Questo lavoro è stato finanziato dal Wellcome Trust (WT 0795880).

Il protocollo seguente descrivere lo studio di invasione delle cellule endoteliali da S. aureus ma può teoricamente essere utilizzato per studiare l&#39;invasione cellulare da qualsiasi batterio coltivabili. Fasi specifiche a S. aureus e cellule endoteliali sono indicati. 1. Preparazione dei batteri <ol><li> Cultura S. ceppi di Staphylococcus aureus per 4-16 ore (a seconda della fase di crescita necessaria) in 10 ml cervello-Heart Infusion (BHI) brodo a 37 ° C in aria con agitazione a 200 rpm. Queste condizioni di crescita sono specifici per S. aureus e potrebbero dover essere adattato per altri batteri. </li><li> Lavare i batteri tre volte in media Dulbecco Modified Eagle (DMEM, Invitrogen) con turni si alternano di centrifugazione a temperatura ambiente (5.000 x g, 10 min), la rimozione del sopranatante cultura e risospensione del batterica pellet in un volume equivalente di DMEM. Misurare la densità ottica della sospensione risultante di batteri che possono poi essere regolato come richiesto. Per S. aureus, prepariamo una sospensione a OD 600 = 1, che corrisponde a ~ 10 9 cfu ml -1. </li></ol> 2. Colture cellulari endoteliali <ol><li> Cultura la linea delle cellule endoteliali EA.hy926 1 in DMEM integrato con siero fetale bovino (FBS; 10%) e L-glutamina (2 mm) a 37 ° C in 5% CO 2. In alternativa, pool primario cellule endoteliali della vena ombelicale (HUVECs) può essere acquistato da Lonza (Basilea, Svizzera) e coltivate in endoteliali medio basale integrato con il 2% FBS, estratto di cervello bovino (compresa l&#39;eparina), umano fattore di crescita endoteliale e idrocortisone a 37 ° C in 5% di CO 2 secondo le istruzioni del produttore (Lonza). Queste condizioni di crescita sono specifici per queste cellule e potrebbero dover essere adattato per altri tipi di cellula ospite. </li><li> Crescere le cellule endoteliali in fiasche T75 per completare la confluenza, verificato da occhi. </li><li> Preparare il 24-pozzetti per l&#39;inserimento del coprioggetto. Una pinza sottile (fiamma sterilizzato) sono necessari per spostare i coprioggetti, che hanno una superficie opaca e una lucida. Coprioggetto posto nei pozzetti del 24 pozzetti con la superficie opaca rivolta verso l&#39;alto per consentire l&#39;attaccamento delle cellule. </li><li> Liberate le cellule dal pallone T75 con 3 ml di tripsina-EDTA (0,25%) e aggiungere 10 ml di terreno di coltura in questione. </li><li> Aggiungere 500 ml di cellule risospese a 24-pozzetti contenenti coprioggetto di vetro Thermanox. Un fiasco T75 confluenti di cellule fornito cellule sufficiente per due piastre da 24 pozzetti, un aumento di circa 5 × 10 5 cellule per pozzetto (in 500 microlitri di media). </li><li> Incubare le piastre per 48 ore come sopra descritto, e verificare 100% confluenza delle cellule al microscopio ottico invertito. </li><li> Dip-wash vetrini in PBS e aggiungere nuove piastre da 24 pozzetti contenente 490 microlitri DMEM contenente 10% FBS in ciascun pozzetto. </li><li> Per esaminare il ruolo di specifici processi metabolici invasione delle cellule, gli inibitori possono essere aggiunti alle cellule in coltura 1 ora prima l&#39;aggiunta di batteri e concentrazione mantenuta durante il test. Ad esempio, per determinare il ruolo di riassetto actina a S. aureus invasione delle cellule endoteliali, 50 mM citocalasina D può essere aggiunto o per il ruolo di caveolae, 5 mm metil-β-ciclodestrina può essere aggiunto. </li></ol> 3. Invasione di analisi <ol><li> Aggiungere 10 ml di batteri lavati (un aumento di circa 2 × 10 7 ufc ml -1 S. aureus) in ogni pozzetto contenente un coprioggetto lavato con uno strato confluente di cellule endoteliali in 490 microlitri DMEM contenente 10% FBS. </li><li> Incubare per 15-90 