Name: use of shigella flexneri to study autophagy-cytoskeleton interactions
Uploaded: 2014-09-09T18:00:00
Duration: 12 min 52 s
Description: Watch this Scientific Journal Video about Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions at JoVE.com

Arbejdet i SM laboratorium understøttes af en Wellcome Trust Research Karriereudvikling Fellowship [WT097411MA].

<ol>
	<li>Ashida, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+are+versatile+mucosal+pathogens+that+circumvent+the+host+innate+immune+system.">Shigella are versatile mucosal pathogens that circumvent the host innate immune system.</a> Curr Opin Immunol. 23, 448-455 (2011).</li><li>Phalipon, A., Sansonetti, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella's+ways+of+manipulating+the+host+intestinal+innate+and+adaptive+immune+system:+a+tool+box+for+survival.">Shigella's ways of manipulating the host intestinal innate and adaptive immune system: a tool box for survival.</a> Immunol Cell Biol. 85, 119-129 (2007).</li><li>Welch, M. D., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arp2/3-mediated+actin-based+motility:+A+tail+of+pathogen+abuse.">Arp2/3-mediated actin-based motility: A tail of pathogen abuse.</a> Cell Host Microbe. 14, 242-255 (2013).</li><li>Haglund, C. M., Welch, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogens+and+polymers:+microbe-host+interactions+illuminate+the+cytoskeleton.">Pathogens and polymers: microbe-host interactions illuminate the cytoskeleton.</a> J Cell Biol. 195, 7-17 (2011).</li><li>Mostowy, S., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septins:+the+fourth+component+of+the+cytoskeleton.">Septins: the fourth component of the cytoskeleton.</a> Nat Rev Mol Cell Biol. 13, 183-194 (2012).</li><li>Saarikangas, J., Barral, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+functions+of+septins+in+metazoans.">The emerging functions of septins in metazoans.</a> EMBO Rep. 12, 1118-1126 (2011).</li><li>Mostowy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrapment+of+intracytosolic+bacteria+by+septin+cage-like+structures.">Entrapment of intracytosolic bacteria by septin cage-like structures.</a> Cell Host Microbe. 8, 433-444 (2010).</li><li>Mostowy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p62+and+NDP52+proteins+target+intracytosolic+Shigella+and+Listeria+to+different+autophagy+pathways.">p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways.</a> J Biol Chem. 286, 26987-26995 (2011).</li><li>Mostowy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septins+regulate+bacterial+entry+into+host+cells.">Septins regulate bacterial entry into host cells.</a> PLoS One. 4 (15), (2009).</li><li>Mostowy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septin+11+restricts+InlB-mediated+invasion+by+Listeria.">Septin 11 restricts InlB-mediated invasion by Listeria.</a> J Biol Chem. 284, 11613-11621 (2009).</li><li>Phan, Q. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+cell+Septin+7+in+the+endocytosis+of+Candida+albicans.">Role of endothelial cell Septin 7 in the endocytosis of Candida albicans.</a> mBio. 4 (e00542-13), (2013).</li><li>Mostowy, S., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septins+as+key+regulators+of+actin+based+processes+in+bacterial+infection.">Septins as key regulators of actin based processes in bacterial infection.</a> Biol Chem. 392, 831-835 (2011).</li><li>Levine, B., Mizushima, N., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+immunity+and+inflammation.">Autophagy in immunity and inflammation.</a> Nature. 469, 323-335 (2011).</li><li>Randow, F., MacMicking, J. D., James, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+self-defense:+how+cell-autonomous+immunity+protects+against+pathogens.">Cellular self-defense: how cell-autonomous immunity protects against pathogens.</a> Science. 340, 701-706 (2013).</li><li>Mostowy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+bacterial+clearance:+a+not+so+clear+picture.">Autophagy and bacterial clearance: a not so clear picture.</a> Cell Microbiol. 2 (12063), (2012).</li><li>Mostowy, S., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+autophagy:+restriction+or+promotion+of+bacterial+replication.">Bacterial autophagy: restriction or promotion of bacterial replication.</a> Trends Cell Biol. 22, 283-291 (2012).</li><li>Kanther, M., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbe+interactions+in+the+developing+zebrafish.">Host-microbe interactions in the developing zebrafish.</a> Curr Opin Immunol. 22, 10-19 (2010).</li><li>Renshaw, S. A., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+450+million+years+in+the+making:+zebrafish+and+vertebrate+immunity.">A model 450 million years in the making: zebrafish and vertebrate immunity.</a> Dis Model Mech. 5, 38-47 (2012).</li><li>Mostowy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+new+model+for+the+in+vivo+study+of+Shigella+flexneri+interaction+with+phagocytes+and+bacterial+autophagy.">The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.</a> Plos Path. 9, e1003588 (2013).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+and+permeabilization+of+cells+and+tissues.">Fixation and permeabilization of cells and tissues.</a> Cold Spring Harb Protoc. , (2008).</li><li>Klionsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy.">Guidelines for the use and interpretation of assays for monitoring autophagy.</a> Autophagy. 8, 445-544 (2012).</li><li>Mizushima, N., Yoshimori, T., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+mammalian+autophagy+research.">Methods in mammalian autophagy research.</a> Cell. 140, 313-326 (2010).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> The Zebrafish Book. , (2000).</li><li>Benard, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> J Vis Exp. 61 (e3781), (2012).</li><li>He, C., Bartholomew, C. R., Zhou, W., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+autophagic+activity+in+transgenic+GFP-Lc3+and+GFP-Gabarap+zebrafish+embryos.">Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos.</a> Autophagy. 5 (4), 520-526 (2009).</li><li>Mizushima, N., Komatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+renovation+of+cells+and+tissues.">Autophagy: renovation of cells and tissues.</a> Cell. 147, 728-741 (2011).</li><li>Komatsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+levels+of+p62+control+cytoplasmic+inclusion+body+formation+in+autophagy-deficient+mice.">Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice.</a> Cell. 131, 1149-1163 (2007).</li><li>Nowicki, B., Coyne, K. E., Lublin, D. M., Nowicki, S., Hart, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+consensus+repeat-3+domain+of+recombinant+decay-accelerating+factor+is+recognized+by+Escherichia+coli+recombinant+Dr+adhesin+in+a+model+of+a+cell-cell+interaction.">Short consensus repeat-3 domain of recombinant decay-accelerating factor is recognized by Escherichia coli recombinant Dr adhesin in a model of a cell-cell interaction.</a> J Exp Med. 178, 2115-2121 (1993).</li><li>Hall, C., Flores, M., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Developmental Biology. 7 (42), (2007).</li><li>Renshaw, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108, 3976-3978 (2006).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish.">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117, 2010-2010 (2011).</li><li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+for+morpholino+use+in+zebrafish.">A primer for morpholino use in zebrafish.</a> Zebrafish. 6 (9), (2009).</li><li>He, C., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+autophagy+in+zebrafish.">Analyzing autophagy in zebrafish.</a> Autophagy. 6, 642-644 (2010).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 25 (496 (7446)), 498-503 (2013).</li><li>Levraud, J. P., Colucci-Guyon, E., Redd, M. J., Lutfalla, G., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+zebrafish+innate+immunity.">In vivo analysis of zebrafish innate immunity.</a> Methods Mol Biol. 415, 337-363 (2008).</li></ol>

