This work was supported by National Health and Medical Research Council (NHMRC) grants (Grant ID 1062383 and 1063646) awarded to HJN. EAL is supported by an Australian Postgraduate Award.

The authors have nothing to disclose.

sistemi di secrezione, e le proteine ​​effettrici batteriche questi sistemi di trasporto nel citoplasma delle cellule ospiti, sono una componente importante di virulenza che molti batteri patogeni utilizzano per stabilire una infezione all&#39;interno di nicchie replicative unici. L&#39;attenzione di molti gruppi di ricerca è stato quello di indagare l&#39;interazione tra effettori batterici e proteine ​​host e l&#39;influenza di questi effettori hanno sui percorsi cellulari ospitanti. la ricerca molto limitata...

Coxiella burnetii è un patogeno intracellulare unico che provoca la febbre Q infezione umana zoonotici. Questa malattia è associata con un ampio spettro di manifestazioni cliniche che si estendono dalla sieroconversione asintomatica a infezione cronica pericolosa per la vita, che spesso si manifesta come anni endocardite dopo l&#39;esposizione 1. L&#39;infezione umana avviene principalmente attraverso l&#39;inalazione di aerosol contaminati con ruminanti il ​​principale serbatoio di infezione, in particolare, vacche da latte, pecore e capre. Anche se l&#39;infezione Coxiella in questi animali è in genere subclinica, l&#39;infezione può innescare l&#39;aborto e la notevole carica batterica all&#39;interno del fluido parto e la placenta può contaminare l&#39;ambiente locale 1. Un esempio di enorme peso tale contaminazione può avere sia la salute pubblica e il settore agricolo è stato recentemente osservato nel focolaio febbre Q che si è verificato nei Paesi Bassi 2. Tra il 2007 eIl 2010, oltre 4.000 casi umani di febbre Q sono stati diagnosticati e questa epidemia era legata alla contaminazione significativa delle aziende agricole di capra 3. Inoltre, Coxiella è un potenziale arma biologica, come classificati dai Centri statunitensi per il controllo e la prevenzione delle malattie, a causa della stabilità ambientale dei batteri e bassa dose infettante necessaria per causare grave morbilità e mortalità 4. Coxiella esiste in due fasi: Fase I microrganismi, isolati da fonti naturali, sono estremamente virulenti e organismi di fase II sono molto attenuate in vivo. Ad esempio, dopo diversi passaggi in vitro degli organismi Coxiella burnetii Nine Mile Fase I, Fase II, i batteri sono stati prodotti che contengono una delezione cromosomica irreversibile conseguente lipopolisaccaride tronco (LPS) 5. Questo ceppo, C. burnetii NMII, è fenotipicamente simile alla Fase I in modelli di coltura di tessuti e fornisce una modalità più sicural per i ricercatori di studiare Coxiella patogenesi nei laboratori 5. Negli ultimi anni molti passi avanti sono rapidamente avanzato il campo della genetica Coxiella. Più in particolare, lo sviluppo dei mezzi di comunicazione axeniche (acidificato medio cisteina citrato - ACCM-2) ha permesso la crescita privo di cellule di Coxiella sia liquido e su supporti solidi 6,7. Ciò ha determinato miglioramenti diretti di strumenti genetici disponibili per Coxiella tra cui un sistema di espressione del gene inducibile, vettori navetta e sistemi di trasposoni casuali 8-11. Più di recente, sono stati sviluppati due metodi per l&#39;inattivazione genica mirata, aprendo la strada per l&#39;esame specifico di virulenza geni candidati 12. A seguito di interiorizzazione da parte dei macrofagi alveolari, Coxiella replica ai numeri elevati all&#39;interno di un vano di membrana definito il Coxiella- contenente vacuolo (CCV). Il CCV richiede traffico endocitico ospite esimogrezzi precoce e tardiva endosomi fino a quando non matura in un organello lisosoma di derivazione 13. In tutto questo processo, il CCV acquisisce fattori dell&#39;ospite che o appaiono transitoriamente