We thank Dr. Antonio Virgilio&#160;Failla for providing the FRET acquisition quantification algorithm and Erwin Bohn for providing the pMK-bla and pMK-ova constructs.

1. picco di emissione e Cross-talk Determinazione (vedi anche figura 2) <ol><li> Al fine di determinare i picchi di emissione e la quantità di cross-talk tra il donatore (cumarina derivato V450) e accettore (fluoresceina) coloranti, eseguire scansioni spettrali di cellule marcate con entrambi i fluorofori individuali a 405 nm di eccitazione. </li><li> Determinare un bandwidth 10 nm entro il picco di ogni colorante che verrà utilizzato per i canali donatore e accettore (455-465 nm qui e 525-535 nm). Tracciare l&#39;intensità normalizzata sulla lunghezza d&#39;onda e calcolare la percentuale media di diafonia del colorante donatore nel canale accettore e viceversa (figura 2). </li></ol> 2. Preparazione di Y. enterocolitica ceppi per Cell Culture infezione <ol><li> Utilizzare i seguenti ceppi: WA-314 PMK-ovuli (WA-314 trasformato con i plasmidi PMK-bla 17 e PMK-ovuli 17, rispettivamente) e WA-314ΔYopE PMK-bla (WA-314ΔYopE trasformato con il plasmide PMK-bla 17) </li><li> Almeno due giorni prima l&#39;infezione striscia Y. enterocolitica ceppi WA-314 PMK-ovuli, WA-314 PMK-bla e WA-314ΔE PMK-BLA da crio scorte su piastre LB-agar contenenti antibiotici necessari (kanamicina per WA-314 PMK-bla e WA-314 PMK-ovuli , kanamicina e tetraciclina per WA-314ΔE PMK-bla) e crescere O / N a 27 ° C. Successivamente mantenere le piastre fino a una settimana a 4 ° C. </li><li> Un giorno prima dell&#39;infezione inoculare 3 ml di LB-mezzo contenente antibiotici richiesti con una colonia dei rispettivi ceppi e incubare O / N a 27 ° C in un agitatore a 180 rpm. </li><li> Il giorno di infezione inoculare 2 ml della / coltura O N in 40 ml di LB fresco-mezzo senza antibiotici e incubare per 90 min a 37 ° C in un agitatore a 180 rpm per indurre l&#39;espressione del sistema di secrezione di tipo III. </li><li> Trasferire le culture in un tubo adatto e mantenere le culture in ghiaccio oa 4 ° CD `ora in poi. Centrifugare le colture per 10 min a 5000 xg e 4 ° C. </li><li> Risospendere il pellet batterico in 2-3 ml di PBS freddo ghiaccio. Diluire la sospensione batterica ad una concentrazione di 8 x 10 8 UFC / ml utilizzando uno spettrofotometro. Determinare la corrispondente densità ottica individualmente. Mantenere questa sospensione in ghiaccio fino infettare le cellule HeLa. </li></ol> 3. Cellule placcatura HeLa <ol><li> Un giorno prima seme infezione 1,5 x 10 4 cellule HeLa in DMEM contenente 200 microlitri di siero 10% inattivato al calore fetale bovino (FCS) in pozzetti di una piastra da 96 pozzetti e coltivate in un incubatore CO 2 5% a 37 ° C. Seed tre pozzi per ogni ceppo (tre diversi tempi di incubazione). </li></ol> 4. L&#39;infezione di cellule HeLa e caricamento con CCF4 <ol><li> Infect cellule HeLa aggiungendo 3,5 ml di sospensione batterica al mezzo e mescolare pipettando delicatamente il mezzo due volte. Consentire ai batteri di sedimento sulle cellule ad una MOI (frazione di infezione) di circa 100 batteri per cellula. Per un corso di tempo iniziare infezioni a -90 min, -60 e -30 min min e incubare a 37 ° C in un incubatore CO 2 5% fino 0 min per raggiungere un punto finale comune. </li><li> Quando l&#39;incubazione è terminata, aspirare accuratamente il medium senza rimuovere le cellule HeLa e aggiungere 50 ml di PBS contenente 20 mg / ml di cloramfenicolo per fermare espressione di effettori