Wir sind dankbar, dass Michael Feiss und Jean Sippy für die Führung auf Phagen Erstellung und Reinigung. Wir danken Michael Elowitz für die Bereitstellung der Zell-Erkennung Software, Schnitzcell. Der Welch Foundation (Grant Q-1759) und Human Frontier Science: Arbeit in der Golding Labor ist durch Zuschüsse aus dem National Institutes of Health (R01GM082837), der National Science Foundation (Center für Physik von lebenden Zellen 082265, PFC) unterstützt Program (RGY 70/2008).

<ol>
	<li>Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., Adhya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switches+in+bacteriophage+lambda+development.">Switches in bacteriophage lambda development.</a> Annu. Rev. Genet. 39, 409-429 (2005).</li><li>Ptashne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A genetic switch : phage lambda revisited. , (2004).</li><li>Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lambda II. , (1983).</li><li>Hershey, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Bacteriophage lambda. , (1971).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decision+making+at+a+subcellular+level+determines+the+outcome+of+bacteriophage+infection.">Decision making at a subcellular level determines the outcome of bacteriophage infection.</a> Cell. 141, 682-691 (2010).</li><li>Edgar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+infection+is+targeted+to+cellular+poles.">Bacteriophage infection is targeted to cellular poles.</a> Mol. Microbiol. , (2008).</li><li>Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in molecular biology. , (1994).</li><li>Sambrook, J., Russell, 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=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical handbook of biochemistry and molecular biology. , (1989).</li><li>Kaiser, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+internal+structure+of+bacteriophage+lambda.">On the internal structure of bacteriophage lambda.</a> J. Gen. Physiol. 49, 171-178 (1966).</li><li>Kobiler, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinetic+analysis+of+the+bacteriophage+lambda+genetic+network.">Quantitative kinetic analysis of the bacteriophage lambda genetic network.</a> Proc Natl Acad Sci. 102, 4470-4475 (2005).</li><li>Shaner, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Personal communication with M. Feiss. , .</li><li>Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adsorption+of+coliphage+lambda+to+its+host:+effect+of+variations+in+the+surface+density+of+receptor+and+in+phage-receptor+affinity.">The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity.</a> J. Mol. Biol. 103, 521-536 (1976).</li></ol>

