La ricerca nel nostro laboratorio è stato sostenuto dalla Max von Pettenkofer Institute, Ludwig-Maximilians University di Monaco di Baviera, il tedesco Research Foundation (DFG, HI 1511/3-1) e il Bundesministerium für Bildung und Forschung (BMBF) iniziativa &quot;Infezione Medical Genomics&quot; ( 0315834C).

1. I preparativi per isolamento LCV <ol><li> Streak out Legionella pneumophila produce DsRed-Express 13,14 dalle scorte di glicerolo su piastre di agar CYE con 5 mcg / ml di cloramfenicolo (Cam) quattro giorni prima di isolamento LCV. Incubare i batteri a 37 ° C. </li><li> Seed out 1 x 10 7 D. discoideum produzione di GFP-calnexina o RAW264.7 macrofagi murini in 75 cm 2 coltura tissutale palloni un&#39;infezione giorno prima. Utilizzare 10 ml HL5 media con 20 ug / mL G418 per D. discoideum e incubare le amebe a 23 ° C. Per utilizzare RAW264.7 macrofagi 10 ml mezzo RPMI 1640 integrato con 10% FCS (inattivato con il calore) e 1% glutammina, e crescere le cellule a 37 ° C e 5% CO 2. Utilizzare almeno tre 75 cm 2 palloni per l&#39;infezione e il campione (minimo 6 x 10 7 cellule). </li><li> Inoculare una cultura notte con L. pneumophila dalla piastra CYE. Prendere una provetta da 15 ml con 3 mL AYE mediae 5 ug / mL Cam. Inoculare con 100 pl di una sospensione batterica per ottenere un OD 600nm di 0,1. Incubare la coltura durante la notte su una ruota rotazione testa a 37 ° C per 21-22 hr. </li></ol> 2. LCV isolamento <ol><li> Cambia il mezzo di D. discoideum cellule per rimuovere il antibiotico, che interferirebbero con la successiva infezione. </li><li> Misurare la densità ottica della cultura durante la notte. I batteri dovrebbe aver raggiunto la loro infettività picco a uno OD 600nm di ≥ 3, che corrisponde a 2 x 10 9 batteri / ml. </li><li> Infettare le cellule aggiungendo circa 500 pl di L. pneumophila coltura durante la notte per le cellule crescono in 10 ml HL5 mezzo (D. discoideum) o 10 ml RPMI 1640 (macrofagi). Questo corrisponde ad una molteplicità di infezione (MOI) di 50. Successivamente, l&#39;infezione è sincronizzato mediante centrifugazione dei batteri sulle celle a 500 g per 10 min. Centrifugazione è seguitada un&#39;incubazione del D. discoideum cellule a 25 ° C ed i macrofagi RAW264.7 a 37 ° C e 5% CO 2, rispettivamente. Il tempo di infezione di 1 ora comprende la fase di centrifugazione. </li><li> Dopo infezione rimuovere il terreno e lavare le cellule una volta per rimuovere i batteri extracellulari. Utilizzare tampone ghiacciato SORC per D. discoideum e gelide PBS per RAW264.7 macrofagi. Aggiungere 3 mL HS buffer, integrato con inibitori della proteasi (Roche) ad ogni pallone e raccogliere le cellule utilizzando un raschietto cellulare. Pool i relativi campioni in una provetta da 15 ml. </li><li> Per la omogeneizzazione delle utilizzare campioni 3 ml di plastica siringhe Luer-Lock e la sfera in acciaio inox Omogeneizzatore. Assicurarsi che si lavora su ghiaccio. Prima di iniziare, lavare l&#39;omogeneizzatore palla con acqua distillata, per evitare qualsiasi contaminazione detergente e sciacquare con tampone ghiacciata HS per sbarazzarsi di bolle d&#39;aria. Utilizzare l&#39;8 ball gioco micron. </li><li> Riempire i primi 3 ml in siringa und montarlo su maggioe omogeneizzatore. Premere il campione avanti e indietro nove volte attraverso l&#39;omogeneizzatore. Scambiarsi le siringhe in seguito per evitare la contaminazione con materiale non omogeneizzato. Raccogliere e mettere in comune il campione omogeneizzato in una provetta da 15 ml e prendere un campione di 150 microlitri per l&#39;analisi microscopica. Prima di procedere con un campione diverso smontare e lavare il omogeneizzatore palla. </li><li> Bloccare l&#39;omogeneizzato con 2% FCS o CS per 30 minuti su un agitatore su ghiaccio o su un overhead filatura ruota (10-20 rpm) a 4 ° C. </li><li> Dopo aver bloccato utilizzare un purificate per affinità anticorpo primario contro il SIDC (diluizione 1:3000) o contro qualsiasi altro marcatore batterica esclusivamente legame alla membrana LCV. Vortex la soluzione di anticorpi prima di aggiungerlo alla omogeneizzato. Incubare il campione per 1 h su un agitatore su ghiaccio o su un overhead filatura ruota (10-20 rpm) a 4 ° C. </li><li> Nel frattempo, preparare il gradiente Histodenz. Utilizzare una provetta da 15 ml per pendenza e tre fiasche 75 cm 2 di un campione. Abetest, aggiungere 5,75 ml di 35% Histodenz al tubo, e la seconda, accuratamente aggiungere 5,75 ml di soluzione al 10% Histodenz sopra. Poi adagiare i tubi in orizzontale verso il basso per 1 ora. </li><li> Dopo incubazione della omogenato con il reagente di bloccaggio e l&#39;anticorpo primario, centrifugare a 600 g per 15 min a 4 ° C per il campione pellet. Eliminare il supernatante e risospendere il pellet in 1,5 mL di tampone HS. Trasferire i campioni in una nuova Provetta da 15 ml e prendere un campione di 40 microlitri per l&#39;analisi microscopica in seguito. </li><li> Incubare il pellet con l&#39;anticorpo secondario - MACS capra anti-IgG di coniglio micro perline (diluizione 1:25) - per 30 minuti su un agitatore a 4 ° C. Nel frattempo, mettere le colonne MACS sul supporto magnetico ed equilibrare con 0,5 mL di tampone HS. </li><li> Successivamente, i campioni applicare alla colonna. Raccogliere 150 pl del flusso passante per la successiva analisi microscopica. Lavare la colonna tre volte con 0,5 mL di tampone HS. </li><li> Rimuovere le colonne da tegli magnete e eluato i veicoli commerciali leggeri con 0,5 mL di tampone HS di immediata squirting. Prendete un campione di 20 microlitri per l&#39;analisi microscopica. La fase di purificazione per immuno-separazione magnetica si prevede di cedere per 1 × 10 7 infettato D. cellule discoideum circa 1 × 10 6 LCV intatte, che sono circa sette volte arricchiti nell&#39;eluato rispetto al flusso passante 13. </li><li> Mettere i tubi gradiente Histodenz indietro in una posizione verticale e accuratamente caricare l&#39;eluato sopra. Centrifugare le provette a 3350 × g per 1 ora a 4 ° C. </li><li> Dopo centrifugazione prendere 1,5 mL frazioni a partire dal fondo della provetta mediante un lungo vetro pipetta Pasteur. Complessivamente raccogliere frazioni otto, ciascuno dal fondo del tubo. Non sono stati osservati livelli visibili nella pendenza costante Histodenz. La massima resa di questi veicoli è tipicamente previsto nella frazione 4 ad un rapporto di circa 2 x 10 5 LCV intatte per 1 × 10 7 ininfetto amebe 13. Minori quantità di veicoli commerciali leggeri sono presenti anche in frazioni 3 e 5, rispettivamente. Pertanto, la resa complessiva di LCV purificate è ~ 1 × 10 6 LCV intatte per 6 × 10 7 infettato D. cellule discoideum 13. Il recupero di veicoli commerciali può essere eseguita a temperatura ambiente (RT), e le LCV appaiono stabile per molte ore. </li></ol> 3. L&#39;analisi microscopica dei campioni raccolti <ol><li> Preparare un 24 pozzetti a fondo piatto piastra di coltura dei tessuti con coprioggetto rivestiti di poli-L-lisina. </li><li> Pipettare ogni campione prelevato durante la purificazione LCV più 150 pl di ciascuna frazione gradiente di densità nella piastra da 24 pozzetti. Aggiungere 0,5 ml di tampone HS ad ogni pozzetto per diluire la concentrazione Histodenz. Successivamente, centrifugare la piastra a 600 g per 10 min a 4 ° C. Rimuovere con cautela il surnatante e fissare i campioni con 4% paraformaldeide (PFA) per 20 minuti a RT. Dopo fissazione lavare i campioni due volte. Noie SORC per D. discoideum e PBS per RAW264.7 macrofagi. </li><li><ol class="lalpha"><li> Montare i campioni di GFP-calnexina esprimono D. discoideum, prese a diverse fasi durante