Recherche dans notre laboratoire a été soutenu par l&#39;Institut Max von Pettenkofer, Ludwig-Maximilians-Universität de Munich, de la recherche allemande (DFG, HI 1511/3-1) et le Bundesministerium für Bildung und Forschung (BMBF) «Génomique des infections médicales&quot; initiative ( 0315834C).

1. Préparatifs pour l&#39;isolement LCV <ol><li> Streak à Legionella pneumophila produire DsRed-Express 13,14 à partir de stocks de glycérol sur gélose CYE avec 5 pg / ml de chloramphenicol (Cam) quatre jours avant l&#39;isolement LCV. Incuber les bactéries à 37 ° C. </li><li> Semences à 1 x 10 7 D. discoideum produire GFP-calnexine ou RAW264.7 macrophages murins dans 75 cm de culture tissulaire 2 flacons une infection jour avant. Utilisez 10 ml HL5 moyenne avec 20 pg / ml G418 pour D. discoideum et incuber les amibes à 23 ° C. Pour utiliser les macrophages RAW264.7 10 ml du milieu RPMI 1640 supplémenté avec 10% de FCS (inactivé par la chaleur) et de la glutamine à 1%, et la croissance des cellules à 37 ° C et 5% de CO 2. Utilisez au moins trois 75 cm 2 flacons par l&#39;infection et l&#39;échantillon (minimum 6 x 10 7 cellules). </li><li> Inoculer une culture de nuit avec L. pneumophila de la plaque de CYE. Prenez un tube à essai 15 ml avec 3 ml AYE moyenneset 5 pg / ml de came. Inoculer avec 100 ul d&#39;une suspension bactérienne pour obtenir une 600nm OD de 0,1. Incuber la culture de la nuit sur une roue de rotation tête à 37 ° C pendant 21-22 h. </li></ol> 2. LCV isolement <ol><li> Changer le support de D. cellules discoideum pour enlever l&#39;antibiotique, ce qui nuirait à la suite d&#39;une infection. </li><li> Mesurer la DO de la culture de la nuit. Bactéries devrait avoir atteint leur infectivité pic à 600 nm une densité optique de ≥ 3, qui correspond à 2 x 10 9 bactéries par ml. </li><li> Infecter les cellules en ajoutant environ 500 pi de la L. pneumophila culture d&#39;une nuit aux cellules en croissance dans 10 ml HL5 moyennes (D. discoideum) ou 10 ml de RPMI 1640 (macrophages). Cela correspond à une multiplicité d&#39;infection (MOI) de 50. Par la suite, l&#39;infection est synchronisé par centrifugation des bactéries sur les cellules à 500 g pendant 10 min. La centrifugation est suiviepar une incubation de la D. discoideum cellules à 25 ° C et les macrophages RAW264.7 à 37 ° C et 5% de CO 2, respectivement. L&#39;infection de temps une h comprend l&#39;étape de centrifugation. </li><li> Après l&#39;infection éliminer le milieu et laver les cellules une fois pour éliminer les bactéries extracellulaires. Utiliser le tampon Sorc glacée pour D. discoideum et PBS glacé pour RAW264.7 macrophages. Ajouter 3 ml de tampon HS, complétées par des inhibiteurs de la protéase (Roche) dans chaque flacon et de recueillir les cellules en utilisant un grattoir à cellules. Piscine des échantillons correspondants dans un tube à essai de 15 ml. </li><li> Pour homogénéisation de l&#39;échantillon de 3 mL en plastique des seringues Luer-Lock et la bille en acier inoxydable homogénéisateur. Assurez-vous que vous travaillez sur de la glace. Avant de commencer, lavez l&#39;homogénéisateur à billes avec de l&#39;eau distillée, afin d&#39;éviter toute contamination du détergent et rincez avec de la glace HS tampon froid pour se débarrasser des bulles d&#39;air. Utilisez la boule de 8 um de dédouanement. </li><li> Remplissez le premier 3 mL dans le und seringue de le monter sur ee homogénéisateur. Appuyez sur l&#39;échantillon avant et en arrière neuf fois à travers l&#39;homogénéisateur. Échanger les seringues après pour éviter toute contamination avec du matériel non-homogénéisé. Recueillir et mettre en commun l&#39;échantillon homogénéisé dans un tube à essai de 15 ml et de prendre un échantillon de 150 microlitres par analyse microscopique. Avant de procéder à un échantillon différent démontons et laver l&#39;homogénéisateur à billes. </li><li> Bloquer l&#39;homogénat avec 2% de CS ou FCS pendant 30 min sur un agitateur de la glace ou sur une roue de tête de filage (10-20 min) à 4 ° C. </li><li> Après le blocage d&#39;utiliser un anticorps purifiés par