Ringraziamo il Dott. Craig Roy e il Dr. Dario Zamboni per fornire un modello per le infezioni delle cellule protozoi. Ringraziamo il Dott. Jagdeep Obhrai, Dr. Georgiana Purdy, il Dr. Fred Heffron e Todd Wisner per le attrezzature e reagenti, il dottor Lulu Cambronne per la revisione critica del manoscritto. La citometria a flusso è stata eseguita presso l&#39;impianto di flusso di risorse OHSU Citometria condivisa. Questo lavoro è stato sostenuto in parte da una sovvenzione della Fondazione per la Ricerca Medica dell&#39;Oregon e una sovvenzione del NIH R21 AI088275 (EDC).

1. Preparazione di Legionella pneumophila Culture per le infezioni della fase di priming <ol><li> Trasforma tutti L. ceppi pneumophila usare nel saggio con le plasmide pAM239, codificante un isopropil β-D-1-tiogalattopiranoside (IPTG) inducibile proteina fluorescente verde (GFP) 28. Streak i ceppi batterici sul ferro e cisteina integrato N-(2-acetammido)-2-acido aminoethanesulfonic (ACES) tamponata agar carbone estratto di lievito (CYEA) contenente 6,25 mg / ml di cloramfenicolo (CM) (per manutenzione plasmide) e incubare per 72 hr a 37 ° C. </li><li> Trasferire un inoculo del ceppo batterico in ACES integrate tamponato estratto di brodo di lievito (AYE) con 6,25 mg / ml CM e 1 mM IPTG e coltivare di fase stazionaria (O / N) su un agitatore orbitale a 37 ° C. </li><li> Confermare espressione GFP in colture in vitro utilizzando la microscopia a fluorescenza. Trasferire 10 microlitri di cultura su un vetrino sotto una coperturaslip e immagine utilizzando un obiettivo 60X con eccitazione GFP / cubo di emissione (AMG EVOS fl). </li><li> Diluire un&#39;aliquota di 100 ml di ciascuna coltura in vitro a 1:10 con sterile H 2 O. Fai il bianco con 100 pl AYE supporti con 1 mM IPTG e 6,25 mg / cm ml e acqua. OD600 prendere misure delle diluizioni utilizzando uno spettrofotometro (Bio-Rad intelligente Spec Plus). Calcolare il volume necessario per infettare un pozzo ad una MOI = 20 per ciascuna coltura in vitro: V = [(ameba seeded) x MOI] / [OD 600 x (fattore di diluizione) x (costante)] = [(1 x 10 6 ) x (20)] / [OD 600 x (10) x (1 x 10 6)] = 2/OD 600. Concentrazione di batteri viene determinato come OD 600 = 1.0 = 1 x 10 9 UFC / ml. </li></ol> 2. Adescamento infezione stage utilizzando Acanthamoeba castellanii <ol><li> Mantenere e coltivare le amebe in ATCC 712 medie PYG in 175 cm 2 flaconi a temperatura ambiente. </li><li> Sostituire media in cul amebeture 24 ore prima di iniziare le colture batteriche liquidi. Lo stesso giorno liquido colture batteriche sono avviati, raccogliere e contare le cellule su un microscopio ottico utilizzando un emocitometro. </li><li> Diluire il amebe con mezzi freschi ad una concentrazione finale di 1 x 10 6 cellule / ml. Seed 12 e piastre di coltura cellulare con aliquote da 1 ml di amebe con un pipettatore a ripetizione ed incubare a RT O / N. </li><li> Dopo l&#39;incubazione, lavare i pozzetti delle piastre di coltura cellulare da 12 pozzetti 