Forfatterne erklærer, at de ikke har nogen konkurrerende finansielle interesser.

Ved overvågning autofagi og cytoskelettet in vitro ved anvendelse vævskulturceller, kan de protokoller, der er beskrevet i afsnit 1 og 2 anvendes til en bred vifte af vævskultur celletyper. Desuden følger autophagy (f.eks ATG8 / LC3 + ve autophagosomes) og cytoskelet (f.eks. Actin haler, septin bure) dynamik i realtid i løbet af Shigella-infektion med levende billeddannelse, kan vævsdyrkningsceller transficeres forbigående hjælp GFP-, RFP- eller FFP-mærkede konstruktioner som tidligere beskrevet 7,8. At øge andelen af celler inficeret med Shigella (dvs. generelt ønskeligt for real-time analyse overvejer at Shigella kan invadere 5-30% af HeLa-celler ved 100:. 1 MOI), direkte tilsættes 400 ul af Shigella (OD600 = 0,3 -0.6) til celler i 2 ml MEM (serum-udsultet), og vent mindst 1,5 timer efter infektion tilstrækkelig bakteriel indrejse, flygte fra fagosomet, replikation, AUtophagy anerkendelse og septin bure. Alternativt kan man anvende Shigella M90T AfaI stamme, der udtrykker adhesin AfaE og har meget højere invasion evner i epitelceller i forhold til M90T stamme 28. Af note, M90T AfaI stamme er endnu ikke blevet testet in vivo ved hjælp af zebrafisk. Plader af Shigella kolonier kan opbevares ved 4 ° C i 2-3 dage og anvendes til flere eksperimenter. Men over tid, kolonier af Shigella, der har mistet virulensplasmidet kan også absorbere Congorødt og synes at have bevaret deres virulensplasmid. Af denne grund anbefaler vi at bruge friske bakterielle bestande, når det er muligt. Ved overvågning af cellebiologi infektion in vivo, protokoller, der er beskrevet i punkt 3 og 4 anvendelse vildtype AB linjen zebrafisk. For at overvåge Shigella -leukocyte interaktioner, kan transgene zebrafisk linjer skal anvendes, f.eks, MPX: GFP eller LYZ: dsRed til visuelize neutrofiler 19,29,30 eller MPEG1: mcherry at visualisere makrofager 19,31. At visualisere autofagi in vivo, kan GFP-Lc3 zebrafisk transgene linje 19,24 bruges som beskrevet i afsnit 3.8. At forstyrre autofagi in vivo, er den effektive morfolino oligonukleotid dosis skal vurderes eksperimentelt baseret på dens effektivitet til at hæmme afskrift splejsning eller protein oversættelse. Det er tilrådeligt at udføre en titrering eksperiment og bekræfte udtømning ved RT-PCR (for splejs morpholino oligonukleotid) eller ved SDS-PAGE (til translationel morpholino oligonukleotid) 32. RNA-isolering fra zebrafisk embryoner eller larver kan udføres under anvendelse af guanidiniumthiocyanat-phenol-chloroform ekstraktion. At udvinde protein fra zebrafisk larver (8 til 15 larver / rør), mekanisk homogeniseres ved hjælp af pistil i 200 pi lysisbuffer (1 M Tris, 5 M NaCl, 0,5 M EDTA, 0,01% oktylphenol ethylenoxid Kondense og proteasehæmmer). Centrifugerør ved 19.000 xg ved 4 ° C i 15 minutter og overføres supernatanten til et nyt rør. Tilføj Laemmli puffer og opvarmes prøven ved 95 ° C i 15 min. Lysater kan opbevares ved -80 ° C indtil brug, og kan evalueres ved Western blotting som beskrevet i afsnit 2.3. Zebrafisken er en fremragende model for in vivo-lægemiddel ansøgning. Analyse ved hjælp af morpholino oligonukleotider kan suppleres med etablerede medicin til at manipulere autofagi (fx rapamycin og bafilomycin). Ikke-inficerede og / eller inficerede larver kan behandles med rapamycin (1,5 uM) eller bafilomycin (80 nM) fortyndet i E2 og autophagic flux kan vurderes ved Western blotting som beskrevet i 25,33. Konsekvensen af ​​autofagi manipulation på resultatet af infektion og overlevelse af de inficerede larver kan vurderes som beskrevet i afsnit 3.5. Ud over at studere værtscelledeterminanter, in vitro og in vivo protokoller kan anvendes til at vurdere bakterielle determinanter, der kræves for autophagy anerkendelse, ved hjælp af bakterielle mutantstammer, der er forskelligt anerkendt af autophagy, f.eks., Shigella ΔicsA (Shigella proteinet ICSA rekrutterer N-WASP og derefter ARP2 / 3 for actin hale og septin bur stiftelse i sit fravær kan der ikke være nogen actin haler, ingen septin bure) og ShigellaΔicsB (Shigella undgår autophagic svar via bakterielle effektor proteiner IcsB, der forhindrer rekruttering af autofagi maskiner til ICSA, i sit fravær Der kan være flere septin bure, mere autofagi) 7,8. Shigella er ikke en naturlig patogen af zebrafisk og vokser optimalt ved 37 ° C. Hidtil er der dog vist, at virulensfaktorer kræves for Shigella invasion, flygte fra den fagocytiske vacuole og replikation i cytosol kan udtrykkes og er funktionelle i zebrafisk larver ved 28 ° C 19. 