Gli autori desiderano ringraziare Richard Riposo per il dono generoso di anthrolysin O, Michael Caparon per il generoso dono dei Franchi SLO plasmide e Jonathon per l&#39;assistenza tecnica. Questo lavoro è stato finanziato dal NIH sovvenzioni T32CA82084 (PAK), e R01AI072083 (RDS).

1. Purificazione di streptolisina O (SLO) <ol><li> Inoculare 20 ml di coltura overnight cellule BL21 ORO contenenti plasmide pBADgIII-SLOhis 9 in 0,5 L di brodo LB e aggiungere 500 microlitri 50 mg / ml ampicillina. Agitare coltura a 225 rpm a 37 ° C fino a OD 600 = 0,6, di solito ~ 1,5 hr. Centrifugare 1 batteri ml (considerato T 0) a 10.000 xg 5 minuti, sciogliere pellet in 140 microlitri 1x tampone campione SDS / 1 OD e sonicare al DNA di taglio per l&#39;analisi delle proteine ​​purezza. </li><li> Indurre i batteri con l&#39;aggiunta di 5 ml arabinosio 20% alla cultura, agitare a 225 rpm a temperatura ambiente per 3 ore. Raccogliere 1 ml di batteri, centrifugare e sciogliere in 1x tampone campione SDS come sopra, per T a 3 punti il tempo per l&#39;analisi della purezza. </li><li> Raccogliere i batteri rimasti in flacone da 500 ml centrifuga, spin 12.000 xg per 12 min a 4 ° C (8.500 rpm Sorvall GS-3 rotore). Decantare il surnatante, negozio di pellet a -80 ° C per una notte o più a lungo se necessario. </li><li> Risospendere il frozen pellet su ghiaccio in 10 ml Lyse / tampone di lavaggio (50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM imidazolo, pH 8,0) supplementato con 400 microlitri 25% Triton X-100, 100 pl 100 mg / ml lisozima, 50 200 microlitri fenilmetilsulfonil fluoruro mM (PMSF). Trasferire il sedimento risospeso a 50 ml di tubo di Oak Ridge. </li><li> Sonicare lisato 5 volte per 30 secondi su ghiaccio con intervalli di 30 sec. Spin sonicata lisato in tubo di Oak Ridge a 39000 xg per 20 minuti a 4 ° C (18.000 min Sorvall SS-34 rotore). </li><li> Mentre il lisato è in rotazione, lavare 1 ml Ni-NTA agarosio con 10 ml Lyse / tampone di lavaggio e centrifugare a 1200 rpm per 5 min a 4 ° C. (Tutti gli altri lavaggi / eluizioni utilizzare la centrifugazione in queste condizioni). Aspirare lavare. </li><li> Aggiungi surnatante batterica dal punto 1.5 a Ni-NTA agarosio, agitare delicatamente per 2,5 ore a 4 ° C. Spin per raccogliere il supernatante. Unire 30 ml di surnatante con 10 μ 4x tampone campione SDS e risparmiare per l&#39;analisi della purezza. Lavare le perline 4 volte in Lyse / Tampone di lavaggio ece in Tris / sale tampone (50 mM Tris, 300 mM NaCl, pH 8,0). </li><li> Eluire 3 volte aggiungendo 1 ml di tampone di eluizione (250 mM imidazolo in tampone Tris / sale buffer) e 5 microlitri 1 M ditiotreitolo (DTT) a perline, incubare su ghiaccio per 10 min, filatura e raccogliere il surnatante. SLO è molto redox-sensibili proteine, e deve essere tenuta in ghiaccio in ogni momento. Una volta ridotta, la proteina si perde rapidamente la sua attività se riscaldato a 37 ° C, anche per un breve periodo di tempo. </li><li> Lavare 500 pl polimixina-coniugato agarosio in tampone di eluizione, centrifugare a 10.000 xg per 1 min a 4 ° C e risospendere in 500 microlitri. Per esaurire endotossina, aggiungere 200 biglie pl ad ogni eluizione e agitare 4 ° C per 30 min. Centrifuga a 10.000 xg per 1 min a 4 ° C, e salvare il surnatante. Combinare 30 microlitri di ogni eluizione con 10 microlitri 4x tampone campione SDS per analisi SDS-PAGE. </li><li> Verificare la purezza eluizioni mediante SDS-PAGE. Campioni bollire salvate per analisi di gel a 95 ° C per 5 min e 10 pl di carico su un gel al 10%. Stain gel con Coomassie per visualizzare proteina (Figura 2A). SLO è di 69 kDa. Eseguire un saggio Bradford per determinare la concentrazione della proteina (resa tipica è di 4 mg / ml per i primi due eluizioni, ma può variare da ceppo batterico e plasmide usato). </li><li> Testare l&#39;attività emolitica di ogni eluizione (vedi sotto). Eluizioni piscina dove l&#39;attività di purezza, concentrazione e emolitica sono soddisfacenti. SLO Aliquotare in monouso aliquote (pl tipicamente 5-10), in ghiaccio secco congelare e conservare a -80 ° C. </li></ol> 2. Saggio emolitica <ol><li> Lavare eritrociti di pecora (RBC) in tampone RBC Assay (0,3% di sieroalbumina bovina (BSA), 2 mM CaCl 2, 10 mM Hepes, pH 7,4), centrifugare a 1200 rpm per 5 min, e risospendere al 2,5% in RBC 10 ml tampone del saggio RBC. </li><li> Aggiungere 10 microlitri tampone di saggio RBC / pozzetto in 96 pozzetti V-parte inferiore piastra su ghiaccio. Serialmente diluire