This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1. Bereiten Sie im Voraus <ol><li> Warme sterile phosphatgepufferte Salzlösung (PBS) auf 37 ° C. </li><li> Warme Dulbecco modifiziertes Eagle-Medium (DMEM) und Minimal Essential Medium (MEM) mit 10% fötalem Rinderserum (FBS), 100 IU / ml Penicillin und 100 ug / ml Streptomycin und 37 ° C. Hinweis: Diese stellen das normale Wachstum Medien (NGM) für RAW und HeLa-Zellen auf. </li></ol> 2. Herstellung von Zellen <ol><li> Wachsen RAW 264.7 Zellen 70-90% Konfluenz in NGM. <ol><li> Pflegen Zellen bei 37 ° C in einer befeuchteten 95% Luft / 5% CO 2 Atmosphäre. </li></ol></li><li> Wachsen HeLa-Zellen zu 70-90% Konfluenz in NGM. <ol><li> Pflegen Zellen bei 37 ° C in einer befeuchteten 95% Luft / 5% CO 2 Atmosphäre. </li></ol></li><li> Vor der Sammlung, waschen Zellmonolayer einmal mit sterilem PBS. </li><li> Sammeln vorge konfluenten Zellen in einem sterilen konischen Röhrchen. <ol><li> Sanft kratzen RAW-Zellenmit einem Gummischaber in PBS, mit wiederholtes Pipettieren zu Zellverbänden zu zerstreuen. </li><li> Nehmen HeLa-Zellen mit 0,25% Trypsin-Lösung, bis die visuelle Untersuchung zeigt Distanzierung von Kulturoberfläche. Verwenden Sie beispielsweise 2 bis 3 ml für eine T-75-Kolben. Stellen Sie die Lautstärke entsprechend zu, um sicherzustellen, Trypsin-Lösung deckt gesamte Wachstumsoberfläche. <ol><li> Stillen Sie Dissoziationsreaktion mit NGM 10% FBS enthielt. </li><li> Verwenden Sie mindestens zwei Mal die Lautstärke der NGM zu Trypsin, mit wiederholtes Pipettieren zu Zellverbänden zu zerstreuen. </li></ol></li></ol></li><li> Leicht pelletisieren Zellen durch Zentrifugation bei 900 × g für 4 min. </li><li> Resuspendieren in demselben Volumen wie Trypsin / Quench-Lösung mit sterilem PBS. </li><li> Zählen von Zellen mit Hämozytometer oder Partikelzähler. </li><li> Leicht pelletisieren Zellen durch Zentrifugation bei 900 × g für 4 min. </li><li> Resuspendieren in ausreichenden Volumen PBS für 5,5 x 10 6 bis 6,0 × 10 6 Zellen / ml. Hinweis: Zum Beispiel wird eine T-75-Kolben approxim AusbeuteLICH 7,5 x 10 6 Zellen 29. </li><li> Halten Zellsuspension auf Eis, bis die Elektroporation. </li></ol> 3. Vorbereitung für die Elektroporation <ol><li> Vorkühlung Elektroporationsküvetten (0,4 cm Abstand) auf Eis. </li><li> Schalten Sie Elektroporationseinrichtung und stellen Sie die Spannung bis 0,3 kV. HINWEIS: Die Kapazität und Widerstand nicht veränderbaren Einstellungen auf unserer Elektroporator (bei 10 &amp; mgr; F und 600 Ω vom Hersteller festgelegt). </li><li> Füllen Erholung Platten mit NGM und äquilibrieren in befeuchteter 95% Luft / 5% CO 2 -Atmosphäre bei 37 ° C </li></ol> 4. Die Elektroporation <ol><li> Platzieren 400 ul Zellsuspension in vorgekühlte Küvetten überführen und 20 ug des ausgewählten Proteins zur Küvette (50 ug / ml). </li><li> Flick Küvette vorsichtig ~ 10 Mal ohne Beschädigung Zellen mischen. Hinweis: Die Küvette kann auch mehrmals umgedreht werden, um gründlich zu mischen, aber nicht auf- und abpipettieren oder Wirbel zu beschädigen Zellen. </li><li&gt; Dry außerhalb der Küvette mit einem