ 40 oeufs dans chaque récipient(Figure 1A). Les oeufs sont séparés de l&#39;alimentation pour empêcher l&#39;humidité provoquée par la contamination fongique. Maintenir les œufs à 26 ° C avec une lumière 16 h: 8 h obscurité photopériode. Oeufs permettra d&#39;alléger d&#39;une couleur blanc-jaune avant l&#39;éclosion. </li><li> Fois l&#39;éclosion terminée, transférez environ 25 larves de chaque dans de nouveaux conteneurs avec un régime alimentaire sur le fond (figure 1B). Il est important de ramasser chaque larve par la longue corne noire à l&#39;aide des pinces pour éviter de les endommager pendant leurs premiers stades de développement fragiles. Incuber pendant 2 jours que ci-dessus. Alors que la mort des larves est inhabituel à ce stade, certains étaient morts ou, plus probablement, un retard de croissance des larves de développement peuvent être observés. Ces insectes devraient être exclus de l&#39;étude plus loin et sacrifiés par le gel. </li><li> Pour éviter les comportements cannibales, les larves transférer dans des contenants individuels avec des petits morceaux de nourriture (figure 1C) et incuber comme ci-dessus. Surveiller le développement des insectes, les excréments remplacer la nourriture et le nettoyage oust de conteneurs tous les deux jours jusqu&#39;à ce qu&#39;ils subissent la mue des larves troisième (habituellement 6-9 jours après l&#39;éclosion). Il s&#39;agit de la 4 ème étape stade larvaire, caractérisé par l&#39;apparition de noir de crochet en forme de crochets de chaque proéminence proleg et l&#39;augmentation de bandes le long du corps de l&#39;insecte (figure 1D). Les larves peuvent varier considérablement en taille, mais pèsent généralement de 0,15 à 0,4 g à l&#39;entrée du stade 4 e stade larvaire. Les larves de taille similaire doit être choisi pour l&#39;injection. </li></ol> 2. Préparation des bactéries pour injection <ol><li> Croître la souche bactérienne (s) pour être testés conformément aux procédures standard de la phase de croissance que vous souhaitez tester. </li><li> Pellet 500 ul de chaque souche bactérienne à tester en faisant tourner pendant 2 min dans une microcentrifugeuse à 13.000 rpm à température ambiante. </li><li> Resuspendre les cellules dans 1 ml de phosphate 1x solution saline stérile tamponnée (PBS) et une pastille de nouveau comme ci-dessus. </li><li> Resuspendre les cellules dans 0,5 ml de PBS stérile 1x et mesurerque la densité optique (DO) de la suspension. Diluer les suspensions à la même OD, si nécessaire. Un milieu de croissance stérile peut également être utilisé pour la remise en suspension des cellules et de dilution en place de PBS 1x, si on le souhaite. </li></ol> 3. L&#39;injection de 4 e stade larvaire <ol><li> Préparer série de 10 dilutions de la première souche bactérienne dans 6 puits d&#39;une microplaque de 96 puits dans du PBS stérile 1x, en utilisant une pointe de pipette propre pour chaque dilution (figure 2A). Le volume final de suspension cellulaire dans chaque puits doit excéder le volume nécessaire pour l&#39;injection (10 pi par les insectes), plus 20 ul de plaque de l&#39;inoculum (voir les étapes 3,2 et 3,7). </li><li> 