Name: purification of pathogen vacuoles from legionella-infected phagocytes
Uploaded: 2012-06-19T13:00:00
Description: Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Forskning i vores laboratorium blev støttet af Max von Pettenkofer Institute, Ludwig-Maximilians Universitet München, det tyske Research Foundation (DFG, HI 1511/3-1) og Bundesministerium für Bildung und Forschung (BMBF) &quot;Medicinske Infektion Genomics&quot;-initiativet ( 0315834C).

1. Forberedelserne til LCV Isolering <ol><li> Streak ud Legionella pneumophila producere DsRed-Express 13,14 fra glycerol bestande på CYE agarplader med 5 mg / ml chloramphenicol (Cam) fire dage før LCV isolation. Inkuber bakterierne ved 37 ° C. </li><li> Seed ud 1 x 10 7 D. discoideum fremstilling GFP-calnexin eller RAW264.7 murine makrofager i 75 cm2 vævskulturkolber en dag før infektion. Anvende 10 ml HL5 medium med 20 ug / ml G418 for D. discoideum o...

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I modsætning til tidligere offentliggjorte fremgangsmåder, er denne protokol baseret på to trin, først til adskillelse af lette erhvervskøretøjer af en immuno-magnetisk metode, og det andet oprensningen af ​​lette erhvervskøretøjer ved densitetsgradientcentrifugering. LCV isolation kan let følges ved hjælp af fluorescensmikroskopi, når enten fluorescensmærkede bakterier og GFP-producerende celler eller antistoffarvning anvendes. Protokollen beskrevet her er en simpel, ligetil fremgangsmåde, hvilket resu...

purification of pathogen vacuoles from legionella-infected phagocytes

Den opportunistisk patogen Legionella pneumophila er en amøbe-resistent bakterie, der også replikerer i alveolære makrofager dermed forårsager den alvorlige lungebetændelse &quot;Legionærsyge&quot; 1. I protozoer og pattedyr fagocytter, L. pneumophila anvender en konserveret mekanisme til dannelse af en specifik, replikations-eftergivende kammer, en &quot;Legionella-holdige vakuolen&quot; (LCV). LCV dannelse kræver bakterielle ICM / Dot type IV sekretionssystem (T4SS), der translokaliserer så mange som 275 &quot;effektor&quot;-proteiner i værtsceller. Effektorerne manipulere vært-proteiner såvel som lipider og kommunikere med sekretorisk, endosomale og mitokondrie organeller 2-4. Dannelsen af ​​lette erhvervskøretøjer repræsenterer en kompleks, robust og overflødige proces, som er vanskeligt at forstå en reduktionistisk måde. En integreret tilgang er påkrævet for at omfattende forstå LCV dannelse, herunder en samlet analyse af stienogen-vært faktor interaktioner og deres tidsmæssige og rumlige dynamik. Som et første skridt hen imod dette mål, er intakte lette erhvervskøretøjer oprenset og analyseret ved proteomics og lipidomics. Sammensætning og dannelse af patogen-holdige vakuoler er blevet undersøgt ved proteomik analyse under anvendelse af væskekromatografi eller 2-D gelelektroforese koblet til massespektrometri. Vakuoler isoleret fra enten sociale jorden amøbe Dictyostelium discoideum eller mammale fagocytter husede Leishmania 5, Listeria 6, Mycobacterium 7, Rhodococcus 8, Salmonella 9 eller Legionella spp. 10. Men, rensning protokoller benyttet i disse undersøgelser er tidskrævende og kedelige, da de kræver fx elektronmikroskopi til at analysere LCV morfologi, integritet og renhed. Derudover har disse protokoller ikke udnytte særlige kendetegn patogenet vacuolen for enrichment. Metoden præsenteres her overvinder disse begrænsninger ved at ansætte D. discoideum frembringelse af en fluorescerende LCV markør og ved at målrette det bakterielle effektorprotein proteinet SidC, som selektivt forankrer til LCV membranen ved at binde til phosphatidylinositol-4-phosphat (PtdIns (4) P) 3,11. Lette erhvervskøretøjer er beriget i et første trin ved immuno-magnetisk separation under anvendelse af et affinitetsoprenset primære antistof mod SidC og et sekundært antistof koblet til magnetiske perler, efterfulgt i et andet trin ved en klassisk Histodenz densitetsgradientcentrifugering 12,13 (fig. 1) . En proteom undersøgelse af isolerede lette erhvervskøretøjer fra D. discoideum afslørede mere end 560 værtscelleproteiner, herunder proteiner associeret med fagocytiske vesikler, mitokondrier, ER og Golgi samt adskillige GTPaser, som ikke er blevet impliceret i LCV dannelse før 13. Lette erhvervskøretøjer beriget og oprenset medprotokollen beskrevet her, kan yderligere analyseres ved mikroskopi (immunfluorescens, elektronmikroskopi), biokemiske metoder (Western blot) og proteom eller lipidomic fremgangsmåder.

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

Denne artikel beskriver en fremgangsmåde til isolering og oprensning af intakt<em&gt; Legionella</em&gt;-Holdige vakuoler (lette erhvervskøretøjer) fra amøbe og makrofager. To-trins protokol omfatter LCV berigelse af immuno-magnetisk separation under anvendelse af et antistof mod et bakterielt LCV markør og yderligere rensning ved densitetsgradientcentrifugering.

Oprensning af Patogen vakuoler fra<em&gt; Legionella</em&gt;-Inficerede Fagocytter

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

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