Les auteurs tiennent à remercier Richard Reste pour le don généreux de anthrolysin O, Michael Caparon pour le don généreux des Francs SLO plasmide et Jonathon d&#39;assistance technique. Ce travail a été financé par des subventions du NIH T32CA82084 (PAK), et R01AI072083 (RDS).

1. Purification de streptolysine O (SLO) <ol><li> Inoculer 20 ml de culture de nuit de cellules BL21 contenant le plasmide GOLD pBADgIII-SLOhis 9 dans 0,5 L de bouillon LB et ajouter 500 ul de 50 mg / ml d&#39;ampicilline. Secouez la culture à 225 rpm à 37 ° C jusqu&#39;à DO600 = 0,6, habituellement ~ 1,5 h. Centrifuger 1 ml (bactéries considérées T 0) à 10.000 xg 5 min, dissoudre le culot dans tampon 140 pi d&#39;échantillon SDS 1x / 1 OD et soniquer à l&#39;ADN de cisaillement pour l&#39;analyse de pureté des protéines. </li><li> Provoquer des bactéries avec l&#39;addition de 5 ml arabinose 20% à la culture, secouez à 225 rpm à température ambiante pendant 3 heures. Prélever 1 ml de bactéries par centrifugation et dissous dans un tampon d&#39;échantillon SDS 1x comme ci-dessus pour le point de temps T 3 pour l&#39;analyse de pureté. </li><li> Recueillir des bactéries restantes dans 500 ml bouteille centrifugeuse, tourner 12.000 xg pendant 12 min à 4 ° C (8.500 rpm Sorvall GS-3 du rotor). Décanter le surnageant, réserve de granulés à -80 ° C pendant la nuit ou plus si nécessaire. </li><li> Reprendre le frozen granulés sur la glace dans 10 ml Lyse / tampon de lavage (50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM imidazole, pH 8,0) additionné de 400 ul de 25% de Triton X-100, 100 pl 100 mg / ml de lysozyme, 50 pl 200 mM de fluorure de phénylméthylsulfonyle (PMSF). Transférer le culot remis en suspension à tube de 50 ml d&#39;Oak Ridge. </li><li> Soniquer lysat 5 fois pendant 30 secondes sur la glace avec des intervalles de 30 sec. Spin soniqué lysat dans un tube Oak Ridge à 39000 xg pendant 20 min à 4 ° C (18.000 rpm Sorvall rotor SS-34). </li><li> Alors que le lysat est en rotation, laver 1 ml de Ni-NTA agarose avec 10 ml Lyse / tampon de lavage et un essorage à 1200 rpm pendant 5 min à 4 ° C. (Tous les autres lavages / ELUTIONS utiliser centrifugation dans ces conditions). Aspirer laver. </li><li> Ajouter surnageant bactérien de l&#39;étape de 1,5 à Ni-NTA agarose, agiter doucement pendant 2,5 heures à 4 ° C. Spin pour ramasser le surnageant. Mélanger 30 ul de surnageant avec 10 μ tampon d&#39;échantillon SDS 4x et d&#39;économiser pour l&#39;analyse de la pureté. Laver les billes 4 fois dans Lyse / tampon de lavage et surce du Tris / sel tampon (50 mM Tris, 300 mM NaCl, pH 8,0). </li><li> Éluer 3 fois en ajoutant 1 ml de tampon d&#39;élution (250 mM d&#39;imidazole dans du Tris / sel tampon) et 5 pi 1 M dithiothréitol (DTT) à des billes, l&#39;incubation sur de la glace pendant 10 min, filature et recueillir le surnageant. SLO est une protéine très redox-sensible, et doit être conservé sur de la glace en tout temps. Une fois réduit, la protéine perdra rapidement son activité si chauffé à 37 ° C, même pour une courte période de temps. </li><li> Lavez 500 pi polymixine conjugué agarose dans un tampon d&#39;élution, spin à 10.000 xg pendant 1 min à 4 ° C et remettre en suspension dans 500 ul. Pour décharger endotoxine, ajouter 200 ul de perles à chaque élution et secouez 4 ° C pendant 30 min. Spin à 10.000 xg pendant 1 min à 4 ° C, puis enregistrez le surnageant. Mélanger 30 ul de chaque élution avec 10 pi de tampon d&#39;échantillon SDS 4x pour analyse SDS-PAGE. </li><li> Tester la pureté de élutions par SDS-PAGE. Faire bouillir les échantillons enregistrés pour analyse sur gel à 95 ° C pendant 5 min et la charge de 10 pi sur un gel à 10%. Stain gel de Coomassie pour visualiser la protéine (figure 2A). SLO est de 69 kDa. Effectuer un test de Bradford afin de déterminer la concentration de protéines (rendement typique est de 4 mg / ml pour les deux premières élutions, mais peut varier selon la souche bactérienne et le plasmide utilisé). </li><li> Tester l&#39;activité hémolytique de chaque élution (voir ci-dessous). Élutions piscine où l&#39;activité pureté, la concentration et hémolytiques sont satisfaisants. SLO aliquote à usage unique aliquotes (ul typiquement 5-10), de geler sur glace sèche et conserver à -80 ° C. </li></ol> 2. Dosage hémolytique <ol><li> Laver globules rouges de mouton (globules rouges) dans RBC dosage tampon (0,3% de sérumalbumine bovine (BSA), 2 mM de CaCl2, 10 mM de HEPES, pH 7,4), spin à 1200 rpm pendant 5 min, et remettre en suspension les globules rouges à 2,5% en 10 ml de tampon de dosage RBC. </li><li> Ajouter 10 ul de tampon d&#39;analyse RBC / puits dans 96 puits à fond en V plat sur la glace. Diluer en série chaque élution en double exemplaire. Gammes de