Name: tractable mammalian cell infections with protozoan-primed bacteria
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Vi takker Dr. Craig Roy og Dr. Dario Zamboni for at tilvejebringe en skabelon for protozo celle infektioner. Vi takker Dr. Jagdeep Obhrai, Dr. Georgiana Purdy, Dr. Fred Heffron og Todd Wisner for udstyr og reagenser, Dr. Lulu Cambronne for kritisk gennemgang af manuskriptet. Flowcytometri blev udført på OHSU Flowcytometri delt ressource facilitet. Dette arbejde blev støttet delvist af en bevilling fra Medical Research Foundation of Oregon og en NIH tilskud R21 AI088275 (EDC).

1. Fremstilling af Legionella pneumophila Cultures for priming Stage Infektioner <ol><li> Omdan alle L. pneumophila stammer, der anvendes i assayet med plasmid pAM239, der koder for et isopropyl β-D-1-thiogalactopyranosid (IPTG) inducerbar grønt fluorescerende protein (GFP) 28. Streak bakteriestammerne over på jern og cystein tilsat N-(2-acetamido)-2-aminoethansulfonsyre (ACES) bufret trækul gærekstraktagar (CYEA) indeholdende 6,25 ug / ml chloramphenicol (CM) (for pl...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+defects+in+intracellular+growth+complemented+by+a+single+genetic+locus+in+Legionella+pneumophila.">Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila.</a> Mol. Microbiol. 7, 7-19 (1993).</li><li>Marra, A., Blander, S. J., Horwitz, M. A., Shuman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Legionella+pneumophila+locus+required+for+intracellular+multiplication+in+human+macrophages.">Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 9607-9611 (1992).</li><li>Segal, G., Purcell, M., Shuman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+cell+killing+and+bacterial+conjugation+require+overlapping+sets+of+genes+within+a+22-kb+region+of+the+Legionella+pneumophila+genome.">Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 1669-1674 (1998).</li><li>Segal, G., Shuman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+new+region+required+for+macrophage+killing+by+Legionella+pneumophila.">Characterization of a new region required for macrophage killing by Legionella pneumophila.</a> Infect Immun. 65, 5057-5066 (1997).</li><li>Vogel, J. P., Andrews, H. L., Wong, S. K., Isberg, R. 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Bakteriel genekspression der kontrolleres nøje ved en kombination af livscyklus progression og reaktion på signaler i det omgivende mikromiljø. Vakuolære patogener, såsom L. pneumophila reagere på en mangfoldighed af værtscelle-afledte signaler, når ruminddelt i en phagosome. Som et samlet resultat af næringsstoffer konsumption i værtscellen, kompenserer bakterien ved at udtrykke faktorer er nødvendige for en vellykket spredning til en efterfølgende værtscelle 25. L. pneumophila...