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Bakterienstämme, Phagen und Plasmide: Stamm LE392 ist supF. Es wurde gewählt, um die Sam7 Mutation im Phagengenoms unterdrücken (siehe Tabelle 1 für weitere Details). So wird induzierten Lysogene schließlich lysieren und lassen Phagenpartikel, ebenso wie infizierte Zellen, die die lytischen Weg gewählt haben. Lysogenen Zellen werden bei 30 ° C aufgrund der Anwesenheit des temperaturempfindlichen cI 857-Allel im Phagengenom. Nach Wärmeinduktion werden gpD-EYFP und Wildtyp gpD aus dem Genom von λ LZ1 und dem Plasmid pPlate * D jeweils coexprimiert. Als Ergebnis enthält das Kapsid des neugeschaffenen Phagen λ LZ2 eine Mischung aus gpD-EYFP und gpD Proteine. Dieses Mosaik Phagen ist strukturell stabil und ausreichend Fluoreszenz auf die Detektion von einzelnen Phagen 5 zu ermöglichen. pP RE - mCherry ist ein Reporterplasmid zur Wahl des lysogenen pathwa erkenneny. Der Promotor P RE wird durch CII beim Aufbau Lysogenie 1,11 aktiviert. pP RE - mCherry 5 wurde aus pE-gfp 11 durch Austausch gfp mit mCherry 12 abgeleitet. Für weitere Details siehe unseren früheren Arbeiten 5. Growth Zustand Parameter: Während Lysogen Induktion (Abschnitt 1), gibt milde Schütteln bei 180 rpm ein guter Virus Ausbeute 13. Verwenden von Glucose im Wachstumsmedium sollte als Glucosemetabolismus vermieden werden erzeugt sauren Stoffwechselprodukte, und reife Lambda Partikel sind instabil bei saurem pH 13. Die Zugabe von MgSO 4 wird zur Stabilisierung des Phagen-Kapsid 3 ab. Für tragende Phagen cI Wildtyp (statt cI 857), kann die induzierte Lysogen Verwendung der DNA-schädigende Mittel Mitomycin C 3 ist. Im Schritt 1.3 wird die Inkubation bei 37 ° C normalerweise nicht länger als 90 Minuten. Es ist usef<html> ul die Zelldichte von OD 600 Check alle 30 min. Für eine gute Lysat sinkt OD 600 auf etwa 0,2 oder weniger, und der verbleibende OD 600 ist ein Ergebnis der Zelltrümmer. Inkubation zu lange in einer niedrigeren Phagen Ertrag führen, da die neu geschaffene Phagen können beginnen, ihre DNA in Zelltrümmer zu injizieren. Um ein sichtbares Phagen Band (mindestens 1 x 10 11 Phagenpartikel) in den Schritten 2.11 und 2,13 zu erhalten, wachsen mindestens 500 ml Kultur in Schritt 1.2. Der Zusatz von 0,2% Maltose in das Wachstumsmedium in den Schritten 5.1 und 5.2 an die Induktion der Expression des LamB, der Rezeptor für den Phagen Lambda-Adsorption 3,14 abzielen. MCherry - das 1000-fache Verdünnung statt 100-fach in Schritt 5.2 wird zur Verringerung der mCherry Hintergrundpegels von der Reporterplasmid pP RE abzielen. In Schritt 5.5 für Phagen DNA-Injektion auslöst, 35 ° C gewählt wird, um die Induktion des temperatursensitiven cI857 Allel vermeiden. Phage Reinigung: jove_content &quot;&gt; Die Phagen Reinigungsschritte (Schritte 2.1 bis 2.11) können mit anderen Aufreinigungsprotokolle 5 ersetzt werden, aber die endgültige Ultrazentrifugation durch CsCl Gradienten Gleichgewicht (Schritte 2.12 und 2.13) ist unvermeidbar. Ausschwingrotoren in den Schritten 2.10 und 2.12 bis benötigten sorgen für scharfe sichtbar Phagen Bands. Erhalt einer reinen Phagen Lager kann leicht bis zu einer Woche, so ist es notwendig, die Phagentiter auf dem Weg zu überprüfen, um sicherzustellen, dass nichts schief geht während der Zwischenschritte. Phage Handhabung: Bei allen Reinigungsverfahren in Abschnitt 2, ist es wichtig zu handhaben Phagen vorsichtig Lysat zu vermeiden Scher Phagen tails aus Phagen Köpfe. Während Zellinfektion in Abschnitt 5 (zB Schritte 5.5 bis 5.7), ist es auch wichtig, um die Scherung von Phagenpartikeln aus der infizierten Zelle zu vermeiden. Beachten Sie, dass, wenn der Phage aus der infizierten Zelle nach dem Injizieren seine DNA geschert wird, das Ergebnis ein &quot;dunkel&quot; Infektion ist, dh der infection Ergebnis wird im Experiment beobachtet werden, aber die Infektion Phagen nicht. Um solche Probleme zu minimieren, verwenden wir eine breite Pipettenspitze Beim Einsatz von Phagen oder Phagen / Zellgemisch. DAPI Testing: Färbung des Phagen Lager mit DAPI (Abschnitt 4) ist eine schnelle und effiziente Methode, um die Reinheit des Phagen Lieferverfügbarkeit überprüfen. Es kann auch verwendet werden, um mögliche Abbau eines bestehenden Phagen Lager über die Zeit zu testen. Lager für ein reines, sollte die Ko-Lokalisation von YFP und DAPI-Signale unter dem Fluoreszenzmikroskop nahe 100% liegen. Wir beobachten, dass typischerweise weniger als 1% der YFP Spots enthalten keine DAPI (repräsentiert Kapside ohne Virusgenom), was bedeutet, dass diese Teilchen nicht erfolgreich verpacken virale DNA anzeigt oder bereits ihre DNA injiziert anderswo. Weniger als 1% der DAPI-Spots enthalten keine YFP (entsprechend nicht fluoreszierenden Phagen). Ist dies nicht der Fall ist, die Schritte 2,12 bis 2,14 Notwendigkeit, in o wiederholt werdenrder wieder zu reinigen. Im Hinblick auf die Bildparameter, ist das Mikroskop Setup in Schritt 4,3 nicht so kritisch wie in Abschnitt 5, da keine