e probenecid 2,5 mm per ridurre l&#39;esportazione di CCF4. </li><li> A questo punto trasferire la piastra a 96 pozzetti in fase di microscopio e definire punti idonei per una successiva analisi in ciascun pozzetto. Terminate questa fase entro 10 minuti (per i dettagli dell&#39;acquisizione vedere paragrafo 5.1). </li><li> Per bene, aggiungere 70 ml di soluzione di CCF4 / AM carico (soluzione 0,24 ml A, 1,2 ml soluzione B, 18,56 ml soluzione C (soluzione A, B, e C fornite entro CCF4 kit / AM carico) e 50 ml di PBS (contenenti 20 ug / ml di cloramfenicolo e probenecid 2,5 mM) usando un multi pipette e incubare per 5 minuti a temperatura ambiente. D&#39;ora in lavoro senza interruzioni. </li><li> Aspirare la soluzione di carico e aggiungere 100 ml di PBS contenente 20 mg / ml cloramfenicolo e probenecid 2,5 mm con una pipetta a più. Poi il trasferimento rapido della piastra 96 ​​bene nella fase di microscopio e iniziare l&#39;acquisizione entro 10 minuti dalla aspirazione della soluzione di carico. </li></ol> 5. Laser Scanning Microscopy e analisi dei dati <ol><li> Acquisizione di immagini <ol><li> Impostare le condizioni di imaging in modo da massimizzare il numero totale di fotoni e minimizzare fototossicità, photobleaching e cross-eccitazione. <ol><li> In un moderno microscopio confocale a scansione laser, usare un numerico alto obiettivo 20X apertura ad immersione, aprire il rilevatore foro stenopeico di 2,5 unità Airy (ad esempio, a livello di sezionamento ottico), scegliere alta sensibilità GaAsP Rivelatori canali donatore e accettore attivi contemporaneamente, profondità 8 bit , ~ 750 nm dimensione dei pixel, 3 volte telaio media e excite con il 10% di un diodo laser 405 nm. </li><li> Per assicurare una corretta quantificazione impostare il guadagno del rivelatore di entrambi i canali sullo stesso valore e per evitare eventuali pixel saturi. Nota: Qui, i guadagni sono stati fissati al 150%. </li></ol></li><li> Assicurare immersione continua diffondendo mezzo di immersione al fondo dei pozzetti, per esempio con una siringa da 1 ml, ed evitare l&#39;intrappolamento di bolle d&#39;aria. </li><li> Attivare la luce trasmessa e trovare un campo rappresentativo di vista in ogni pozzetto. Utilizzare una fase automatico tarato correttamente e marcare la posizione nel software. </li><li> Dopo la rimozione della piastra per la colorazione (vedere paragrafo 4.3), reinserire la piastra e aggiungere un peso sopra per evitare spostamenti focale. Rivedere le posizioni salvate con la modalità di scansione laser e regolare il fuoco e posizione laterale correttamente. </li><li> Impostare il lasso di tempo di 2 minuti, la durata di 2 ore e iniziare l&#39;acquisizione. </li></ol></li><li> Quantificazione Immagine (esempi sarà dato per Bitplane Softwsono tali da Imaris) <ol><li> Importare il set di dati in un software che permette di calcoli aritmetici di matrici di pixel. A tale scopo, trascinando il file di origine che contiene sia il donatore e il canale accettore sull&#39;icona del software. </li><li> Sottrarre il fondo in entrambi i canali con lo stesso offset. Per fare questo, fare clic su Menu e quindi fare clic su Immagine&gt; produzione&gt; Baseline sottrazione. Selezionare un canale e immettere il valore di base. </li><li> Correggere lo sfiato-through del canale donatore (Ch1) nel canale accettore (Ch2) con il predeterminato correzione del fattore f (vedi 1.1), creando un nuovo canale: Ch2 &#39;= Ch2 - Ch1 * f NOTA: A spurgo attraverso del accettore