l&#39;isolamento LCV, direttamente sul vetro diapositive utilizzando Vectashield ad es. </li><li> Incubare i campioni da RAW264.7 macrofagi, fatte a passi differenti durante l&#39;isolamento LCV, con una soluzione bloccante (1% BSA in PBS) per 10 minuti a RT. Utilizzare un anticorpo anti-SIDC primaria a macchiare i veicoli commerciali e incubare per 1 ora in una camera umida a temperatura ambiente (purificato per affinità di coniglio anti-SIDC; 1:1000 in tampone di bloccaggio). Successivamente, lavare i campioni tre volte con PBS. Incubare con un anticorpo secondario (per esempio anti-coniglio Cy5, 1:200 in soluzione bloccante) per 30 minuti in una camera umida e buio, a RT. Infine, lavare i campioni tre volte con PBS e li montano sul vetro diapositive utilizzando Vectashield ad es. </li></ol></li><li> Morfologia, l&#39;integrità e la quantità dei veicoli commerciali leggeri isolati da GFP-calnexina esprimere D. DIScoideum o da RAW264.7 macrofagi vengono analizzati mediante un microscopio a fluorescenza equipaggiato con i filtri adeguati. </li></ol> 4. Risultati rappresentativi La qualità e la resa di purificazione LCV mediante immuno-affinità separazione e centrifugazione in gradiente di densità può essere seguito mediante microscopia a fluorescenza (Fig. 1). Una panoramica dei campioni raccolti durante l&#39;isolamento LCV dal D. discoideu m o macrofagi viene mostrato (Fig. 2). L&#39;omogenato di L. pneumophila con infezione da fagociti mostra intatte veicoli commerciali leggeri, ma anche un sacco di detriti cellulari e batteri extracellulari. Dopo pellet il campione, l&#39;immagine è simile al omogenato, a volte un po &#39;più densa. Poiché LCV intatte dovrebbe aderire alla colonna, il flusso attraverso lo contiene batteri extracellulari ed i detriti cellulari. Dopo eluizione del campione dalla colonna, l&#39;eluato contiene LCV intatte in grandi quantità. Nelle nostre mani, la LCV purificati<html> sulla sembra essere più efficace per D. discoideum che per macrofagi. In D. discoideum i vacuoli patogeno appaiono più rotondo e la resa dei veicoli commerciali leggeri isolati più di 10 volte superiore rispetto ai macrofagi (Fig. 3). In macrofagi intatti LVCs colorati con anticorpi diversi appaiono anche più di forma irregolare. <img alt="Figura 1" src="/files/ftp_upload/4118/4118fig1.jpg" /> Figura 1. Schema di isolamento LCV. A) Isolamento di veicoli commerciali leggeri da immuno-separazione magnetica utilizzando un anticorpo purificato per affinità contro il SIDC batterica proteine ​​effettrici, la localizzazione esclusivamente alla membrana LCV. Un secondo MACS micro perline accoppiato anticorpo e un magnete vengono utilizzati per isolare i veicoli commerciali da detriti cellulari. B) Dopo immuno-magnetiche veicoli commerciali leggeri di separazione sono ulteriormente purificati mediante centrifugazione in gradiente di densità Histodenz. Veicoli commerciali leggeri sono arricchiti in un&#39;aliquota del 4. upload/4118/4118fig2.jpg &quot;/&gt; I campioni Figura 2. Raccolte durante la purificazione LCV. Immagini da omogenato, pellet, a flusso continuo e eluato da D. discoideum o macrofagi sono raffigurate. I campioni mostrano L. pneumophila produce DsRed-Express (rosso) in D. discoideum produzione di GFP-calnexina segnale (verde, pannello superiore) o in macrofagi colorato con un anticorpo anti-SIDC (ciano, pannello inferiore). Bar, 10 pm. <img alt="Figura 3" src="/files/ftp_upload/4118/4118fig3.jpg" /> Figura 3. Purificato veicoli commerciali leggeri da D. discoideum o macrofagi. I campioni mostrano un&#39;aliquota del 4, dopo centrifugazione in gradiente di densità Histodenz di L. pneumophila produce DsRed-Express (rosso) (A) D. discoideum produzione di GFP-calnexina segnale (verde) o (B) in macrofagi colorato con un anticorpo anti-SIDC (ciano). Bar, 10 pm.

