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

Nota: Durante l&#39;intero esperimento, macrofagi vengono incubate a 37 ° C in un incubatore CO 2 ad aria forzata 5% e il prelievo delle dell&#39;incubatore solo per manipolazioni sperimentali, che vengono eseguite in una cappa di sicurezza biologica Classe II. Lavorare con L. batteri monocytogenes è in base al livello di sicurezza biologica 2 regolamenti. 1. Preparazione e cellulare infezione batterica (giorno 1 e 2) <ol><li> Giorno 1 <ol><li> Cellule Seed 2.0 x 10 7 midollo osseo derivate macrofagi (BMDM) su un piatto 145 millimetri in 30 ml BMDM + Pen-Strep supporti (Tabella 2). Seed 3 piastre per ogni ceppo batterico da analizzare. Incubare per una notte a 37 ° C in un incubatore CO 2 ad aria forzata 5%. </li><li> Avviare una coltura batterica durante la notte di wild-type (WT) L. monocytogenes 10403S deformazione in 10 ml di terreno, cuore, cervello infusione (BHI), a 30 ° C in un incubatore standard. tubi porre la cultura inclinati senza agitare. Nota:Queste condizioni di crescita up-regolare l&#39;espressione dei geni flagelli, che promuove l&#39;infezione efficiente. </li></ol></li><li> Giorno 2 <ol><li> Medio Pre-caldo BMDM (senza antibiotici Pen-Strep) (Tabella 2) e tampone fosfato salino (PBS) a 37 ° C. </li><li> Lavare monostrato macrofagi due volte con 25 ml di PBS preriscaldata per rimuovere antibiotici. Aggiungere 30 ml di media BMDM fresco. </li><li> Lavare 1,5 ml di coltura batterica durante la notte con PBS mediante centrifugazione dei batteri a&gt; 14000 g per 1 minuto, scartando il surnatante, e risospendere i batteri delicatamente in 1,5 ml di PBS pipettando. Ripetere due volte. </li><li> Infettare ogni piatto macrofagi con 0,5 ml di una cultura durante la notte lavata di wild-type L. monocytogenes. Se si utilizzano più piatti, infettare ogni piatto 15 min a parte. Questo intervallo di tempo consentirà raccolta ciascuna piastra singolarmente al termine dell&#39;infezione. Nota: la molteplicità di infezione (MOI) è dosati con placcatura diluizione in series di coltura batterica usati per l&#39;infezione su piastre di agar BHI e contando unità formanti colonie (CFU) dopo 24 ore di incubazione delle piastre a 37 ° C. Utilizzando 0,5 ml di WT L. monocytogenes ceppo risultati 10403S in un MOI di ~ 100 (100 CFU di batteri per cella macrofagi). Nell&#39;analizzare mutanti batterici difettosi in crescita intracellulare, un più alto MOI dovrebbe essere considerato. </li><li> Dopo 0,5 ore di incubazione a 37 ° C, lavare le cellule infette due volte con PBS per rimuovere i batteri senza legami, e aggiungere 30 ml di pre-riscaldato medie BMDM. batteri attaccati saranno interiorizzare durante il prossimo 0,5 ore. </li><li> A 1 dall&#39;infezione hr, aggiungere 30 ml di gentamicina (1: 1.000 a raggiungere una concentrazione finale di 50 mg / ml) per uccidere i batteri extracellulari. </li><li> Montare l&#39;apparecchiatura filtrante inserendo una testa filtro su un pallone di liquido di raccolta con una luce di uscita del vuoto. Poi posto un filtro (0,45 micron) su una testa del filtro, seguito da un imbuto cilindro e fissare le varie parti con un metallo clampada. Preparare acqua priva di RNasi ghiacciata. </li><li> Al 6 ore dopo l&#39;infezione, batteri raccolto dalle cellule infette come segue. Trattare ogni piatto individualmente. Nota: Di solito, L. monocytogenes completa 1-log di crescita durante 6 ore di infezione in cellule macrofagi. incubazione più lunghi potrebbero causare la proliferazione batterica e la morte cellulare dei macrofagi, mentre periodi di incubazione più brevi potrebbero causare cariche batteriche sensibilmente inferiori. Il MOI e momento dell&#39;infezione può essere modificato per raggiungere le condizioni ottimali. <ol><li> Lavare le cellule infettate con PBS una volta. Aggiungere 20 ml di acqua priva di RNasi ghiacciata per la lisi delle cellule macrofagi. Utilizzando un raschietto cellulare, raschiare le cellule dalla piastra rapidamente, ma con attenzione. </li><li> Raccogliere cellule lisate in una provetta conica da 50 ml. Vortex per 30 sec. Centrifugare a 800 xg per 3 min a 4 ° C. </li><li> Passare il surnatante attraverso l&#39;apparecchio filtro con un sistema a vuoto. Usando le pinzette, rotolare il filtro e rapidamente trasferirla a 15 ml CONItubo cal. Snap-congelare i tubi con i filtri in azoto liquido. </li><li> Montare l&#39;apparecchiatura di filtrazione con un nuovo filtro per il campione successivo, come descritto in 1.2.7. </li><li> Conservare i filtri congelati a -80 ° C per il giorno successivo. In alternativa, procedere direttamente alla estrazione degli acidi nucleici. </li></ol></li></ol></li></ol> 2. Estrazione acidi nucleici (3 ° giorno) Nota: eseguire tutte le manipolazioni con soluzioni di fenolo e cloroformio in una cappa aspirante. <ol><li> Preparare una 1: 1 miscela di fenolo acidificati: cloroformio, 400 microlitri per ogni campione. Mescolare in tubi separati, e quindi ritirare la fase acquosa risultante per aspirazione. Aggiungere 40 ml di 10% SDS. </li><li> Scongelare i tubi del filtro contenente il ghiaccio per tenerli freddo. Per ogni tubo-filtro contenente aggiungere 650 ml di acetato-EDTA (AE) tampone (Tabella 2). </li><li> Effettuare le seguenti operazioni il più rapidamente possibile. <ol><li> Vigorosamente vortice la provetta contenente il filtro in modoche il filtro conduce allo periferia del tubo e il buffer lava completamente il filtro. Tenere sempre il freddo tubo ponendolo di nuovo sul ghiaccio. Nota: Potrebbe essere necessario inoltre vortice tubo mentre invertito per lavare completamente i batteri il filtro. </li><li> Ripetere l&#39;operazione per tutte le provette. Può essere utile spin-giù la sospensione breve (1 min a 120 xg) alla fine del processo. </li></ol></li><li> Trasferire il buffer di batteri contenenti in una provetta da 1,5 ml microcentrifuga contenente SDS e il mix di fenolo / cloroformio (come preparato in 2.1). Ripetere l&#39;operazione per tutte le provette. Può essere necessario girare i tubi del filtro contenente (1 min a 120 xg) per ottenere tampone residuo dal filtro. </li><li> Mettere tutti i tubi da 1,5 ml in un dispositivo a vortice multi-tubo, e vortex a pieno ritmo per 10 minuti. </li><li> Incubare le provette in un blocco di calore a 65 ° C per 10 minuti. Centrifugare alla massima velocità (&gt; 14.000 xg) per 5 min. </li><li> Trasferire la fase acquosa (circa 400 ml) per ogni tube in una nuova provetta da 1,5 ml contenente 40 ml di 3 M acetato di sodio (pH 5,2) e 1,0 ml di etanolo al 100%. Vortice accuratamente ogni provetta. Nota: Glicogeno può essere aggiunto in questa fase per migliorare la visualizzazione della pastiglia precipitato. </li><li> Incubare i campioni a -80 ° C per 1 ora. In alternativa, incubare a -20 ° C per una notte. Poi, centrifugare i campioni a 4 ° C per 20 min alla massima velocità (&gt; 14.000 xg). </li><li> Aspirare con cautela il etanolo (strato superiore) da ogni provetta. Aggiungere 500 ml freddo etanolo al 70% per ogni campione e miscelare accuratamente. Centrifugare a 4 ° C per 20 min alla massima velocità (&gt; 1 4.000 xg). </li><li> Aspirare con cautela il etanolo da ogni provetta. Essiccare i campioni per circa 2 minuti usando un evaporatore sotto vuoto senza riscaldamento. Non over-asciugare i campioni. </li><li> Aggiungere acqua priva di RNasi 25 microlitri di ciascun campione. Incubare a temperatura ambiente per 20 min. Attenzione vortice e spin-down. </li><li> Misurare la concentrazione di RNA in tegli campioni utilizzando un microvolumi UV-Vis spettrofotometro. Aspettatevi di estrarre circa 0,5-1 mg di acidi nucleici per piastra. Nota: repliche tecniche possono essere combinati in questa fase. </li></ol> 3. Trattamento DNase <ol><li> Impostare la reazione in base alla tabella 1 in una provetta per microcentrifuga. Incubare a 37 ° C per 45 min. Aggiungere 450 acqua priva di RNasi ml e 500 ml di fenolo-cloroformio-IAA mix (Tabella 2), fasi separate mediante centrifugazione per 2 minuti a velocità massima (&gt; 14000 g). </li><li> Trasferire la fase acquosa in una nuova provetta e aggiungere 500 microlitri cloroformio-IAA mix (Tabella 2), vortice e fasi separate da 2 min centrifugazione a velocità massima (&gt; 14.000 xg). </li><li> Trasferire la fase acquosa in una nuova provetta e aggiungere 1 ml di etanolo e 50 ml di 3 M acetato di sodio (pH 5,2). Vortice. Nota: Glicogeno può essere aggiunto in questa fase per migliorare la visualizzazione della pastiglia precipitato. </li><li&gt; incubare per 1 ora a -80 ° C. In alternativa, incubare a -20 ° C per una notte. Centrifugare a 4 ° C per 20 min a velocità massima. </li><li> Aspirare con cautela il etanolo da ogni provetta. Aggiungere 500 ml freddo etanolo al 70% per ogni campione e miscelare accuratamente. Centrifugare a 4 ° C per 20 min alla massima velocità. Aspirare con cautela il etanolo da ogni provetta. </li><li> Essiccare i campioni per circa 2 minuti usando un evaporatore sotto vuoto senza riscaldamento. Non over-asciugare i campioni. </li><li> Aggiungere 12 ml di acqua RNase-free, incubare per 2 minuti a temperatura ambiente, vortice e spin-down. I campioni contengono RNA purificato, tenerli su ghiaccio. In alternativa, sciogliere RNA in RNase-free H 2 O con 0,1 mM EDTA o tampone TE (10 mM Tris, 1 mM EDTA). </li><li> Misurare le concentrazioni di RNA utilizzando un microvolumi UV-Vis spettrofotometro. Aspettatevi ~ 100 ng di RNA per campione (se replicati tecniche sono combinate). Nota: RNA estratto in base a questo protocollo è adatto foR analisi del gene trascrizione con tecniche multiple. Noi di solito utilizziamo l&#39;analisi RT-qPCR per studiare L. monocytogenes trascrizione genica durante l&#39;infezione delle cellule macrofagi 1-4. </li></ol>