ogni eluizione in duplicato. Campi di diluizione tipici sono 1:1.000 a 1:512,000. Include 3 pozzi senza tossina e 3 pozzi con 10microlitri 2% Triton X-100 come pozzi minimo e massimo, rispettivamente. </li><li> Coprire la piastra ed incubare a 37 ° C 30 min. Centrifugare a 1200 rpm per 5 min. Trasferire 70 ml di surnatante in un fondo piatto da 96 pozzetti. Leggi A 405. Una unità è la diluizione della tossina necessaria per il 50% di lisi dei globuli rossi. Attività di tossina è 1 unità * diluizione factor/0.01 ml. </li></ol> 3. Cella saggio litico <ol><li> Cellule Harvest, conteggio, spin. Risospendere a 2x10 6 / ml in tampone di R / B (RPMI con 2 mM CaCl 2, 0,5% BSA) e 20 ug / ml di ioduro di propidio. Aggiungere 100 ul di cellule a 96 pozzetti V-piastra inferiore. </li><li> Diluire serialmente SLO a 2x concentrazione finale in tampone R / B, aggiungere 100 microlitri tossina o 100 microlitri di buffer R / B per le cellule. Incubare 5 min 37 ° C. Distanza tipica finale concentrazioni da 2.000 U / ml a 31,25 U / ml. </li><li> Eseguire le cellule al citofluorimetro e raccogliere i dati con filtri per ficoeritrina (PE). Un&#39;unica log turno rappresenta cel transitoriamente permeabilizzateLS mentre un 3 turni registro indica le cellule morte 4. Calcolare la lisi specifica delle cellule sottraendo la percentuale di cellule morte nel controllo (% PI alta ctl) dalla (exp% PI alta) sperimentale, come segue: lisi specifica = (% exp PI alta -% PI alta ctl) / (100 -% PI alta ctl) * 100 </li></ol> 4. Micropipetta Consegna di tossina di cellule in coltura <ol><li> Un giorno prima dell&#39;esperimento, piastra 2x10 5 macrofagi in un collagene rivestito con fondo di vetro piastra da 35 mm. </li><li> Accendere il microscopio e microiniettore. Lasciare il tempo per riscaldarsi fase riscaldata a 37 ° C. La scelta del microscopio dipende di solito su ciò che è localmente disponibile. Il microscopio richiede una fase invertita, una fase in grado di riscaldare i piatti a 37 ° C, eccitazione / emissione cubi filtro appropriato per il colorante scelto, e lo spazio collegare fisicamente il microiniettore. Una lente Bertrandè utile per microiniezione, ma non obbligatorio. Il computer guida il microscopio ha bisogno di memoria sufficiente per raccogliere e memorizzare i dati. </li><li> Cellule etichette per 30 minuti con la tintura a 37 ° C. A seconda del dosaggio, etichettatura può essere fatto con 5 Fura2 pl AM in 1 ml di PBS o 2 microlitri calceina AM in 1 ml mezzi completi. Per Fura2, eccitazione / emissione viene raccolta per 340/510 nm e 380/510 nm, e il rapporto di 340/380 determina segnali flusso di calcio. Per calceina, eccitazione / emissione è 495/515 nm, mentre etidio omodimero è da 525/620 nm e APC è 650/660 nm. Etichette possono essere scelti altri pure. </li><li> Lavare le cellule con PBS e posto in 1 ml RPMI supplementato con 2 mM CaCl 2. Montare il microscopio. </li><li> Diluire tossina e destrano in acqua, e centrifugare a 20.000 xg per 10 min a 4 ° C. Tipicamente, 1 pl SLO e 4 microlitri 10 mg / ml destrano-555 viene diluito in acqua 6 microlitri. Destrano fluorescente o un&#39;altra fase fluida molecola viene utilizzato per verificare che il femto-punta non è ostruito, e injects come desiderato. </li><li> Caricare femto-punta dalla parte posteriore con 0,07 ml di tossina diluito utilizzando un Microloader. </li><li> Caricare femto-punta su microiniettore. Regolare l&#39;angolo di iniettore in modo che la punta si siede sul centro delle cellule con spazio per muoversi in tutte le direzioni. Cancella z-limite. Impostazioni di iniezione devono essere iniezione per 0,5 secondi a 120 psi a 20 psi di pressione posteriore. Abbassare punta fino a quando non entra nel terreno. </li><li> Utilizzando la lente di Bertrand, centro della punta e seguire la punta come viene abbassato più vicino alle cellule. Una volta perdere la concentrazione, tornare a ottiche normali. L&#39;ombra ago deve essere evidente nel campo. Concentrarsi sopra le celle e abbassare l&#39;ago finché non è a fuoco. Concentrarsi indietro sulle cellule e accuratamente portare l&#39;ago adiacente a una cella. Impostare il z-limite per l&#39;iniezione. Iniziare immagini, spostare punta nella posizione desiderata, iniettare la tossina di rilasciare al punto di tempo desiderato. Sollevare l&#39;ago, passare a una nuova regione di celle e iniettare. Spostare l&#39;ago alla posizione iniziale per evitare indesiderateperdite tossina dall&#39;ago. </li></ol>