nel canale donatore non era rilevabile rispetto al livello di rumore. <ol><li> Fare clic su Menu&gt; Elaborazione immagini&gt; Canale Aritmetica e inserire abs (CH2- (ch1 * 0.33)). Successivamente eliminare Canale 2 facendo clic su Menu&gt; Modifica&gt; Elimina Canali. Assicurarsi che ilspurgo attraverso il canale accettore corretto rimane come il nuovo canale 2. Opzionale: Modificare il colore del canale dal grigio al colore originale andando su Menu&gt; Proprietà Modifica&gt; Immagine. Selezionare Channel 4&gt; mappata colore&gt; colore di base e modificarlo, ad esempio,, verde. </li></ol></li><li> Creare un nuovo canale con la seguente operazione per determinare il coefficiente relativo FRET: Ch3 = Ch2 &#39;/ (Ch1 Ch2 +&#39;) <ol><li> Vai a Menu&gt; Elaborazione immagini&gt; Canale Aritmetica e inserire ch2 ./ (ch1 + ch2) </li></ol></li><li> Per una corretta visualizzazione del canale FRET in un&#39;immagine a 8 bit, creare un quarto canale: Ch4 = 255 * Ch3 <ol><li> Vai a Menu&gt; Elaborazione immagini&gt; Canale Aritmetica e immettere 255 * ch3. Cambiare il colore del canale andando su Menu&gt; Proprietà Modifica&gt; Immagine. Selezionare Channel 4&gt; mappata File Table colori&gt; Colore&gt; Jet. </li></ol></li><li> Applicare un filtro mediano 3x3 ai canali CH3 e CH4 that ridurrà il rumore nelle immagini. Per fare questo, fare clic su Menu&gt; Elaborazione immagini&gt; Smoothing&gt; Filtro mediano. Selezionare entrambi i canali e applicare un formato del filtro &quot;3x3x1&quot;. </li><li> Per la quantificazione dei segnali cellulari, rilevare le cellule in Ch4 impostando correttamente l&#39;offset e filtrare le strutture in base alla dimensione di escludere troppo piccole strutture. <ol><li> Selezionare il Surpass Visualizza e fare clic sull&#39;icona &quot;Aggiungi nuove superfici&quot;. Definire i parametri dell&#39;algoritmo di rilevamento nella finestra di superficie. Per l&#39;elaborazione più veloce, attivare la due caselle di controllo &quot;Segmento solo una regione di interesse&quot; e &quot;Processo intera immagine finalmente&quot;. Definire la regione di interesse, modificando le sue dimensioni. </li><li> Selezionare Channel 4 come canale sorgente e impostare la soglia da Thresholding&gt; Sfondo Sottrazione (Contrasto Locale) e impostare un diametro di 10 micron. Impostare la soglia minima intensità manualmente a 0 e la soglia massima al massimo intensity. Filtrare le strutture per area e impostare il valore minimo per esempio, 187 μm². Finire la rilevazione della superficie. </li></ol></li><li> Estrarre i valori medi per intensità di fluorescenza del donatore (Ch1) e il coefficiente relativo FRET (Ch3) e loro deviazioni standard nell&#39;entità cellulare. Per fare questo, andare alle proprietà di superficie. Modificare l&#39;aspetto delle strutture selezionando Surfaces Style / Qualità&gt; Surface. Selezionare tutte le strutture facendo clic sulla scheda filtri. Unificare le strutture rilevate andando a processo di selezione&gt; Unify. Esportare le statistiche di superficie in MS Excel facendo clic sulla scheda statistica&gt; Esporta tutte le statistiche su file. </li><li> Tracciare questi valori nel tempo. Identificare l&#39;intervallo 10 min di pendenza massima di aumento della fluorescenza canale donatore per ogni condizione come un parametro ragionevole per rappresentare la quantità di effettore traslocato. Calcolare la pendenza come m = Δ intensità di fluorescenza del donatore / tempo Δ (min). </li></ol></li&gt; </ol>

