Gli autori ringraziano il Ellison Medical Foundation per il loro sostegno. Desideriamo anche ringraziare i membri del laboratorio Abby Dernburg per l&#39;assistenza con l&#39;utilizzo del wormsorter.

1. Ottenere una cultura sincronizzata di giovane adulto animali <ol><li> Crescere vermi in diverse piastre NGM seminato con OP50-1 E. coli, batteri (10x concentrata da una cultura satura) fino a molti worm hanno raggiunto la fase gravida. </li><li> Trattare gli animali gravide con la soluzione di uovo-prep per ottenere una coltura sincronizzata (uova). </li><li> Uova Piatto su diversi 60 mm piastre NGM seminato con 10x OP50-1 concentrato ad una densità di circa 150-200 uova / piastra. </li><li> Incubare le piastre a 25 ° C per 2 giorni (fino vermi hanno raggiunto la L4 - Giovani stadio adulto). </li></ol> 2. Preparazione Pseudomonas aeruginosa Piatti e infettare Worms <ol><li> Il giorno dell&#39;uovo-prep (passo 1.1) inoculare una coltura PA14 :: GFP in 2 ml di B supporti contenenti rifampicina re ad una concentrazione finale di 100 mg / ml. Incubare la coltura a 37 ° C con agitazione O / N. </li><li> Piatto il PA14 :: GFP culTure su altrettanti piatti Uccidere lenta (SKP) come necessario per il saggio. Su ogni 150 millimetri SKP Petri dish pipetta 75-100 ml di cultura satura e diffondere uniformemente con una spatola di vetro sterile. Incubare le piastre a 37 ° C per 20-24 ore. </li><li> Rimuovere le piastre da incubatore e lasciare raffreddare a temperatura ambiente (~ 20 min). Utilizzando tampone M9, lavare i vermi dal passaggio 1.4 su PA14 :: piastre GFP in un volume minimo di liquido. Quando si utilizzano piastre da 150 mm, diverse centinaia di vermi possono essere posizionati su un unico piatto. </li><li> Incubare le piastre a 25 ° C per 18-21 ore. Per P. vermi adulti giovani aeruginosa esposte, questa è la finestra temporale visualizza la distribuzione ottimale delle colonizzato vs animali non colonizzato. </li></ol> 3. Impostazione del Wormsorter Prima di eseguire il campione tipo, assicurarsi che la macchina sta funzionando correttamente. I dettagli possono essere ottenuti presso http://www.unionbio.com/support/documents.aspx?id=43, e THe passaggi essenziali sono riportate qui di seguito. <ol><li> Impostare la pressione della valvola guaina a circa 5 psi (4,5-5,5 range). </li><li> Per verificare che la portata del liquido guaina è opportuno posizionare un tubo da 15 ml sotto l&#39;erogatore, attivare la valvola di guaina, e spegnere la valvola selezionatrice. Tenere il tubo sotto il dispenser per 60 sec. Il volume del tubo deve essere 9-10 ml. Se è fuori di questo intervallo, regolare la pressione della valvola guaina di conseguenza. </li><li> Per garantire che la portata del liquido guaina (impostato come nel passaggio 