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 ...

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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

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Trattabili infezioni cellula di mammifero con protozoo-innescate batteri

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

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Immunologia e infezioni

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Uso di<em&gt; Galleria mellonella</em&gt; Come organismo modello per studiare<em&gt; Legionella pneumophila</em&gt; Infezione

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Studio di fagolisosoma Biogenesis nei macrofagi in diretta

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Quantificazione dei citosolico vs. vacuolare<em&gt; Salmonella</em&gt; In primarie macrofagi di Permeabilization differenziale

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila membrana esterna Vescicole: Isolamento e analisi del loro potenziale pro-infiammatori sui macrofagi

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Quantificazione della crescita intracellulare all'interno di macrofagi è un veloce e affidabile metodo per valutare la virulenza dei parassiti di Leishmania

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Applicazione di Live Cell Imaging e tomografia Cryo-Electron a risolvere le caratteristiche spatiotemporali del sistema di secrezione Legionella pneumophila Dot/Icm

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Studiare la fagocitosi della Leishmania usando la microscopia confocale

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Utilizzo di LPS batterici per eliminare i batteriofagi dalle colture di Salmonella

Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Autore in primo piano: Eliminazione efficiente dei batteriofagi dalle colture di Salmonella infette utilizzando i lipopolisaccaridi

Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.

Purificazione di Vacuoli patogeno da

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