Papiertuch oder einem anderen Wischer um elektrische Überschläge in der Elektroporator vermeiden. </li><li> Elektroporieren Probe bei 0,3 kV für 1,5 bis 1,7 ms. Hinweis: Das war typisch für diese Studie. </li><li> Unmittelbar nach der Elektroporation Flick Küvette vorsichtig ~ 10-mal zu durchmischen. </li></ol> 5. Überzug von Zellen <ol><li> Shop Küvette mit elektroporierten Zellen auf Eis, bis Sie sie in stabile Seitenplatten zu platzieren. </li><li> Für die meisten Downstream-Analysen, waschen Sie die Zellen 1x mit vorgewärmter NGM auf fremde Effektor-Protein zu entfernen. <ol><li> Entfernen Zellen für die Analyse und Aussetzung in 3-5 ml NGM. </li><li> Pelle Zellen durch Zentrifugation bei 900 × g für 4 min. </li><li> Resuspendieren in angemessenen Volumens NGM für gewünschte Plattengröße (zB 2 ml für 35-mm-Schale). </li></ol></li><li> Entfernen Sie entsprechende Menge an Zellen für nachfolgende Analyse. <ol><li> Beispiel 1: Platte in Glasbodenschalen für die Mikroskopie Analyse. </li><li> Beispiel 2: Platte into Zellkultur-Kunststoff für die Proteinanalyse wie Affinitätsreinigungen. </li></ol></li><li> Sodass die Zellen im äquilibriertem Platten in befeuchteter 95% Luft / 5% CO 2 -Atmosphäre bei 37 ° C für mindestens 4 Stunden zu erholen. </li></ol> 6. Mikroskopie Analyse 6.1) Befestigung / Immunfluoreszenzfärbungen <ol><li> Waschen Sie die Zellen 1x mit sterilem PBS nach 4 Stunden Erholungsphase. </li><li> Fix-Zellen in 100% Methanol für 2 min bei Raumtemperatur. Verwenden Sie ausreichend Methanol vollständig zu bedecken Zellen (zB 2 ml für 35-mm-Schale). </li><li> Jeweils 3x mit sterilem PBS. </li><li> Permeabilisieren Zellen mit 0,4% Triton X-100 in PBS für 15 min. Verwenden Sie beispielsweise 1 ml für eine 35 mm Durchmesser Teller. <ol><li> Einzustellen Länge Permeabilisierung und Stärke von Triton X-100 nach Epitop und Lage des Zielproteins. Hinweis: Die besten Ergebnisse müssen empirisch für jedes Ziel festgelegt werden, aber die oben genannten Bedingungen sollte für die meisten c seinytosolic Ziele. </li></ol></li><li> Blockieren mit 5% Rinderserumalbumin (BSA) in PBS für 1 h bei RT. Verwenden Sie beispielsweise 1 ml für eine 35 mm Durchmesser Teller. </li><li> Jeweils 3x mit PBS. </li><li> Inkubieren primären Antikörper in Antikörperbindungslösung (0,1% Triton X-100 und 1% BSA in PBS) über Nacht bei 4 &#160; ° C unter leichtem Schütteln. Verwenden Sie beispielsweise 0,5 ml für eine 35 mm Durchmesser Teller. <ol><li> Folgen Sie den Empfehlungen des Herstellers für die Antikörperverdünnung. Anmerkung: Beispielsweise wurde die Streptavidin-Bindungspeptid-Tag (SBP-tag) Antikörper in einer 1: 1000 Verdünnung, während die PKN1 Antikörper wurde in 1: 200-Verdünnung. </li></ol></li><li> Waschen Sie 4x mit PBS. </li><li> Inkubieren mit entsprechenden fluoreszenz konjugierten sekundären Antikörpers in Antikörper-Bindungslösung für 1 h bei Raumtemperatur und vor Licht geschützt. <ol><li> Beispielsweise verwenden Alexa 488 oder Alexa 647 für 1 h bei Raumtemperatur. <ol><li> Folgen Sie den Empfehlungen des Herstellers für antibody Verdünnung. (Beispielsweise