10 ul de chaque tache de 5 dilutions (10 -2 à travers 10 -6) le long de la partie supérieure d&#39;une boîte de Pétri contenant un milieu de croissance approprié. Incliner la plaque pour que les taches s&#39;étendent vers le centre (figure 2B). </li><li> Retirez l&#39;alimentation des insectes et des excréments d&#39;insectes et placer dans des contenants sur la glace pendantenviron 5 min. </li><li> Stériliser l&#39;aiguille de la seringue par un rinçage 3 fois chacune dans 2 tubes d&#39;éthanol 70-100%, suivis par un tube stérile de H 2 O (figure 2C). Le volume de liquide doit être suffisant pour submerger l&#39;aiguille. </li><li> Quantifier les suspensions de cellules diluées au microscope en utilisant un hémocytomètre. Selon l&#39;inoculum désiré, dessiner 10 pl de la microplaque convient bien (figure 2D). </li><li> Écouvillon la surface d&#39;insecte avec de l&#39;éthanol 95%, et injecter la suspension dans 10 ml de cellules de l&#39;insecte à un angle (inférieure à 45 °) l&#39;une derrière des fausses pattes abdominales. Prenez soin de ne pas percer le tube digestif et injecter le contenu juste sous la couche épidermique (figure 2E). Alors que certains la perte d&#39;hémolymphe (clair, vert-bleu liquide) est normal, les particules et le liquide jaune émergeant du site d&#39;injection sont révélateurs de la perforation intestinale. Dans ce cas, sacrifier l&#39;insecte et obtenir une nouvelle larve à nouveaule placer. </li><li> Répéter l&#39;étape pour chaque insecte 3,6 restante dans le groupe d&#39;injection premier. Aiguille stérilisation n&#39;est pas nécessaire entre chaque insecte injecte avec la même dilution et la souche de bactéries à moins de contamination. Alternativement, un distributeur de répéter peut être utilisé pour une plus grande cohérence dans le volume d&#39;injection entre les échantillons. </li><li> Après l&#39;injection chaque insecte avec la première souche de bactéries, répéter l&#39;étape 3,2, cette fois spotting suspensions cellulaires au milieu de la plaque et de les laisser s&#39;écouler vers le bas (figure 2F). Incuber les plaques à la température appropriée et compter les colonies d&#39;énumérer l&#39;inoculum. Cette deuxième étape de placage est utilisé pour vérifier que les échantillons n&#39;étaient pas contaminés pendant le processus d&#39;injection, comme cela se traduirait par le nombre de colonies disparates entre les premier et deuxième placages. </li><li> Répétez les étapes 3.1 à 3.8 pour chaque souche de bactéries dans l&#39;expérience. Enfin, injecter 3-5 insectes stériles avec 1x PBS à l&#39;aide d&#39;un monstreaiguille ile comme un groupe témoin négatif. Incuber les insectes à 26 ° C avec une lumière 16 h: 8 h obscurité photopériode. </li><li> Surveiller la survie des insectes au cours du temps après l&#39;injection. Mort de l&#39;insecte se caractérise par un manque de mouvement volontaire lors de la stimulation. Insectes présentent souvent une perte d&#39;appétit, diarrhée (aqueuse, excréments jaune), et / ou de perte d&#39;eau («transpiration») au cours de l&#39;infection avant son décès; ces caractéristiques est intéressant de noter que bien. </li></ol>