3x con 1 ml di PBS sterile utilizzando una pipetta 10 ml sierologica e un aiuto pipetta manuale. </li><li> Dopo l&#39;aspirazione di PBS, aggiungere 1 ml di infezione supporti (ATCC 712 supporti PYG meno glucosio, peptone, estratto di lievito e) con IPTG 1 mM e 6,25 mcg / ml cm a ciascun pozzetto. Incubare le piastre a RT per 1 ora. </li><li> Infettare pozzetti ad una MOI = 20. Centrifugare la piastra a 400 xg per 5 min (Eppendorf 5810R) e galleggiare in un bagno di acqua 37 ° C per 5 min. Trasferire la piastra ad un 37 ° C, 5% CO 2 incubatore per 18 ore. </li><li> Confermare le infezioni utilizzando l&#39;imaging dal vivo cella di un microscopio a fluorescenza prima di ospitare la lisi cellulare e la raccolta di batteri. </li></ol> 3. Semina cellule THP-1 per l&#39;infezione fase cella obiettivo <ol><li> (Inizia questo processo 24 ore prima di impostare liquidi colture batteriche). Coltivare cellule THP-1 in un cm 75 due fiasche per colture a quasi confluenza in RPMI 1640 con 10% di siero siero fetale bovino (FBS). </li><li> Contare le cellule in sospensione utilizzando un emocitometro, diluire le cellule THP-1 in RPMI 1640 con 10% di FBS e 100 ng / ml forbol-12-miristato-13-acetato (PMA) ad una concentrazione di 1 x 10 6 cellule / ml , e la piastra in aliquote da 1 ml su piastre di coltura cellulare da 12 pozzetti. Incubare le piastre per 48 ore a 37 ° C, 5% di CO 2. </li></ol> 4. Lavorazione dei batteri per infezione fase cella obiettivo dopo l&#39;infezione stage adescamento <ol><li> Aspirare il supporto dal amebe innescato. </li><li> Lyse l&#39;unamoebae utilizzando 500 pl di ghiaccio freddo, sterile ultra-filtrata (UF) H 2 O ed incubare a temperatura ambiente per 10 min. </li><li> Pool I lisati secondo ceppo e prendere una misura E 512 nm per ciascuno dei lisati aggregati utilizzando un lettore di piastra di fluorescenza (Molecular Devices Spectramax Gemini EM). </li><li> Calcolare un misura 600 DO per tutti i lisati pool: calcOD 600 = 0,0008 (E 512 - sfondo lisato) + 0,0019. La formula è stata precedentemente determinate dal grafico di un confronto diretto sia la DO 600 e la E 512 nm misure di diluizioni di L. pneumophila GFP di tipo esprimendo fase stazionaria da coltura in vitro (Figura 1 *). I lisati di ameba non infetto, alle stesse condizioni sperimentali l&#39;ameba infetta, sono usati come bianco e fornito un valore di correzione (lisato es sfondo), che è stato accolto nell&#39;equazione. La t volume necessarioo infettare un pozzo ad una MOI = 20 viene calcolato per ciascun pool lisato: V = [(THP-1 seminate) x MOI] / [calcOD 600 x (costante)] = [(1 x 10 6) x (20)] / [calcOD 600 x (1 x 10 6)] = 20/600 calcOD. </li><li> Lavare le cellule THP-1 3x con PBS e aggiungere 1 ml fresco RPMI 1640 (10% FBS) in ciascun pozzetto. Incubare le cellule THP-1 per 1 ora a 37 ° C, 5% di CO 2. </li><li> Infettare le cellule THP-1 al MOI calcolato utilizzando i lisati pool. Permettono una serie di pozzi di rimanere non infetto, serve