etwa 1: 1.000 für diese Studie). </li></ol></li><li> Andere Verschmutzungen wie erforderlich, beispielsweise 5 &amp; mgr; M Wheat germ agglutinnin (WGA) Alexa 647 oder DAPI konjugiert gemäß der Empfehlung des Herstellers, für 1 Stunde bei Raumtemperatur. </li></ol></li><li> Wash 5x mit PBS und bei 4 ° C, vor Licht geschützt, bis Sie sie Bild. </li></ol> 6.2) Die konfokale Mikroskopie und Bildanalyse <ol><li> Bild Proben auf einem invertierten konfokalen Mikroskop mit einem 63x Ölimmersionsobjektiv. <ol><li> Bild Grün-Kanal mit dem 488 nm-Linie eines Argon-Laser, mit einer Bandbreite von 492 bis 542 nm Emission zwischen. </li><li> Bild Rot-Kanal mit einem 633 nm Diodenlaser, mit einer Bandbreite von 640 bis 718 nm Emission zwischen. </li><li> Bild Blau-Kanal mit einem 405 nm Diodenlaser, mit einer Bandbreite von 407 bis 453 nm Emission zwischen. </li><li> Sicherstellen, dass die Mehrkanal-Z-Stapel umfassen die Gesamtheit der Zellvolumen. </li></ol></li><li> Prozessbilder mit entsprechenden Bildbearbeitungssoftware. </li></ol> 7. Affinitätsreinigung <ol><li> Nach 4 Stunden Erholungsphase waschen elektroporierten Zellen zweimal mit 4 ° C PBS. </li><li> Lyse mit ~ 1,0 ml Lysepuffer (1% Triton X-100 mit einem Protease-Inhibitor-Cocktail und Phosphatase-Inhibitor in PBS) auf Eis. Verwenden Inhibitoren gemäß Herstellerangaben. Hinweis: Zum Beispiel können sowohl die Phosphatase und Protease-Inhibitoren in dieser Studie verwendet wurden 100x zugeführt wird, aber auch andere Formulierungen sollten gleich gut funktionieren. </li><li> Prompt kratzen Zellen mit Gummiwischer und sammeln in konische Röhrchen. </li><li> Lyse durch kräftiges Vortexen und Ultraschall. Hinweis: Andere Lyse-Verfahren kann genauso gut funktionieren, aber die Effizienz müssen empirisch über die Eignung der Testanforderungen bestimmt werden. <ol><li> Beschallen 3 x 30 sec mit intermittierendem Vortexen. </li></ol></li><li> Zentrifuge bei 10.000 g für 10 min bei 4 ° C zu sammelnZelltrümmer und unlösliche Aggregate; speichern Sie den Überstand. </li><li> Kombinieren gleicher Volumina (~ 1,0 ml) von elektroporiert Zelllysats mit 50 ul Streptavidin-Agarose-Harz Suspension bei 4 ° C über Nacht mit end-over-end-Rotation. Hinweis: Dieses Harz fängt die elektroporierten Protein (und die damit verbundenen Komplexe) über seine Streptavidin-Bindungspeptid Affinitätstag. </li><li> Zentrifugieren bei 2.500 g für 2 min und Überstand verwerfen. </li><li> Zweimal waschen mit 40 Bettvolumen (~ 1 ml) PBS. </li><li> Zugabe von 30 &amp; mgr; l 4x LDS-Ladepuffer (141 mM Tris-Base, 2% LDS, 10% Glycerol, 0,51 mM EDTA, 0,22 mM SERVA Blau G, 0.175 mM Phenolrot, pH 8,5), 20 ul DIH 2 O und 1 ul 0,5 M Tris (2-carboxyethyl) phosphin (TCEP), gleichzeitig für eine gewisse Volumen in Kügelchen bleiben. </li><li> Wärme bei 95 ° C für 10 Minuten und kühle auf Eis. </li><li> Spin&gt; 10,000 × g bei 4 ° C für 5 min. </li><li> Sammeln Stand. </li><li> Führen Sie eine Western-Blot unter Verwendung geeigneter Antikörper Hinweis: Erre, Anti-PKN1 primären Antikörper verwendet. </li></ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