affinité primaire contre SIDC (dilution 1:3000) ou contre tout autre marqueur bactérienne exclusivement la liaison à la membrane LCV. Vortex la solution d&#39;anticorps avant de l&#39;ajouter à l&#39;homogénat. Incuber l&#39;échantillon pendant 1 heure sur un agitateur de la glace ou sur une roue de tête de filage (10-20 min) à 4 ° C. </li><li> Dans le même temps, préparer le gradient Histodenz. Utilisation d&#39;un tube à essai de 15 ml par gradient et trois 75 cm 2 flacons d&#39;un échantillon. Sapiner, ajouter 5,75 ml de Histodenz 35% sur le tube, et le second, ajouter prudemment 5,75 ml de solution Histodenz 10% sur le dessus. Ensuite posez avec soin les tubes horizontalement pendant 1 heure. </li><li> Après une incubation de l&#39;homogénat avec le réactif de blocage et de l&#39;anticorps primaire, centrifuger à 600 xg pendant 15 min à 4 ° C pour obtenir un culot de l&#39;échantillon. Jeter le surnageant et remettre en suspension le culot dans 1,5 ml de tampon HS. Transférer les échantillons dans un nouveau tube de 15 ml de test et de prendre un échantillon de 40 ul pour une analyse microscopique plus tard. </li><li> Incuber le culot avec l&#39;anticorps secondaire - MACS de chèvre anti-IgG de lapin microbilles (dilution 1:25) - pour 30 min sur un agitateur à 4 ° C. Dans le même temps, mettre les colonnes sur le support magnétique MACS et les équilibrer avec 0,5 mL de tampon HS. </li><li> Ensuite, appliquer les échantillons à la colonne. Recueillir 150 pi de l&#39;écoulement continu pour l&#39;analyse microscopique ultérieure. Laver la colonne trois fois avec 0,5 mL de tampon HS. </li><li> Supprimez les colonnes de til l&#39;aimant et les véhicules utilitaires légers avec éluat de 0,5 mL de tampon par HS immédiate gicler. Prenez un échantillon de 20 ul pour l&#39;analyse microscopique. L&#39;étape de purification par immuno-séparation magnétique est prévu de céder pour 1 × 10 7 infectées D. cellules discoideum environ 1 × 10 6 véhicules utilitaires légers, qui sont intactes environ sept fois enrichis dans l&#39;éluat par rapport à la 13 accréditives. </li><li> Placer les tubes de gradient Histodenz revenir à une position verticale et soigneuse au chargement de l&#39;éluat sur le dessus. Centrifuger les tubes à 3350 × g pendant 1 h à 4 ° C. </li><li> Après centrifugation prendre fractions de 1,5 mL à partir du bas du tube à essai en utilisant une longue pipette Pasteur en verre. Recueillir total huit fractions, chacune de la partie inférieure du tube. Pas de calques visibles sont observées dans le gradient continu Histodenz. Le rendement le plus élevé de véhicules utilitaires est généralement prévu dans la fraction 4 à un ratio de ~ 2 × 10 5 véhicules utilitaires intactes par 1 × 10 7 àinfectés amibes 13. Des quantités mineures de véhicules utilitaires sont également présents dans les fractions 3 et 5, respectivement. Ainsi, le rendement global de véhicules utilitaires légers purifiés est d&#39;environ 1 × 10 6 véhicules utilitaires intactes par 6 × 10 7 infectées D. cellules discoideum 13. La récupération de véhicules utilitaires peuvent être réalisées à température ambiante (RT), et les véhicules utilitaires semblent stables pendant plusieurs heures. </li></ol> 3. Analyse microscopique des échantillons prélevés <ol><li> Préparer un 24 bien à fond plat plaque de culture tissulaire avec de la poly-L-lysine lamelles recouvertes. </li><li> Introduire à la pipette à chaque échantillon prélevé lors de la purification et 150 LCV ul de chaque fraction de gradient de densité dans la plaque de 24 puits. Ajouter 0,5 mL de tampon HS à chaque puits pour diluer la concentration élevée Histodenz. Ensuite, centrifuger la plaque à 600 × g pendant 10 min à 4 ° C. Retirez délicatement le surnageant et fixer les échantillons avec du paraformaldéhyde 4% (PFA) pendant 20 min à température ambiante. Après fixation laver les échantillons à deux reprises. Nouse Sorc pour D. discoideum et PBS pour RAW264.7 macrophages. </li><li><ol class="lalpha"><li> Montez les échantillons provenant de la GFP-calnexine exprimant D. discoideum, prises à différentes étapes