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The authors have nothing to disclose.

Il protocollo qui descritto rappresenta un metodo ottimizzato per l&#39;isolamento di RNA batterico da L. monocytogenes batteri che crescono intracellulare nelle cellule macrofagi. Questo protocollo si basa su lisi differenziale cellulare e comprende due fasi principali per l&#39;arricchimento di RNA batterico: macrofago sedimentazione nuclei mediante centrifugazione ed un rapido accumulo di batteri per filtrazione. Questi passaggi sono seguiti da un procedimento di estrazione di RNA standard. Mentre questo pro...

Intracellulari batteri patogeni ─ batteri che causano malattie infettive e in grado di crescere e riprodursi all&#39;interno delle cellule di ospiti umani ─ sono un grave problema di salute in tutto il mondo 5. Per invadere e replicarsi all&#39;interno di una cellula di mammifero, patogeni intracellulari hanno acquisito sofisticati meccanismi di virulenza e fattori. Mentre questi meccanismi sono fondamentali per la capacità di causare una malattia, si sa poco circa la loro regolazione e la dinamica. Dal momento che i profili di espressione genica di batteri coltivate in mezzi liquidi non riflettono l&#39;ambiente reale all&#39;interno di cellule ospiti, vi è una crescente necessità per le analisi del trascrittoma di batteri coltivati ​​nelle loro nicchie intracellulari. Tali analisi consentiranno la decifrazione di specifici adattamenti batteriche innescati dal conduttore e contribuiranno a identificare nuovi bersagli terapeutici per la progettazione. analisi del trascrittoma di batteri intracellulare coltivate è molto impegnativo dal momento che l&#39;RNA dei mammiferi più numerosi RNA batterica da ATalmeno dieci volte. In questo manoscritto, descriviamo un metodo sperimentale per isolare l&#39;RNA batterica da Listeria monocytogenes batteri che crescono all&#39;interno delle cellule macrofagi murini. L&#39;RNA estratto può essere usato per studiare adattamenti intracellulari e meccanismi di virulenza dei batteri patogeni di varie tecniche di analisi della trascrizione, come RT-PCR, RNA-Seq, microarray e altre tecnologie di ibridazione base. L. monocytogenes è l&#39;agente eziologico della listeriosi negli esseri umani, una malattia con manifestazioni cliniche di targeting principalmente individui immunocompromessi, anziani e donne incinte 6. esso è un agente patogeno intracellulare facoltativo Gram-positivi che invade una vasta gamma di cellule di mammiferi che sono stati utilizzati per decenni come un modello in interazioni ospite-patogeno studi 7. Dopo l&#39;invasione, risiede inizialmente in un vacuolo o phagosome (nel caso delle cellule fagocitiche), da cui deve fuggire in tegli ospitare citoplasma delle cellule al fine di replicare. Diversi fattori di virulenza hanno dimostrato di mediare il processo di fuga, soprattutto l&#39;emolisina pore-forming, listeriolysin O (LLO) e due fosfolipasi ulteriori 8. Nel citosol i batteri utilizzano macchinari polimerizzazione ospitante per spingersi sui filamenti di actina all&#39;interno della cellula e di diffondersi da cellula a cellula (Figura 1). Tutti i principali fattori di virulenza di L. monocytogenes coinvolti nella invasione, la sopravvivenza intracellulare e la replica, vengono attivati ​​dal regolatore di trascrizione maestro virulenza, PrfA. 8-10. Negli ultimi dieci anni, diversi studi condotti da noi e altri hanno applicato con successo metodi per l&#39;analisi del trascrittoma di batteri intracellulare cresciuti all&#39;interno delle cellule ospiti 2,11-15. Due approcci principali sono utilizzati per separare l&#39;RNA batterica da host-RNA che si basano su: 1) l&#39;arricchimento selettivo di RNA batterico e 2) l&#39;isolamento di RNA da diffelisi cellulare renziale. Il primo approccio si basa sulla ibridazione sottrattiva del totale estratti di RNA di molecole di RNA di mammiferi (ad esempio utilizzando kit disponibili in commercio) o la cattura selettiva di sequenze di trascritti batteriche (Scozia) 11. Il secondo approccio si basa sulla lisi differenziale di batteri e cellule ospiti, in cui le cellule ospiti vengono lisate mentre le cellule batteriche rimangono intatte. Le cellule batteriche sono poi separati dal lisato cellula ospite, di solito per centrifugazione, e l&#39;RNA è estratto utilizzando tecniche standard. Il