Die Autoren möchten letzten Mitglieder der Goodrich-Blair-Labor danken: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard und Youngjin Park für ihre Beiträge zur Entwicklung dieses Protokolls. Diese Arbeit wurde von der National Science Foundation IOS-0950873 und der National Institutes of Health NRSA Gemeinschaft FAI084441Z finanziert.

Ein. Insect Egg Sterilisation und Aufzucht <ol><li> Bereiten Ernährung durch erste Autoklavieren 15 g der bereitgestellten Agar in 900-1.000 ml H 2 O. Unmittelbar nach dem Autoklavieren Mischung mit 166 g Weizenkeime Ernährung und Mischung gut in einem Labor Mixer. Gießen Sie in einer Schüssel (oder Geschirr) zu kühlen, dann Transfer Ernährung auf eine Aluminiumfolie, fest wickeln, und bei 4 ° C. </li><li> Nach der Ankunft, sterilisieren M. sexta Eier mit 250 ml von 0,6% Bleichlösung für 2-3 min in einem Glas Filterhalter und Thermoskanne, die mit einer 90 mm ​​Filterpapier, dabei gelegentlich umrühren. </li><li> Schalten im Vakuum bis Bleichlösung und waschen Eier 3-4 mal mit 250 ml sterilem destilliertem H 2 O pro Waschgang abtropfen lassen. </li><li> Übertragen Eier an die frische 90 mm Filterpapier in einer Petrischale Deckel gelegt und trocknen lassen, bis sie nicht mehr verkleben (ca. 20-30 min). </li><li> Übertragen Eier an der Unterseite des Kunststoff-Behälter mit Insekt Diät, dem Einbringen von etwa 40 Eiern in jedem Behälter(Abbildung 1A). Die Eier werden aus der Nahrung getrennt, um Feuchte-Pilzbefall zu verhindern. Pflegen Sie Eier bei 26 ° C mit einem 16 Stunden Licht: 8 Std. dunklen Photoperiode. Eier werden zu einem gelb-weißer Farbe vor aufzuhellen bis zum Schlüpfen. </li><li> Wenn Schlupf abgeschlossen ist, transferieren es vorsichtig in etwa 25 Larven jeden neuen Container mit einer Diät auf der Unterseite (Abbildung 1B). Es ist wichtig zu holen jede Larve durch den langen schwarzen Horn mit einer Pinzette zu schaden sie während ihrer fragilen frühen Entwicklungsstadien. Inkubieren für 2 Tage wie oben. Während zum Tod der Larve ist in diesem Stadium ungewöhnlich ist, kann einige tot oder, noch wahrscheinlicher, entwicklungspolitisch verkümmerte Larven beobachtet werden. Solche Insekten von der weiteren Untersuchung ausgeschlossen werden und opferte durch Einfrieren. </li><li> Um kannibalischen Verhaltensweisen, Transfer Larven einzelnen Behältern mit kleinen Stücken von Lebensmitteln (1C) zu vermeiden und inkubieren wie oben. Überwachen Insekten Entwicklung, ou ersetzen Lebensmittel-und Reinigungsmittel Kott von Containern jeden zweiten Tag, bis sie das dritte Larvenstadium häuten (in der Regel 6-9 Tage nach dem Schlüpfen) unterziehen. Dies ist die 4. Larvenstadium Larvenstadium, gekennzeichnet durch das Auftreten von schwarzen hakenartigen crochets auf jeder Proleg und erhöhte Bedeutung von Streifen entlang der Insektenkörper (1D). Die Larven können erheblich variieren in der Größe, aber wiegen in der Regel 0,15 bis 0,4 g beim Betreten des 4. Larvenstadium Bühne. Larven von ähnlicher Größe sollte zur Injektion gewählt werden. </li></ol> 2. Vorbereitung der Bakterien zur Injektion <ol><li> Wachsen die Bakterienstamm (en) gemäß den Standardverfahren der Wachstumsphase Sie testen möchten getestet werden. </li><li> Pellet 500 ul jeder Bakterienstamm durch Spinnen für 2 min in einer Mikrozentrifuge bei 13.000 rpm bei Raumtemperatur getestet. </li><li> Die Zellen in 1 ml sterilem 1x phosphatgepufferter Kochsalzlösung (PBS) und Pellet erneut wie oben. </li><li> Die Zellen in 0,5 ml sterile 1x PBS und meadass die optische Dichte (OD) der Suspension. Verdünnte Suspensionen alle