<ol>
	<li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+regulates+IL-1+beta+processing+via+calcium-independent+phospholipase+A2.">Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.</a> J. Immunol. 164, 5120-5124 (2000).</li><li>Scolding, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+removal+by+oligodendrocytes+of+membrane+attack+complexes+formed+by+activated+complement.">Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.</a> Nature. 339, 620-622 (1989).</li><li>Keyel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+O+clearance+through+sequestration+into+blebs+that+bud+passively+from+the+plasma+membrane.">Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.</a> J. Cell. Sci. 124, 2414-2423 (2011).</li><li>MacKenzie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+secretion+of+interleukin-1beta+by+microvesicle+shedding.">Rapid secretion of interleukin-1beta by microvesicle shedding.</a> Immunity. 15, 825-835 (2001).</li><li>Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+IL-1+beta+secretion+stimulated+by+P2X7+receptors+is+dependent+on+inflammasome+activation+and+correlated+with+exosome+release+in+murine+macrophages.">Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.</a> J. Immunol. 179, 1913-1925 (2007).</li><li>Andrei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+C+and+A2+control+lysosome-mediated+IL-1+beta+secretion:+Implications+for+inflammatory+processes.">Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).</li><li>Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat. Immunol. 10, 241-247 (2009).</li><li>Magassa, N., Chandrasekaran, S., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+cytolysin-mediated+translocation+does+not+require+pore+formation+by+streptolysin+O.">Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.</a> EMBO Rep. 11, 400-405 (2010).</li><li>Pinkney, M., Beachey, E., Kehoe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiol-activated+toxin+streptolysin+O+does+not+require+a+thiol+group+for+cytolytic+activity.">The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.</a> Infect. Immun. 57, 2553-2558 (1989).</li><li>Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+immune+cells+mediated+by+tunneling+nanotubules.">Functional connectivity between immune cells mediated by tunneling nanotubules.</a> Immunity. 23, 309-318 (2005).</li><li>McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patching+plasma+membrane+disruptions+with+cytoplasmic+membrane.">Patching plasma membrane disruptions with cytoplasmic membrane.</a> J. Cell. Sci. 113, 1891-1902 (2000).</li><li>Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anthrolysin+O,+the+Bacillus+anthracis+cholesterol-dependent+cytolysin.">Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.</a> Infect. Immun. 71, 3183-3189 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