28 ° C er den mest almindeligt anvendte temperatur for zebrafisk opdræt og standard temperatur for at sikre normal zebrafisk udvikling 23. Bemærkelsesværdigt er de vigtigste patogene begivenheder, der fører til shigellose i mennesker (dvs. makrofag celledød, invasion og multiplikation i epitelceller, celle-til-celle-spredning, inflammatorisk ødelæggelse af værtens epitel) trofast gengivet i zebrafisk model af Shigella infektion 19. Autofagi og Cytoskeleton gener ubikvitært udtrykt og har et bredt spektrum af biologiske funktioner. Museforsøg har vist, at knockout af væsentlig autofagi 26 eller septin gener 5 er embryonale dødbringende, og det er sandsynligt, at nogle af disse gener også vil være afgørende for zebrafisk udvikling (selv om dette problem kan reduceres ved, at zebrafisk har flereparalogous gener 33). Hvis ja, er der flere alternativer til at løse dette problem, herunder (i) brug af farmakologiske reagenser til at regulere autofagi og cytoskelettet, (ii) morpholinos kan titreres ned, kan (iii) knockout af gener være konstrueret til kun bestemt celle typer, og / eller (iv) gener involveret i autophagic erkendelse af, at der ikke er afgørende for udvikling af dyr (f.eks., p62) kan målrettes. Mens zebrafisk er en ideel model for at undersøge autofagi og cytoskelettet under Shigella-infektion, er molekylære værktøjer øjeblikket mangler. Feltet har brug for at generere nye værktøjer og drev celle konkret udtryk for de proteiner af interesse. At banke ned udtryk for autofagi / Cytoskeleton gener er nye morpholinogrupper sekvenser, der kræves, og nye metoder til genom teknik (f.eks Talen, CRISPR / Cas9) kan også anvendes. I mellemtiden, tidligere flere værktøjer genereret for mennesker eller museforsøgkan ligeledes arbejde for zebrafisk. De intracellulære bakterier S. flexneri har vist sig som en ekstraordinær modelorganisme at tage fat på centrale spørgsmål i biologi, herunder evnen af bakterier til at blive genkendt af immunsystemet 1,2. Værtscellen anvender septins at begrænse motilitet S. flexneri og målrette dem til autophagy, en kritisk komponent i celle autonom immunitet 7,8. Disse observationer tyder på en ny molekylær rammer at studere autofagi og dets evne til at nedbryde cytosoliske bakterier. Et stort problem er nu fuldt ud at dechifrere de underliggende molekylære og cellulære begivenheder, og at validere disse begivenheder analyseret in vitro under bakteriel infektion in vivo anvendelse af relevante dyremodeller. Til dette formål har zebrafisk blevet etableret som en værdifuld ny vært til analyse af S. flexneri infektion 19. Interaktioner mellem bakterier og værtsceller kan afbildes ved høj opløsning, ogzebrafisk modellen skal vise sig nyttig for forståelsen af cellebiologi af Shigella-infektion in vivo. Zebrafisk larver kan bruges til at undersøge, hvilken rolle af bakteriel autofagi i vært forsvar, og arbejde har vist, at der den forstyrrelse af autofagi negativt kan påvirke vært overlevelse som reaktion på Shigella-infektion 19. De bemærkninger, der genereres fra studiet af Shigella, septin anbringelse i bur og autofagi in vitro ved anvendelse vævsdyrkningsceller og in vivo ved hjælp af zebrafisk larver kan give betydelige fremskridt i forståelsen vært forsvar. De kunne også foreslå udviklingen af ​​nye strategier til bekæmpelse af smitsomme sygdomme. En kritisk Formålet med rapporten er at give mening for de molekylære og cellulære begivenheder analyseret in vitro (dvs. autofagi, aktin haler, septin anbringelse i bur) under bakteriel infektion in vivo i forbindelse med en entire organisme ved hjælp af zebrafisk larver. Hvis ikke bekendt med zebrafisk biologi og håndtering, kan man henvise til i dybden protokoller for korrekt zebrafisk dyrehold 23 og in vivo analyser af zebrafisk infektion 19,35.