auf den gleichen Außendurchmesser, falls notwendig. Steriler Nährmedien können auch zur Resuspension und Verdünnung Zelle anstelle von 1x PBS verwendet werden, falls gewünscht. </li></ol> 3. Injektion von 4 th Larvenstadium <ol><li> Planen seriell 10-fach-Verdünnungen des ersten Bakterienstamm in 6 Vertiefungen einer 96-Well-Mikrotiterplatte in sterilem PBS 1x, unter Verwendung einer frischen Pipettenspitze für jede Verdünnung (Abbildung 2A). Das Endvolumen der Zellsuspension in jede Vertiefung überschreiten sollte das Volumen für die Injektion (10 ul pro Insekt) plus 20 ul der Platte das Inokulum (siehe Schritte 3.2 und 3.7) erforderlich. </li><li> Spot 10 ul jeweils für 5 Verdünnungen (10 -2 bis 10 -6) entlang der Oberseite einer Petrischale enthält einen geeigneten Wachstumsmedium. Schwenk die Platte, so dass die Flecken auf den Mittelpunkt (2B) zu verbreiten. </li><li> Entfernen Insektennahrung und Kot und Ort Insekten in Containern auf Eisca. 5 min. </li><li> Sterilisieren die Nadel der Spritze durch Spülen jeweils 3 Mal in 2 Röhren von 70-100% Ethanol, gefolgt von einem Rohr aus sterilem H 2 O (Abbildung 2C). Das Volumen der Flüssigkeit muss ausreichend sein, um die Nadel eintauchen. </li><li> Quantifizierung der verdünnten Zellsuspensionen unter einem Mikroskop mit einer Zählkammer. Je nach gewünschter Inokulum, zeichnen 10 ul aus der entsprechenden Mikrotiterplatte (2D). </li><li> Tupfer das Insekt Oberfläche mit 95% Ethanol und 10 ml injizieren die Zellsuspension in das Insekt in einem Winkel (kleiner 45 °) hinter der einen der abdominalen prolegs. Achten Sie darauf, nicht zu durchstechen den Darm und injizieren die Inhalte direkt unter der Epidermis (Abb. 2E). Während einige Verlust von Hämolymphe (klar, grün-blaue Flüssigkeit) normal ist, sind Feinstaub und gelbe Flüssigkeit, die aus der Injektionsstelle indikativ für gut Punktion. Wenn dies geschieht, zu opfern das Insekt und eine neue Larve wiederplatzieren Sie es. </li><li> Wiederholen Sie Schritt 3,6 für jede weitere Insekt in der ersten Injektion Gruppe. Needle Sterilisation ist nicht notwendig, zwischen den einzelnen Insekt mit der gleichen Verdünnung und Bakterienstamm injiziert, wenn Verunreinigungen auftreten. Alternativ kann ein Repetierpipette für größere Kohärenz Injektionsvolumen zwischen den Proben verwendet werden. </li><li> Nach der Injektion jeder Insekt mit dem ersten Bakterienstamm, Schritt 3.2, diesmal Spotting Zellsuspensionen in der Mitte der Platte und ließ sie Strömung in Richtung des Bodens (2F). Inkubieren Platten bei entsprechender Temperatur und zählen Kolonien, um das Inokulum aufzuzählen. Diese zweite Plattierungsschritt verwendet, um zu überprüfen, dass die Proben nicht während des Einspritzvorgangs kontaminiert, wie dies in unterschiedlichen Kolonie Zahlen zwischen der ersten und zweiten Beschichtungen führen würde. </li><li> Die Schritte 3.1 bis 3.8 für jeden Bakterienstamm im Experiment. Schließlich injizieren 3-5 Insekten mit sterilen 1x PBS mit einem sterile Nadel als negative Kontrollgruppe. Inkubieren Insekten bei 26 ° C mit einem 16 Stunden Licht: 8 Std. dunklen Photoperiode. </li><li> Überwachen Insekten das Überleben im Laufe der Zeit nach der Injektion. Insect Tod wird als ein Mangel an freiwilligen Bewegung nach Stimulation aus. Insekten weisen oft Appetitlosigkeit, Durchfall (wässrig, gelb Kot), und / oder Wasser Verlust (&quot;Schwitzen&quot;) während der Infektion vor dem Tod; diese Eigenschaften können erwähnenswert als gut. </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>

Keine Interessenskonflikte erklärt.