Talrige bakterielle patogener har tilpasset generelle strategier til at udnytte værtsceller til overlevelse og replikation i en intracellulær rum. I mange tilfælde er patogene mekanismer ens mellem protozoisk og metazo celler. Disse to mikromiljøer er meget forskellige og kan resultere i forskellig ekspression af virulensfaktorer 1-4. Den legionærsyge bakterie Legionella pneumophila er ubikvitært forbundet med ferskvandsområder verdensplan 5. Vigtigere, L. pneumophila dyrket i protozo-celler før infektion af humane monocytter opnå en patogen fordel, hvilket antyder, at globale genekspressionsprofiler af bakterien forlader en protozo celle er anderledes end det in vitro dyrkede organisme 6-8. I naturen tilvejebringe ferskvand amøber næringsrige begrænset til hurtig amplifikation af en indtrængende bakterie. Menneskelig overtagelse af L. pneumophila er oftest tilskrives inhalation af forurenet vanddråber, der indeholder bakterien. Det er sandsynligt, at disse dråber harbor protozo celleassocierede bakterier, hvor protozo celler er mere resistente over for konventionelle vandbehandling praksis 9,10. Infektion af lunge alveolære makrofager gør dette på en måde, der næsten identisk med den intracellulære livscyklus af bakterien i protozoan værtsceller 11-13. For at overleve og replikere i eukaryote celler, L. pneumophila anvender en specialiseret form IVb sekretionssystem betegnes Dot / Icm at levere næsten 300 &quot;effektor&quot;-proteiner i cytosolen af værtscellen 14-16. Disse effektor proteiner fungere samlet for at nedbryde cellulære processer for at frembringe en replikations eftergivende kammer for bakterien 17,18. Deletioner i nogen af ​​de 26 gener, der omfatter Dot / ICM transporter resultat i stammer defekte for intracellulær multiplication 19-23. Historisk set sletning af individuelle effektor kodende gener sjældent resulteret i stammer svækkede for intracellulær vækst. Dette fænomen er blevet tilskrevet flere hypoteser, herunder redundant funktion og paralogous kopier af effektorer. Nogle virulensfaktorer kun udtrykkes i forbindelse med værtscelle-associeret intracellulær vækst 24. Vi rationaliseres, at hvis en bestemt effektor kun blev udtrykt i forbindelse med protozo-infektion, da bidraget fra effektoren ikke kunne sammenlignes med en vildtypestamme, når begge blev dyrket in vitro. L. pneumophila overgange fra et replikativt til en transmissivt fase da den går ind stationær fase i kultur 25. Fasen skift fænotype repræsenterer næringsstof udtynding optræder under intracellulær vækst og er eksemplificeret gennem samling af flageller for motilitet 26. Fordi L. pneumophila er mere invasiontende og ondartet når de er høstet fra protozo celler, vi søgt at udvikle en analyse, som mere trofast repræsenterede patogen tilstand af bakterien, da denne løb vært makrofager. Til dette formål har vi udviklet en alsidig protozo priming assay, der kan rumme en egnet vært for både den første (priming celle) og den anden (målcelle) fase infektioner. Infektionsprocessen er medgørlig ved anvendelse af bakterier, der stabilt udtrykker grønt fluorescerende protein (GFP). Infektionen model for protozo Acanthamoeba castellanii følger en metode almindeligt anvendt inden for området 27. For pennen, L. pneumophila-stammer dyrkes in vitro til stationær fase i flydende medier for at fremstille mærkede &quot;transmissive &#39;bakterier (figur 1A). Bakterier næste anvendt til at inficere monolag af A. castellanii i 18 timer for at opnå et sent tidspunkt af det intracellulære livscyklus. Store vakuoler indeholderING bakterier kan visualiseres på dette tidspunkt ved hjælp af fluorescensmikroskopi (Figur 1A). Protozo-celler lyseres derefter, og bakterier udvundet fra lysatet måles for emission ved 512 nm under anvendelse af en fluorescenspladelæser. Fluorescens er korreleret med optisk tæthed til at beregne multiplicitet-af-infektion (MOI) for infektionen af målceller (fig. 1, * korrelationskurve). Efter invasion (T 0) og 18 timer efter invasion (T 18), er målceller kvantificeret for fluorescens, der repræsenterer intracellulære bakterier. Fluorescens kan overvåges ved mikroskopi og flowcytometri, og levedygtighedstællinger kan måles ved hjælp af koloni udpladning. Den priming analysen er altid ledsaget af infektioner med vildtype L. pneumophila og en stamme defekt i Dot / Icm type IV secerneringssystem (Δ DOTA) (figur 1A). Dette vigtigere giver interne kontroller for direkte sammenligninger mellem vildtype ennd eventuelle isogene mutante stammer, der anvendes i infektionsprocessen. Medtagelsen af den avirulente Δ DOTA-stamme under priming fase fastsætter en tærskelværdi for observation af svækkede vækst fænotyper associeret med isogene mutante stammer, der er dyrket in vitro.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>chloramphenicol</td>
 <td>Fisher Scientific</td>
 <td>BP904-100</td>
 <td>antibiotic</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Fisher Scientific</td>
 <td>BP1755-10</td>
 <td></td>
 </tr>
 <tr>
 <td>ACES</td>
 <td>Sigma</td>
 <td>A9758-1KG</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>ATCC medium: 712 PYG</td>
 <td>ATCC</td>
 <td></td>
 <td>growth media for protozoans</td>
 </tr>
 <tr>
 <td>1X PBS</td>
 <td>Fisher Scientific</td>
 <td>SH30256FS</td>
 <td>phosphate buffered saline</td>
 </tr>
 <tr>
 <td>activated charcoal</td>
 <td>Fisher Scientific</td>
 <td>C272-212</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>yeast extract</td>
 <td>Fisher Scientific</td>
 <td>BP1422-500</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>peptone</td>
 <td>BD Diagnostics</td>
 <td>211677</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>Fisher Scientific</td>
 <td>BP1423-2</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>L-cysteine, 99%+</td>
 <td>Acros organics</td>
 <td>173601000</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td>Ferric nitrate nonahydrate</td>
 <td>Fisher Scientific</td>
 <td>I110-100</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>EVOS fl</td>
 <td>AMG</td>
 <td>EVOS fl</td>
 <td>fluorescence microscope</td>
 </tr>
 <tr>
 <td>Smart Spec Plus</td>
 <td>Bio-Rad</td>
 <td>170-2525</td>
 <td>spectrophotometer</td>
 </tr>
 <tr>
 <td>5810 R centrifuge</td>
 <td>Eppendorf</td>
 <td>22627023</td>
 <td>bench top centrifuge</td>
 </tr>
 <tr>
 <td>Repeater plus</td>
 <td>Eppendorf</td>
 <td>22230201</td>
 <td>repeating pipette</td>
 </tr>
 <tr>
 <td>SpectraMax Gemini EM</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>microplate reader</td>
 </tr>
 <tr>
 <td>Softmax Pro 5.3</td>
 <td>Molecular Devices</td>
 <td>0200-310</td>
 <td>microplate reader software</td>
 </tr>
 <tr>
 <td>5424 microfuge</td>
 <td>Eppendorf</td>
 <td>22620401</td>
 <td>table top microcentrifuge</td>
 </tr>
 <tr>
 <td>Fast-release pipette pump II</td>
 <td>Scienceware</td>
 <td>379111010</td>
 <td>pipette aid</td>
 </tr>
 <tr>
 <td>FACS Calibur</td>
 <td>BD Bioscience</td>
 <td></td>
 <td>flow cytometer</td>
 </tr>
 <tr>
 <td>FlowJo 7.6.1</td>
 <td>FlowJo</td>
 <td>license</td>
 <td>flow cytometery software</td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>polypropylene conical tube</td>
 </tr>
 <tr>
 <td>1.6 ml microfuge tube</td>
 <td>Neptune</td>
 <td>3745.X</td>
 <td>microcentrifuge tubes</td>
 </tr>
 </tbody>
</table>

