Name: rna purification from intracellularly grown listeria monocytogenes in macrophage cells
Uploaded: 2016-06-04T14:00:00.000+00:00
Duration: 10 min 42 s
Description: Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.

Anmerkung: Während des gesamten Versuchs werden Makrophagenzellen bei 37 ° C in einem 5% CO 2 Umluft Inkubator inkubiert und nur aus dem Inkubator genommen für experimentelle Manipulationen, die in einer Klasse - II biologischen Sicherheitswerkbank durchgeführt werden. Arbeiten mit L. monocytogenes Bakterien ist nach biologischen Sicherheitsstufe 2 Vorschriften. 1. Zellpräparation und bakterielle Infektion (Tag 1 und 2) <ol><li> Tag 1 <ol><li> Seed 2,0 x 10 7 Knochenmark stammMakrophagenZellen (BMDM) auf einer 145 mm - Schale in 30 ml BMDM + Pen-Strep - Medien (Tabelle 2). Seed 3 Platten für jeden Bakterienstamm zu analysieren. Über Nacht bei 37 ° C inkubieren in einem 5% CO 2 Inkubator Gebläseluft. </li><li> Starten Sie eine Nacht Bakterienkultur von Wildtyp (WT) L. monocytogenes Stamms 10403S in 10 ml Hirn - Herz - Infusion (BHI) -Medium bei 30 ° C in einem Standard - Inkubator. Platz Kulturröhrchen schräg ohne Schütteln. Hinweis:Diese Wachstumsbedingungen bis regulieren die Expression der Flagellen Gene, die effiziente Infektion fördert. </li></ol></li><li> Tag 2 <ol><li> Vorwärmen BMDM Medium (ohne Pen-Strep - Antibiotika) (Tabelle 2) und Phosphat - gepufferter Salzlösung (PBS) bei 37 ° C. </li><li> Makrophagen-Monoschicht wasche zweimal mit 25 ml vorgewärmtes PBS Antibiotika zu entfernen. 30 ml frisch BMDM Medium. </li><li> Waschen wurden 1,5 ml Bakterienkultur über Nacht mit PBS durch Bakterien bei&gt; 14,000 × g für 1 min, Verwerfen des Überstandes und Resuspendieren Bakterien sanft in 1,5 ml PBS durch Pipettieren Zentrifugieren. Zweimal wiederholen. </li><li> Infect jede Makrophagen Platte mit 0,5 ml einer gewaschen Nacht - Kultur von Wildtyp L. monocytogenes. Wenn mehrere Platten verwenden, infizieren auseinander jede Platte 15 min. Dieses Zeitintervall ermöglicht jede Platte einzeln am Ende der Infektion erntet. Hinweis: Die Vielzahl der Infektion (MOI) durch Plattierung serielle Verdünnung getestet wirds Bakterienkultur für eine Infektion auf BHI-Agarplatten und Zählen koloniebildenden Einheiten (CFU) nach 24-stündiger Inkubation der Platten bei 37 ° C verwendet. Mit 0,5 ml WT L. monocytogenes Stamm 10403S Ergebnisse in einer MOI von ~ 100 (100 Bakterien KBE pro Makrophagen - Zelle). Bei bakteriellen Mutanten in das intrazelluläre Wachstum zu analysieren, sollte eine höhere MOI in Betracht gezogen werden. </li><li> Nach 0,5 Stunden Inkubation bei 37 ° C, waschen Sie die infizierten Zellen zweimal mit PBS, ungebunden Bakterien zu entfernen, und 30 ml vorgewärmten BMDM Medium. Befestigt Bakterien werden bei der nächsten 0,5 Stunden internalisieren. </li><li> Bei 1 h nach Infektion, fügen Sie 30 ul Gentamicin (1: 1000 bis zu einer Endkonzentration von 50 ug / ml erreichen) extrazelluläre Bakterien zu töten. </li><li> Bauen Sie die Filtervorrichtung durch einen Filterkopf auf einem Sammelflüssigkeit Kolben, der mit einem Vakuumaustrittsöffnung platzieren. Dann legen Sie einen Filter (0,45 &amp; mgr; m) auf einem Filterkopf, durch einen Zylinder Trichter gefolgt und sichern die verschiedenen Teile mit