dilutions typiques sont 1:1,000 à 1:512,000. Inclure les 3 puits avec aucune toxine et 3 puits avec 10pi 2% de Triton X-100 comme puits minimale et maximale, respectivement. </li><li> Couvrir la plaque et incuber à 37 ° C 30 min. Centrifuger à 1200 rpm pendant 5 min. Transfert 70 pi de surnageant dans un fond plat plaque de 96 puits. Lire A 405. Une unité est la dilution de la toxine requis pour la lyse de 50% des globules rouges. Activité de la toxine est de 1 unité de dilution * factor/0.01 ml. </li></ol> 3. Essai cellule lytique <ol><li> Cellules de récolte, de comptage, de spin. Remettre en suspension à 2x10 6 / ml dans un tampon R / B (RPMI avec 2 mM de CaCl2, 0,5% de BSA) et 20 pg / ml d&#39;iodure de propidium. Ajouter 100 ul de cellules à un 96-V et plaque à fond. </li><li> Diluer en série SLO 2x concentration finale dans le tampon R / B, ajouter 100 ul toxine ou 100 pi de tampon R / B pour les cellules. Incuber 5 min à 37 ° C. Portée typique concentrations finales de 2.000 U / ml à 31,25 U / ml. </li><li> Exécuter sur les cellules cytomètre de flux et de recueillir des données avec des filtres pour la phycoérythrine (PE). Un décalage d&#39;une cel journal représente transitoirement perméabiliséesls tandis qu&#39;un changement de 3 log indique les cellules mortes 4. Calculer la lyse spécifique des cellules en soustrayant le pourcentage de cellules mortes dans le contrôle (PI% haute ctl) de la (PI exp% élevé) expérimental, comme suit: lyse spécifique = (PI% exp haute -% PI haute ctl) / (100 -% PI haute ctl) * 100 </li></ol> 4. Micropipette de livraison de la toxine aux cellules dans des boîtes de culture <ol><li> Un jour avant l&#39;expérience, les macrophages plaque 2x10 5 sur un revêtue de collagène à fond de verre boîte de 35 mm. </li><li> Allumez le microscope et microinjecteur. Prévoyez du temps Platine chauffante pour réchauffer à 37 ° C. Le choix d&#39;un microscope dépend généralement de ce qui est disponible localement. Le microscope inversé a besoin d&#39;un stade, un stade capable de chauffer les plats à 37 ° C, blocs de filtres d&#39;excitation / émission appropriées pour le colorant choisi, et l&#39;espace pour connecter physiquement le micro-injecteur. Lentille de Bertrandest utile pour la micro-injection, mais pas obligatoire. L&#39;ordinateur de conduite du microscope a besoin de mémoire suffisant pour recueillir et stocker des données. </li><li> Marquer les cellules pendant 30 min avec de la teinture à 37 ° C. Selon le dosage, l&#39;étiquetage peut être fait avec 5 pl Fura2 AM dans 1 ml de PBS ou 2 pi calcéine AM dans 1 ml de milieu complet. Pour Fura2, excitation / émission est recueillie pour 340/510 nm et 380/510 nm, et le rapport de 340/380 signaux détermine le flux de calcium. Pour calcéine, excitation / émission est 495/515 nm tandis que l&#39;éthidium homodimère est 525/620 nm et APC est 650/660 nm. D&#39;autres marqueurs peuvent être choisis aussi bien. </li><li> Laver les cellules avec du PBS et placer dans 1 ml de RPMI supplémenté avec 2 mM de CaCl 2. Montez le microscope. </li><li> Diluer la toxine et le dextrane dans l&#39;eau, et centrifugation à 20.000 xg pendant 10 min 4 ° C. Typiquement, 1 pl SLO et 4 pl 10 mg / ml de dextran-555 est dilué dans 6 pl d&#39;eau. Dextrane ou un autre fluide fluorescent en phase molécule est utilisé pour vérifier que le femto-pointe n&#39;est pas bouché, et injects comme vous le souhaitez. </li><li> Chargez femto-pointe de l&#39;arrière avec 0,07 l de toxine diluée à l&#39;aide d&#39;un Microloader. </li><li> Chargez femto-pointe sur microinjecteur. Ajustez l&#39;angle de l&#39;injecteur de sorte que la pointe sera assis sur le centre des cellules avec espace pour se déplacer dans toutes les directions. Effacer z-limite. Paramètres d&#39;injection doivent être injecter plus de 0,5 seconde à 120 psi à 20 psi pression. Abaissez la pointe jusqu&#39;à ce qu&#39;elle entre dans le milieu. </li><li> En utilisant la lentille de Bertrand, centre de la pointe et la pointe suivre lorsqu&#39;il est abaissé au plus près des cellules. Une fois que vous perdre le focus, revenez à l&#39;optique normales. L&#39;ombre aiguille doit être visible sur le terrain. Concentrez-dessus des cellules et abaisser l&#39;aiguille jusqu&#39;à ce qu&#39;elle soit mise au point. Concentrer arrière sur les cellules et soigneusement amener l&#39;aiguille adjacente à une cellule. Réglez le z-limite pour l&#39;injection. Commencer imagerie, déplacer la pointe à la position désirée, injecter pour libérer la toxine à l&#39;instant souhaité. Relevez l&#39;aiguille, passer à une nouvelle région de cellules et injecter. Déplacer l&#39;aiguille en position initiale pour éviter toute indésirablefuite de l&#39;aiguille de la toxine. </li></ol>