lors de l&#39;isolement LCV, directement sur ​​verre diapositives à l&#39;aide Vectashield par exemple. </li><li> Incuber les échantillons à partir des macrophages, des prises RAW264.7 à différentes étapes lors de l&#39;isolement LCV, avec une solution de blocage (1% de BSA dans du PBS) pendant 10 min à température ambiante. Utilisez un anticorps anti-SIDC primaire pour colorer les véhicules utilitaires et incuber pendant 1h dans une chambre humide à température ambiante (purifiée par affinité de lapin anti-SIDC; 1:1000 dans le tampon de blocage). Par la suite, se laver les échantillons trois fois avec du PBS. Incuber avec un anticorps secondaire (par exemple anti-lapin Cy5; 1:200 dans une solution de blocage) pendant 30 min dans une chambre humide et sombre à la température ambiante. Enfin, se laver les échantillons trois fois avec du PBS et les monter sur des diapositives à l&#39;aide de verre par exemple Vectashield. </li></ol></li><li> Morphologie, l&#39;intégrité et la quantité des véhicules utilitaires légers isolés à partir de la GFP-calnexine exprimer D. DIScoideum ou de macrophages RAW264.7 sont analysés par un microscope à fluorescence équipé des filtres adéquats. </li></ol> 4. Les résultats représentatifs La qualité et le rendement de la purification par immuno-TR affinité de séparation et de centrifugation en gradient de densité peut être suivie par microscopie de fluorescence (fig. 1). Un aperçu des échantillons prélevés au cours de l&#39;isolement LCV de D. discoideu m ou des macrophages est montré (Fig. 2). L&#39;homogénat de L. pneumophila-infectés par le phagocytes montre VU intactes, mais aussi beaucoup de débris cellulaires et les bactéries extracellulaires. Après la granulation de l&#39;échantillon, l&#39;image est similaire à l&#39;homogénat, parfois un peu plus dense. Depuis VU intactes devrait s&#39;en tenir à la colonne, le flux continu contient principalement des bactéries extracellulaires et les débris cellulaires. Après élution de l&#39;échantillon de la colonne, l&#39;éluat contient VU intactes en grandes quantités. Dans nos mains, le LCV Purificati<html> sur qui semble être plus efficace pour D. discoideum que pour les macrophages. Dans D. discoideum les vacuoles des agents pathogènes apparaissent plus rond et le rendement des véhicules utilitaires isolées de plus de 10 fois plus élevé comparativement à macrophages (Fig. 3). Dans les macrophages intactes PFV colorées avec des anticorps différents semblent également plus de forme irrégulière. <img alt="Figure 1" src="/files/ftp_upload/4118/4118fig1.jpg" /> Figure 1. Vue d&#39;ensemble schématique de l&#39;isolement LCV. A) L&#39;isolement des véhicules utilitaires par immuno-séparation magnétique en utilisant un anticorps purifiés par affinité contre le SIDC protéine effectrice bactérienne, la localisation exclusivement à la membrane LCV. Un MACS secondaire micro-billes couplé anticorps et un aimant sont utilisés pour isoler véhicules utilitaires à partir des débris cellulaires. B) Après véhicules utilitaires de séparation immuno-magnétique sont en outre purifié par centrifugation Histodenz gradient de densité. VU sont enrichis dans la fraction 4. upload/4118/4118fig2.jpg &quot;/&gt; Les échantillons Figure 2. Recueillies lors de la purification LCV. Images de homogénat, granulés, en écoulement continu et éluat de D. discoideum ou les macrophages sont représentés. Les échantillons montrent L. pneumophila produire DsRed-Express (en rouge) dans D. discoideum produire GFP-calnexine signal (vert, panneau supérieur) ou dans les macrophages colorées avec un anticorps anti-SIDC (cyan, panneau inférieur). Bars, 10 um. <img alt="Figure 3" src="/files/ftp_upload/4118/4118fig3.jpg" /> Figure 3. Purifiée véhicules utilitaires à partir de D. discoideum ou les macrophages. Les échantillons montrent fraction correspondant à 4 après centrifugation Histodenz gradient de densité de L. pneumophila produire DsRed-Express (en rouge) dans (A) D. discoideum produire GFP-calnexine signal (vert) ou (B) dans les macrophages colorées avec un anticorps anti-SIDC (cyan). Bars, 10 um.