einem Metall-cLampe. Bereiten Sie eiskalten RNase-freies Wasser. </li><li> Nach 6 Stunden nach der Infektion, Ernte Bakterien aus infizierten Zellen wie folgt. Behandeln Sie jeden einzeln Platte. Hinweis: In der Regel L. monocytogenes abgeschlossen 1-log des Wachstums während 6 Stunden der Infektion in Makrophagenzellen. Längere Inkubationen in bakterielle Überwucherung und Zelltod der Makrophagen führen könnte, während kürzere Inkubationszeiten deutlich niedriger Bakterienbelastungen führen könnte. Die MOI und die Zeit der Infektion kann modifiziert werden, um optimale Bedingungen zu erreichen. <ol><li> Waschen infizierten Zellen einmal mit PBS. 20 ml eiskaltem RNase-freies Wasser zu Makrophagenzellen lysieren. Mit einem Zellschaber abkratzen die Zellen von der Platte schnell, aber sorgfältig. </li><li> Sammeln lysierten Zellen auf eine 50 ml konischen Röhrchen. Vortex für 30 Sekunden. Zentrifuge bei 800 × g für 3 min bei 4 ° C. </li><li> Führen Sie den Überstand durch das Filtergerät über ein Vakuumsystem. Mit einer Pinzette, den Filter rollen und es schnell übertragen zu einem 15 ml conical Rohr. Snap-gefrieren die Rohre mit den Filtern in flüssigem Stickstoff. </li><li> Bauen Sie die Filtrationsvorrichtung mit einem neuen Filter für die nächste Probe, wie in 1.2.7 beschrieben. </li><li> Lagern Sie die gefrorenen Filter bei -80 ° C für den nächsten Tag. Alternativ gehen Sie direkt zur Nukleinsäure-Extraktion. </li></ol></li></ol></li></ol> 2. Nucleic Acids Extraction (Tag 3) Hinweis: Führen Sie alle Manipulationen mit Phenol und Chloroform-Lösungen in einem Abzug. <ol><li> Bereiten Sie eine 1: 1-Mischung aus angesäuerten Phenol: Chloroform, 400 &amp; mgr; l für jede Probe. Mischen Sie in getrennten Röhren, und dann ziehen Sie die resultierende wässrige Schicht durch Absaugen. In 40 ul 10% SDS. </li><li> Tauen Sie die Filter enthaltenden Röhrchen auf Eis sie kalt zu halten. Jedem Filter enthaltende Röhre fügen 650 ul Acetat-EDTA (AE) Puffer (Tabelle 2). </li><li> Führen Sie die folgenden Schritte so schnell wie möglich. <ol><li> Energisch verwirbeln das Rohr den Filter so enthältdass der Filter Handrührer zum Umfang des Rohres und der Puffer voll wäscht den Filter. Halten Sie das Rohr kalt, indem sie es wieder auf Eis gestellt. Hinweis: Es kann notwendig sein, um zusätzlich das Rohr verwirbeln, während umgekehrt, um vollständig die Bakterien aus dem Filter zu waschen. </li><li> Wiederholen Sie dies für alle Röhrchen. Es kann nützlich sein, kurz die Suspension auf Spin-down (1 min bei 120 xg) am Ende des Prozesses. </li></ol></li><li> Übertragung der bakterienhaltigen Puffer in ein 1,5 ml Mikrozentrifugenröhrchen, die SDS und Phenol / Chloroform-Mischung, enthaltend (hergestellt wie in 2.1). Wiederholen Sie dies für alle Röhrchen. Es kann notwendig sein, die Filter enthalten, Rohre (1 min bei 120 · g) wieder zu spinnen Restpuffer aus dem Filter zu erhalten. </li><li> Legen Sie alle 1,5-ml-Röhrchen in einem Mehrrohrwirbelvorrichtung und Wirbel bei voller Geschwindigkeit für 10 min. </li><li> Inkubieren der Röhrchen in ein 65 ° C Heizblock für 10 min. Zentrifuge bei maximaler Geschwindigkeit (&gt; 14.000 x g) für 5 min. </li><li> Übertragen Sie die wässrige Schicht (etwa 400 ul) von jedem tube in ein neues 1,5-ml-Röhrchen 40 ul 3 M