o rimanere connessi con il vacuolo, compresi, ma non limitatamente a, Rab5, Rab7, CD63 e LAMP-gennaio 13-15. La replica di Coxiella all&#39;interno di cellule ospiti è interamente dipendente da un Dot / Icm Tipo IVB secrezione sistema completamente funzionante (T4SS) 8,16,17. Questo sistema di secrezione è una struttura multi-proteina ancestrally relative ai sistemi di coniugazione e si estende su entrambi membrane batteriche e vacuolari per fornire proteine ​​batteriche, definito effettori, nel citoplasma ospitante 18. Il Coxiella T4SS è funzionalmente molto simile al ben caratterizzato tipo IVB Dot / Icm secrezione Sistema di Legionella pneumophila 19,20. È interessante notare che l&#39;attivazione del T4SS e successiva effettore traslocazione avviene solo Coxiella raggiunge l&#39;acido lisosoma derivatoorganello, circa 8 ore dopo l&#39;infezione 17,21. Ad oggi, più di 130 effettori Dot / ICM sono stati identificati 9,17,22-24. Molti di questi effettori probabilmente svolgono un ruolo importante durante la replica di Coxiella all&#39;interno di cellule ospiti; tuttavia, solo pochi effettori sono stati funzionalmente caratterizzata 25-29. In questo studio utilizziamo un saggio di traslocazione di fluorescenza base che si basa sulla scissione del substrato CCF2-AM FRET (di seguito denominato il substrato blam) attraverso attività di β-lattamasi all&#39;interno del citoplasma della cellula ospite (Figura 1). Il gene di interesse è fuso TEM-1 β-lattamasi (blam) su un plasmide giornalista che fornisce espressione costitutiva. Il substrato BLAM è composto da due fluorofori (cumarina e fluoresceina) che formano una coppia FRET. Eccitazione dei risultati cumarinici in FRET della fluoresceina e l&#39;emissione di fluorescenza verde in assenza di effettore traslocazione; Tuttavia, se il BlaM-effector proteina di fusione viene traslocato nel citoplasma ospite, la risultante β-lattamasi attività fende l&#39;anello β-lattamici del substrato Blam, che separa la coppia FRET produrre blu emissione di fluorescenza a seguito di eccitazione. Questo test traslocazione è stato ben dimostrato come un approccio per identificare le proteine ​​effettrici da una gamma di diversi batteri intracellulari ed extracellulari, tra cui C. burnetii, L. pneumophila, L. longbeachae, Chlamydia trachomatis, enteropatogena E. coli, Salmonella e Brucella 17,30-35. Per determinare il ruolo dei fattori di accoglienza specifici su Coxiella effettrici traslocazione utilizziamo un metodo consolidato per silenziamento gene noto come interferenza dell&#39;RNA, in particolare piccoli RNA interferenti (siRNA). Originariamente identificato nel Caenorhabditis elegans, interferenza dell&#39;RNA è un processo cellulare endogena conservato utilizzato per defe innataNSE contro i virus così come gene regolamentazione 36,37. Dopo il legame di specifiche sequenze siRNA, degradazione del mRNA avviene attraverso RISC (RNA-indotta complesso tacere) con conseguente specifico silenziamento genico o atterramento 38. In questo studio, siRNA è stato utilizzato per indirizzare due proteine ​​ospitanti, Rab5A e Rab7A, che sono importanti regolatori della via endocitico. L&#39;impatto di tacere Rab5A e Rab7A su effettori traslocazione è stata determinata mediante C. burnetii pBlaM-CBU0077. CBU0077 è stato scelto in quanto è stato precedentemente dimostrato di essere traslocata dal sistema di secrezione Dot / Icm di Coxiella 17. Utilizzando sia siRNA silenziamento genico e il saggio di traslocazione fluorescencebased descritto qui, stiamo cominciando a definire un ruolo per fattori di accoglienza nella traslocazione di proteine ​​effettrici da Coxiella. Questo approccio può essere applicato a una vasta gamma di entrambi batteri intracellulari ed extracellulari che possiedono secretio similesistemi n responsabili della traslocazione di proteine ​​effettrici.