minuti a 37 ° C in 5% di CO 2. </li><li> Per misurare il numero totale dei batteri associati con le cellule (aderenti e interiorizzato), dip-lavare i coprioggetti tre volte in PBS e aggiungere ai pozzi fresco contenente 500 microlitri 0,5% Triton X-100 in PBS. Per garantire le cellule completamente lisi e rilasciare tutti i batteri interiorizzato, la pipetta più volte, puntando la punta della pipetta direttamente sulla superficie del vetrino coprioggetto. </li><li> Enumerare i batteri dal rivestimento della sospensione liquida (o diluizioni di questo se necessario) sulla superficie delle piastre TSA. Dal TX-100 sarà lyse molti batteri Gram-negativi, saponina può essere usato al posto 2. </li><li> Per misurare il numero di batteri internalizzati, rimuovere il supernatante della coltura di ogni batterio legato pozzetti contenenti e sostituirlo con 500 microlitri DMEM/10% FBS integrato con 200 mg ml -1 gentamicina. Noi abitualmente uso gentamicina piuttosto che lysostaphin qui come è più conveniente e ci permette di passare da esperimenti utilizzando diversi tipi di batteri (stafilococchi e lattococchi ad esempio) senza dover modificare il protocollo sperimentale. </li><li> Incubare le piastre a 37 ° C in 5% di CO 2 per 60 minuti per uccidere tutti i batteri extracellulari. </li><li> Lavare coprioggetto 3 volte in PBS,Lyse ed enumerare da piastrato su TSA come descritto per il test di adesione di cui sopra. </li><li> In alcuni casi può essere preferibile contare visivamente il numero di batteri mediante microscopia ottica. In questo caso, e specifico a S. invasione delle cellule aureus, l&#39;uso lysostaphin (10 mg ml -1), invece di gentamicina per distruggere fisicamente i batteri extracellulari. </li><li> Incubare lamelle per 20min a 37 ° C in CO 2 nella soluzione lysostaphin, quindi risciacquare e fissare con Cytopath (Cellpath). </li><li> Inondare il coprioggetto con cristallo viola (0,5% w / v) per 5 min. </li><li> Dip-risciacquo in acqua, asciugare e montare su vetrini. Il numero di batteri per mm 2 di confluenti cellule endoteliali può essere quantificata mediante microscopia ottica. </li></ol> 4. Rappresentante dei risultati: Espressione delle proteine ​​leganti la fibronectina (FnBPA e FnBPB) sulla superficie di S. aureus conferisce la capacità di invadere le cellule endoteliali. Un recente lavoro ha ridefinito la fibronectina dominio di legame FnBPA 3. Wild-type (WT) S. aureus 8325,4 invade le cellule endoteliali con alta efficienza, mentre un ceppo privo sia FnBPA e B (Δ FNB) hanno mostrato livelli significativamente più ridotto di internalizzazione (Figura 1A). Complementazione del mutante con FNB codifica plasmide A meno la fibronectina dominio di legame (pFnR0) non ha promosso l&#39;invasione (Figura 1A). Al contrario, complementazione del mutante con una codifica plasmide FNB intero Un gene (pFnBA4) restaurato invasione a livelli WT (Figura 1A). Il ruolo della FnBPA nell&#39;invasione delle cellule endoteliali può essere dimostrata utilizzando l&#39;espressione eterologa Lactococcus lactis host. Espressione di plasmidica FnBPA in L. lactis (pRM9 9) non ha significativamente migliorare l&#39;adesione alle cellule endoteliali rispetto ai batteri non esprimono una FnBPA (CTL) (bar aperti, Figura 1B). Al contrario, FnBPA che esprimono L. lactis invaso le cellule endoteliali a livelli significativamente più alto rispetto il ceppo non-espressione (bar chiuso, Figura 1B). Gli esperimenti sono stati effettuati quattro volte in duplice copia e la media ± deviazione standard è presentato. * Rappresentano dati che sono significativamente differenti (p = &lt;0,05) da WT o valori CTL. <img src="/files/ftp_upload/2693/2693fig1.jpg" alt="Figura 1" /> Figura 1. Invasione di EA. Hy926 cellule endoteliali da S. aureus (A) o L. lactis (B).