Shigella flexneri, en Gram-negativ invasiv enteropatogene bakterie, kan flygte fra phagosomes til cytosolen, og polymerisere værten actincytoskelettet at unddrage cytosoliske immunreaktioner og fremmer intern og intercellulære bevægelse 1,2. På trods af den forståelse af actin-baserede motilitet in vitro 3,4, de mekanismer, der begrænser bakteriel spredning i vivo er ikke fuldstændigt klarlagt. Dette er afgørende for en mere fuldstændig forståelse af medfødte immunitet og vært forsvar. Septins, en højkonserveret familie af proteiner blandt metazoans er guanosintriphosphat (GTP) -bindende proteiner, der samler ind i hetero-oligomert komplekser og danner polære filamenter, der forbinder med cellemembraner og cytoskelettet 5,6. Nyligt arbejde har opdaget, at inficerede værtsceller kan forhindre Shigella actin baseret motilitet ved afskaerme bakterier målrettet til autophAGY indenfor &#39;septin bure «, afslører den første cellulære mekanisme, der modvirker actin baseret motilitet 7,8. En bred åben mark efterforskning ligger nu i &#39;septin biologi og infektion. Septin samling, fremkaldt af en række forskellige patogener (f.eks, Listeria monocytogenes 7,9,10, Mycobacterium marinum 7,8, Candida albicans 11) kan fremstå som et centralt emne i værtsforsvaret 5,12. Autophagy, en stærkt bevaret intracellulær nedbrydning proces, ses som et centralt element i celle-autonom immunitet på grund af sin evne til at levere cytosoliske bakterier til lysosomet 13,14. Men den rolle af bakteriel autofagi in vivo begrænse eller fremme bakteriereplikation stadig dårligt forstået 15,16. Den zebrafisk (Danio rerio) er dukket op som et hvirveldyr model for studiet af infektioner, fordi det er optisk tilgængeligpå larvestadier når det medfødte immunsystem er allerede funktionelle 17,18. Nyligt arbejde har præget modtagelighed zebrafisk larver til S. flexneri, et paradigme for bakteriel autofagi 15, og har brugt Shigella -zebrafish infektion model til at studere manipulation af autophagy til antibakteriel behandling in vivo 19. Denne rapport indeholder nye værktøjer og analyser for at studere S. flexneri interaktioner med autofagi og cytoskelettet. I et første skridt, at protokoller overvåge autofagi-cytoskelettet interaktioner beskrives ved hjælp af Shigella-infektion af den humane epitelcellelinie HeLa. For at vurdere den rolle, autofagi-Cytoskeleton interaktioner på Shigella-infektion proces in vitro metoder manipulere autofagi og Cytoskeleton komponenter (ved hjælp af siRNA eller farmakologiske reagenser) er forudsat. Nyt arbejde har vist, at ved hjælp af Shigella-infektion of zebrafisk larver, kan lignende analyser anvendes til at studere celle biologi infektion in vivo. Protokoller til at forberede og inficere zebrafisk larver er detaljeret, og at vurdere værtens respons på Shigella-infektion in vivo, protokoller til at bestemme vært overlevelse og bakteriel byrde af inficeret larver er forudsat. Metoder til at overvåge ansættelsen af septin og autofagi markører til Shigella (ved hjælp af enten fast eller levende zebrafisk larver), og metoder til at teste rollen af disse processer in vivo [hjælp morpholinogrupper oligonukleotider (injiceret i 1-4 celle embryoer) eller farmakologiske reagenser ( tilsættes direkte til zebrafisk badevandet)] er også diskuteret. Dette program forventes arbejde at give indsigt i de mekanismer, der er nødvendige til kontrol af infektion af cytosole værtsresponser.