tractable mammalian cell infections with protozoan-primed bacteria

Mange intracellulære bakterielle patogener anvender ferskvand protozoer som et naturligt reservoir for proliferation i miljøet. Legionella pneumophila, det agens af legionærsyge lungebetændelse, får en patogen fordel i forhold til in vitro-dyrkede bakterier, når først høstet fra protozo-celler før infektion af mammale makrofager. Dette tyder på, at vigtige virulensfaktorer muligvis ikke korrekt udtrykkes in vitro. Vi har udviklet en medgørlig system til priming L. pneumophila gennem sin naturlige protozo host Acanthamoeba castellanii før pattedyrcelle infektion. Bidraget af en virulensfaktor kan undersøges ved at sammenligne intracellulær vækst af en mutantstamme med vildtype-bakterier efter protozoisk priming. GFP-udtrykkende vildtype og mutant L. pneumophila-stammer anvendes til at inficere protozo-monolag i en pennen og tillades at nå sene trin af intracellulær vækst. Fluorescerende bakterier høstes derefter fra disse inficerede celler og normaliseret ved spektrofotometri til at generere tilsvarende antal bakterier for en efterfølgende infektion i mammale makrofager. Til kvantificering, er levende bakterier overvåges efter infektion ved hjælp af fluorescensmikroskopi, flow-cytometri, og ved koloni udpladning. Denne teknik fremhæver og bygger på bidrag fra værtscellen-afhængig genekspression ved at efterligne miljøet, som ville blive mødt i en naturlig erhvervelse rute. Denne fremgangsmåde kan modificeres til at passe til enhver bakterie, der anvender et mellemled vært som et middel til at få en patogen fordel.

Et typisk resultat for hele infektionen er skitseret i figur 1.. Levende celler fluorescens mikrografier skildrer monolag af A.castellanii inficeret med vildtype L. pneumophila under priming fase er vist i figur 1A. En vellykket mål af pennen vil føre til en population af omkring 90% af værtscellerne indeholdende store vakuoler befolket med GFP-mærkede bakterier på denne MOI. Ved 18 timer efter infektion, A. mest castellanii celler er opnået mak...

Watch this Scientific Journal Video about Tractable Mammalian Cell Infections with Protozoan-primed Bacteria at JoVE.com

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Denne teknik tilvejebringer en fremgangsmåde til at høste, normaliseres og kvantificere intracellulær vækst af bakterielle patogener, der er præ-dyrket i naturlige protozoer værtsceller inden infektioner i pattedyrceller. Denne fremgangsmåde kan modificeres til at rumme en lang række værtsceller til priming fase samt målcelletyper.

Medgørlige pattedyrscellelinier Infektioner med protozo-primede Bakterier

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

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