<ol>
	<li>Galan, J. E., Lara-Tejero, M., Marlovits, T. C., Wagner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Type+III+Secretion+Systems:+Specialized+Nanomachines+for+Protein+Delivery+into+Target+Cells.">Bacterial Type III Secretion Systems: Specialized Nanomachines for Protein Delivery into Target Cells.</a> Annu Rev Microbiol. 68, 415-438 (2014).</li><li>Aepfelbacher, M., Trasak, C., Ruckdeschel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+functions+of+pathogenic+Yersinia+species.">Effector functions of pathogenic Yersinia species.</a> Thromb Haemost. 98 (3), 521-529 (2007).</li><li>Heesemann, J., Sing, A., Trulzsch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia's+stratagem:+targeting+innate+and+adaptive+immune+defense.">Yersinia's stratagem: targeting innate and adaptive immune defense.</a> Curr Opin Microbiol. 9 (1), 55-61 (2006).</li><li>Viboud, G. I., Bliska, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+outer+proteins:+role+in+modulation+of+host+cell+signaling+responses+and+pathogenesis.">Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis.</a> Annu Rev Microbiol. 59, 69-89 (2005).</li><li>Dewoody, R. S., Merritt, P. M., Marketon, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+Yersinia+type+III+secretion+system:+traffic+control.">Regulation of the Yersinia type III secretion system: traffic control.</a> Front Cell Infect Microbiol. 3, 10-3389 (2013).</li><li>Pettersson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+virulence+factor+expression+by+pathogen+target+cell+contact.">Modulation of virulence factor expression by pathogen target cell contact.</a> Science. 273 (5279), 1231-1233 (1996).</li><li>Schweer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytotoxic+necrotizing+factor+of+Yersinia+pseudotuberculosis+(CNFY)+enhances+inflammation+and+Yop+delivery+during+infection+by+activation+of+Rho+GTPases.">The cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by activation of Rho GTPases.</a> PLoS Pathog. 9 (11), e1003746 (2013).</li><li>Wolters, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+necrotizing+factor-Y+boosts+Yersinia+effector+translocation+by+activating+Rac+protein.">Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein.</a> J Biol Chem. 288 (32), 23543-23553 (2013).</li><li>Mejia, E., Bliska, J. B., Viboud, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+controls+type+III+effector+delivery+into+host+cells+by+modulating+Rho+activity.">Yersinia controls type III effector delivery into host cells by modulating Rho activity.</a> PLoS Pathog. 4 (1), e3 (2008).</li><li>Aili, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Yersinia+Yop-effector+delivery+by+translocated+YopE.">Regulation of Yersinia Yop-effector delivery by translocated YopE.</a> Int J Med Microbiol. 298 (3-4), 183-192 (2008).</li><li>Gaus, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Destabilization+of+YopE+by+the+ubiquitin-proteasome+pathway+fine-tunes+Yop+delivery+into+host+cells+and+facilitates+systemic+spread+of+Yersinia+enterocolitica+in+host+lymphoid+tissue.">Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue.</a> Infect Immun. 79 (3), 1166-1175 (2011).</li><li>Nordfelth, R., Wolf-Watz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YopB+of+Yersinia+enterocolitica+is+essential+for+YopE+translocation.">YopB of Yersinia enterocolitica is essential for YopE translocation.