Natriumacetat (pH 5,2) und 1,0 ml 100% Ethanol enthält. Vortex jedes Röhrchen gründlich. Hinweis: Glycogen kann in diesem Schritt zugegeben werden Sichtbarmachung des ausgefällten Pellet zu verbessern. </li><li> Inkubieren der Proben bei -80 ° C für 1 Stunde. Alternativ brüten Proben bei -20 ° C über Nacht. zentrifugieren Dann werden die Proben bei 4 ° C für 20 min bei maximaler Geschwindigkeit (&gt; 14.000 xg). </li><li> absaugen vorsichtig die Ethanol (obere Schicht) aus jedem Röhrchen. In 500 &amp; mgr; l kaltem 70% Ethanol zu jeder Probe und Vortex gründlich. Zentrifuge bei 4 ° C für 20 min bei maximaler Geschwindigkeit (&gt; 1 4,000 xg). </li><li> absaugen vorsichtig das Ethanol aus jedem Röhrchen. Trocknen Sie die Proben für ca. 2 min einen Vakuumverdampfer ohne Wärme. Nicht zu trocknen die Proben. </li><li> Zugeben von 25 &amp; mgr; l RNase-freies Wasser zu jeder Probe. für 20 min bei Raumtemperatur inkubieren. Sorgfältig Wirbel und Spin-down. </li><li> Messen Sie RNA-Konzentration in ter Proben unter Verwendung eines Mikrovolumens UV-Vis-Spektralphotometer. Erwarten Sie etwa 0,5 zu extrahieren - 1 ug Nukleinsäuren pro Platte. Anmerkung: Technical Replikaten kann auf dieser Stufe kombiniert werden. </li></ol> 3. DNase-Behandlung <ol><li> Eingerichtet , um die Reaktion gemäß Tabelle 1 in einem Mikrozentrifugenröhrchen. für 45 min bei 37 ° C inkubieren. In 450 &amp; mgr; l RNase-freiem Wasser und 500 ul Phenol-Chloroform-IAA - Mix (Tabelle 2), getrennte Phasen von 2 min bei maximaler Geschwindigkeit zentrifugiert (&gt; 14.000 xg). </li><li> Übertragen Sie die wässrige Phase in ein neues Röhrchen und fügen 500 ul Chloroform-IAA - Mix (Tabelle 2), Wirbel und getrennte Phasen von 2 min Zentrifugation bei maximaler Geschwindigkeit (&gt; 14.000 xg). </li><li> Übertragen die wässrige Schicht in ein neues Röhrchen und Zugabe von 1 ml Ethanol und 50 ul 3 M Natriumacetat (pH 5.2). Wirbel. Hinweis: Glycogen kann in diesem Schritt zugegeben werden Sichtbarmachung des ausgefällten Pellet zu verbessern. </li><li&gt; Inkubieren für 1 Stunde bei -80 ° C. Alternativ brüten Proben bei -20 ° C über Nacht. Zentrifuge bei 4 ° C für 20 min bei maximaler Geschwindigkeit. </li><li> absaugen vorsichtig das Ethanol aus jedem Röhrchen. In 500 &amp; mgr; l kaltem 70% Ethanol zu jeder Probe und Vortex gründlich. Zentrifuge bei 4 ° C für 20 min bei maximaler Geschwindigkeit. absaugen vorsichtig das Ethanol aus jedem Röhrchen. </li><li> Trocknen Sie die Proben für ca. 2 min einen Vakuumverdampfer ohne Wärme. Nicht zu trocknen die Proben. </li><li> In 12 ul RNase-freies Wasser, Inkubation für 2 min bei Raumtemperatur, Wirbel und Spin-down. Die Proben enthalten gereinigte RNA, halten sie auf dem Eis. Alternativ lösen sich RNA in RNase-freiem H 2 O mit 0,1 mM EDTA oder TE - Puffer (10 mM Tris, 1 mM EDTA). </li><li> Messen Sie RNA-Konzentrationen mit Hilfe eines Mikrovolumens UV-Vis-Spektralphotometer. Erwarten Sie ~ 100 ng RNA pro Probe (wenn technische Replikate kombiniert werden). Anmerkung: RNA extrahiert gemäß diesem Protokoll ist geeignet for Gen-Transkription Analyse von mehreren Techniken. Wir in der Regel RT-qPCR - Analyse beschäftigen L. zu studieren monocytogenes Gen - Transkription während der Infektion von Makrophagen - Zellen 1-4. </li></ol>