<table border="0" cellpadding="0" cellspacing="0" width="771">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Name of Material/ Equipment</td>
			<td style="width:240px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Bafilomycin A1</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td>B1793&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Blebbistatin</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td>B0560</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass microcapillars&#160;</td>
			<td>Harvard Apparatus</td>
			<td>30038</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coarse manual manipulator&#160;</td>
			<td>Narishige</td>
			<td style="width:119px;">M-152</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytochalasin D</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6762</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Dumont #5 fine tweezers</td>
			<td style="width:240px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forchlorfeneuron&#160;</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">32974</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Goat anti-mouse IgG (H+L) antibody</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">N/A&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Goat ant-rabbit IgG (H+L) antibody</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">JetPEI transfection reagent</td>
			<td style="width:240px;">Polyplus transfection</td>
			<td style="width:119px;">101-01N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Latrunculin B</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5288</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">LC3 antibody</td>
			<td style="width:240px;">Novus Biologicals</td>
			<td style="width:119px;">NB100-2220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Low melting agarose</td>
			<td style="width:240px;">Promega</td>
			<td style="width:119px;">V2111</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">MatTek glass bottom dish</td>
			<td style="width:240px;">MatTek corporation</td>
			<td style="width:119px;">P35G-1.0-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM plus L-alanyl-L-glutamine&#160;</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">41090028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">MEM non-essential amino acids solution</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">11140-035</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjector&#160;</td>
			<td>Narishige</td>
			<td>IM-300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller device&#160;</td>
			<td>Sutter Instrument Co., Novato,</td>
			<td>P-87&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Mineral oil</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">P35G-1.0-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Monoclonal anti-tubulin, acetylated antibody&#160;</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">T6793</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nocodazole</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1404</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-phenylthiourea&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Paraformaldehyde</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">158127</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phalloidin&#160;</td>
			<td style="width:240px;">Molecular Probes</td>
			<td>A12379</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Phenol red solution</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0290</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Protease inhibitor cocktail</td>
			<td style="width:240px;">Roche</td>
			<td style="width:119px;">4693116001</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:412px;">p62/SQSTM1 antibody</td>
			<td style="width:240px;">Cliniscience</td>
			<td style="width:119px;">PM045</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapamycin</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">R8781</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Sodium pyruvate</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">11360039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Transfection reagent</td>
			<td style="width:240px;">Life Technologies</td>
			<td style="width:119px;">12252-011</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield hard set mounting medium containing DAPI&#160;</td>
			<td style="width:240px;">Vector Laboratories</td>
			<td style="width:119px;">H-1500&#160;</td>
		</tr>
	</tbody>
</table>