<ol>
	<li>Bintrim, S. B., Ensign, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+inactivation+of+genes+encoding+the+crystalline+inclusion+proteins+of+Photorhabdus+luminescens+results+in+mutants+with+pleiotropic+phenotypes.">Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes.</a> J. Bacteriol. 180, 1261-1269 (1998).</li><li>Schesser, J. H., Kramer, K. J., Bulla, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioassay+for+homogeneous+parasporal+crystal+of+Bacillus+thuringiensis+using+the+tobacco+hornworm,+Manduca+sexta.">Bioassay for homogeneous parasporal crystal of Bacillus thuringiensis using the tobacco hornworm, Manduca sexta.</a> Appl. Environ. Microbiol. 33, 878-880 (1977).</li><li>P&#233;chy-Tarr, M., Bruck, D. J., Maurhofer, M., Fischer, E., Vogne, C., Henkels, M. D., Donahue, K. M., Grunder, J., Loper, J. E., Keel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+a+novel+gene+cluster+encoding+an+insect+toxin+in+plant-associated+strains+of+Pseudomonas+fluorescens.">Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens.</a> Environ. Microbiol. 10, 2368-2386 (2008).</li><li>Nu&#241;ez-Valdez, M. E., Calder&#243;n, M. A., Aranda, E., Hern&#225;ndez, L., Ram&#237;rez-Gama, R. M., Lina, L., Rodr&#237;guez-Segura, Z., Guti&#233;rrez Mdel, C., Villalobos, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+putative+Mexican+strain+of+Serratia+entomophila+pathogenic+against+root-damaging+larvae+of+Scarabaeidae+(Coleoptera).">Identification of a putative Mexican strain of Serratia entomophila pathogenic against root-damaging larvae of Scarabaeidae (Coleoptera).</a> Appl. Environ. Microbiol. 74, 802-810 (2008).</li><li>Forst, S. A., Tabatabai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+histidine+kinase,+EnvZ,+in+the+production+of+outer+membrane+proteins+in+the+symbiotic-pathogenic+bacterium+Xenorhabdus+nematophilus.">Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.</a> Appl. Environ. Microbiol. 63, 962-968 (1997).</li><li>Kanost, M. R., Jiang, H., Yu, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+a+lepidopteran+insect,+Manduca+sexta.">Innate immune responses of a lepidopteran insect, Manduca sexta.</a> Immunol. Rev. 198, 97-105 (2004).</li><li>Yu, X. Q., Zhu, Y. F., Ma, C., Fabrick, J. A., Kanost, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+proteins+in+Manduca+sexta+plasma.">Pattern recognition proteins in Manduca sexta plasma.</a> Insect Biochem. Mol. Biol. 32, 1287-1293 (2002).</li><li>Eleftherianos, I., ffrench-Constant, R. H., Clarke, D. J., Dowling, A. J., Reynolds, S. E. <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+immune+response+to+the+entomopathogen+Photorhabdus.">Dissecting the immune response to the entomopathogen Photorhabdus.</a> Trends Microbiol. 18, 552-560 (2010).</li><li>Herbert, E. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friend+and+foe:+the+two+faces+of+Xenorhabdus+nematophila.">Friend and foe: the two faces of Xenorhabdus nematophila.</a> Nat. Rev. Microbiol. 5, 634-646 (2007).</li><li>D'Argenio, D. A., Gallagher, L. A., Berg, C. A., Manoil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+host+for+Pseudomonas+aeruginosa+Infection.">Drosophila as a model host for Pseudomonas aeruginosa Infection.</a> J. Bacteriol. 183, 1466-1471 (2001).</li><li>Waterfield, N., Dowling, A., Sharma, S., Daborn, P. J., Potter, U., Ffrench-Constant, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+toxicity+of+Photorhabdus+luminescens+W14+toxin+complexes+in+Escherichia+coli.">Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.</a> Appl. Environ. Microbiol. 67, 5017-5024 (2001).</li><li>Park, Y., Herbert, E. E., Cowles, C. E., Cowles, K. N., Menard, M. L., Orchard, S. S., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+variation+in+Xenorhabdus+nematophila+virulence+and+suppression+of+Manduca+sexta+immunity.">Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity.</a> Cell. Microbiol. 9, 645-656 (2007).</li><li>Park, Y., Kim, Y., Putnam, S. M., Stanley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterium+Xenorhabdus+nematophilus+depresses+nodulation+reactions+to+infection+by+inhibiting+eicosanoid+biosynthesis+in+tobacco+hornworms,+Manduca+sexta.">The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms, Manduca sexta.</a> Arch. Insect Biochem. Physiol. 52, 71-80 (2003).</li><li>Cowles, K. N., Cowles, C. E., Richards, G. R., Martens, E. C., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+regulator+Lrp+contributes+to+mutualism,+pathogenesis+and+phenotypic+variation+in+the+bacterium+Xenorhabdus+nematophila.">The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila.</a> Cell. Microbiol. 9, 1311-1323 (2007).</li><li>Cowles, K. N., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+activity+of+a+Xenorhabdus+nematophila+haemolysin+required+for+full+virulence+towards+Manduca+sexta+insects.">Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects.</a> Cell. Microbiol. 7, 209-219 (2005).</li><li>Goodrich-Blair, H., Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutualism+and+pathogenesis+in+Xenorhabdus+and+Photorhabdus:+two+roads+to+the+same+destination.">Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination.</a> Mol. Microbiol. 64, 260-268 (2007).</li><li>Eleftherianos, I., Baldwin, H., ffrench-Constant, R. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+modulation+of+immunity:+changes+within+the+feeding+period+of+the+fifth+larval+stage+in+the+defence+reactions+of+Manduca+sexta+to+infection+by+Photorhabdus.">Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.</a> J. Insect Physiol. 54, 309-318 (2008).</li><li>Kavanagh, K., Reeves, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+potential+of+insects+for+in+vivo+pathogenicity+testing+of+microbial+pathogens.">Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens.</a> FEMS Microbiol. Rev. 28, 101-112 (2004).</li></ol>