</a> Infect Immun. 69 (5), 3516-3518 (2001).</li><li>Radics, J., Konigsmaier, L., Marlovits, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+pathogenic+type+3+secretion+system+in+action.">Structure of a pathogenic type 3 secretion system in action.</a> Nat Struct Mol Biol. 21 (1), 82-87 (2014).</li><li>Sory, M. P., Cornelis, 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=Translocation+of+a+hybrid+YopE-adenylate+cyclase+from+Yersinia+enterocolitica+into+HeLa+cells.">Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells.</a> Mol Microbiol. 14 (3), 583-594 (1994).</li><li>Schlumberger, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+type+III+secretion:+Salmonella+SipA+injection+into+host+cells.">Real-time imaging of type III secretion: Salmonella SipA injection into host cells.</a> Proc Natl Acad Sci U S A. 102 (35), 12548-12553 (2005).</li><li>Charpentier, X., Oswald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+secretion+and+translocation+domain+of+the+enteropathogenic+and+enterohemorrhagic+Escherichia+coli+effector+Cif,+using+TEM-1+beta-lactamase+as+a+new+fluorescence-based+reporter.">Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter.</a> J Bacteriol. 186 (16), 5486-5495 (2004).</li><li>Koberle, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+enterocolitica+targets+cells+of+the+innate+and+adaptive+immune+system+by+injection+of+Yops+in+a+mouse+infection+model.">Yersinia enterocolitica targets cells of the innate and adaptive immune system by injection of Yops in a mouse infection model.</a> PLoS Pathog. 5 (8), e1000551 (2009).</li><li>Geddes, K., Cruz, F., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cells+targeted+by+Salmonella+type+III+secretion+in+vivo.">Analysis of cells targeted by Salmonella type III secretion in vivo.</a> PLoS Pathog. 3 (12), e196 (2007).</li><li>Marketon, M. M., DePaolo, R. W., DeBord, K. L., Jabri, B., Schneewind, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plague+bacteria+target+immune+cells+during+infection.">Plague bacteria target immune cells during infection.</a> Science. 309 (5741), 1739-1741 (2005).</li><li>Mills, E., Baruch, K., Aviv, G., Nitzan, M., Rosenshine, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+type+III+secretion+system+activity+of+enteropathogenic+Escherichia+coli.">Dynamics of the type III secretion system activity of enteropathogenic Escherichia coli.</a> MBio. 4 (4), (2013).</li><li>Mills, E., Baruch, K., Charpentier, X., Kobi, S., Rosenshine, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+effector+translocation+by+the+type+III+secretion+system+of+enteropathogenic+Escherichia+coli.">Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli.</a> Cell Host Microbe. 3 (2), 104-113 (2008).</li><li>Heesemann, J., Laufs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+mobilizable+Yersinia+enterocolitica+virulence+plasmid.">Construction of a mobilizable Yersinia enterocolitica virulence plasmid.</a> J Bacteriol. 155 (2), 761-767 (1983).</li><li>Zumbihl, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytotoxin+YopT+of+Yersinia+enterocolitica+induces+modification+and+cellular+redistribution+of+the+small+GTP-binding+protein+RhoA.">The cytotoxin YopT of Yersinia enterocolitica induces modification and cellular redistribution of the small GTP-binding protein RhoA.</a> J Biol Chem. 274 (41), 29289-29293 (1999).</li></ol>