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In contrasto con i metodi pubblicati in precedenza, questo protocollo si basa su due fasi, prima la separazione dei veicoli commerciali leggeri da parte di un approccio immuno-magnetico, e la seconda, la purificazione dei veicoli commerciali leggeri di centrifugazione in gradiente di densità. L&#39;isolamento LCV può essere facilmente seguita da microscopia a fluorescenza, quando sia i batteri fluorescente GFP e cellule produttrici di anticorpi o di colorazione viene utilizzato. Il protocollo qui descritto è un sempl...

purification of pathogen vacuoles from legionella-infected phagocytes

L&#39;agente patogeno opportunistico Legionella pneumophila è un ameba-batterio resistente, che replica anche nei macrofagi alveolari causando la grave polmonite &quot;&#39;malattia del legionario&quot; 1. In fagociti protozoi e mammiferi, L. pneumophila si avvale di un meccanismo conservato in modo da formare uno specifico, la replica-permissive vano, la &quot;Legionella contenente vacuolo&quot; (LCV). Richiede la formazione di LCV batterica Icm / Dot sistema di secrezione di tipo IV (T4SS), che trasloca ben 275 &quot;effettrici&quot; proteine ​​in cellule ospiti. Gli effettori manipolare proteine ​​di accoglimento, nonché lipidi e comunicare con secretoria, organelli endosomiali e mitocondriale 2-4. La formazione di veicoli commerciali leggeri rappresenta un processo complesso, robusto e ridondante, che è difficile da afferrare in maniera riduzionista. Un approccio integrato è necessario per comprendere esaurientemente la formazione di LCV, compresa un&#39;analisi globale del percorsoplasminogeno interazioni ospite-fattori e le loro dinamiche temporali e spaziali. Come primo passo verso questo obiettivo, veicoli commerciali leggeri intatte vengono purificati e analizzati dalla proteomica e lipidomica. La composizione e la formazione di patogeno contenenti vacuoli è stata studiata mediante analisi proteomica mediante cromatografia liquida o 2-D elettroforesi su gel accoppiato a spettrometria di massa. Vacuoli isolati sia dal suolo un&#39;ameba sociale Dictyostelium discoideum o fagociti di mammifero ospitato Leishmania 5, 6 Listeria, Mycobacterium 7, Rhodococcus 8, 9 o Salmonella Legionella spp. Dieci. Tuttavia, i protocolli di purificazione impiegati in questi studi sono tempo e noiosa, in quanto richiedono ad esempio microscopia elettronica per analizzare morfologia LCV, l&#39;integrità e purezza. Inoltre, questi protocolli non sfruttare le caratteristiche specifiche del vacuolo patogeno per l&#39;enrichment. Il metodo qui presentato supera queste limitazioni impiegando D. discoideum produrre un marcatore fluorescente LCV e prendendo di mira il SIDC batterica proteine ​​effettrici, che selettivamente ancoraggio alla membrana LCV legandosi alla fosfatidilinositolo 4-fosfato (PtdIns (4) P) 3,11. LCV sono arricchite in una prima fase di immuno-separazione magnetica utilizzando un purificati per affinità anticorpo primario contro SIDC e un anticorpo secondario accoppiato a biglie magnetiche, seguita in una seconda fase dalla classica centrifugazione in gradiente di densità Histodenz 12,13 (Fig. 1) . Uno studio del proteoma di veicoli commerciali leggeri isolati dal D. discoideum rivelato più di 560 proteine ​​della cellula ospite, comprese le proteine ​​associate con vescicole fagocitica, mitocondri, ER e Golgi, nonché GTPasi diversi, che non sono stati implicati nella formazione LCV prima 13. LCV arricchito e purificato con ilprotocollo descritto qui può essere ulteriormente analizzati al microscopio (immunofluorescenza, microscopia elettronica), metodi biochimici (Western Blot) e gli approcci di proteomica o lipidomic.

Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Questo articolo descrive un metodo per l&#39;isolamento e la purificazione di intatto<em&gt; Legionella</em&gt; Contenenti vacuoli (LCV) da amebe e macrofagi. Le due fasi protocollo comprende arricchimento LCV mediante immuno-separazione magnetica utilizzando un anticorpo contro un marcatore batterica LCV e ulteriore purificazione mediante centrifugazione a gradiente di densità.

Purificazione di Vacuoli patogeno da<em&gt; Legionella</em&gt;-Infetti fagociti

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

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Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K Views.  Ludwig-Maximilians-Universität. Questo articolo descrive un metodo per l'isolamento e la purificazione di intatto Legionella Contenenti vacuoli (LCV) da amebe e macrofagi. Le due fasi protocollo comprende arricchimento LCV mediante immuno-separazione magnetica utilizzando un anticorpo contro un marcatore batterica LCV e ulteriore purificazione mediante centrifugazione a gradiente di densità.

Video: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

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. ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificazione per affinità di Complessi virus influenzali ribonucleoproteina dalla cromatina delle cellule infettate

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

Live-cellula Microscopia Video di funghi patogeni fagocitosi

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

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isotermico di amplificazione (lampada) Assays Loop-mediata per rilevamento delle specie-specifica<em&gt; Eimeria</em&gt; Che infettano Polli

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA Purificazione da nella cellula Grown<em&gt; Listeria monocytogenes</em&gt; In macrofagi

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Sviluppo e identificazione di una sottopopolazione romanzo di Human neutrofili di derivazione fagociti Giant<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

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 ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.