3.2) consentirà un&#39;accurata selezione, verificare il tasso di ordinamento mediante 40 particelle micron di controllo: </li><li> Spegnere la valvola guaina. Aggiungere circa 25 ml di soluzione di particella controllo al serbatoio. </li><li> Aprire la valvola guaina, e la valvola di campionamento. </li><li> Sul software per computer, passare alla modalità &quot;particella di controllo&quot; e fare clic acquisire. </li><li> Assicurarsi che il flusso di particelle è ~ 5-8 particelle / sec, con una TOF (Time Of Flight) approssimare la larghezzadella particella, 40 pm. Tale TOF indica che solo un singolo 40 pm oggetto attraversa alla volta. Se i valori sono al di fuori di questo intervallo, regolare le impostazioni (seguendo le indicazioni di cui sopra) di conseguenza. </li></ol> 4. Ordinando Colonizzato vs non colonizzato Worms <ol><li> Utilizzando M9, lavare i vermi in tubi conici da 15 ml. Consentire vermi adulti depositarsi sul fondo della provetta per gravità e rimuovere il surnatante. Riempire il tubo con fresche M9. Ripetere questo processo in 3-4x. Ciò elimina gran parte delle larve e batteri in eccesso e dura circa 10 min. </li><li> Aggiungi vermi al serbatoio wormsorter con un dolcemente mescolando piccola ancoretta. </li><li> Regolare il guadagno iniziale del segnale impostando la PMT verde a 600 e il rosso e giallo PMT a 200. </li><li> Iniziare l&#39;acquisizione di dati per regolare le impostazioni. </li><li> Obiettivo per un tasso sorta di 25-30 eventi (vermi) al secondo. Se il tasso di numero è troppo alta, diluire i vermi nel serbatoio con M9 conseguenza. Se il tasso è quelloo bassa, aggiungere più vermi in un piccolo volume di M9. </li><li> Se l&#39;esperimento richiede una separazione molto rigorosa colonizzato vs vermi non colonizzato, impostare il &quot;controllo di coincidenza&quot; a &quot;puro&quot;. Ciò garantisce che, in caso si verificano due oggetti troppo vicini l&#39;uno all&#39;altro oppure nella stessa caduta, la macchina rifiuta la goccia anziché raccogliendola. </li><li> Per ordinare la popolazione di interesse, disegnare cancelli intorno agli assi che indicano le dimensioni (per ottenere vermi adulti) e intensità di fluorescenza (cioè alto per colonizzata, basso per non colonizzato). I valori di fluorescenza gating devono essere determinati empiricamente. </li><li> Posizionare un piatto multi-pozzo o una capsula di Petri sotto l&#39;erogatore per raccogliere i vermi quindi aprire la valvola di ordinamento per iniziare l&#39;ordinamento vermi. </li><li> Raccogliere una piccola popolazione di animali per verificare al microscopio che la popolazione filtrate è la popolazione di interesse. In caso contrario, regolare i parametri di gating di conseguenza e continuare con la raccolta di campioni. </li></ol>