come controllo negativo per l&#39;analisi citofluorimetrica. Elaborazione dello stadio primer deve essere completato in meno di 30 minuti. I lisati sono tenuti su ghiaccio per limitare eventuali cambiamenti nell&#39;espressione genica batterica prima dell&#39;infezione. </li><li> Centrifugare la piastra, galleggiare per aumentare la temperatura, e incubare come nella fase di adescamento. </li><li> Un&#39;ora dopo l&#39;infezione, rimuovere il supporto mediante aspirazione e lavare i pozzetti 3 volte con PBS per rimuovere i batteri extracellulari. </li><li> Aggiungere 1 ml di fresh RPMI 1640 (10% FBS) ai pozzetti e restituire la piastra al incubatore. Il tempo subito dopo la sostituzione dei supporti serve come tempo zero (T 0). Incubare le cellule THP-1 per 14-16 ore a 37 ° C, 5% di CO 2. </li></ol> 5. Analisi sperimentale 5,1 imaging cellulare in diretta Immagine i pozzetti infetti utilizzando un microscopio a fluorescenza (AMG EVOS fl) a 10X o 20X di ingrandimento (Figure 1A-D). Le immagini possono essere confrontati qualitativa o quantitativa per determinare i livelli di infezione sia in fase di innesco e di destinazione infezioni stadio cellulari. 5,2 Citometria a flusso <ol><li> I picchi di emissione di diverse infezioni possono essere confrontati mediante citometria a flusso (BD FACS Calibur). Tripsinizzare gli infetti cellule THP-1 e delicatamente di lavaggio dai pozzetti mescolando in PBS con una pipetta. </li><li> Piscina le cellule tipo versare in 15 ml Falcon tubi e pellet a 1.800 xg per 2min nella centrifuga piano del tavolo. </li><li> Sospendere il pellet in 1 ml di PBS e trasferire ai tubi microcentrifuga da 1,5 ml. </li><li> Centrifugare le sospensioni a 1.800 x g (Eppendorf 5424) e risospendere il pellet in 1 ml di PBS. Se le sospensioni risultanti sono altamente torbidi (OD 600 ≥ 1,0) devono essere diluiti in PBS supplementare (1:3) per impedire l&#39;intasamento delle linee del citofluorimetro. </li><li> Utilizzare sterile filtrata PBS per l&#39;equilibramento della citometria a flusso e per le fasi di lavaggio tra le iniezioni del campione. Tracciare l&#39;avanti / lato dispersione di cellule bersaglio infettate prima iniezione di campioni di cellule bersaglio di infezione. </li><li> Raccogliere 20.000 eventi totali per ogni condizione con 488 nm di eccitazione laser e il canale FL1. </li><li> Porta post-acquisizione popolazioni cellulari di escludere le cellule non infette e l&#39;intensità trama fluorescenza in un istogramma (FlowJo) (Figura 1E). </li></ol> 5,3 CFU placcatura <ol><li> Esaminare l&#39;efficienza of infezione mediante placcatura CFU delle cellule raccolte per citometria a flusso. Preparare diluizioni seriali dei campioni in ultra-filtrata H 2 O a 10 -1, 10 -2 e 10 -3. </li><li> Piastra 20 pl di ciascuna diluizione su 1/3 di una piastra CYEA con 6,25 mg / ml CM. </li><li> Incubare le piastre a 37 ° C per 72 ore. </li><li> Contare le colonie con uno stuzzicadenti e contatore di cellule. </li></ol>