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The authors have nothing to disclose.

Das hier beschriebene Protokoll stellt ein optimiertes Verfahren zur Isolierung von bakteriellen RNA aus L. monocytogenes Bakterien intrazellulär in Makrophagen - Zellen wachsen. Dieses Protokoll basiert auf Zell Differential-Lyse und umfasst zwei Hauptschritte zur Anreicherung von bakterieller RNA: Makrophagen-Kerne Sedimentation mittels Zentrifugation und eine schnelle Ansammlung von Bakterien durch Filtration. Diese Schritte werden durch eine Standard-RNA-Extraktionsverfahren gefolgt. Während dieses Protok...

Die intrazelluläre bakterielle Erreger ─ Bakterien verursachen Infektionskrankheiten und in der Lage zu wachsen und in den Zellen von menschlichen Wirten Wiedergabe ─ sind ein großes Gesundheitsproblem weltweit 5. Um eindringen und zu replizieren in einer Säugerzelle, haben intrazelluläre Erreger anspruchsvolle Virulenzmechanismen und Faktoren erworben. Während diese Mechanismen grundlegender Bedeutung für die Fähigkeit sind, eine Krankheit zu verursachen, wissen wir wenig über ihre Regulation und Dynamik. Da Genexpressionsprofile von in flüssigen Medien gezüchtet Bakterien nicht die tatsächliche Umgebung, in Wirtszellen beziehen, gibt es einen wachsenden Bedarf an Transkriptom in ihren intrazellulären Nischen gewachsen von Bakterien analysiert. Solche Analysen werden die Entzifferung von spezifischen bakteriellen Anpassungen durch den Host ausgelöst ermöglichen und wird dazu beitragen, neue Ziele für therapeutische Design zu identifizieren. Transkriptom-Analyse von intrazellulär gewachsen Bakterien ist sehr anspruchsvoll, da Säuger RNA durch an bakterielle RNA-Überzahlmindestens das Zehnfache. In diesem Manuskript beschreiben wir eine experimentelle Methode bakterielle RNA von Listeria monocytogenes Bakterien zu isolieren innerhalb murine Makrophagen - Zellen wachsen. Die extrahierte RNA kann verwendet werden, intrazelluläre Anpassungen und Virulenzmechanismen von pathogenen Bakterien durch verschiedene Techniken von Transkriptionsanalyse, wie beispielsweise RT-PCR, RNA-Seq, Mikroarray und andere Hybridisierungs basierten Technologien zu studieren. L. monocytogenes ist der Erreger der Listeriose beim Menschen eine Krankheit mit klinischen Manifestationen Targeting in erster Linie Personen mit geschwächtem Immunsystem, ältere Menschen und schwangere Frauen 6. Es ist ein Gram-positive fakultativ intrazelluläre Erreger , die eine breite Palette von Säugetierzellen befällt, die seit Jahrzehnten als ein Modell in der Wirt-Pathogen - Interaktionen 7 Studien verwendet wurden. Nach invasion, liegt es zunächst in einer Vakuole oder einem phagosome (im Falle von Phagozyten), von dem es in t entweichen müssener Gastgeber um Zellzytosol zu replizieren. Mehrere Virulenzfaktoren wurden 8 das Entweichen Prozess zu vermitteln gezeigt, in erster Linie die porenbildende Hämolysin, Listeriolysin O (LLO) und zwei zusätzliche Phospholipasen. Im Cytosol nutzen die Bakterien die Host - Aktin - Polymerisation Maschinen selbst auf Aktin - Filamenten innerhalb der Zelle zu treiben und von Zelle zu Zelle (1) zu verteilen. Alle wichtigen Virulenzfaktoren von L. monocytogenes beteiligt Invasion, intrazellulären Überleben und die Replikation, werden vom Master Virulenz Transkriptionsregulator aktiviert, PrfA. 8-10. In den letzten zehn Jahren mehrere Studien , die von uns durchgeführt und haben andere Methoden zur Transkriptom - Analyse von intrazellulär gewachsenen Bakterien in Wirtszellen 2,11-15 erfolgreich angewendet. Zwei Hauptansätze sind verwendet, um bakterielle RNA von Wirt-RNA zu trennen, die basieren auf: 1) Selektive Anreicherung von bakterieller RNA und 2) RNA-Isolierung durch differenz Zell-Lyse. Der erste Ansatz beruht auf subtraktive Hybridisierung von Gesamt - RNA - Extrakten zu Säuger - RNA - Moleküle (beispielsweise kommerziell erhältlichen Kits) oder selektive Erfassung von bakteriellen transkribierte Sequenzen (SCOTS) 11. Der zweite Ansatz beruht auf differentiellen Lysis von Bakterien und Wirtszellen, in denen die Wirtszellen lysiert werden, während Bakterienzellen intakt bleiben. Bakterienzellen werden dann aus der Wirtszelle Lysat, üblicherweise durch Zentrifugation abgetrennt und die RNA Standardtechniken extrahiert werden. Das Hauptproblem mit diesem Ansatz ist, dass zusammen mit der intakten Bakterien-Wirtszellen Zellkerne sind ebenfalls isoliert, so dass die RNA-Präparationen noch Säuger RNA enthalten. Eine Möglichkeit, dieses Problem zu überwinden, ist intakten Bakterien aus Wirtszellen Zellkerne unter Verwendung differentieller Zentrifugation zu trennen, obwohl dieses Verfahren in der Regel Zeit in Anspruch nimmt während der Extraktion das Anliegen von Veränderungen in Genexpressionsprofil zu erhöhen. In diesem Papier stellen wir eine verbesserte und schnelle bacteria RNA-Extraktion-Protokoll, das auf die Zelldifferential Lyse-Ansatz basiert. Zuerst L. monocytogenes Makrophagenzellen infiziert sind , mit kaltem Wasser lysiert. Als nächstes werden Makrophagen-Kerne werden durch eine kurze Zentrifugation entfernt und intakten Bakterien schnell auf Filtern gesammelt, aus dem RNA heißem Phenol-SDS-Extraktion bakterieller Nukleinsäuren isoliert werden.