langfristigen live-cell imaging hier erforderlich ist. Allerdings halten die gleichen Mikroskopie Einstellungen wie in Abschnitt 5 ist nützlich, wenn man wünscht, die Fluoreszenzintensität eines einzelnen Phagenpartikel zu kalibrieren. Falls der PBS-Agarose Bramme nicht sehr sauber oder zu sehr DAPI Farbstoff verwendet wird, können einige DAPI Stellen entsprechend Phagen-DNA mit einem &quot;Halo&quot; umgeben sein. Wenn zu wenig DAPI Farbstoff verwendet wird, kann das Signal von der DAPI-Kanal sehr schwach sein. Microscope System: Für die Bildgebung in Abschnitt 6, verwenden wir eine kommerzielle invertierten Epifluoreszenzmikroskop (Eclipse TE2000-E, Nikon) mit einer 100x-Objektiv (Plan Fluo, numerische Apertur 1,40, Öl-Immersion) und Standard-Filter-Sets (Nikon). Die Fluoreszenz Lichtquelle eine Bogenlampe mit Steuerung der Lichtintensität. Die folgenden Funktionen sind computergesteuert: x, y und z position; Hellfeld-und Fluoreszenz-Shutter und Fluoreszenz-Filter Wahl. Eine Autofokus-Funktion erforderlich ist. Andernfalls kann der Fokus leicht abdriften während der Zeitraffer-Film (in der Regel 4 Stunden lang). Die Möglichkeit, mehrere (x, y)-Positionen zu jedem Zeitpunkt zu erwerben ist nützlich, da sie mehrere Infektionsereignissen parallel verfolgen können. Wir in der Regel erwerben 8 Bühne Positionen in jedem Film, nach bis zu 100 Infektionen Veranstaltungen. Die Kamera, die wir verwenden, ist eine gekühlte 512x512 CCD mit 16x16 um Pixel-Kamera mit einem Dynamikbereich von 16 Bit (Cascade512, Photometrics). Akquisition unter Verwendung MetaMorph Software (Molecular Devices). Das Mikroskop sollte in einem temperierten Raum gestellt werden, alternativ der Mikroskoptisch sollte durch eine temperaturgesteuerte Kammer umgeben sein. Image Acquisition: Für Live-Cell-Imaging, ist es wichtig, um unnötige Belastung der Probe, die Bleich-und pho führen könntetotoxicity. Daher ist es am besten, zuerst zu charakterisieren Ihr System, um eine optimale Belichtung, die für die Fluoreszenz-Detektion ermöglicht zwar nicht was zu übermäßigen Bleichen oder Hemmung des Zellwachstums zu finden. Um eine gute Fluoreszenz-Bild zu erhalten, mit dem spannenden Lichtintensität, Belichtungszeit und Kamera Gewinn spielen. In den Schritten von 6,2 bis 6,3, wird das Gestell 10 min Intervall für den Zweck der Minimierung Belichtung gewählt. In jedem Rahmen ist nur ein einziger In-Fokus-Bild im Phasenkontrastmikroskop (für Zellerkennung) und fluoreszierenden Kanäle (zum Bestimmen Zellschicksals) benötigt. In dem ersten Zeitpunkt sind jedoch mehrere Bilder z-Position durch den Kanal YFP erforderlich, um alle infizierenden Phagen an der Zelloberfläche zu erfassen. Die YFP Belichtungszeit im Anfangs-Rahmen müssen gegebenenfalls auch höher sein als die für den Zeitraffer-Film in den späteren Zeitrahmen verwendet. Image Analysis: Sehr genau rechnen Phagenpartikel Umgebung der Zelloberfläche in Schritt 7.1. Alsbereits erwähnt, nehmen wir eine Reihe von Z-Stapel durch YFP-Kanal in Schritt 6.2. Dies kann jedoch noch verlassen einige fluoreszierende Phagenpartikel out-of-focus, die die Zählung herausfordert. Die Zellenlänge in der anfänglichen Zeitspanne wird gemessen unter Verwendung des Metamorph Software. Die Zelle kann auch durch Länge ImageJ oder andere Software-Tools zu messen. Zusätzlich kann ein automatisiertes Haus gebaut Matlab Programm sehr nützlich sein, Informationen zu erhalten, wie Fluoreszenz-Änderung über die Zeit entlang Zelllinien.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Chloroform </td><td> Fisher Scientific </td><td> C298-500 </td></tr><tr><td> NaCl </td><td> Fisher Scientific </td><td> S271-3 </td></tr><tr><td> DNase I </td><td> Sigma </td><td> D4527-10KU </td></tr><tr><td> RNase </td><td> Sigma </td><td> R4642-10MG </td></tr><tr><td> PEG8000 </td><td> Fisher Scientific </td><td> BP233-1 </td></tr><tr><td> SM-Puffer </td><td> Teknova </td><td> S0249 </td></tr><tr><td> NZYM </td><td> Teknova </td><td> N2062 </td></tr><tr><td> CsCl </td><td> Sigma </td><td> C3011-250G </td></tr><tr><td> Spritze </td><td> Becton Dickinson </td><td> 309585 </td></tr><tr><td> Nadel </td><td> Becton Dickinson </td><td> 305176 </td></tr><tr><td> Dialysekassette </td><td> Thermo Scientific </td><td> 66333 </td></tr><tr><td> Objektträger </td><td> Corning </td><td> 2947-75x50 </td></tr><tr><td> Agarose </td><td> Fisher Scientific </td><td> BP160-100 </td></tr><tr><td> SW40Ti ultra-durchsichtigen Schlauch </td><td> Beckman Coulter </td><td> 344060 </td></tr><tr><td> SW60Ti ultra-durchsichtigen Schlauch </td><td> Beckman Coulter </td><td> 344062 </td></tr><tr><td> SW40Ti Rotors </td><td> Beckman Coulter </td><td> 331302 </td></tr><tr><td> SW60Ti Rotors </td><td> Beckman Coulter </td><td> 335649 </td></tr><tr><td> Refraktometer </td><td> Fisher Scientific </td><td> 13-947 </td></tr><tr><td> Epifluoreszenzmikroskop </td><td> Nikon </td><td> Eclipse TE2000-E </td></tr></tbody></table> Tabelle 2. Reagenzien und Ausrüstung.