Le tecniche qui descritte consentono l&#39;esame delle risposte delle cellule immunitarie di tossine batteriche. La fase più critica è la manipolazione e somministrazione della tossina. Attività tossina può essere estremamente variabile, anche tra differenti aliquote del preparato stesso, per la sua fragilità. Ciò richiede sia la prova di ogni aliquota di tossina contro una linea cellulare di riferimento o globuli rossi o utilizzando gradienti tossina. Gradienti di tossina, come espresso dal micropipetta, permetto...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Ni-NTA agarosio </td><td> Qiagen </td><td> 30210 </td><td></td></tr><tr><td> polimixina-agarosio </td><td> Sigma </td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba desalt Spin col </td><td> Pescatore </td><td> PI-89891 </td><td></td></tr><tr><td> globuli rossi di pecora </td><td> Pescatore </td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> vedi rif 9 </td></tr><tr><td> Cy5 colorante monoreactive </td><td> GE Healthcare </td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies </td><td> F1221 </td><td></td></tr><tr><td> Calcein AM / Ethidium omodimero </td><td> Life Technologies </td><td> L3224 </td><td></td></tr><tr><td> Anti-CD11c-APC </td><td> BD Biosciences </td><td> 550261 </td><td></td></tr><tr><td> collagene rivestito con fondo di vetro piatto </td><td> Mattek </td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td> femto-tip II </td><td> Pescatore </td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td> Pescatore </td><td> E5242956003 </td><td></td></tr><tr><td> destrano Alexa 555 </td><td> Life Technologies </td><td> D34679 </td><td></td></tr><tr><td> InjectMan NI 2 </td><td> Eppendorf </td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf </td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 1. Elenco e fonte di reagenti specifici e equipment necessario. Attrezzature e reagenti utilizzati in questo protocollo, insieme con l&#39;azienda e il numero di catalogo sono elencati. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Buffer </td><td> Composizione </td><td> Passo Usato </td></tr><tr><td> Lyse / lavaggio </td><td> 50 mM NaH 2 PO 4 300 mM NaCl Imidazolo 10 mM, pH 8,0 </td><td> 1,4 </td></tr><tr><td> Tris / sale </td><td> 50 mM Tris, pH 8,0 300 mM NaCl </td><td> 1,7 </td></tr><tr><td> Tampone di eluizione </td><td> 50 mM Tris, pH 8,0 300 mM NaCl 250 mM imidazolo </td><td> 1,8 </td></tr><tr><td> RBC Assay Buffer </td><td> 0,3% BSA 2 mM CaCl 2 HEPES 10 mM, pH 7,4 </td><td> 2,1 </td></tr><tr><td> buffer di R / B </td><td> Mezzo di coltura cellulare RPMI 2 mM CaCl 2 0,5% BSA </td><td&gt; 3.1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> Tabella 2. Lista di buffer utilizzati in questo protocollo. I tamponi usati, la loro composizione e il primo passo in cui sono utilizzati nel protocollo sono elencati. </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Tossine batteriche legano al colesterolo nelle membrane, formando pori che permettono la fuoriuscita di contenuto cellulare e afflusso di materiali dall&#39;ambiente esterno. La cella può recuperare da questo insulto, che richiede processi di riparazione membrane attive, oppure muoiono a seconda della quantità di esposizione tossina e tipo cellulare 1. Inoltre, queste tossine indurre forti risposte infiammatorie in host infetti attraverso l&#39;attivazione di cellule immunitarie, compresi i macrofagi, che producono una matrice di citochine pro-infiammatorie 2. Molti batteri Gram-positivi producono colesterolo vincolante tossine che hanno dimostrato di contribuire alla loro virulenza attraverso meccanismi in gran parte non ancora caratterizzati. Alterazioni morfologiche della membrana plasmatica di cellule esposte a tali tossine includere il sequestro in colesterolo arricchiti protuberanze superficiali, che può essere versato nello spazio extracellulare, suggerendo una difesa intrinseca cellulareMeccanismo 3,4. Questo processo si verifica in tutte le cellule in assenza di attività metabolica, e possono essere visualizzati utilizzando EM dopo 4 chimica fissazione. In cellule immunitarie quali macrofagi che mediano l&#39;infiammazione in risposta all&#39;esposizione tossina, vescicole di membrana indotte sono suggeriti per contenere citochine della famiglia IL-1 e può essere responsabile sia spargimento tossina e diffondere queste citochine pro-infiammatorie 5,6,7. Un collegamento tra IL-1β rilascio e un tipo specifico di morte cellulare, pyroptosis chiamato è stato suggerito, in quanto entrambi sono processi caspasi-1 8 dipendenti. Per risolvere la complessità di questa risposta dei macrofagi, che comprende vincolante tossina, spargimento di vescicole di membrana, rilascio di citochine, e la morte delle cellule potenzialmente, abbiamo sviluppato le tecniche di etichettatura e metodi di microscopia a fluorescenza che consentono la visualizzazione in tempo reale della tossina cellule interazioni, tra cui misurazioni di disfunzioni e morte (Figura 1). Utilizzaredi imaging cellulare dal vivo è necessario a causa di limiti di altre tecniche. Approcci biochimici non possono risolvere effetti che si verificano in cellule individuali, mentre citofluorimetria non offre alta risoluzione, visualizzazione in tempo reale delle singole celle. I metodi qui descritti possono essere applicati ad analisi cinetica delle risposte indotte da altri stimoli implicano complesse variazioni fenotipiche nelle cellule.