The authors declare that they have no competing financial interests.

Siamo qui applicato con successo un saggio basato TEM-1 beta-lattamasi giornalista per l&#39;analisi quantitativa dei effettori traslocazione di Y. enterocolitica. Molte diverse varianti di questa tecnica sensibile, specifico e relativamente semplice sono state descritte in letteratura. In questo studio microscopia a scansione laser è stato condotto per il rilevamento più sensibile e preciso dei segnali di fluorescenza. Specificamente la correzione per diafonia tra i coloranti donatore e accettore e ...

III sistemi di secrezione di tipo sono macchine proteina-export specializzata utilizzati da diversi generi di batteri gram-negativi per fornire direttamente le proteine ​​effettrici batteri codificate all&#39;interno delle cellule bersaglio eucariotiche. Mentre il meccanismo secretorio stessa è altamente conservata, insiemi specializzati di proteine ​​effettrici sono evoluti tra le diverse specie batteriche per manipolare percorsi di segnalazione cellulare e facilitare specifiche strategie di virulenza batterica 1. In caso di Yersinia, fino a sette proteine ​​effettrici, i cosiddetti (proteine ​​esterne Yersinia) YOPS, sono traslocato al contatto della cellula ospite e agire insieme per sovvertire le risposte delle cellule immunitarie come la fagocitosi e la produzione di citochine, vale a dire, per permettere la sopravvivenza extracellulare dei batteri 2-4. Il processo di traslocazione è strettamente controllata in diverse fasi 5. E &#39;stabilito che l&#39;attivazione primaria del T3SS è innescata dal suo contatto con il ce bersaglioll 6. Tuttavia, il meccanismo preciso di questa iniziazione deve ancora essere chiarito. In Yersinia un secondo livello di cosiddetta messa a punto di traslocazione si ottiene up- o down-regolare l&#39;attività delle proteine ​​cellulari Rho GTP-binding Rac1 o RhoA. L&#39;attivazione di Rac1 ad esempio invasina o citotossico necrotizzante fattore Y (CNF-Y) porta ad un aumento traslocazione 7-9, mentre la funzione del GAP traslocato YopE (GTPasi attivando proteina) down-regola l&#39;attività Rac1 e diminuisce di conseguenza traslocazione da un tipo di feedback negativo Meccanismo di 10,11. Metodi validi e precisi sono il presupposto per indagare come traslocazione è regolata durante l&#39;interazione cellula ospite Yersinia. Molti sistemi differenti sono stati utilizzati per questo scopo, ciascuno con vantaggi e svantaggi specifici. Alcuni approcci si basano su lisi delle cellule infette, ma non i batteri da diversi detergenti seguita da western blot. The svantaggio comune di questi metodi è che la lisi batterica minore ma potenzialmente inevitabile contamina il lisato cellulare con batteri associati proteine ​​effettrici. Tuttavia, il trattamento di cellule con proteinasi K per degradare proteine ​​effettrici extracellulari e successivo utilizzo di digitonina per lisi selettiva delle cellule eucariotiche sono state proposte per minimizzare questo problema 12. È importante sottolineare che questi test cruciale dipendono anticorpi anti-effector alta qualità, che non sono per lo più disponibili in commercio. I tentativi di utilizzare fusioni traduzionali delle proteine ​​effettrici e proteine ​​fluorescenti come la GFP per monitorare traslocazione non hanno avuto successo probabilmente a causa della struttura terziaria delle proteine ​​globulari fluorescenti e l&#39;incapacità dell&#39;apparato di secrezione a svolgersi prima secrezione 13. Tuttavia, diverse etichette giornalista diversi, come il cya (calmodulina-dipendente ciclasi) dominio del tossina della pertosse Bordetella cyclolysin 14 o Flashtag sono stati utilizzati con successo per analizzare traslocazione. Nel primo saggio di attività enzimatica di cya viene utilizzato per amplificare il segnale della proteina di fusione intracellulare, mentre il tag Flash, un tag brevissimo tetracysteine ​​(4Cys) motivo, permette per l&#39;etichettatura con il colorante Flash biarsenical senza disturbare il processo della secrezione 15. L&#39;approccio applicato qui è stato riportato per la prima volta da Charpentier et al. E si basa sulla conversione intracellulare del Förster Resonance Energy Transfer (FRET) dye CCF4 da traslocati effettore TEM-1 beta-lattamasi fusioni 16 (Figura 1A). CCF4 / AM è un composto cellulare permeanti in cui un derivato cumarina (donatore) e un residuo fluoresceina (accettore) sono collegati da un nucleo cefalosporina. All&#39;entrata passivo nella cellula eucariotica, il non fluorescente esterificati composto CCF4 / AM viene elaborato dalle esterasi cellulari per il CCF4 carica e fluorescente e quindi intrappolatoall&#39;interno della cellula. Eccitazione della porzione cumarina a 405 nm risultati in FRET alla frazione fluoresceina, che emette un segnale di fluorescenza verde a 530 nm. Dopo la scissione del nucleo cefalosporina dal FRET beta-lattamasi è interrotto e l&#39;eccitazione della porzione cumarina porta a blu fluorescenza at460 nm. Differenti applicazioni di questo metodo sono stati descritti in letteratura evidenziando la sua versatilità. Il metodo consente di analizzare traslocazione in vitro e in vivo anche, ad esempio, la tecnica è stata utilizzata in un modello di infezione mouse per individuare le popolazioni leucocitarie mirati per traslocazione in vivo 17-19. Lettura dei segnali può essere condotta utilizzando lettori di piastre, FACS o microscopia a fluorescenza. Da notare, il metodo prevede anche la possibilità di monitorare traslocazione in tempo reale mediante microscopia live-cellulare durante il processo di infezione 20,21. Qui microscopia a scansione laser a fluorescenza è stato applicato per readout di segnali di fluorescenza in quanto fornisce massima sensibilità e precisione. In particolare, la capacità di regolare la finestra di emissione con precisione nanometrica in combinazione con rivelatori ad alta sensibili facilita ottimizzato rivelazione a fluorescenza e minimizzato cross-talk. Inoltre questa configurazione microscopia può essere adattato per il monitoraggio in tempo reale di traslocazione e potenzialmente consente per l&#39;analisi simultanea di interazione ospite-patogeno a livello cellulare. In questo studio i tassi di traslocazione di un Y. enterocolitica ceppo selvatico e una delezione mutante YopE esibendo un fenotipo hypertranslocator 10,11 sono stati analizzati in modo esemplare.