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L&#39;injection directe de M. larves avec des bactéries entomopathogènes sexta, comme décrit ici, sert comme un moyen simple et efficace pour analyser la virulence bactérienne. La méthode est également très adaptable à différents sujets de l&#39;expérience et / ou des conditions. Les bactéries peuvent être préparés de diverses façons avant l&#39;injection. Dans le cas de X. nematophila, les cellules de type sauvage cultivées dans riche en nutriments Luria-Bertani (LB) à la mi-...

<table border="1"><tbody><tr><td> Réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> 90 Papier filtre mm </td><td> Whatman </td><td> 1001 090 </td><td></td></tr><tr><td> Porte-filtre en verre </td><td> Millipore </td><td> XX1004700 </td><td></td></tr><tr><td> Manduca sexta oeufs </td><td> Caroline biologique Alimentation </td><td> 143880 </td><td></td></tr><tr><td> Diet spongieuse + agar </td><td> MP Biomédical </td><td> 0296029301 </td><td></td></tr><tr><td> 5,5 oz contenants et couvercles de plastique </td><td> Solo Cup Company </td><td> URC55-0090-0090 PL4 </td><td></td></tr><tr><td> 1 oz contenants et couvercles de plastique </td><td> DART Container Corporation </td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1x PBS </td><td></td&gt; <td></td><td> 137 mm de NaCl, 2,7 mM de KCl, 8 mM Na 2 HPO 4, 1,46 mM KH 2 PO 4, pH 7,4 </td></tr><tr><td> Seringue </td><td> Hamilton </td><td> 80208 </td><td> Calibre 30, 0,375 &quot;style de point de longueur, 2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

Manduca sexta, communément connu sous le sphinx du tabac, est considéré comme un ravageur agricole important, se nourrissant de plantes solanacées, dont le tabac et la tomate. La susceptibilité de M. larves sexta à une grande variété d&#39;espèces bactériennes entomopathogènes 1-5, ainsi que la richesse des informations disponibles sur le système immunitaire de l&#39;insecte 6-8, et la séquence du génome attente 9 rendent un organisme bon modèle pour une utilisation dans l&#39;étude des interactions hôte-microbe au cours de la pathogenèse. En outre, M. larves sexta sont relativement grands et faciles à manipuler et à entretenir dans le rapport de laboratoire sensibles à d&#39;autres espèces d&#39;insectes. Leur grande taille facilite aussi efficace tissus / hémolymphe d&#39;extraction pour l&#39;analyse de la réponse de l&#39;hôte à l&#39;infection. La méthode présentée ici décrit l&#39;injection directe de bactéries dans le hémocèle (cavité sang) de M. larves sexta. Cette approchepeut être utilisé pour analyser et comparer les caractéristiques de virulence de diverses espèces bactériennes, les souches, ou des mutants par simple surveillance du temps de mort de l&#39;insecte après l&#39;injection. Cette méthode a été développée pour étudier la pathogénicité de Xenorhabdus et des espèces Photorhabdus qui associent généralement avec les nématodes vecteurs comme un moyen de gagner l&#39;entrée dans l&#39;insecte. Les nématodes entomopathogènes infectent généralement les larves via digestive naturelle ou orifices respiratoires, et de libérer leurs contenus symbiotiques bactériens dans l&#39;hémolymphe des insectes (sang) peu de temps après 10. Le procédé d&#39;injection décrits ici contourne la nécessité d&#39;un nématode vecteur, ainsi découpler les effets des bactéries et des nématodes sur les insectes. Cette méthode permet à l&#39;énumération précise des matières infectieuses (cellules ou protéines) dans l&#39;inoculum, ce qui n&#39;est pas possible à l&#39;aide d&#39;autres méthodes existantes pour l&#39;analyse entomopathogenesis, y compris entaille 11 et des essais de toxicité orale 12 <em&gt;. Aussi, les essais de toxicité orale face à la virulence des toxines sécrétées introduites dans le système digestif des larves, alors que la méthode d&#39;injection directe porte sur la virulence de la cellule entière inoculums. L&#39;utilité de la méthode d&#39;injection directe comme décrit ici est d&#39;analyser la pathogenèse bactérienne en surveillant la mortalité des insectes. Cependant, cette méthode peut facilement être étendu pour une utilisation dans l&#39;étude des effets de l&#39;infection sur la M. sexta système immunitaire. L&#39;insecte réagit à l&#39;infection par les deux réponses humorales et cellulaires. La réponse humorale comprend la reconnaissance de bactéries associées aux modèles et la production subséquente de plusieurs peptides anti-microbiens 7; l&#39;expression de gènes codant pour ces peptides peut être surveillée à la suite de l&#39;infection directe par l&#39;intermédiaire d&#39;extraction d&#39;ARN et PCR quantitative 13. La réponse cellulaire à l&#39;infection implique la nodulation, l&#39;encapsulation et la phagocytose des agents infectieux par les hémocytes 6 </sup&gt;. Pour analyser ces réponses, les insectes injectés peuvent être disséqués et visualisées par microscopie 13, 14.