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	<li>Edgell, C. J., McDonald, C. C., Graham, 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=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc. Natl. Acad. Sci. U. S. A. 80, 3734-3737 (1983).</li><li>Makino, S., van Putten, J. P., Meyer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+variation+of+the+opacity+outer+membrane+protein+controls+invasion+by+Neisseria+gonorrhoeae+into+human+epithelial+cells.">Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.</a> EMBO J. 10, 1307-1315 (1991).</li><li>Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Gurusiddappa, S., Kim, J. H., Pilka, E. S., Briggs, J. A., Gough, T. S., Höök, M., Campbell, I. D., Potts, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+bacteria+attach+to+human+fibronectin+through+a+tandem+beta-zipper.">Pathogenic bacteria attach to human fibronectin through a tandem beta-zipper.</a> Nature. 423, 177-181 (2003).</li><li>Magoffin, R. L., Spink, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protection+of+intracellular+Brucella+against+streptomycin+alone+and+in+combination+with+other+antibiotics.">The protection of intracellular Brucella against streptomycin alone and in combination with other antibiotics.</a> J. Lab. Clin. Med. 37, 924-930 (1951).</li><li>Mandell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+intraleukocytic+bacteria.">Interaction of intraleukocytic bacteria.</a> Antimicrob. Agents Chemother. 52, 1673-1679 (1973).</li><li>Vaudaux, P., Waldvogel, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+antibacterial+activity+in+the+presence+of+human+polymorphonuclear+leukocytes.">Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes.</a> Antimicrob. Agents Chemother. 16, 743-749 (1979).</li><li>De Melo, M. A., Pechere, J. 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Nessun conflitto di interessi dichiarati.