Tipicamente 10 7 -10 8 U / ml SLO può essere ottenuta con una concentrazione proteica di 4 mg / ml. La quantità di tossina necessaria per lisi cellulare varia con il tipo cellulare, ma di solito è 125-500 U / ml SLO (Figura 2B). Tipi di cellule, come macrofagi può essere più resistenti (4000 U / ml) se altri (linee cellulari T specialmente) sono più sensibili. Queste sensibilità corrispondono SLO disponibile in commercio. Tossina attività diminuisce di circa 2 volte con ogn...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Metodi per purificare il colesterolo vincolante tossina streptolisina O ricombinante da<em&gt; E. coli</em&gt; E visualizzazione di tossina legarsi a vivere cellule eucariotiche sono descritti. Erogazione localizzata di tossina induce cambiamenti rapidi e complessi nelle cellule bersaglio che rivelano nuovi aspetti della biologia tossina.

Visualizzazione delle risposte tossina batterica provocata con diretta microscopia a fluorescenza cellulare

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

visualization-bacterial-toxin-induced-responses-using-live-cell

Research

JoVE Journal

Immunology and Infection

11.7K Views.  University of Pittsburgh School of Medicine. Metodi per purificare il colesterolo vincolante tossina streptolisina O ricombinante da E. coli E visualizzazione di tossina legarsi a vivere cellule eucariotiche sono descritti. Erogazione localizzata di tossina induce cambiamenti rapidi e complessi nelle cellule bersaglio che rivelano nuovi aspetti della biologia tossina.

Video: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizzazione Cell-per-cell trasferimento del virus HIV usando cloni fluorescenti di HIV e Live Microscopia confocale

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Ricerca

Immunologia e infezioni

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Valutazione della risposta stomatica a Live cellule batteriche utilizzando Imaging Whole Leaf

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Cellulare Imaging in diretta di Bacillus subtilis E Streptococcus pneumoniae Utilizzando automatico Time-lapse Microscopia

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Singola cella Analisi Bacillus subtilis I biofilm usando la microscopia a fluorescenza e citometria a flusso

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-cellula Microscopia Video di funghi patogeni fagocitosi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Applicazione di lungo termine colta interferone-γ Enzyme Linked immunospot Assay per la valutazione effettori e memoria delle cellule T risposte a Bovino

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Misurare risposte fisiologiche di<em&gt; Drosophila</em&gt; Neuroni sensoriali a Lipid feromoni utilizza Live Calcio Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantificazione del fagosoma da microscopia di fluorescenza di cella singola

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

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

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

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