Die direkte Injektion von M. sexta Larven mit entomopathogene Bakterien, wie hier beschrieben, dient als einfaches und wirksames Mittel, um bakteriellen Virulenz analysieren. Das Verfahren ist auch sehr anpassungsfähig an unterschiedliche Versuchspersonen und / oder Bedingungen anzupassen. Bakterien können auf verschiedene Weise vor der Injektion hergestellt werden. Im Fall von X. nematophila sind Wildtyp-Zellen in nährstoffreichen Luria-Bertani (LB)-Medium bis zur mittleren logarithmischen Phase ge...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 90 mm Filterpapier </td><td> Whatman </td><td> 1001 090 </td><td></td></tr><tr><td> Glass Filterhalter </td><td> Millipore </td><td> XX1004700 </td><td></td></tr><tr><td> Motteneier </td><td> Carolina Biological Supply </td><td> 143880 </td><td></td></tr><tr><td> Gypsy Moth Diet + Agar </td><td> MP Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5,5 Unzen Kunststoff-Behälter und Deckel </td><td> Solo Cup Company </td><td> URC55-0090 PL4-0090 </td><td></td></tr><tr><td> 1 Unze Kunststoff-Behälter und Deckel </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 NaCl, 2,7 mM KCl, 8 mM Na 2 HPO 4, 1,46 mM KH 2 PO 4, pH 7,4 </td></tr><tr><td> Spritze </td><td> Hamilton </td><td> 80208 </td><td> 30 Gauge, 0,375 &quot;Länge, zeigen style 2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

Manduca sexta, die gemeinhin als Tabakschwärmer bekannt ist, wird als wichtiger landwirtschaftlicher Schädling, Fütterung auf Solanaceen einschließlich Tabak und Tomaten. Die Anfälligkeit von M. sexta-Larven zu einer Vielzahl von entomopathogenen Bakterienarten 1-5, sowie die Fülle von Informationen über das Insekt, das Immunsystem 6-8, und die anstehende Genomsequenz 9 machen es zu einem guten Modell für den Einsatz in Studium Host-Mikroben-Interaktionen während der Pathogenese. Außerdem M. sexta-Larven sind relativ groß und leicht zu manipulieren und im Labor im Vergleich zu anderen anfälligen Insektenarten halten. Ihre Größe erleichtert auch effizient Gewebe / Hämolymphe Extraktion zur Analyse des Wirts auf eine Infektion. Die hier vorgestellte Methode beschreibt die direkte Injektion von Bakterien in die Hämocoel (Blut Hohlraum) M. sexta Larven. Dieser Ansatzkann verwendet werden, um zu analysieren und vergleichen die Virulenz Eigenschaften verschiedener Bakterienarten, Stämme oder Mutanten durch einfaches Überwachen der Zeit, um Insekten Tod nach der Injektion werden. Dieses Verfahren wurde entwickelt, um die Pathogenität von Xenorhabdus und Photorhabdus Spezies, denen in der Regel mit Nematoden-Vektoren als Mittel zum Eintrag in das Insekt gewinnen studieren. Entomopathogene Nematoden Regel infizieren Larven über natürliche Verdauungs-oder Atemöffnungen, und lassen ihre symbiotische Bakterien Inhalte in das Insekt Hämolymphe (Blut) kurz danach 10. Die Injektion hier beschriebene Verfahren umgeht den Bedarf an einem Nematoden-Vektor, wodurch Entkopplung der Wirkungen der Bakterien und Nematoden auf das Insekt. Dieses Verfahren ermöglicht eine genaue Zählung von infektiösem Material (Zellen oder Protein) innerhalb des Inokulums, was nicht möglich ist mit anderen bestehenden Methoden zur Analyse entomopathogenesis einschließlich Nicking 11 und 12 orale Toxizität Assays <em&gt;. Auch adressieren orale Toxizität Assays die Virulenz von sekretiertem Toxine in das Verdauungssystem der Larven eingeführt, während die direkte Injektion Verfahren behandelt die Virulenz von Ganzzell-Inokula. Die Nützlichkeit der Direkteinspritzung Verfahren wie hier beschrieben ist, um bakterielle Pathogenese durch Überwachen Insektenmortalität analysieren. Jedoch kann dieses Verfahren leicht für die Verwendung bei der Untersuchung der Auswirkungen der Infektion auf der M. erweiterbar sexta Immunsystems. Das Insekt reagiert auf eine Infektion über beide humorale und zelluläre Reaktionen. Die humorale Antwort enthält Erkennung von bakterieller-assoziiertes Muster und die anschließende Herstellung von verschiedenen antimikrobiellen Peptiden 7; die Expression von Genen, die diese Peptide können überwacht anschließende direkte Infektion durch RNA-Extraktion und quantitative PCR 13 werden. Die zelluläre Reaktion auf eine Infektion beinhaltet Nodulation, Kapselung und Phagozytose von Erregern durch Hämocyten 6 </sup&gt;. Um diese Reaktionen zu analysieren, können injiziert Insekten seziert und visualisiert werden durch Mikroskopie 13, 14.