use of shigella flexneri to study autophagy-cytoskeleton interactions

Shigella flexneri er et intracellulært patogen, som kan flygte fra phagosomes at nå cytosolen, og polymerisere værten actincytoskelettet at fremme sin motilitet og formidling. Nyt arbejde har vist, at proteiner involveret i actin-baserede motilitet også er knyttet til autophagy, en intracellulær nedbrydning proces afgørende for celle autonom immunitet. Bemærkelsesværdigt, kan værtsceller forhindre actin-baserede motilitet S. flexneri ved afskaerme bakterier indenfor &#39;septin bure «og målrette dem til autofagi. Disse observationer tyder på, at en mere fuldstændig forståelse af septins, en familie af trådformede GTP-bindende proteiner, vil give ny indsigt i processen med autophagy. Denne rapport beskriver protokoller til at overvåge autofagi-Cytoskeleton interaktioner forårsaget af S. flexneri in vitro under anvendelse vævskulturceller og in vivo ved hjælp af zebrafisk larver. Disse protokoller muliggøre undersøgelse af intracellulære mechanisms, der styrer bakteriel udbredelse på molekylært, cellulært og hele organismen niveau.

Efter infektion af vævskulturceller in vitro, S. flexneri kan undslippe fra fagosomet og invadere cytosolen. I cytosolen, kan værtsceller forhindre actin-baserede motiliteten af Shigella ved afskaerme bakterier inde septin bure (figur 1A). Bakterier indesluttet af septin bure kan også mærkes ved autofagi markører p62 (figur 1B), og LC3 (figur 1C). Disse observationer fremhæve en ny virkningsmekanisme vært forsvar, der begrænser udbredelsen af ​​invasive patogener, og også afsløre nye forbindelser mellem autofagi og cytoskelettet. Påfaldende, udtømningen af autofagi markører reducerer septin anbringelse i bur af bakterier (figur 2A), og arbejde har også vist, at nedbrydningen af septin anbringelse i bur reducerer rekruttering af autofagi 8 markører betydeligt. I det mindste i tilfælde af Shigella septin huskonstruktion og autophagosome FORMATIOn kan ses som indbyrdes afhængige processer. Andre cellulære krav til opdeling af Shigella efter septin bure omfatter actinpolymerisation og actomyosin aktivitet (figur 2B). Der er ikke nogen naturlig musemodel af shigellose, og efterforskning af Shigella patogenese, septin biologi og bakteriel autofagi in vivo kan drage fordel af en ny dyremodel for infektion, zebrafisk larver 19. Det er muligt at inficere zebrafisk larver ved at injicere bakterier i forskellige anatomiske steder såsom kaudale intravenøse injektioner for overlevelse eksperimenter og hale muskel injektioner til in vivo-mikroskopi (figur 3A). Afhængig af den dosis, S. flexneri injiceret i zebrafisk larver kan enten blive ryddet inden 48 timer efter infektion, eller kan resultere i en progressiv og i sidste ende dødelig infektion (figur 3B - 3D). Shigella virulens factors udtrykkes ved 28 ° C, den optimale væksttemperatur af zebrafisk, og zebrafisk infektion af Shigella er strengt afhængig af en type III sekretionssystem (T3SS) 19, en vigtig virulens determinant i human sygdom. Tilsammen disse observationer tyder på, at zebrafisk larve repræsenterer en værdifuld ny vært til in vivo analyser af Shigella-infektion. Den optiske tilgængelighed zebrafisk larver muliggør visualisering af septin bure in vivo (figur 4A), en præstation, som aldrig før er blevet opnået under anvendelse af pattedyr-modeller. Som supplement til dokumentation for, at septin bure indfange bakterier målrettet til autophagy in vivo, kan man inficere transgene zebrafisk larver udtrykker GFP-Lc3 og observere autofagi markør rekruttering til Shigella (figur 4B). Til ultrastrucutral analyse af Shigella autophagosomes in vivo, electron mikroskopi kan bruges til tydeligt at vise cytosoliske beslaglæggelse af bakterier ved dobbelte membran vakuoler 19. Autofagi ses som et centralt element i celle-autonom immunitet og en afgørende forsvarsmekanisme mod intracytosolic bakterier 14-16. For at karakterisere autofagi funktion in vivo, kan p62 morpholino-behandlede zebrafisk larver anvendes. I modsætning til kernen autofagi maskiner [f.eks. De 36 autofagi relaterede proteiner (ATG&#39;er) 26], p62 ikke er essentielt for vertebrat udvikling 27 og dermed zebrafisk larver kan udvikle sig normalt før infektion. Bemærkelsesværdigt, p62-udtømte larver inokuleret med S. flexneri resultat betydeligt øget dødelighed og øget bakteriel byrde 19. Efter aftale med in vitro-arbejde viser, at septin bur samling er indbyrdes afhængige med autophagosome dannelse 7,8 er septin rekruttering til Shigella klart reduceret i p62-udtømte larver ( <strong&gt; figur 4C). Disse data viser, at zebrafisk overlevelse afhænger af p62-medieret autofagi at styre intracellulær bakterieinfektion. <img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig1highres.jpg" width="500" /> Figur 1. septin bur in vitro. (A) HeLa-celler blev inficeret med S. flexneri i 4 timer 40 min, faste, mærket med antistoffer mod SEPT9 og phalloidin, og afbildet ved konfokal mikroskopi. Scale bar, blev en um. (B) HeLa-celler inficeret med S. flexneri i 4 timer 40 min, faste, mærket med antistoffer mod p62 og SEPT2, og afbildet af fluorescerende lys mikroskopi. Scale bar, blev en um. (C) HeLa-celler transficeret med GFP-ATG8 / LC3, inficeret med S. flexneri i 4 timer 40 minutter, fikseret, mærket med antistoffer mod SEPT2 og afbildet af fluorescerende lys kmcroscopy. Målestok, 1 um. Disse tal er blevet ændret fra Mostowy m.fl. 7. <img alt="Figur 2" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig2highres.jpg" width="500" /> Figur 2. Cellular krav til Shigella -septin bur dannelse. (A) HeLa-celler blev behandlet med kontrol (CTRL), P62, ATG5, ATG6 eller ATG7 siRNA. Helcellelysater af siRNA-behandlede celler blev immunoblottet til GAPDH, p62, ATG5, ATG6 eller ATG7 at vise effektiviteten af ​​siRNA udtømning (øverst). siRNA-behandlede celler blev inficeret med S. flexneri i 4 timer 40 min, faste, og mærket til kvantitativ mikroskopi. Grafer (nederst) repræsenterer middelværdien% ± SEM af Shigella inde SEPT2 bure fra n ≥3 eksperimenter pr behandling. (B) HeLa-celler blev inficeret med S. flexneri, behandlet med DMSO, cytochalasin D (CytD), latrunculin B (LatB), nocodazol (NOCO) eller Blebbistatin (Bleb), og efter 4 timer 40 min blev fikseret og mærket til kvantitativ mikroskopi. Grafer repræsenterer middelværdien% ± SEM af Shigella inde SEPT2 bure fra to uafhængige forsøg per behandling. Disse tal er blevet ændret fra Mostowy m.fl. 7. <img alt="Figur 3" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig3highres.jpg" width="500" /> Figur 3. zebrafisk model af Shigella-infektion. (A), der illustrerer orienteringen af zebrafisk larve under stereomikroskop. (Venstre panel) zebrafisk larver 72 timer efter befrugtning var placeret sideværts i injektionen plade med deres dorsalsiden vender kanylen. (Midterste panel) infektion i blodet blev udført ved injecting bakterierne (rød opløsning) i halevenen, posteriort for urogenitale åbning. (Højre panel) Infektion i halen musklen blev udført ved injektion af bakterier (rød opløsning) over et somit. (B) Overlevelseskurver af 72 timer efter befrugtning larver injiceret med forskellige doser af S. flexneri og inkuberet ved 28 ° C i 48 timer efter infektion. Den effektive inokulum blev klassificeret som lav (&lt;10 3 CFU, åbne cirkler), medium (~ 4 x 10 3 CFU, åbne trekanter) eller høj (~ 10 4 CFU, åbne pladser). Mean% ± SEM (vandrette stænger) fra n ≥3 eksperimenter pr inokulum klasse. (C) Tælling af levende bakterier i homogenater fra de enkelte larver på forskellige tidspunkter efter infektion målt ved udpladning på LB Bemærk, kun larver har overlevet infektionen er inkluderet i optællingen analyse. Middel ± SEM (vandrette stænger), også vist. (D) Fordeling af GFP- Shigella bestemt vedlevende billeddannelse ved hjælp af et fluorescerende stereomikroskop på forskellige tidspunkter efter infektion med en lav, medium eller høj dosis inokulum (caudale intravenøse injektioner). Overlay for overførsel billede (grå) og GFP-fluorescens (grøn) (B) -. (D) Disse tal er blevet ændret fra Mostowy m.fl. 19. <img alt="Figur 4" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig4highres.jpg" width="500" /> Figur 4. cellebiologi af Shigella-infektion in vivo. (A) zebrafisk larver blev inficeret i halen musklen med GFP- Shigella (lav dosis) i 24 timer, fikseret, mærket med antistoffer mod SEPT7 (rød) og GFP (grønt ) og afbildet ved konfokal mikroskopi. Målestokken blev 5 um. (B) GFP-Lc3 zebrafisk larver inficeret med mCherry- Shigella (medium dosis) i4 timer, faste, mærket med antistoffer mod mCherry (rød) og til GFP (grøn) og afbildet ved konfokal mikroskopi. . Målestokken, 1,5 um (C) zebrafisk larver behandlet med enten kontrol (Ctrl; billedet til venstre) eller P62 (højre billede) morpholinos blev inficeret med GFP- Shigella i 4 timer (middel dosis), fast, mærket med antistoffer mod SEPT7 ( rød) og GFP (grønt) og afbildet ved konfokal mikroskopi. Pile fremhæve nogle eksempler på Shigella indesluttet i septin bure (CTRL) eller ej (P62 forarmet) en 4 timer efter infektion. Scale bar, 5 um. Disse tal er blevet ændret fra Mostowy m.fl. 19.