<table><tbody><tr><td>Listeria monocytogenes 10403S</td><td></td><td></td><td>&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Bone marrow derived macrophages prepared from C57B/6 female mice</td><td></td><td></td><td>&lt;sup&gt;21&lt;/sup&gt;</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, RNAse free</td><td>Thermo Scientific</td><td>10977-015</td><td>DEPC-treated water can be used</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41965039</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-088</td><td></td></tr><tr><td>&amp;#946;-Mercaptoethanol</td><td>Gibco</td><td>31350010</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Brain heart infusion (BHI)</td><td>Merckmillipore</td><td>1104930500</td><td></td></tr><tr><td>Phenol saturated pH 4.3</td><td>Fisher</td><td>BP1751I-400</td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>BP1145-1</td><td></td></tr><tr><td>Iso-amyl alcohol</td><td>Sigma-Aldrich</td><td>W205702</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>W302406</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>DNaseI</td><td>Fermentas</td><td>EN0521</td><td></td></tr><tr><td>SDS 10%</td><td>Sigma-Aldrich</td><td>L4522</td><td></td></tr><tr><td>Ethanol absolute</td><td>Merck Millipore</td><td>1070174000</td><td></td></tr><tr><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; forced-air incubator</td><td>Thermo Scientific</td><td>Model 3111</td><td></td></tr><tr><td>Cell scrapers</td><td>Nunc</td><td>179693</td><td></td></tr><tr><td>Kontes glass holder for 45 mm filters</td><td>Fisher</td><td>K953755-0045</td><td></td></tr><tr><td>MF-Millipore filters 45&amp;nbsp;mm, 0.45&amp;nbsp;&amp;micro;m</td><td>Merck Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>SpeedVac system</td><td>Thermo Scientific</td><td>SPD131DDA</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries</td><td>Model G560E</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>145 mm cell culture dishes</td><td>Greiner</td><td>639 160</td><td></td></tr><tr><td>1.7 ml tubes, RNase-free</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>30 &amp;deg;C incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>65 &amp;deg;C heat block</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>4 &amp;deg;C table centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Sterile pipettes, 25 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Falcon tubes, 50 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr></tbody></table>