Un exemple représentatif d&#39;un essai mortalité des insectes est représenté dans la figure 3. Dans cette expérience, les insectes ont été injectés avec environ 50 unités formant colonies (UFC) de l&#39;une de type sauvage (ATCC19061) ou une souche mutante atténuée (lrp 13) de Xenorhabdus nematophila grandi au milieu de la phase logarithmique (n = 6 insectes par souche). Les insectes ont été observés pendant environ 72 heures, et le pour cent des insectes inje...

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Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

La méthode décrite ici utilise l&#39;injection directe de bactéries entomopathogènes dans le hémocèle de<em&gt; Manduca sexta</em&gt; Les larves d&#39;insectes.<em&gt; M. sexta</em&gt; Est un insecte disponible dans le commerce et bien étudié. Ainsi, cette méthode représente une approche simple pour analyser les interactions hôte-bactéries du point de vue de l&#39;un ou des deux partenaires.

L&#39;élevage et de l&#39;injection de<em&gt; Manduca sexta</emLes larves&gt; pour évaluer la virulence bactérienne

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K vues.  University of Wisconsin-Madison. La méthode décrite ici utilise l'injection directe de bactéries entomopathogènes dans le hémocèle de Manduca sexta Les larves d'insectes. M. sexta Est un insecte disponible dans le commerce et bien étudié. Ainsi, cette méthode représente une approche simple pour analyser les interactions hôte-bactéries du point de vue de l'un ou des deux partenaires.

Vidéo: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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

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

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

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

La salive, les glandes salivaires, et Collection hémolymphe de tiques Ixodes scapularis

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

Recherche

Immunologie et infection

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

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.

Génération et multi-phénotypique criblage à haut contenu de<em&gt; Coxiella burnetii</em&gt; Mutants de transposon

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

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

L&#39;application d&#39;un système d&#39;expression inductible pour étudier les interférences de facteurs de virulence bactérienne avec signalisation intracellulaire

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

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

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

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

Le Madagascar blatte comme Alternative non-mammifère modèle Animal pour étudier la Virulence, pathogenèse et l’efficacité du médicament

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

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

Identification de marqueurs de Virulence de Mycobacterium abscessus de réplication intracellulaire dans les Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

Évaluer la Virulence et la pathogenèse de l’Infection à Aeromonas dans un modèle de Caenorhabditis elegans

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modèle de souris pour évaluer réponse immunitaire innée à l’infection par Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

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.

Un haut débit de Shigella-test bactéricide spécifique

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.

Opsonophagocytic meurtre dosage pour évaluer les réponses immunologiques contre les bactéries pathogènes

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