<ol>
	<li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+regulates+IL-1+beta+processing+via+calcium-independent+phospholipase+A2.">Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.</a> J. Immunol. 164, 5120-5124 (2000).</li><li>Scolding, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+removal+by+oligodendrocytes+of+membrane+attack+complexes+formed+by+activated+complement.">Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.</a> Nature. 339, 620-622 (1989).</li><li>Keyel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+O+clearance+through+sequestration+into+blebs+that+bud+passively+from+the+plasma+membrane.">Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.</a> J. Cell. Sci. 124, 2414-2423 (2011).</li><li>MacKenzie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+secretion+of+interleukin-1beta+by+microvesicle+shedding.">Rapid secretion of interleukin-1beta by microvesicle shedding.</a> Immunity. 15, 825-835 (2001).</li><li>Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+IL-1+beta+secretion+stimulated+by+P2X7+receptors+is+dependent+on+inflammasome+activation+and+correlated+with+exosome+release+in+murine+macrophages.">Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.</a> J. Immunol. 179, 1913-1925 (2007).</li><li>Andrei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+C+and+A2+control+lysosome-mediated+IL-1+beta+secretion:+Implications+for+inflammatory+processes.">Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).</li><li>Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, 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+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat. Immunol. 10, 241-247 (2009).</li><li>Magassa, N., Chandrasekaran, S., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+cytolysin-mediated+translocation+does+not+require+pore+formation+by+streptolysin+O.">Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.</a> EMBO Rep. 11, 400-405 (2010).</li><li>Pinkney, M., Beachey, E., Kehoe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiol-activated+toxin+streptolysin+O+does+not+require+a+thiol+group+for+cytolytic+activity.">The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.</a> Infect. Immun. 57, 2553-2558 (1989).</li><li>Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+immune+cells+mediated+by+tunneling+nanotubules.">Functional connectivity between immune cells mediated by tunneling nanotubules.</a> Immunity. 23, 309-318 (2005).</li><li>McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patching+plasma+membrane+disruptions+with+cytoplasmic+membrane.">Patching plasma membrane disruptions with cytoplasmic membrane.</a> J. Cell. Sci. 113, 1891-1902 (2000).</li><li>Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anthrolysin+O,+the+Bacillus+anthracis+cholesterol-dependent+cytolysin.">Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.</a> Infect. Immun. 71, 3183-3189 (2003).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Les techniques décrites ici permettent l&#39;examen des réponses des cellules immunitaires à des toxines bactériennes. L&#39;étape la plus critique est la manipulation et le dosage de la toxine. Activité de la toxine peut être extrêmement variable, même entre différentes aliquotes de la même préparation, en raison de sa fragilité. Cela nécessite soit l&#39;essai de chaque aliquote de la toxine contre une lignée cellulaire de référence ou globules rouges ou en utilisant des gradients de toxines. Gradient...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Ni-NTA agarose </td><td> Qiagen </td><td> 30210 </td><td></td></tr><tr><td> polymixine-agarose </td><td> Sigma </td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba dessaler Spin col </td><td> Pêcheur </td><td> PI-89891 </td><td></td></tr><tr><td> hématies de mouton </td><td> Pêcheur </td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> voir ref 9 </td></tr><tr><td> Cy5 colorant monoréactif </td><td> GE Healthcare </td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies </td><td> F1221 </td><td></td></tr><tr><td> Calcéine AM / Ethidium homodimère </td><td> Life Technologies </td><td> L3224 </td><td></td></tr><tr><td> Anti-CD11c-APC </td><td> BD Biosciences </td><td> 550261 </td><td></td></tr><tr><td> revêtu de collagène à fond de verre plat </td><td> Mattek </td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td> femto-tip II </td><td> Pêcheur </td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td> Pêcheur </td><td> E5242956003 </td><td></td></tr><tr><td> dextrane Alexa 555 </td><td> Life Technologies </td><td> D34679 </td><td></td></tr><tr><td> InjectMan NI 2 </td><td> Eppendorf </td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf </td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tableau 1. Liste et source de réactifs spécifiques et des équipement nécessaire. Equipements spécifiques et les réactifs utilisés dans ce protocole, ainsi que la société et le numéro de catalogue sont répertoriés. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Tampon </td><td> Composition </td><td> Étape usagé </td></tr><tr><td> Lyse / Wash </td><td> 50 mM de NaH 2 PO 4 300 mM NaCl 10 mM d&#39;imidazole, pH 8,0 </td><td> 1,4 </td></tr><tr><td> Tris / sel </td><td> Tris 50 mM, pH 8,0 300 mM NaCl </td><td> 1,7 </td></tr><tr><td> Tampon d&#39;élution </td><td> Tris 50 mM, pH 8,0 300 mM NaCl 250 mM d&#39;imidazole </td><td> 1,8 </td></tr><tr><td> Tampon de dosage RBC </td><td> 0,3% de BSA 2 mM de CaCl2 HEPES 10 mM, pH 7,4 </td><td> 2,1 </td></tr><tr><td> tampon R / B </td><td> Milieu de culture RPMI cellule 2 mM de CaCl2 0,5% de BSA </td><td&gt; 3,1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> Tableau 2. Liste des tampons utilisés dans ce protocole. Les tampons utilisés, leur composition et de la première étape au cours de laquelle ils sont utilisés dans le protocole sont énumérés. </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Toxines bactériennes se lier au cholestérol dans les membranes, en formant des pores qui permettent une fuite du contenu cellulaire et l&#39;afflux de matériaux provenant de l&#39;environnement extérieur. La cellule peut soit se remettre de cette insulte, ce qui nécessite actifs des processus de réparation des membranes, ou mourir en fonction de la quantité d&#39;exposition aux toxines et 1 type de cellule. En outre, ces toxines induisent de fortes réactions inflammatoires chez les hôtes infectés par l&#39;activation des cellules immunitaires, dont les macrophages, qui produisent une gamme de cytokines pro-inflammatoires 2. De nombreuses bactéries Gram-positives produisent des toxines cholestérol contraignants qui ont été montrés pour contribuer à leur virulence en grande partie grâce à des mécanismes non caractérisés. Des changements morphologiques dans la membrane plasmique des cellules exposées à ces toxines comprennent leur séquestration dans les protubérances de surface cholestérol enrichis, qui peuvent être versées dans l&#39;espace extracellulaire, ce qui suggère une défense cellulaire intrinsèqueMécanisme 3,4. Ce processus se produit sur ​​toutes les cellules en l&#39;absence de l&#39;activité métabolique, et peuvent être visualisées à l&#39;aide EM au bout de 4 fixation chimique. Dans les cellules immunitaires comme les macrophages, qui interviennent dans l&#39;inflammation en réponse à l&#39;exposition aux toxines, des vésicules membranaires induits sont suggérées pour contenir cytokines de la famille IL-1 et peut être responsable à la fois pour l&#39;excrétion des toxines et la diffusion de ces cytokines pro-inflammatoires 5,6,7. Un lien entre l&#39;IL-1β libération et un type spécifique de mort cellulaire, appelée pyroptosis a été suggéré, car les deux sont des processus caspase-1 dépendantes 8. Pour démêler les complexités de cette réponse des macrophages, ce qui inclut la liaison toxine, l&#39;excrétion de vésicules membranaires, la libération de cytokines et la mort cellulaire potentiellement, nous avons développé des techniques d&#39;étiquetage et les méthodes de microscopie par fluorescence qui permettent de visualiser en temps réel des toxines cellulaires des interactions, y compris mesures de dysfonctionnement et la mort (figure 1). Utiliserd&#39;imagerie des cellules vivantes est nécessaire en raison des limitations dans d&#39;autres techniques. Approches biochimiques ne peut pas résoudre effets qui se produisent dans des cellules individuelles, tout en cytométrie en flux n&#39;offre pas de haute résolution, visualisation en temps réel des cellules individuelles. Les méthodes décrites ici peuvent être appliqués à l&#39;analyse cinétique des réactions induites par des stimuli complexes impliquant d&#39;autres changements phénotypiques dans les cellules.