<ol>
	<li>Hilbi, H., Hoffmann, C., Harrison, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+spp.+outdoors:+colonization,+communication+and+persistence.">Legionella spp. outdoors: colonization, communication and persistence.</a> Environ. Microbiol. Rep. 3, 286-296 (2011).</li><li>Zhu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+identification+of+protein+substrates+of+the+Dot/Icm+type+IV+transporter+of+Legionella+pneumophila.">Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.</a> PLoS ONE. 6, e17638 (2011).</li><li>Hilbi, H., Weber, S., Finsel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anchors+for+effectors:+subversion+of+phospho-inositide+lipids+by+Legionella.">Anchors for effectors: subversion of phospho-inositide lipids by Legionella.</a> Front Microbiol. 2, 91 (2011).</li><li>Hubber, A., Roy, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+host+cell+function+by+Legionella+pneumophila+type+IV+effectors.">Modulation of host cell function by Legionella pneumophila type IV effectors.</a> Annu. Rev. Cell Dev. Biol. 26, 261-283 (2010).</li><li>Kima, P. E., Dunn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+calnexin+expression+on+phagosomes+to+isolate+Leishmania+parasitophorous+vacuoles.">Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles.</a> Microb. Pathog. 38, 139-145 (2005).</li><li>L&#252;hrmann, A., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+purify+bacteria-containing+phagosomes+from+infected+macrophages.">A method to purify bacteria-containing phagosomes from infected macrophages.</a> Methods Cell Sci. 22, 329-341 (2000).</li><li>Sturgill-Koszycki, S., Haddix, P. L., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+Mycobacterium+and+the+macrophage+analyzed+by+two-dimensional+polyacrylamide+gel+electrophoresis.">The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis.</a> Electrophoresis. 18, 2558-2565 (1997).</li><li>Fernandez-Mora, E., Polidori, M., L&#252;hrmann, A., Schaible, U. E., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+Rhodococcus+equi-containing+vacuoles+is+arrested+after+completion+of+the+early+endosome+stage.">Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage.</a> Traffic. 6, 635-653 (2005).</li><li>Mills, S. D., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+Salmonella+typhimurium+and+Yersinia+pseudotuberculosis-containing+phagosomes+from+infected+mouse+macrophages:+Y.+pseudotuberculosis+traffics+to+terminal+lysosomes+where+they+are+degraded.">Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded.</a> Eur. J. Cell Biol. 77, 35-47 (1998).</li><li>Shevchuk, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+Legionella-containing+phagosomes+isolated+from+Dictyostelium.">Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium.</a> Int. J. Med. Microbiol. 299, 489-508 (2009).</li><li>Brombacher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab1+guanine+nucleotide+exchange+factor+SidM+is+a+major+phosphatidylinositol+4-phosphate-binding+effector+protein+of+Legionella+pneumophila.">Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.</a> J. Biol. Chem. 284, 4846-4856 (2009).</li><li>Urwyler, S., Finsel, I., Ragaz, C., Hilbi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Legionella-containing+vacuoles+by+immuno-magnetic+separation.">Isolation of Legionella-containing vacuoles by immuno-magnetic separation.</a> Curr. Protoc. Cell Biol. Chapter 3, Unit 3 (2010).</li><li>Urwyler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Legionella+vacuoles+purified+by+magnetic+immunoseparation+reveals+secretory+and+endosomal+GTPases.">Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases.</a> Traffic. 10, 76-87 (2009).</li><li>Mampel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planktonic+replication+is+essential+for+biofilm+formation+by+Legionella+pneumophila+in+a+complex+medium+under+static+and+dynamic+flow+conditions.">Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions.</a> Appl. Environ. Microbiol. 72, 2885-2895 (2006).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Contrairement aux méthodes déjà publiées, ce protocole est basé sur deux étapes, d&#39;abord la séparation des véhicules utilitaires par une approche immuno-magnétique, et la seconde, la purification de véhicules utilitaires par centrifugation en gradient de densité. L&#39;isolement LCV peut être facilement suivie par microscopie à fluorescence, soit quand les bactéries marquées par fluorescence et GFP-cellules productrices ou coloration anticorps est utilisé. Le protocole décrit ici est un simple, stra...