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Gli autori non hanno nulla da rivelare.

Il metodo descriviamo approfitta marcatura fluorescente di entità esterna della vite senza fine, di seguire le interazioni tra la vite e il suo ambiente. Nel caso che presentiamo, la separazione è basata sull&#39;etichettatura di un agente patogeno ed è stato impiegato per vermi separati con carico agente patogeno pesante da quelli senza (o luce) del carico. Successiva analisi di espressione genica trovato alcuna differenza nella risposta immunitaria tra i due gruppi suggerendo che fossero indipendenti carico patogen...

Smistamento automatico verme è molto simile FACS, operando misurando un segnale fluorescente in una vite senza fine (tipicamente fornita dai transgenici espressione di proteine ​​reporter) che passa raddrizzato in un tubo, consentendo il reindirizzamento sia ad un tubo di raccolta / pozzetto o ad un contenitore per rifiuti secondo di gating parametri fissati dal ricercatore 1. Il wormsorter può facilitare la ricerca in molti modi, un esempio di impiegare come uno strumento analitico è uno studio che ha seguito i modelli spazio-temporali di attività del promotore per quasi 1.000 geni 2. Tuttavia, il principale uso del wormsorter è in schermi genetici, seguendo bersaglio livelli di espressione di geni o localizzazione di proteina fluorescente lungo l&#39;asse del verme 3-5. Qui, descriviamo una nuova applicazione per la wormsorter, nel seguire la colonizzazione del worm da un agente patogeno fluorescente tag. Con questo come uno strumento ci siamo concentrati sul rapporto tra pathogen colonizzazione / carico e la risposta immunitaria, per acquisire nuove conoscenze sui meccanismi responsabili per l&#39;inizio delle risposte immunitarie nel verme. In quasi tutti gli organismi studiati fino ad oggi, l&#39;inizio delle risposte immunitarie innate di agenti patogeni microbici dipende dal riconoscimento di pattern molecolari associati ai patogeni (PAMP), e / o modelli molecolari di pericolo / danno-associato (smorza) 6,7. Le strutture microbiche primi sono conservati che includono componenti della parete cellulare microbica, il suo flagello, o il suo doppio strato lipidico 6, la seconda, comprendono sia molecole rilasciate (ad esempio ATP 8), proteine ​​alterate o altri marcatori di processi cellulari alterati 9,10. Entrambi i tipi di segnali sono riconosciuti da proteine ​​designati come recettori di pattern recognition (PRR), che su specifica di legame di una molecola modello di attivare una catena di eventi che conducono ad una risposta protettiva. C. elegans è stato estremamente utile come tractamodello bile sviscerare i vari aspetti delle interazioni ospite-patogeno, ma una cosa che non è ben compreso è come risposte immunitarie sono iniziati nel verme. Nessuno dei recettori putativi che sono orthologous ai recettori pattern recognition (PRR) in altri organismi hanno dimostrato di legare PAMPs, e molti degli ortologhi di PRRs che sono cardine per le risposte immunitarie in altri organismi mostrare un contributo sorprendentemente limitato a risposte verme patogeni e resistenza. Per esempio, il recettore Toll Drosophila, che è essenziale per resistere patogeni Gram positivi, è rappresentato in C. elegans da una suola omologo, tol-1, che contribuisce alla protezione dal patogeno Gram-negativo Salmonella Typhimurium 11, ma non da altre testate Gram-negativi, patogeni o-positivi 11,12. Queste osservazioni, combinato con i dati che indicano che le risposte immunitarie potrebbe essere indotta da interrompendo proteina cellulare traduzione hcome indotto alcuni a suggerire che C. elegans rileva principalmente smorza 9,13,14. Tuttavia, i rapporti capacità degli agenti patogeni morti per indurre risposte immunitarie descrivono suggerire che PAMP legame può essere ha un ruolo importante nel riconoscimento patogeno in C. elegans 15,16. Il lavoro precedente concentrandosi sulle risposte immunitarie in età sincronizzato geneticamente identici C. elegans popolazioni, hanno dimostrato grande variabilità individuale nella colonizzazione intestinale da parte dei batteri Gram-negativi patogeno Pseudomonas aeruginosa. Tuttavia, gli studi di profilatura trascrizionale trattate queste popolazioni variabile colonizzati come un&#39;unica entità 17,18. Approfittando di questa variabilità, abbiamo sviluppato un protocollo di messa a fuoco di un wormsorter automatizzato per separare le popolazioni colonizzate differenziale di Caenorhabditis elegans esposti a GFP che esprimono P. aeruginosa. Esaminando l&#39;espressione genica in popolazioni diversamente colonizzate FACIla valutazione litated del rapporto tra carico patogeno (e il danno associato) e le risposte immunitarie e nuove intuizioni fornite circa il riconoscimento dei patogeni in C. elegans 19. Qui di seguito descriviamo il protocollo, che potrebbe essere applicato per ordinare i vermi infettati con qualsiasi agente patogeno fluorescente. Per utenti potenziali occorre notare che il numero di vermi che devono essere risolti dipende dalla natura delle analisi successive e protocolli in uso. Ad esempio, nel caso di analisi di espressione genica microarray,&gt; 1.000 vermi dovranno avere abbastanza RNA, se si utilizzano protocolli standard, ma ~ 100 vermi sarebbero sufficienti se l&#39;amplificazione è impiegato, consentendo veloce raccolta di materiale e riducendo al minimo lo stress a i vermi.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">M9 Buffer</td>
			<td style="width:139px;">Prepared in house</td>
			<td style="width:527px;">Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin&#160;</td>
			<td>Sigma</td>
			<td>R3501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg prep solution&#160;</td>
			<td>Prepared in house</td>
			<td>50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGM plates&#160;</td>
			<td>Prepared in house</td>
			<td>Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKP plates</td>
			<td>Prepared in house</td>
			<td>Recipe same as NGM only 0.35% peptone instead of 0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control test particles&#160;</td>
			<td>Union Biometrica&#160;</td>
			<td>310-5071-001</td>
		</tr>
	</tbody>
</table>