<html> Il test che descriviamo si basa sul test di protezione gentamicina, che è stato ampiamente utilizzato per studiare l&#39;invasione delle cellule ospite da batteri. Osservazioni della incapacità degli antibiotici gentamicina e di altri per uccidere i batteri intracellulari sono state utilizzate nei primi studi l&#39;invasione delle cellule ospiti per 4-8 batteri. L&#39;uso di 24, 48 o anche 96-pozzetti permette la generazione di grandi quantità di dati quantitativi e riproducibile in un breve periodo di tempo ea costi relativamente bassi. Il saggio è interessante anche perché non richiede attrezzature specialistiche, può essere adattata per lavorare con vari batteri e cellule, e può essere utilizzato per studiare il ruolo dei processi cella sia batterica e host in invasione 9. Infatti, sin dai primi esperimenti, l&#39;analisi di protezione gentamicina è stato ampiamente utilizzato per misurare la cellula ospite invasione da una serie di diversi batteri o anche combinazioni di batteri 10,11. Mentre i principi fondamentali sono gli stessi, molte sottili variazioni nel metodo sono stati segnalati. In questo articolo abbiamo descritto la nostra versione e indicato dove le modifiche possono essere necessarie per altri batteri o cellule ospiti. Quando si progetta un esperimento per misurare la cellula invasione host utilizzando questo approccio ci sono una serie di punti importanti da considerare: Sensibilità batterica alla gentamicina. Questo può sembrare ovvio, ma è importante assicurarsi che il batterio è in fase di studio sensibili alla gentamicina alla temperatura concentrazione, e per tutta la durata di tempo da utilizzare. Nel caso di insensibilità, potrebbe essere possibile utilizzare altri antibiotici 5. Un approccio alternativo può essere quello di utilizzare enzimi litici (lysostaphin, mutanolysin, lisozima) 12. Incubazione e l&#39;inoculo. Quando si esegue questo test per la prima volta che è importante stabilire i tempi ottimali di incubazione, e l&#39;inoculo dei batteri da utilizzare. Pertanto, esperimenti iniziali dovrebbe esaminare sia l&#39;adesione e l&#39;invasione nel tempo (5 minuti a un massimo di 6 ore). E &#39;anche importante valutare come l&#39;invasione è influenzata dalla molteplicità di infezione (MOI; numero di batteri per cella). In generale è meglio usare il minor numero possibile di batteri in quanto riduce i danni alle cellule in coltura. Importanti differenze tra ceppi possono essere perse se i periodi di incubazione prolungato o di grandi dimensioni vengono utilizzati inoculi 9,13. Danno cellulare. E &#39;importante lavare i batteri prima dell&#39;uso. Tuttavia, il danno cellulare non si limita alla produzione di tossine. Invasione batterica, induzione di apoptosi e di interruzione delle normali funzioni cellulari possono causare danni cellulari. Questo può portare alla penetrazione di gentamicina nella cellula, uccidendo i batteri interiorizzato e dando l&#39;impressione che l&#39;invasione non si è verificato 14. Condizioni di incubazione. La temperatura e la composizione del terreno di coltura può avere un impatto significativo sulla invasione. Per esempio, l&#39;invasione delle cellule HeLa da Streptococcus pyogenes dipende dalla presenza di fibronectina solubile, che è tipicamente presente nel siero 15. Un&#39;altra considerazione è la crescita dei batteri nel terreno di coltura nel corso dell&#39;esperimento. Cultura e quantificazione dei batteri intracellulari. Sebbene TX-100 non può uccidere i batteri intracellulari, può inibire la loro crescita. Come tale, è importante controllare la replicazione batterica su terreno solido, in presenza del detergente. Qualora interessati, può essere possibile usare concentrazioni più basse di detersivo o di utilizzare in alternativa come saponina. Un vantaggio del test di protezione gentamicina sulla colorazione viola cristallo e analisi di microscopia è che è meno laborioso, e consente il rilevamento e la differenziazione di sub-popolazioni di batteri interiorizzato. Per esempio, S. aureus può produrre sia un tipo normale colonia (NCT) o la variante piccola colonia (SCV) 16, che non sarebbero distinguibili al microscopio di singole cellule batteriche. Un vantaggio della colorazione viola cristallo e analisi di microscopia sui test di protezione gentamicina è aggregazione delle cellule possono essere considerati e che i batteri intracellulari che entrano in un &#39;vitale ma non coltivabili&#39; stato verrà rilevato 17. Esaminando il ruolo dei processi nella cellula ospite invasione batterica. Molti studi hanno impiegato inibitori della funzione della cellula ospite come citocalasina D, che interferisce con riarrangiamento actina. Tali studi possono includere anche gli anticorpi che colpiscono i recettori della superficie cellulare e knockdown siRNA di specifici geni 18. È di vitale importanza per garantire che questi trattamenti non influiscono sulla vitalità o attaccamento di cellule in coltura o avere effetti tossici sui batteri. Direzioni future: CONTENUTO &quot;&gt; Perché questo test è di solito eseguita in lastre multi-cellula e cultura, è teoricamente possibile adattare ad un high-throughput operazione di screening, che potrebbe comportare l&#39;esame di librerie di potenziali cellule funzione inibitori, anti-infettivi o antibiotici progettate per colpire i batteri intracellulari. In alternativa, un tale approccio potrebbe essere utilizzato per lo screening di librerie di mutanti identificare i geni coinvolti nella adesione, invasione o la sopravvivenza intracellulare. In alcuni casi può essere opportuno inserire forze di taglio (flusso) nel sistema modello, per esempio quando la modellazione l&#39;endotelio, per imitare più da vicino l&#39;ambiente in vivo. attuali progressi nella progettazione e nella produzione di celle di flusso e microfluidica sistemi di colture cellulari offrono la possibilità di impiegare il dosaggio protezione gentamicina in ospite-patogeno modelli che incorporano forze di taglio. In sintesi, il protocollo qui descritto si basa su approcci simili utilizzati per molti anni. È ampiamente applicabile a molti tipi di cellule in coltura e specie batteriche e in grado di generare grandi quantità di dati in modo rapido ed a costi relativamente poco.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31885-023</td>
<td>&nbsp;</td>
<tr>
<td>Brain heart infusion</td>
<td>&nbsp;</td>
<td>BioChemika</td>
<td>53286</td>
<td>&nbsp;</td>
<tr>
<td>Foetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>HUVECs</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-2519</td>
<td>&nbsp;</td>
<tr>
<td>Endothelial basal medium</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3121</td>
<td>&nbsp;</td>
<tr>
<td>Bovine brain extract (including heparin)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092</td>
<td>&nbsp;</td>
<tr>
<td>Human endothelial growth factor</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4017C</td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4035C</td>
<td>&nbsp;</td>
<tr>
<td>GA-1000</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092C</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Lysostaphin</td>
<td>&nbsp;</td>
<td>AMBI</td>
<td>LSPN-50</td>
<td>&nbsp;</td>
<tr>
<td>Thermanox coverslips</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>TKT-210-330P</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Cytopath</td>
<td>&nbsp;</td>
<td>TCS Biosciences</td>
<td>HC8595</td>
<td>&nbsp;</td>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3886</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4049</td>
<td>&nbsp;</td>
<tr>
<td>T75 flasks</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>430641</td>
<td>&nbsp;</td>
<tr>
<td>Cytochalasin D</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2618</td>
<td>&nbsp;</td>
<tr>
<td>Methyl-B-cyclodextrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>332615</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