Watch this Scientific Journal Video about Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions at JoVE.com

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

At modvirke patogen udbredelse, værtsceller reorganisere deres cytoskelet at inddeler bakterier og fremkalde autofagi. Brug af Shigella-infektion af vævskulturceller, vært og patogen determinanter bag denne proces er identificeret og karakteriseret. Brug af zebrafisk modeller af Shigella-infektion, er den rolle, som opdagede molekyler og mekanismer undersøgt in vivo.

Anvendelse af<em&gt; Shigella flexneri</em&gt; For at studere autofagi-Cytoskeleton interaktioner

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

use-of-shigella-flexneri-to-study-autophagy-cytoskeleton-interactions

Research

JoVE Journal

Immunology and Infection

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

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.

Anvendelse af<em&gt; Galleria mellonella</em&gt; Som en model organisme til at studere<em&gt; Legionella pneumophila</em&gt; Infektion

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

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Evaluering af effekt og toksicitet af RNA Målretning HIV-1 Produktion til brug i Gene eller Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Oprettelsen af en tætte Transposon indsættelse bibliotek ved hjælp af bakteriel konjugation i Enterobacterial stammer som Escherichia Coli eller Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Imaging Ca2 + svar under Shigella infektionen af epitelceller

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

En høj overførselshastighed Platform for Screening af Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Supplerende anvendelse af mikroskopiske teknikker og fluorescens aflæsning i studiet af Cryptococcus-Amoeba interaktioner

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

En høj overførselshastighed Shigella-specifikke bakteriedræbende Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Menneskelige Colonoid encellelag at studere samspillet mellem patogener, latente og vært intestinale epitel

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...