purification of pathogen vacuoles from legionella-infected phagocytes

Le pathogène opportuniste Legionella pneumophila est une bactérie résistante aux amibes, qui reproduit également dans les macrophages alvéolaires causant ainsi la pneumonie sévère &quot;la maladie du légionnaire&quot; 1. Dans les phagocytes protozoaires et les mammifères, L. pneumophila emploie un mécanisme conservé pour former un particulier, la réplication permissive compartiment, la &quot;Legionella contenant vacuole&quot; (LCV). La formation exige que le LCV bactérienne Icm / Dot système de sécrétion de type IV (T4SS), qui translocation jusqu&#39;à 275 &quot;effecteurs&quot; protéines dans les cellules hôtes. Les effecteurs de manipuler protéines de l&#39;hôte ainsi que des lipides et de communiquer avec sécrétoire, organites endosomes et mitochondrial 2-4. La formation de véhicules utilitaires légers représente un processus complexe, robuste et redondant, ce qui est difficile à saisir d&#39;une manière réductionniste. Une approche intégrative est nécessaire pour complètement comprendre la formation des LCV, y compris une analyse globale de la trajectoireles interactions de facteurs fibrinogène-hôtes et de leurs dynamiques temporelles et spatiales. Comme une première étape vers cet objectif, véhicules utilitaires intactes sont purifiés et analysés par la protéomique et la lipidomique. La composition et la formation de micro-organismes pathogènes contenant des vacuoles a été étudié par analyse protéomique par chromatographie en phase liquide ou 2-D électrophorèse sur gel couplée à la spectrométrie de masse. Vacuoles isolées soit de la discoideum sol social amibe Dictyostelium ou phagocytes de mammifères nourrissait Leishmania 5, 6 Listeria, Mycobacterium 7, Rhodococcus 8, 9 ou Salmonella Legionella spp. 10. Cependant, les protocoles de purification utilisées dans ces études sont longues et fastidieuse, car elles nécessitent par exemple la microscopie électronique pour analyser la morphologie des LCV, l&#39;intégrité et la pureté. En outre, ces protocoles ne pas exploiter les caractéristiques spécifiques de la vacuole pathogène pour enenrichissement par. La méthode présentée ici permet de surmonter ces limitations en utilisant D. discoideum production d&#39;un marqueur fluorescent TR et en ciblant la protéine bactérienne SIDC effecteur, qui sélectivement ancres à la membrane par TR se lier à la phosphatidylinositol-4-phosphate (PtdIns (4) P) 3,11. VU sont enrichis dans une première étape par immuno-magnétique de séparation en utilisant un anticorps purifié par affinité primaire contre SIDC et un anticorps secondaire couplé à des billes magnétiques, suivie d&#39;une deuxième étape d&#39;une centrifugation en gradient de densité classique Histodenz 12,13 (fig. 1) . Une étude du protéome de véhicules utilitaires légers isolés de D. discoideum a révélé plus de 560 protéines de la cellule hôte, y compris les protéines associées à des vésicules phagocytaires, les mitochondries, ER et appareil de Golgi, ainsi que plusieurs GTPases qui n&#39;ont pas été impliqués dans la formation avant le 13 LCV. VU enrichi et purifié avec leprotocole décrit ici peut être encore analysés par microscopie (immunofluorescence, microscopie électronique), des méthodes biochimiques (Western blot) et des approches protéomiques ou lipodimique.