<table><tbody><tr><td>LB-Agar</td><td>Roth</td><td>X969.2</td><td></td></tr><tr><td>LB-Medium</td><td>Roth</td><td>X968.2</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8662-500ML</td><td></td></tr><tr><td>96-well plate (black, clear bottom)</td><td>Greiner Bio One</td><td>655087</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td><td>Gibco/Life Technologies</td><td>31966-047</td><td></td></tr><tr><td>Probenecid</td><td>Sigma-Aldrich</td><td>P8761-25G</td><td></td></tr><tr><td>LiveBLAzer FRET-B/G Loading Kit with CCF4-AM</td><td>Life Technologies</td><td>K1095</td><td></td></tr><tr><td>Lectin from Triticum vulgaris (wheat) FITC conjugate</td><td>Sigma-Aldrich</td><td>L4895</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>V450 Rat anti-Mouse CD8a</td><td>BD Bioscience</td><td>560469</td><td>Used for peak emission and cross-talk determination</td></tr><tr><td>Immersol W immersion oil&amp;nbsp;</td><td>Zeiss</td><td>444969-0000-000</td><td>Refractive index = 1.3339 @ 23 &amp;deg;C</td></tr><tr><td>TCS SP5 II confocal laser scanning microscope</td><td>Leica microsystems</td><td></td><td>2x GaAsP-Hybrid detectors, 4 channel spectrometer, acusto optical beam spliter, motorized XY stage, adjustable pinhole, objective 20X HC PL APO CS IMM/CORR, 405&amp;#160;nm diode laser 50&amp;#160;mW</td></tr><tr><td>Imaris 7.6 software</td><td>Bitplane</td><td></td><td>Plugins included ImarisXT and MeasurementPro</td></tr><tr><td>MatLab compiler runtime</td><td>MathWorks</td><td></td><td></td></tr><tr><td>Prism 5</td><td>GraphPad software</td><td></td><td></td></tr></tbody></table>

analysis of yersinia enterocolitica effector translocation into host cells using beta-lactamase effector fusions