automated separation of c. elegans variably colonized by a bacterial pathogen

Il wormsorter è uno strumento analogo ad una macchina FACS che viene utilizzato in studi di Caenorhabditis elegans, tipicamente per ordinare vermi basato su espressione di un reporter fluorescente. Qui, si evidenzia un uso alternativo di questo strumento, per l&#39;ordinamento vermi in base al loro grado di colonizzazione da un agente patogeno-GFP che esprimono. Questo nuovo utilizzo ci ha permesso di affrontare il rapporto tra la colonizzazione dell&#39;intestino verme e induzione di risposte immunitarie. Mentre C. elegans risposte immunitarie a diversi agenti patogeni sono stati documentati, non si sa ancora che cosa li avvia. Le due possibilità principali (che non si escludono a vicenda) sono il riconoscimento di pattern molecolari associati ai patogeni, e la rilevazione dei danni causati da infezione. Per distinguere tra le due possibilità, l&#39;esposizione al patogeno deve essere dissociata dai danni che provoca. Il permesso wormsorter separazione dei vermi che sono stati ampiamente-colonizzata dai Gram-npatogeno egative Pseudomonas aeruginosa, con danni probabilmente causato da un carico patogeno, da vermi che sono stati esposti in modo simile, ma non, o marginalmente, colonizzato. Queste popolazioni distinte sono stati usati per valutare il rapporto tra carico patogeno e l&#39;induzione di risposte immunitarie trascrizionali. I risultati suggeriscono che i due sono dissociate, supportando la possibilità di riconoscimento patogeno.

Quando pari età, geneticamente identici C. elegans sono esposti a P. aeruginosa, un&#39;ampia distribuzione si osserva nei livelli di colonizzazione (Figura 1A). Con l&#39;aiuto del protocollo qui descritto separazione efficiente di non colonizzato da vermi colonizzati può essere raggiunto (Figura 1B). A differenza dei worm non colonizzato, worm colonizzati mostrano segni di danni, come la lentezza e ridotto defecazione 19. Quest&#39;ultimo può essere un ...

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

Il wormsorter facilita schermi genetiche in Caenorhabditis elegans di classificare vermi secondo l&#39;espressione di giornalisti fluorescenti. Qui, descriviamo un nuovo utilizzo: ordinamento secondo la colonizzazione da un agente patogeno-GFP che esprimono, e ci avvaliamo di esaminare il ruolo poco compreso del riconoscimento patogeno in avvio risposte immunitarie.

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 ...

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JoVE Journal

Immunology and Infection

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

8.3K Views.  University of California, Berkeley.  Il wormsorter facilita schermi genetiche in Caenorhabditis elegans di classificare vermi secondo l'espressione di giornalisti fluorescenti. Qui, descriviamo un nuovo utilizzo: ordinamento secondo la colonizzazione da un agente patogeno-GFP che esprimono, e ci avvaliamo di esaminare il ruolo poco compreso del riconoscimento patogeno in avvio risposte immunitarie. 

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

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

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 ...

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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. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

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

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

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 ...

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 ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Arricchimento delle lipoproteine batteriche e preparazione del N-terminale lipopeptidi per determinazione strutturale mediante spettrometria di massa

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Un ad alta velocità, ad alto contenuto, liquido a base di c. elegans patosistema come contributo

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Analisi del comportamento di grande c. elegans popolazioni utilizzando un ampio campo di vista Monitoraggio piattaforma automatizzata

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...