rna purification from intracellularly grown listeria monocytogenes in macrophage cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

Das Modellsystem ist in Figur 1 und enthält Makrophagenzellen mit L. infiziert gezeigten monocytogenes Bakterien, die in den Makrophagen Cytosol replizieren. 2 sind die Versuchsschema darstellt. Abbildung 3 stellt typische Ergebnisse solcher RT-qPCR - Analyse von Virulenzgenen bei WT L. monocytogenes Wachstum in Makrophagen im Vergleich zum Wachstum in den reichen Labor Medium BHI. Die Ergebnisse zeigen die Tr...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA-Reinigung aus intrazellulär Grown<em&gt; Listeria monocytogenes</em&gt; In Makrophagenzellen

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

rna-purification-from-intracellularly-grown-listeria-monocytogenes

Research

JoVE Journal

Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

9.1K Aufrufe.  Tel Aviv University. Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Serie: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Eine einfache und effiziente Methode, um Makrophagen aus Mixed primären Kulturen von adulten Leber Zellen zu isolieren

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Forschung

Immunologie und Infektion

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Mess-bakterielle Belastung und Immunreaktionen in Mäuse Infizierte mit Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

Die Reinigung von Pathogen Vakuolen aus<em&gt; Legionellen</em&gt;-Infizierten Phagozyten

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Mündliche Übertragung von<em&gt; Listeria monocytogenes</em&gt; In Mäusen über die Aufnahme von kontaminierten Speisen

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC Sekretion to Cellular-Infektion durch den bakteriellen Erreger untersuchen<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Mit RNA-Interferenz, um die angeborene Immunantwort in Makrophagen der Maus Untersuchen

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila der äußeren Membran - Vesikeln: Isolierung und Analyse ihrer Pro-inflammatorische Potential auf Makrophagen

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Verwenden eine bakterielle Erreger, Sonde für zelluläre und organismische Ebene Host Antworten

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

Etikettenfreier quantitativer Proteomik-Workflow für Discovery-gesteuerte Host-Pathogen-Interaktionen

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

Durchflusszytometrische Analyse von intrazellulären und emergenten extrazellulären Listeria monocytogenes in vitro

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...

RNA-Reinigung aus intrazellulär Grown

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Eine einfache und effiziente Methode, um Makrophagen aus Mixed primären Kulturen von adulten Leber Zellen zu isolieren

Mess-bakterielle Belastung und Immunreaktionen in Mäuse Infizierte mit Listeria monocytogenes

Die Reinigung von Pathogen Vakuolen aus

Mündliche Übertragung von

Imaging InlC Sekretion to Cellular-Infektion durch den bakteriellen Erreger untersuchen

Mit RNA-Interferenz, um die angeborene Immunantwort in Makrophagen der Maus Untersuchen

Legionella pneumophila der äußeren Membran - Vesikeln: Isolierung und Analyse ihrer Pro-inflammatorische Potential auf Makrophagen

Verwenden eine bakterielle Erreger, Sonde für zelluläre und organismische Ebene Host Antworten

Etikettenfreier quantitativer Proteomik-Workflow für Discovery-gesteuerte Host-Pathogen-Interaktionen

Durchflusszytometrische Analyse von intrazellulären und emergenten extrazellulären Listeria monocytogenes in vitro