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Gli autori dichiarano di non avere conflitto di interessi finanziari.

Espressione genica batterico è strettamente controllata mediante una combinazione di progressione del ciclo di vita e di risposta ai segnali nel microambiente circostante. Vacuolare patogeni come L. pneumophila rispondere a una moltitudine di cellula ospite derivati ​​spunti quando compartimenti stagni in un phagosome. Come risultato collettivo di esaurimento nutrienti nella cellula ospite, il batterio compensa esprimendo fattori richiesti per diffusione successo ad una cellula ospite successivo 25.</...

Numerosi batteri patogeni si sono adattati strategie generalizzate per sfruttare cellule ospiti per la sopravvivenza e la replicazione in un compartimento intracellulare. In molti casi, i meccanismi patogenetici sono simili tra protozoi e metazoi le cellule. Tuttavia, questi due microambienti sono molto diversi e possono provocare un&#39;espressione differenziale di fattori di virulenza 1-4. Il batterio della malattia del legionario &#39;Legionella pneumophila è ubiquitariamente associati agli ambienti d&#39;acqua dolce in tutto il mondo 5. È importante sottolineare che, L. pneumophila coltivato in cellule protozoarie prima infezione dei monociti umani acquisire un vantaggio patogeno, suggerendo che globali profili di espressione genica del batterio uscita una cella protozoo sono diverse da quella del coltivati ​​in vitro dell&#39;organismo 6-8. In natura, amebe acqua dolce forniscono nutrienti confini ricchi per l&#39;amplificazione rapida di un batterio invasore. Acquisizione umana della L. pneumophila è spesso attribuita a inalazione di goccioline di acqua contaminata che contengono il batterio. È probabile che queste goccioline porto protozoi cellula batteri associati; dove cellule protozoarie sono più resistenti alle pratiche convenzionali di trattamento delle acque 9,10. Infezione di macrofagi alveolari polmonari procede in modo quasi identico al ciclo di vita del batterio intracellulare in cellule ospiti protozoi 11-13. Per sopravvivere e replicarsi in cellule eucariotiche, L. pneumophila utilizza uno speciale sistema di secrezione di tipo IV ter chiamato Dot / Icm per fornire circa 300 &quot;effettrici&quot; proteine ​​nel citosol della cellula ospite 14-16. Queste proteine ​​effettrici collettivamente funzionamento per sovvertire processi cellulari per generare un vano permissive per la replicazione del batterio 17,18. Delezioni in qualsiasi dei 26 geni che costituiscono il Dot / Icm risultato trasportatore in ceppi difettosi per mult intracellulareiplication 19-23. Storicamente, cancellazione di singoli geni codificanti effettori raramente portato in ceppi attenuati di crescita intracellulare. Questo fenomeno è stato attribuito a diverse ipotesi tra cui la funzione ridondante e copie paralogous di effettori. Alcuni fattori di virulenza sono espressi solo nel contesto della cellula ospite associata crescita intracellulare 24. Abbiamo razionalizzato che se un effettore particolare è stato espresso soltanto nel contesto di infezione protozoo, quindi il contributo del effector non può essere confrontato con un ceppo selvatico quando entrambi sono stati coltivati ​​in vitro. L. pneumophila transizioni da un replicativa a una fase trasmissivo, che entra nella fase stazionaria cultura 25. Il fenotipo di commutazione fase rappresenta l&#39;esaurimento degli elementi nutritivi incontrato durante la crescita intracellulare ed è esemplificata attraverso il montaggio di flagelli per la motilità 26. Perché L. pneumophila è più invaSIVE e virulenta quando raccolto da cellule protozoi, abbiamo cercato di sviluppare un metodo che più fedelmente rappresentato lo stato patogeno del batterio quando ha incontrato macrofagi host. A tal fine, abbiamo sviluppato un saggio di adescamento protozoo versatile in grado di ospitare qualsiasi host adatto sia per i primi (cella priming) e la seconda (cella di destinazione) infezioni stadio. Il processo di infezione è trattabile mediante uso di batteri che esprimono stabilmente la proteina fluorescente verde (GFP). Il modello di infezione per il protozoo Acanthamoeba castellanii segue una metodologia ampiamente