Typiquement 10 7 -10 8 U / ml SLO peut être obtenue avec une concentration en protéines de 4 mg / ml. La quantité de toxine nécessaire pour la lyse des cellules varie selon le type cellulaire, mais elle est généralement de 125 à 500 U / ml SLO (figure 2B). Types de cellules comme les macrophages peuvent être plus résistantes (4000 U / ml) alors que d&#39;autres (en particulier des lignées de cellules T) sont plus sensibles. Ces sensibilités correspondre avec SLO disponib...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Méthodes de purification du cholestérol contraignant toxine streptolysine O de recombinaison<em&gt; E. coli</em&gt; Et la visualisation de liaison de la toxine à vivre cellules eucaryotes sont décrits. Application locale de la toxine induit des changements rapides et complexes dans les cellules ciblées révélant de nouveaux aspects de la biologie toxine.

Visualisation des réponses toxine bactérienne induite en utilisant direct microscopie de fluorescence cellulaire

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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JoVE Journal

Immunology and Infection

11.7K vues.  University of Pittsburgh School of Medicine. Méthodes de purification du cholestérol contraignant toxine streptolysine O de recombinaison E. coli Et la visualisation de liaison de la toxine à vivre cellules eucaryotes sont décrits. Application locale de la toxine induit des changements rapides et complexes dans les cellules ciblées révélant de nouveaux aspects de la biologie toxine.

Vidéo: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualisation cellule à cellule de transfert du VIH en utilisant des clones fluorescents du VIH et Live microscopie confocale

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

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Immunologie et infection

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Évaluer la réponse stomatique à Live cellules bactériennes en utilisant l&#39;imagerie feuilles entières

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imagerie de cellules vivantes de la<emBacillus subtilis> Et Streptococcus pneumoniae À l&#39;aide automatisée de microscopie time-lapse

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Analyse unicellulaire des<emBacillus subtilis> Les biofilms par microscopie à fluorescence et cytométrie de flux

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

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 ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Application de long terme de culture interféron-γ-enzymatique immunospot Essai d&#39;évaluation de mémoires et effectrices réponses des cellules T chez les bovins

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Génération et multi-phénotypique criblage à haut contenu de<em&gt; Coxiella burnetii</em&gt; Mutants de transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Mesurer les réponses physiologiques de<em&gt; Drosophila</em&gt; Neurones sensoriels à Lipid Phéromones Utilisation en direct d&#39;imagerie de calcium

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantification des Efferocytosis en microscopie par Fluorescence des cellules individuelles

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.