Molti batteri gram-negativi, tra cui patogeno Yersinia spp. Impiegano tipo III sistemi di secrezione di traslocare le proteine ​​effettrici all&#39;interno delle cellule bersaglio eucariotiche. All&#39;interno della cellula ospite proteine ​​effettrici manipolare funzioni cellulari a beneficio dei batteri. Per meglio comprendere il controllo di tipo III secrezione durante l&#39;interazione cellula ospite, dosaggi precisi e sensibile per misurare la traslocazione sono obbligatori. Abbiamo qui descriviamo l&#39;applicazione di un saggio basato sulla fusione di un frammento di proteina effettore Yersinia enterocolitica (Yersinia proteina esterna; YopE). Con TEM-1 beta-lattamasi di analisi quantitativa di traslocazione Il test si basa sulla scissione di una cella permeante FRET dye (CCF4 / AM) da traslocata fusione di beta-lattamasi. Dopo la scissione del nucleo cefalosporine CCF4 dalla beta-lattamasi, FRET da cumarina a fluoresceina viene interrotto e l&#39;eccitazione della porzione cumarina porta a blu emissione di fluorescenza.Differenti applicazioni di questo metodo sono stati descritti in letteratura evidenziando la sua versatilità. Il metodo consente di analizzare traslocazione in vitro e in vivo anche, ad esempio, in un modello di topo. Rilevazione dei segnali di fluorescenza può essere eseguita utilizzando lettori di piastre, analisi FACS o microscopia a fluorescenza. Nella configurazione descritta qui, in vitro traslocazione di fusioni effettrici in cellule HeLa da diversi mutanti Yersinia è monitorato mediante microscopia a scansione laser. Registrazione conversione intracellulare del reporter FRET dalla beta-lattamasi effettori fusione in tempo reale fornisce risultati quantitativi affidabili. Siamo qui Mostriamo dati esemplificativi, dimostrando una maggiore traslocazione da un Y. enterocolitica YopE mutante rispetto al ceppo selvatico.

Per dimostrare la capacità del metodo descritto per analizzare quantitativamente effettrici traslocazione in cellule bersaglio, sono stati studiati due ceppi Yersinia con differenti cinetiche traslocazione: Y. enterocolitica ceppo selvatico WA-314 (sierogruppo O8, l&#39;ospitare il plasmide di virulenza pYVO8 22) e la sua derivata WA-314ΔYopE (WA-314 ospitare pYVO8ΔYopE 23). Lavoro precedente ha mostrato che Y. mutanti enterocolitica privi funzionale mostra Yo...

Watch this Scientific Journal Video about Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions at JoVE.com

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 ...

analysis-yersinia-enterocolitica-effector-translocation-into-host

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

8.5K Views.  University Medical Center Hamburg-Eppendorf. 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.

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

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Ricerca

Immunologia e infezioni

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Identificazione dei percorsi host mirati dalle proteine degli effetti batterici utilizzando la tossicità del lievito e gli schermi soppressori

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Genetica

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

Monitoraggio parte dell&#39;Assemblea di una Secreted batterica Virulence Factor Uso site-specific reticolazione

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Elettroporazione di effettori batterici funzionali in cellule di mammifero

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identificazione dei fenotipi inibizione della crescita indotta da Espressione di effettori batterica di tipo III nel lievito

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Biologia

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during 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 replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

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

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

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 ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Svelare la funzione di un batterica Effector da un non-coltivabili fitopatogeni Utilizzando un lievito schermo doppio ibrido

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Generazione di<em&gt; Enterobacter</em&gt; Sp. YSU auxotrofi Utilizzando Transposon Mutagenesi

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Proteina fluorescente verde sistema di visualizzare gli effettori consegnati dai batteri durante l'infezione Split

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging. 
Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM) 
Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

Imaging a super-risoluzione di proteine secrete da batteri utilizzando l'espansione del codice genetico

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

Analisi di

Riepilogo

Esplora Altri Video

Capitoli in questo video