invasion of human cells by a bacterial pathogen

Qui si descrive il modo in cui studiare l&#39;invasione di cellule endoteliali umane da Staphylococcus aureus batteri patogeni. Il protocollo generale può essere applicata allo studio di invasione delle cellule da qualsiasi batterio coltivabili. Le fasi in cui aspetti specifici di invasione possono essere studiati, come il ruolo di riassetto actina o caveolae, sarà evidenziata. Cellule ospiti sono coltivate in fiaschi e quando è pronto per l&#39;uso sono seminate in piastre da 24 pozzetti contenenti coprioggetto Thermanox. Utilizzando coprioggetto permette successiva rimozione delle cellule dai pozzi per ridurre le interferenze da proteine ​​del siero depositato sui lati dei pozzi (a cui S. aureus sarebbe allegare). I batteri sono cresciuti alla densità richiesta e lavate per eliminare ogni proteine ​​secrete (ad esempio tossine). Coprioggetto con strati confluenti di cellule endoteliali vengono trasferiti al nuovo 24-pozzetti contenenti mezzo fresco cultura prima che l&#39;aggiunta di batteri. I batteri e le cellule vengono poi incubate insieme per la quantità di tempo in 5% CO 2 a 37 ° C. Per S. aureus questo è in genere tra 15-90 minuti. Coprioggetto Thermanox vengono rimossi da ogni pozzetto e dip-lavati in PBS per rimuovere i batteri separati. Se il totale dei batteri associati (aderente e interiorizzate) devono essere quantificati, lamelle vengono poi poste in un pozzo fresco contenente 0,5% Triton X-100 in PBS. Pipettaggio dolce porta alla completa lisi delle cellule e batteri sono enumerate dalla diluizione seriale e piastrato su agar. Se il numero di batteri che hanno invaso le cellule è necessario, coprioggetto sono aggiunti ai pozzetti contenenti 500 microlitri terreno di coltura di tessuto integrato con gentamicina e incubazione continuato per 1 ora, che uccidono tutti i batteri esterni. Coprioggetto possono poi essere lavate, le cellule lisate e batteri enumerate dalla piastrato su agar come descritto sopra. Se l&#39;esperimento richiede una visualizzazione diretta, coprioggetto possono essere fissate e colorate per la microscopia ottica, fluorescenza o confocale o preparati per microscopia elettronica.

Watch this Scientific Journal Video about Invasion of Human Cells by a Bacterial Pathogen at JoVE.com

Invasion of Human Cells by a Bacterial Pathogen

Un protocollo generale per lo studio di invasione delle cellule ospiti da un agente patogeno batterico, concentrandosi su<em&gt; Staphylococcus aureus</em&gt; E cellule endoteliali umane.

Invasione di cellule umane da un agente patogeno batterico

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

invasion-of-human-cells-by-a-bacterial-pathogen

Research

JoVE Journal

Immunology and Infection

34.6K Views.  University of Bath. Un protocollo generale per lo studio di invasione delle cellule ospiti da un agente patogeno batterico, concentrandosi su Staphylococcus aureus E cellule endoteliali umane.

Video: Invasion of Human Cells by a Bacterial Pathogen

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Applicazione di un mouse legatura Patch Peyer Assay intestinale Loop per valutare assorbimento da parte delle cellule batteriche M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Ricerca

Immunologia e infezioni

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Un giorno Scheme flusso di lavoro per batterica Rilevazione dei patogeni e test di resistenza antimicrobica dalle emocolture

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Enterico invasione batterica della cellule epiteliali intestinali<em&gt; In Vitro</em&gt; È drammaticamente migliorata utilizzando una camera di diffusione verticale Modello

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC secrezione per Indagare infezione Cellular dalla batterica patogeni<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Separazione automatica di<em&gt; C. elegans</em&gt; Variabilmente Colonizzato da un batterio patogeno

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

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

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

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

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

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

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 ...

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 ...

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.