Die Autoren haben nichts zu offenbaren.

Sezerniert Effektoren aus pathogenen Bakterien entwickelt haben, um innerhalb des Wirtszellenumgebung funktionieren und somit ist es hilfreich, sie in situ in dem Wirt zu detektieren. Einführung spezifischer Effektoren von Interesse in die Wirtszellen können die relevanten Pathogen-Wirt-Wechselwirkungen, in Isolation, ohne Störung von anderen bakteriellen Proteinen untersucht werden. Das Ziel war es, die Elektroporation als Mittel zur bakteriellen Effektorproteine ​​in eukaryotische Wirtszellen einzubrin...

Viele Gram-negative Bakterien zu verwenden spezialisierte Sekretionssysteme zur Virulenz-verwandte Proteine ​​(die als Effektoren bezeichnet) direkt in die Wirtszellen 1-5 injizieren. Diese Effektoren haben eine breite Palette von biologischen Funktionen, einschließlich: Unterdrückung der Wirtsimmunität, Zytoskelett-Änderungen, Änderung der intrazellulären Transport und Signalisierung, Transkriptionsänderungen und Gastgeber Proteasom Änderungen 9.6. Die Funktionen einiger Effektoren sind bekannt, aber die Wirts Ziele und biochemische Wirkung (en) von vielen anderen noch bestimmt werden. Beim Vergleich Wildtyp und rekombinante bakterielle Infektionen ist ein gültiger Ansatz für die intrazelluläre Effektor Virulenzmechanismen studieren, ist es oft von Vorteil, ein individuelles Effektor in die Wirtszelle einzuführen. Somit ist eine einfache Verfahren zum Einbringen und zur Charakterisierung bakterieller Effektor-Proteine ​​im Rahmen von Wirtszellen sehr wünschenswert. Die Vereinfachung des experimentellen Analyse with eine einzige Effektor ist kritisch, da andere Effektoren können gegnerische oder redundante Funktionen haben. Um diese Vereinfachung zu erreichen, haben die Forscher bereits vorgestellten Makromolekülen in Zellen durch viele verschiedene Methoden, einschließlich der Virusübertragung 10, die Mikroinjektion 11, kratzen Laden 12,13, Zellfusion mit chemisch induzierten Mikroinjektion 14, proprietären Protein &quot;Transfektion&quot; Reagenzien 15, Calciumphosphat Niederschläge 16, 17-20 und Elektroporation. Die eingeführten Moleküle reichen von Nukleinsäuren, einschließlich DNA, RNA, &amp; RNAi-Spezies auf Proteine, zellundurchlässigen Farbstoffen und Antikörpern für die intrazelluläre Targets 21,22. Einige Methoden haben ihre Grenzen, einschließlich der Art von Makromolekülen, die eingeführt werden können, und insbesondere Downstream-Analysen können durch hohe Zelltoxizität, schädlichen Wirkungsmechanismen, geringe Wirksamkeit oder Einführung Effizienz beschränkt. Transfektion, eine often-Methode zur Expression bakterieller Gene in Säugerzellen, leidet auch die Einschränkung, dass einige wichtige Wirtszelltypen, wie beispielsweise Makrophagen und primäre Zellen, sind besonders resistent gegenüber der Transfektion. Darüber hinaus ist es schwierig, das Niveau der Bakterienprotein bei Einführung von Fremd-DNA hergestellt steuern. Viel Arbeit wurde die Elektroporation von Nukleinsäuren in beiden bakteriellen und Säugerzellen als gemeinsame Labortechnik etablierten; jedoch gibt es ständige Suche nach der besten Methoden für die Bereitstellung Proteine ​​in Zellen unter physiologischen Bedingungen. Berichte über Protein Transfektion sind vielversprechend, aber teure Reagenzien und Optimierung. Der Wunsch, die potentiell toxischen Bakterien Effektoren in eine Vielzahl von Zell Ziele mit minimalen Kosten einführen führte uns zur Elektroporation als Mittel zur Untersuchung dieser Proteine ​​in vivo zu prüfen. Protein Elektroporation 23-25 ​​ist ein eingehaltenhod, Proteine ​​in lebenden Zellen über Elektropermeabilisierung, auch als elektro-Transfektion oder elektro-Injektion 26 bekannt vor. Diese Technik verwendet hoher Intensität elektrischer Impulse an Poren in den Zellmembranen zu erstellen. Diese reversible Poren ermöglichen Makromoleküle, die normalerweise von intrazellulären Raum ausgeschlossen sind die Zelle zu gelangen. Nach dem Entfernen des äußeren elektrischen Feldes kann die Membran verschließen, so dass die Zelle auf Moleküle, die durch die Poren 27,28 geführt zu halten. Ein Standard-Elektroporator in dieser Studie verwendet, um bakterielle Effektoren in beiden Maus Makrophagen-ähnlichen Zellen und humane Epithelzellen konsequent einzuführen. Das Verfahren ist schnell, effizient und kostengünstig ist, ohne nennenswerte Verringerung der zellulären Lebensfähigkeit. Die eingebrachten Proteine ​​durch Immunfluoreszenzmikroskopie sichtbar oder für funktionelle Assays verwendet werden. Dies wurde unter Verwendung von grün fluoreszierendem Protein (GFP) als einen nicht-toxischen Standard gezeigt, sowiezwei Salmonella-Effektor-Proteine, SspH1 und GtgE. Wir schlagen Protein Elektroporation als zusätzliches Werkzeug in der Literatur für die Untersuchung der bakteriellen Virulenz-Proteine ​​und ihre Funktionen in eukaryotischen Wirtszellen.