problema principale di questo approccio è che, insieme con i batteri intatti, cellule ospiti nuclei sono anche isolati, in tal modo i preparativi di RNA contengono ancora RNA mammiferi. Un modo per superare questo problema è quello di separare i batteri intatti da cellule ospiti nuclei mediante centrifugazione differenziale, se questa procedura richiede solitamente tempo aumentare la preoccupazione delle variazioni del profilo di espressione genica durante l&#39;estrazione. In questo articolo presentiamo un ba migliore e rapidaprotocollo di estrazione cteria RNA, che si basa sulla differenza di cellule approccio lisi. Primo, L. monocytogenes infetti macrofagi sono lisate con acqua fredda. Successivamente, i nuclei dei macrofagi vengono rimossi da una breve centrifugazione e batteri intatti sono rapidamente raccolti su filtri, da cui l&#39;RNA è isolato tramite hot estrazione con fenolo-SDS degli acidi nucleici batterici.

<table>
	<tbody>
		<tr>
			<td rowspan="2">Listeria monocytogenes 10403S</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">20</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Bone marrow derived macrophages prepared from C57B/6 female mice</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">21</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>H2O, 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>b-Mercaptoethanol&#160;&#160;</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&#8217;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&#176;C, 5% CO2 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 mm, 0.45 &#181;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&#176;C incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>65 &#176;C heat block</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 &#176;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.

Il sistema modello è illustrato nella figura 1 e comprende cellule di macrofagi infettati con L. monocytogenes batteri, che replicano nel citoplasma dei macrofagi. La figura 2 rappresenta lo schema sperimentale. Figura 3 rappresenta i risultati tipici di tale analisi RT-qPCR di geni di virulenza durante WT L. monocytogenes crescita nei macrofagi in confronto alla crescita ricca di laboratorio medio BHI. I risultati mos...

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 Purificazione da nella cellula Grown<em&gt; Listeria monocytogenes</em&gt; In macrofagi

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 Views.  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.

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

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-coltura Modelli di Pseudomonas aeruginosa Biofilm Cresciuto in diretta cellule delle vie aeree umane

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Ricerca

Immunologia e infezioni

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.

Misurazione della carica batterica e Risposte immunitarie in topi infettati con Listeria monocytogenes</em

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

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificazione per affinità di Complessi virus influenzali ribonucleoproteina dalla cromatina delle cellule infettate

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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.

Purificazione di Vacuoli patogeno da<em&gt; Legionella</em&gt;-Infetti fagociti

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.

Trasmissione orale di<em&gt; Listeria monocytogenes</em&gt; Nei topi tramite ingestione di alimenti contaminati

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

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Derivate dal midollo osseo macrofagi Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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 secrezione per Indagare infezione Cellular dalla batterica patogeni<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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Valutazione dell'invasione batterica delle cellule cardiache in coltura e colonizzazione cardiaca nei topi infetti utilizzando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Infezione sperimentale con<em&gt; Listeria monocytogenes</em&gt; Come modello per lo studio Host Interferone-gamma Risposte

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...