following cell-fate in e. coli after infection by phage lambda

Das System umfasst Bakteriophagen (Phage) Lambda und das Bakterium E. coli ist seit langem als ein Paradigma für die Zell-Schicksal Bestimmung 1,2 serviert. Nach der simultanen Infektion der Zelle durch eine Anzahl von Phagen wird einer der zwei Pfade ausgewählt lytischen (virulenten) oder lysogenen (ruhenden) 3,4. Wir haben kürzlich eine Methode entwickelt, Fluoreszenzmarkierung einzelnen Phagen und konnten die nach der Infektion Entscheidung in Echtzeit unter dem Mikroskop zu untersuchen, auf der Ebene der einzelnen Phagen und Zellen 5. Hier beschreiben wir die ganze Prozedur für die Durchführung der Infektion Experimente in unseren früheren Arbeiten 5 beschrieben. Dazu gehören die Schaffung von fluoreszierenden Phagen, Infektion der Zellen, Bildgebung unter dem Mikroskop und Datenanalyse. Das fluoreszierende Phage ist ein &quot;Hybrid&quot;-, Co-exprimierenden Wildtyp und YFP-fusion Versionen des Kapsids gpD Protein. Eine grobe Phagenlysat wird zunächst durch Induktion einer Lysogen der gpD-EYFP (Enh erhaltenWEITERFÜHRENDE Yellow Fluorescent Protein) Phagen, ein Plasmid, die Wildtyp-gpD. Eine Reihe von Reinigungsschritte werden dann durchgeführt, gefolgt von DAPI-Kennzeichnung und Bildgebung unter dem Mikroskop. Dies wird getan, um die Gleichmäßigkeit, DNA Verpackungseffizienz, Fluoreszenzsignal und strukturelle Stabilität des Phagen Lager verifizieren. Die anfängliche Adsorption der Phagen an Bakterien wird auf Eis durchgeführt, dann gefolgt von einer kurzen Inkubation bei 35 ° C, um virale DNA-Injektion 6 auslösen. Die Phagen / Bakterien Gemisch wird dann auf die Oberfläche einer dünnen Nähr-Agar-Platte, bedeckt mit einem Deckglas und abgebildet unter einem Epifluoreszenzmikroskop bewegt. Die nach der Infektion Prozess wird für 4 Stunden folgte, bei 10 min Intervall. Mehrstufige Positionen nachgeführt, so dass ~ 100 Zellen Infektionen in einem einzigen Experiment zurückgeführt werden kann. An jeder Position und Zeitpunkt werden die Bilder in der Phasenkontrast-und rot und grün fluoreszierendes Kanäle erfasst. Die Phasenkontrast-Bild wird später für die automatisierte cel verwendetl Anerkennung während die fluoreszierenden Kanäle verwendet werden, um die Infektion zu charakterisieren Ergebnis: Herstellung neuer fluoreszierenden Phagen (grün) durch Zelllyse oder Expression Lysogenie Faktoren (rot) durch Wiederaufnahme Zellwachstum und-teilung folgt. Die erworbenen Zeitraffer-Filme verarbeitet werden mit einer Kombination aus manuellen und automatisierten Methoden. Datenanalyse führt zur Identifizierung einer Infektion Parameter für jeden Infektion Ereignis (z. B. Anzahl und Positionen der infizierenden Phagen) sowie Infektion Ergebnis (Lyse / Lysogenie). Zusätzliche Parameter extrahiert werden, falls gewünscht.