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

Cet article décrit une méthode pour l&#39;isolement et la purification de intacts<em&gt; Legionella</em&gt; Contenant des vacuoles (VU) de l&#39;amibe et les macrophages. Le protocole en deux étapes comprend l&#39;enrichissement TR par immuno-magnétique séparation utilisant un anticorps dirigé contre un marqueur bactérien TR et la purification en outre par centrifugation en gradient de densité.

Purification de vacuoles agent pathogène à partir<em&gt; Legionella</em&gt; Infectées par les phagocytes

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

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

Research

JoVE Journal

Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K vues.  Ludwig-Maximilians-Universität. Cet article décrit une méthode pour l'isolement et la purification de intacts Legionella Contenant des vacuoles (VU) de l'amibe et les macrophages. Le protocole en deux étapes comprend l'enrichissement TR par immuno-magnétique séparation utilisant un anticorps dirigé contre un marqueur bactérien TR et la purification en outre par centrifugation en gradient de densité.

Vidéo: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

L&#39;invasion des cellules humaines par une bactérie pathogène

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Recherche

Immunologie et infection

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Schéma de flux de travail d&#39;une journée pour la détection de pathogènes et des bactéries tests de résistance antimicrobienne à partir d&#39;hémocultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

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

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

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

Purification et visualisation de lipopolysaccharide de bactéries à Gram négatif par Hot-phénol aqueux d&#39;extraction

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

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

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

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

Purification par affinité des complexes virus de la grippe ribonucléoprotéiques de la chromatine des cellules infectées

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

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

Video Microscopy cellules vivantes de la phagocytose des pathogènes fongiques

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

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

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

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

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

amplification isotherme (LAMP) Essais de boucle médiation pour la détection des espèces spécifiques de<em&gt; Eimeria</em&gt; Qui infectent les poulets

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

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

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

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

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

ARN Purification de Intracellulairement Grown<em&gt; Listeria monocytogenes</em&gt; Dans les cellules macrophages

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

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

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

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

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

Développement et identification d&#39;une sous-population Novel of Human neutrophiles dérivé phagocytes géant<em&gt; In vitro</em

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

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Démêler la fonction d&#39;un effecteur bactérien à partir d&#39;un non-cultivable Pathogen usine utilisant une levure de l&#39;écran à deux hybrides

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

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

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

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