<table border="0" cellpadding="0" cellspacing="0" width="760">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Reagents</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Citric acid</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C0759</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium citrate</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">S4641</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Potassium phosphate</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">60218</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Magnesium chloride</td>
			<td style="width:87px;">Calbiochem</td>
			<td style="width:119px;">442611</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Calcium chloride</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C5080</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Iron sulfate</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:119px;">S93248</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium chloride</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">S9625</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">L-cysteine</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C6852</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bacto-neopeptone</td>
			<td style="width:87px;">BD</td>
			<td style="width:119px;">211681</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Casamino acids</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:119px;">BP1424</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Methyl beta cyclodextrin</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C4555</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">RPMI + Glutamax</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">61870-036</td>
			<td style="width:211px;">ACCM-2 medium component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Chloramphenicol</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">C0378</td>
			<td style="width:211px;">For bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ON-TARGETplus Non-targeting Control Pool (OTP)</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">D-001810-10-05</td>
			<td style="width:211px;">Non-targeting control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">siGENOME Human PLK1 (5347) siRNA - SMARTpool</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">M-003290-01-005</td>
			<td style="width:211px;">Causes cell death; measure of transfection efficiency</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">siGENOME Human RAB5A (5968) siRNA - SMARTpool</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">M-004009-00-0005</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">siGENOME Human RAB7A (7879) siRNA - SMARTpool</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">M-010388-00-0005</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:343px;">5X siRNA buffer</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">B-002000-UB-100</td>
			<td style="width:211px;">Use sterile RNase-free water to dilute 5X siRNA buffer to 1X siRNA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DharmaFECT-1 Transfection Reagent</td>
			<td style="width:87px;">Dharmacon</td>
			<td style="width:119px;">T-2001-01</td>
			<td style="width:211px;">For transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Opti-MEM + GlutaMAX</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">51985-034</td>
			<td style="width:211px;">Reduced-serum medium used for transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">DMEM + GlutaMAX</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10567-014</td>
			<td style="width:211px;">For cell culture and infection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Heat-inactivated fetal calf serum (FCS)</td>
			<td style="width:87px;">Thermo Scientific</td>
			<td style="width:119px;">SH30071.03</td>
			<td style="width:211px;">Can use alternate equivalent product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DMSO</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td style="width:211px;">For storage of Coxiella strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PBS</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">0.05% Trypsin + EDTA</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25300-054</td>
			<td style="width:211px;">For cell culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:343px;">dH2O</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For dilution of samples and standards for qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Quick-gDNA Mini Prep</td>
			<td style="width:87px;">ZYMO Research</td>
			<td style="width:119px;">D3007</td>
			<td style="width:211px;">Can use alternate equivalent product to extract gDNA</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:343px;">SensiFAST SYBR No-ROX Kit</td>
			<td style="width:87px;">Bioline</td>
			<td style="width:119px;">BIO-98020</td>
			<td style="width:211px;">Can use alternate equivalent qPCR master mix product for qPCR reaction</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:343px;">LiveBLAzer FRET-B/G Loading kit with CCF2-AM</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:119px;">K1032</td>
			<td style="width:211px;">For measurement of translocation using fluorescence and generation of the 6X loading solution (contains Solution A, B, C and DMSO)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium hydroxide</td>
			<td style="width:87px;">Merck</td>
			<td style="width:119px;">106469</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium phosphate monobasic</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">71505</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">di-Sodium hydrogen orthophosphate</td>
			<td style="width:87px;">Merck</td>
			<td style="width:119px;">106586</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Probenicid</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">P8761</td>
			<td style="width:211px;">Probenicid solution component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:343px;">DRAQ5 Fluorescent Probe Solution (5 mM)</td>
			<td style="width:87px;">ThermoFisher Scientific</td>
			<td style="width:119px;">62251</td>
			<td style="width:211px;">Nuclei stain to determine cell viablity. Use 1:4000 diluted in PBS.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:343px;">4% PFA (paraformaldehyde) solution in PBS</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:211px;">Fixing solution for HeLa 229 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">25cm2 tissue culture flask with vented cap</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">430639</td>
			<td style="width:211px;">For growth of bacterial strain</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:343px;">96 Well Flat Clear bottom Black Polystyrene TC-Treated Microplates, Individually wrapped, with Lid, Sterile</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">3603</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">75cm2 tissue culture flask with vented cap</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">430641</td>
			<td style="width:211px;">For growth of HeLa 229 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">175cm2 tissue culture flask with vented cap</td>
			<td style="width:87px;">Corning</td>
			<td style="width:119px;">431080</td>
			<td style="width:211px;">For growth of HeLa 229 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Haemocytometer</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For quantification of HeLa 229 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Tear-A-Way 96 Well PCR plates</td>
			<td style="width:87px;">4titude</td>
			<td style="width:119px;">4ti-0750/TA</td>
			<td style="width:211px;">For qPCR reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">8-Lid chain, flat</td>
			<td style="width:87px;">Sarstedt</td>
			<td style="width:119px;">65.989.002</td>
			<td style="width:211px;">For qPCR reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Equipment</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bench top microfuge</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bench top vortex</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Orbital mixer</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Centrifuge</td>
			<td style="width:87px;">Eppendorf</td>
			<td style="width:119px;">5810 R</td>
			<td style="width:211px;">For pelleting bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Nanodrop</td>
			<td style="width:87px;"></td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For gDNA quantification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:343px;">Mx3005P QPCR machine</td>
			<td style="width:87px;">Aligent Technologies</td>
			<td style="width:119px;">401456</td>
			<td style="width:211px;">For quantification of Coxiella genomes in 7 day culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">ClarioSTAR microplate reader</td>
			<td style="width:87px;">BMG LabTech</td>
			<td style="width:119px;"></td>
			<td style="width:211px;">For measurement of fluorescence</td>
		</tr>
	</tbody>
</table>

applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing

Coxiella burnetii, l&#39;agente eziologico della febbre Q, è un patogeno intracellulare che si basa su un tipo IV Dot / Icm secrezione di sistema per stabilire una nicchia replicativa. Una coorte di effettori sono traslocato attraverso questo sistema nella cellula ospite per manipolare i processi di accoglienza e consentire la creazione di un vacuolo lisosoma di derivazione unica per la replica. Il metodo qui presentato comporta la combinazione di due tecniche ben consolidate: specifica silenziamento genico con siRNA e misura di traslocazione effettore utilizzando un substrato FRET-based che si basa sull&#39;attività β-lattamasi. L&#39;applicazione di questi due approcci, possiamo cominciare a comprendere il ruolo dei fattori di accoglienza in batterico funzione del sistema di secrezione e traslocazione effettrici. In questo studio abbiamo esaminato il ruolo di Rab5A e Rab7A, entrambi importanti regolatori del percorso traffico endocitico. Abbiamo dimostrato che il silenziamento dell&#39;espressione di una proteina risultati in una diminuzione della effettori translocatioefficienza n. Questi metodi possono essere facilmente modificati per esaminare altri agenti patogeni intracellulari ed extracellulari che utilizzano anche sistemi di secrezione. In questo modo, un quadro globale dei fattori di accoglienza che partecipano batterica traslocazione effettrici può essere rivelata.

Per questo studio, la C. burnetii pBlaM-CBU0077 ceppo è stato selezionato come CBU0077 è stato precedentemente dimostrato di essere un effettore traslocato del sistema di secrezione coxiella Dot / Icm 17. Prima di infezione, il numero totale dei genomi / ml in i sette giorni d&#39;C. cultura burnetii pBlaM-CBU0077 è stato enumerato utilizzando qPCR. La figura 4 dimostra un esempio della soglia di ciclo valori (Ct) attesi ...

Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Indagare le interazioni tra batteri patogeni ei loro ospiti è un&#39;area importante della ricerca biologica. Qui, descriviamo le tecniche necessarie per misurare effettrici traslocazione da Coxiella burnetii durante siRNA silenziamento genico mediante substrato Blam.

Applicando Fluorescence Resonance Energy Transfer (FRET) per esaminare Effector Translocation efficienza da<em&gt; Burnetii Coxiella</em&gt; Durante siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

applying-fluorescence-resonance-energy-transfer-fret-to-examine

Research

JoVE Journal

Immunology and Infection

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

10.6K Views.  University of Melbourne.  Indagare le interazioni tra batteri patogeni ei loro ospiti è un'area importante della ricerca biologica. Qui, descriviamo le tecniche necessarie per misurare effettrici traslocazione da Coxiella burnetii durante siRNA silenziamento genico mediante substrato Blam. 

Video: Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purificazione della popolazione di cellule specifiche per fluorescenza separazione delle cellule attivate (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Ricerca

Immunologia e infezioni

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

Rilevamento di Traslocazione tossina nel citosol Host Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated diabete di tipo 1 nei topi induzione trasferimento adottivo di cellule CD4 + T Cells diabetogenici

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 e Multi-fenotipica Screening-Alto contenuto di<em&gt; Coxiella burnetii</em&gt; Mutants transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

L&#39;applicazione di un sistema di espressione inducibile per studiare interferenza di fattori batterici di virulenza con segnalazione intracellulare

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analisi di<em&gt; Yersinia enterocolitica</em&gt; Effector traslocazione in cellule ospiti Utilizzando beta-lattamasi Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Misurare phagosome pH Raziometrico microscopia a fluorescenza

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Lo sviluppo del liofilizzato Loop-mediate isotermici amplificazione reagenti per la rilevazione di<em&gt; Burnetii Coxiella</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...