Ein repräsentatives Beispiel eines Insektenmortalität Assay ist in Abbildung 3 dargestellt. In diesem Experiment wurden die Insekten mit etwa 50 Kolonie-bildenden Einheiten (CFU) von entweder Wildtyp (ATCC19061) oder eines abgeschwächten Mutantenstamm (lrp 13) von Xenorhabdus nematophila gezüchtet bis zur mittleren logarithmischen Phase (n = 6 Tieren pro Stamm) injiziert. Insekten wurden für ungefähr 72 h beobachtet und die Prozent der injizierten Insekten noch am Lebe...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Die hier beschriebene Methode nutzt Direkteinspritzung entomopathogener Bakterien in die Hämocoel der<em&gt; Manduca sexta</em&gt; Insektenlarven.<em&gt; M. sexta</em&gt; Ist ein im Handel erhältlich und gut untersuchten Insekten. Somit stellt diese Methode einen einfachen Ansatz zur Analyse Host-bakterielle Wechselwirkungen aus der Perspektive eines oder beider Partner.

Aufzucht und Injektion von<em&gt; Manduca sexta</em&gt; Larven zu bakteriellen Virulenz beurteilen

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

rearing-injection-manduca-sexta-larvae-to-assess-bacterial

Research

JoVE Journal

Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K Aufrufe.  University of Wisconsin-Madison. Die hier beschriebene Methode nutzt Direkteinspritzung entomopathogener Bakterien in die Hämocoel der Manduca sexta Insektenlarven. M. sexta Ist ein im Handel erhältlich und gut untersuchten Insekten. Somit stellt diese Methode einen einfachen Ansatz zur Analyse Host-bakterielle Wechselwirkungen aus der Perspektive eines oder beider Partner.

Serie: 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.

Speichel, Speicheldrüse, und Hämolymphe Abholung Ixodes scapularis Zecken

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

Forschung

Immunologie und Infektion

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.

Überwachung der Montage einer sezernierte bakterielle Virulenz-Faktor mit Site-spezifische Vernetzung

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.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

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.

Anlegen eines induzierbaren Expression System zu Störungen der bakteriellen Virulenzfaktoren mit Intrazelluläre Signal Studieren

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.

Der Madagaskar Zischen Schabe als Alternative nicht-Säugetier Tiermodell, Virulenz, Pathogenese und Wirksamkeit von Arzneimitteln zu untersuchen

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.

Identifikation der Virulenz Marker von Mycobacterium Abscessus für die intrazelluläre Replikation in Phagozyten

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.

Bewertung von Virulenz und Pathogenese der Aeromonas Infektion in einem Caenorhabditis Elegans -Modell

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.

Ein Mausmodell zu beurteilen, angeborene Immunantwort auf Staphylococcus Aureus -Infektion

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.

Ein High-Throughput- Shigella-spezifischen bakterizide Assay

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 Tötung Assay Immunreaktionen gegen bakterielle Krankheitserreger zu bewerten

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