Watch this Scientific Journal Video about Following Cell-fate in E. coli After Infection by Phage Lambda at JoVE.com

Following Cell-fate in E. coli After Infection by Phage Lambda

Dieser Artikel beschreibt das Verfahren zur Herstellung eines fluoreszenzmarkierten Version des Bakteriophagen Lambda, Infektion<em&gt; E. coli</em&gt; Bakterien nach der Infektion Ergebnis unter dem Mikroskop, und die Analyse von Infektion führt.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

following-cell-fate-in-e-coli-after-infection-by-phage-lambda

Research

JoVE Journal

Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

23.2K Aufrufe.  University of Illinois at Urbana-Champaign. Dieser Artikel beschreibt das Verfahren zur Herstellung eines fluoreszenzmarkierten Version des Bakteriophagen Lambda, Infektion E. coli Bakterien nach der Infektion Ergebnis unter dem Mikroskop, und die Analyse von Infektion führt.

Serie: Following Cell-fate in E. coli After Infection by Phage Lambda

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Überwachung Immunzellen Trafficking Fluorescent Prion Rods Stunden nach intraperitonealer Infektion

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Forschung

Immunologie und Infektion

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Einsatz von künstlichen Sputum Mittlere bis antibiotische Wirkung gegen die getestet<em&gt; Pseudomonas aeruginosa</em&gt; AGB in einen engeren Bezug zu den Mukoviszidose-Lunge

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Gesamtproteinextraktion und 2-D Gelelektrophorese-Methoden für<em&gt; Burkholderia</em&gt; Arten

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Skalierbare High Throughput Auswahl aus Phagen-Antikörperbibliotheken angezeigt Synthetische

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Nichtinvasive Modell Neuropathogenic<em&gt; Escherichia coli</em&gt; Infektion in der neonatalen Ratten

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Phage Phenomics: Physiologische Ansätze zur Charakterisierung Novel virale Proteine

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Die Transformation von probiotischen Hefe und ihre Erholung von Magen-Darm-Immun Geweben nach orale Gabe bei Mäusen

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Foodborne Krankheitserreger Screening mit Magneto-fluoreszierende Nanosensor: Schnellnachweis von E. Coli O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Erkennung von Enterohemorrhagic Escherichia Coli Besiedlung in murinen Host durch nicht-invasive In Vivo Biolumineszenz-System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Schaffung einer dichten Transposon Insertion Bibliothek verwenden bakterielle Konjugation in Enterobacterial Stämme wie Escherichia Coli oder Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Methods Article

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