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			<td>25-050-Cl</td>
			<td></td>
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			<td height="20" style="height:20px;">0.4cm Gap-disposable electroporation cuvettes</td>
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			<td>165-2088</td>
			<td></td>
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			<td height="20" style="height:20px;">100% Methanol</td>
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			<td>Flammable, Toxic</td>
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			<td>A4919</td>
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			<td height="20" style="height:20px;">Fluorescent confocal microscope&#160;</td>
			<td>Ziess</td>
			<td>Model&#160; LSM 710&#160;</td>
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			<td>HBSt-3522</td>
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			<td height="20" style="height:20px;">HALT Protease Inhibitor Cocktail</td>
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			<td height="20" style="height:20px;">Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
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			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-Cl</td>
			<td></td>
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			<td height="20" style="height:20px;">RAW 264.7 Cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
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			<td height="20" style="height:20px;">Primary Antibody Against Target of Interest</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Chill to 4 &#176;C&#160;</td>
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			<td height="20" style="height:20px;">Sterile Phosphate Buffered Saline</td>
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			<td>Warm to 37 &#176;C&#160;</td>
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			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
			<td align="right">20353</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
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electroporation of functional bacterial effectors into mammalian cells

Die Untersuchung von Protein-Interaktionen in Zusammenhang mit lebenden Zellen können wichtige Informationen über die Lokalisierung, Dynamik und Interaktionspartner zu erzeugen. Diese Informationen sind im Rahmen der Wirt-Pathogen-Interaktionen besonders wertvoll. Viele Erregerproteine ​​Funktion innerhalb von Wirtszellen in einer Vielzahl von Art und Weise, wie ermöglichen Umgehung des Immunsystems des Wirts und das Überleben in der intrazellulären Umgebung. Um diese Pathogen-Protein-Host-Zell-Wechselwirkungen zu untersuchen, werden verschiedene Ansätze häufig verwendet, unter anderem: in vivo-Infektion mit einem Stamm ein markiertes oder mutierte Protein, oder Einführung von Pathogen-Gene mittels Transfektion oder Transduktion exprimieren. Jeder dieser Ansätze hat Vorteile und Nachteile. Wir suchten ein Mittel, um exogene Proteine ​​direkt in die Zellen einzuführen. Die Elektroporation wird üblicherweise verwendet, um Nukleinsäuren in Zellen einzuführen, wurde jedoch seltener auf Proteine ​​angewandt, obwohl die biophysikalischen Basis ist genau die gleiche.Ein Standard Elektroporator wurde verwendet, um affinitätsmarkierten