usato nel campo 27. Per la fase di caricamento, L. ceppi pneumophila sono coltivati ​​in vitro alla fase stazionaria in mezzi liquidi per produrre etichettati &#39;trasmissive&#39; batteri (Figura 1A). I batteri vengono quindi utilizzati per infettare monostrati di A. castellanii per 18 ore per ottenere una fase avanzata del ciclo di vita intracellulare. Vacuoli di grandi dimensioni contengonobatteri zione può essere visualizzata a questo punto di tempo utilizzando microscopia a fluorescenza (Figura 1A). Protozoi cellule vengono lisate e batteri recuperati dal lisato sono misurati per l&#39;emissione a 512 nm usando un lettore di fluorescenza piastra. Fluorescenza è correlata con la densità ottica per calcolare molteplicità d&#39;infezione (MOI) per l&#39;infezione di cellule bersaglio (Figura 1, Correlazione * Curve). Dopo ore invasione (T 0) e 18 post-invasione (T 18), le cellule bersaglio sono quantificati per la fluorescenza, che rappresenta i batteri intracellulari. Fluorescenza può essere monitorato mediante microscopia e citometria a flusso, e la conta può essere misurata mediante placcatura colonia. Il test di adescamento è sempre accompagnato da infezioni da wild-type L. pneumophila e un ceppo difettosa in Dot / Icm sistema secrezione di tipo IV (Δ DOTA) (Figura 1A). Ciò fornisce soprattutto controlli interni per i confronti diretti tra wild-type di unnd eventuali ceppi mutanti isogenici utilizzati nel processo di infezione. L&#39;inclusione del ceppo virulento Δ DOTA durante la fase di adescamento imposta una soglia per l&#39;osservazione della crescita attenuati fenotipi associati isogeniche ceppi mutanti che sono coltivate in vitro.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>chloramphenicol</td>
 <td>Fisher Scientific</td>
 <td>BP904-100</td>
 <td>antibiotic</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Fisher Scientific</td>
 <td>BP1755-10</td>
 <td></td>
 </tr>
 <tr>
 <td>ACES</td>
 <td>Sigma</td>
 <td>A9758-1KG</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>ATCC medium: 712 PYG</td>
 <td>ATCC</td>
 <td></td>
 <td>growth media for protozoans</td>
 </tr>
 <tr>
 <td>1X PBS</td>
 <td>Fisher Scientific</td>
 <td>SH30256FS</td>
 <td>phosphate buffered saline</td>
 </tr>
 <tr>
 <td>activated charcoal</td>
 <td>Fisher Scientific</td>
 <td>C272-212</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>yeast extract</td>
 <td>Fisher Scientific</td>
 <td>BP1422-500</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>peptone</td>
 <td>BD Diagnostics</td>
 <td>211677</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>Fisher Scientific</td>
 <td>BP1423-2</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>L-cysteine, 99%+</td>
 <td>Acros organics</td>
 <td>173601000</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td>Ferric nitrate nonahydrate</td>
 <td>Fisher Scientific</td>
 <td>I110-100</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>EVOS fl</td>
 <td>AMG</td>
 <td>EVOS fl</td>
 <td>fluorescence microscope</td>
 </tr>
 <tr>
 <td>Smart Spec Plus</td>
 <td>Bio-Rad</td>
 <td>170-2525</td>
 <td>spectrophotometer</td>
 </tr>
 <tr>
 <td>5810 R centrifuge</td>
 <td>Eppendorf</td>
 <td>22627023</td>
 <td>bench top centrifuge</td>
 </tr>
 <tr>
 <td>Repeater plus</td>
 <td>Eppendorf</td>
 <td>22230201</td>
 <td>repeating pipette</td>
 </tr>
 <tr>
 <td>SpectraMax Gemini EM</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>microplate reader</td>
 </tr>
 <tr>
 <td>Softmax Pro 5.3</td>
 <td>Molecular Devices</td>
 <td>0200-310</td>
 <td>microplate reader software</td>
 </tr>
 <tr>
 <td>5424 microfuge</td>
 <td>Eppendorf</td>
 <td>22620401</td>
 <td>table top microcentrifuge</td>
 </tr>
 <tr>
 <td>Fast-release pipette pump II</td>
 <td>Scienceware</td>
 <td>379111010</td>
 <td>pipette aid</td>
 </tr>
 <tr>
 <td>FACS Calibur</td>
 <td>BD Bioscience</td>
 <td></td>
 <td>flow cytometer</td>
 </tr>
 <tr>
 <td>FlowJo 7.6.1</td>
 <td>FlowJo</td>
 <td>license</td>
 <td>flow cytometery software</td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>polypropylene conical tube</td>
 </tr>
 <tr>
 <td>1.6 ml microfuge tube</td>
 <td>Neptune</td>
 <td>3745.X</td>
 <td>microcentrifuge tubes</td>
 </tr>
 </tbody>
</table>