bakterielle Effektoren in Säugetierzellen einzuführen. Humanen epithelialen und Maus-Makrophagen-Zellen wurden durch traditionelle Methoden kultiviert, freistehend, und in 0,4 cm Abstand Elektroporationsküvetten mit einem exogenen bakteriellen Erreger Protein von Interesse (zB Salmonella Typhimurium GtgE) platziert. Nach der Elektroporation (0,3 kV) und einem kurzen (4 h) Erholungsperiode wurde die intrazelluläre Protein durch Fluoreszenzmarkierung, die das Protein über seine Affinitätsmarkierung und, die räumliche und zeitliche Verteilung von konfokaler Mikroskopie verifiziert. Die elektroporierten Protein wurde auch gezeigt funktionellen innerhalb der Zelle und in der Lage, korrekte subzelluläre Handel und Protein-Protein-Wechselwirkung zu sein. Während die exogenen Proteine ​​eher auf der Oberfläche der Zellen zu akkumulieren, war die elektroporierten Proben große Erhöhungen der intrazellulären Effektors Konzentration bezogen auf allein Inkubation. Das Protokoll ist einfach und schnell genug, um in ap geschehenarallel Mode, so dass für Hochdurchsatz-Charakterisierung der Erreger Proteine ​​in Wirtszellen einschließlich subzelluläre Targeting und Funktion Virulenzproteine.

Als ersten Beweis des Konzeptes wurde gereinigtes grün fluoreszierende Protein erfolgreich in Säugerzellen unter Verwendung Elektroporation eingeführt. GFP, eine ungefähre 27 kD ein Molekulargewicht Protein wird üblicherweise in Säugerzellen (in der Regel aus dem Plasmid DNA exprimiert) als Molekularbiologie Werkzeug ohne signifikante zelluläre Toxizität eingeführt. HeLa-Zellen wurden inkubiert (Figur 1A) oder durch Elektroporation (1B) mit 25 ug / ml GFP, gefolgt von Immunfluo...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Elektroporation von Functional bakterielle Effektoren in Säugerzellen

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K Aufrufe.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

Serie: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Invasion von menschlichen Zellen durch einen bakteriellen Erreger

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Forschung

Immunologie und Infektion

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

In-vitro-Assay der bakteriellen Adhäsion auf Mammalian Epithelzellen

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolierung von Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Echtzeit-Imaging von myeloischen Zellen in Dynamics<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Darmtumoren durch Spinning-Disk-konfokale Mikroskopie

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Erkennen von Glykogen in peripheren mononukleären Blutzellen mit Perjodsäure-Schiff-Färbung

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Bakterielle künstliche Chromosomen: A Functional Genomics-Werkzeug für das Studium der Positiv-Strang-RNA-Viren

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Implementierung eines Systems durchlässige Membran Insert-basierten Infektion der Auswirkungen von sekretierten bakteriellen Toxins zur Untersuchung auf Säugetierwirtszellen

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Die Entwirrung der Funktion eines bakteriellen Effektor aus einem nicht-kultivierter Pflanzenschädling eine Hefe mit Zwei-Hybrid-Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...