tractable mammalian cell infections with protozoan-primed bacteria

Molti batteri patogeni intracellulari utilizzare protozoi d&#39;acqua dolce come un serbatoio naturale per la proliferazione nell&#39;ambiente. Legionella pneumophila, l&#39;agente eziologico della legionario &#39;polmonite, guadagna un vantaggio su di batteri patogeni in vitro in coltura al momento della prima raccolti da cellule protozoi prima infezione di macrofagi mammiferi. Questo suggerisce che i fattori di virulenza importanti non possono essere adeguatamente espresse in vitro. Abbiamo sviluppato un sistema per l&#39;adescamento trattabile L. pneumophila attraverso il suo naturale ospite protozoo Acanthamoeba castellanii prima dell&#39;infezione cellula di mammifero. Il contributo di ogni fattore di virulenza può essere esaminata confrontando la crescita intracellulare di un ceppo mutante di wild-type batteri dopo aver riempito protozoo. GFP che esprimono L. wild-type e mutante ceppi pneumophila sono utilizzati per infettare monostrati protozoi in fase di caricamento, e ha permesso di raggiungere ultime fasi della crescita intracellulare. Batteri fluorescenti vengono quindi raccolte da queste cellule infette e normalizzati per spettrofotometria per generare numeri comparabili di batteri per una successiva infezione in macrofagi mammiferi. Per la quantificazione, batteri vivi sono monitorati dopo l&#39;infezione usando la microscopia a fluorescenza, citometria a flusso, e mediante placcatura colonia. Questa tecnica mette in evidenza e si avvale del contributo della cellula ospite-dipendente l&#39;espressione del gene imitando l&#39;ambiente che si può incontrare in un percorso di acquisizione naturale. Questo approccio può essere modificato per ospitare qualsiasi batterio che utilizza un host intermediario come un mezzo per ottenere un vantaggio patogeno.

Un risultato tipico per l&#39;intero processo di infezione è descritto nella Figura 1. Vivo delle cellule al microscopio a fluorescenza che raffigurano monostrati di A.castellanii infettati con wild-type L. pneumophila durante la fase di adescamento è mostrato nella Figura 1A. Una misura di successo della fase di caricamento porterebbe ad una popolazione di circa il 90% delle cellule ospiti contenenti grandi vacuoli popolate con batteri GFP etichettati in questo MOI....

Watch this Scientific Journal Video about Tractable Mammalian Cell Infections with Protozoan-primed Bacteria at JoVE.com

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Questa tecnica fornisce un metodo per raccogliere, normalizzare e quantificare la crescita intracellulare di batteri patogeni che sono pre-coltivate in cellule naturali ospiti protozoi prima di infezioni di cellule di mammifero. Questo metodo può essere modificato per accogliere una grande varietà di cellule ospiti per la fase di adescamento e tipi di cellule bersaglio.

Trattabili infezioni cellula di mammifero con protozoo-innescate batteri

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

tractable-mammalian-cell-infections-with-protozoan-primed-bacteria

Research

JoVE Journal

Immunology and Infection

10.4K Views.  Oregon Health & Science University. Questa tecnica fornisce un metodo per raccogliere, normalizzare e quantificare la crescita intracellulare di batteri patogeni che sono pre-coltivate in cellule naturali ospiti protozoi prima di infezioni di cellule di mammifero. Questo metodo può essere modificato per accogliere una grande varietà di cellule ospiti per la fase di adescamento e tipi di cellule bersaglio.

Video: Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Utilizzando luciferasi di infezioni batteriche immagine in topi

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Ricerca

Immunologia e infezioni

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generazione di Multivirus-cellule T specifiche per prevenire / curare infezioni virali dopo trapianto allogenico di cellule staminali ematopoietiche

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D imaging multimodale di<em&gt; Citrobacter rodentium</em&gt; Infezioni in topi

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Microscopia a fluorescenza metodi per determinare la vitalità di batteri in associazione con cellule di mammifero

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

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 ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis come organismo modello per la Valutazione Interazione di batteri anaerobici con cellule ospiti

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Utilizzando modelli di Zebrafish di umana virus infezioni allo schermo farmaci antivirali e caratterizzare host immune cellulo Risposte

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

L&#39;analisi dei linfociti stravaso utilizzo di un<em&gt; In Vitro</em&gt; Modello del Sangue-Cervello umano Barriera

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Il WinCF Modello - Un Microcosmo Economico e Tractable di un muco bronchiale Plugged per studiare la Microbiologia del polmone Infezioni

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...