P. Cossart Araştırma laboratuvarında Pasteur Enstitüsü, Institut National de la Sante et de la Recherche Médicale, Institut National de la Recherche Agronomique, ERC Advanced Grant (233.348), Agence Nationale de la Recherche (Grant Mie-tarafından desteklenmektedir SignRupVac), Louis-Jeantet Vakfı ve Fondation Le Roch Les Mousquetaires. AK Pasteur-Paris Üniversitesi Uluslararası Doktora Programı / Institut Carnot İlletler Infectieuses bir burs bir alıcı. Biz hücresel transfeksiyon protokolü optimize Jason Mercer teşekkür ederim.

1.. Hücresel hazırlanması ve Bakteriyel Kültürleri, Transfeksiyonu Araçlar ve Primer Antikorlar <ol><li> Bireysel L. izole etmek için taze bir agar plaka hazırlayın -80 ° C&#39;de tutulan bir bakteri gliserol stoku (bakteriyel sıvı kültürü bir gece boyunca, doymuş glycerol/50% 50%) dan koloniler monocytogenes <ol><li> Bir Beyin Kalp İnfüzyon (BHI) agar plaka üzerinde donmuş bir bakteri gliserol stok, çizgi bakteri taşımak için (-80 ° C&#39;de muhafaza) bir alüminyum raf kullanma. </li><li> 48 saat (ya da bireysel bakteriyel koloniler izole edilebilir ye kadar) içinde 37 ° C&#39;de inkübe edin. </li><li> (Sıvı tohum kültürleri için kullanılacaktır), 1 aydan fazla bir süre boyunca 4 ° C &#39;de sonra bu çalışma plakası tutun. </li><li> Bizim protokol, L. kullanın serotip 1/2ã EGDe.PrfA * suşu monocytogenes, ancak Şekil 1 &#39;de gösterildiği gibi L. P14.PrfA 4b monocytogenes serotip * gerginlik benzer sonuçlar verir. </li></ol></li><li> OLa ATCC CCL2 hücreleri, antibiyotik olmadan,% 10 fetal sığır serumu (FBS) ile takviye edilmiş Dulbecco Modified Eagle Medium (DMEM) içinde büyütülür. </li><li> Karıştırılmış (kontrol) ve anti-Met siRNA&#39;lar 384 oyuklu siyah mikroskopi hücre kültürü plakaları içinde yüklenir bağımsız havuzlar. <ol><li> RNase-içermeyen su içinde 500 ul siRNA 160 pmol seyreltin ve bu çözeltinin oyuk başına 5 ul (nihai konsantrasyon: 1.6 pmol) ekleyin. </li><li> 1 yıllık bir süre için -20 ° C &#39;de bu plaka tutun. </li></ol></li><li> 25, daha önce tarif edildiği gibi bir inlc-GST rekombinant proteini kullanılarak aşılama ile elde edilmiştir inlc bakteriyel proteine ​​karşı poliklonal tavşan antikorlarının seviyelerini yükseltmiştir. </li></ol> 2. SiRNA hücre transfeksiyonu Ters <ol><li> Enfeksiyondan önce 72 saat oda sıcaklığında her bir hazne içinde RNase içermeyen su içinde 5 ul 1.6 pmol siRNA içeren siyah 384 oyuklu mikroskopi hücre kültürü plakası getirmek. </li><li> 300 3 dakika boyunca plaka santrifüjkuyuların duvarlarında biriken olabilirdi siRNA&#39;yı aşağı getirmek için oda sıcaklığında rcf. </li><li> % 0.4 Lipofectamine RNAiMAX ile takviye edilmiş DMEM oda sıcaklığı hazırlayın. </li><li> (SiRNA eklemeden önce uzun süre 20 dakika daha inkübe transfeksiyon ortamı yok), her bir oyuğa DMEM / RNAiMAX transfeksiyon ortamı 25 ul ekle. </li><li> Transfeksiyon solüsyonu ile siRNA karıştırmak için ileri ve geri hareket plaka, daha sonra siRNA Lipofectamine kompleksleri oluşturmak için izin vermek için, oda sıcaklığında, 1 saat boyunca levha tutun. </li><li> Bir kere, 10 ml 37 ° C önceden ısıtılmış PBS ile birleşen veya alt konfluent hücre kültürü şişesinden yıkama HeLa hücreleri. </li><li> 1 ml 37 ° eklenerek HeLa hücreleri ayırın C hücre kültürü şişesine tripsin önceden ısıtılmış ve 37 ° C&#39;de 3-5 dakika boyunca inkübe </li><li> 10 ml 37 ° C &#39;de yeniden askıya hücreler% 16 FBS ile takviye edilmiş DMEM önceden ısıtılmış. </li><li> Hücre sayımı ve hazırlanması DMEM içinde, ml başına 12,000 hücre, bir hücre süspansiyonu ile desteklenmiş% 16 FBS. </li><li> 384-kuyulu bir levhadaki her kuyuya, hücre süspansiyonu, 50 ul ekle. </li><li> Hücreleri dağıtmak ve hücreler oda sıcaklığında 10 dakika boyunca yerleşmek izin hızla ileri ve geri plakasını taşıyın. </li><li> Parafilm ile plaka yalıtılır ve 37 ° C&#39;de nemlendirilmiş% 5 CO2 içeren bir atmosferde 72 saat boyunca tutmak </li></ol> 3. Hücresel Enfeksiyon ve Boyama <ol><li> Enfeksiyondan bir gün önce, L. tek bir koloni almak BHI agar plakasından monocytogenes ve bir 15 ml tüp içinde polistiren sıvı BHI ortamı içinde, 5 ml içinde tekrar süspansiyon haline getirin. </li><li> Bakteri büyümesi için izin vermek için bir shacking cihazı içerisinde 37 ° C&#39;de gece boyunca inkübe edin. </li><li> Enfeksiyonun gün, L., gece boyunca 1 ml yıkama bir masa üstü santrifüj 10.600 RCF 2 dakika santrifüj edilerek kültür monocytogenes. </li><li> (Salgılanmış sitotoksin listeriolisin O içerir) Süpernatant atılır ve 1 ml PBS (repea pelet yeniden askıya) yıkama aşaması 3 kez t. </li><li> 600 nm&#39;de optik yoğunluk bakteriyel okuma ve bakteri sayısını (OD = 1 1E9 bakteri / ml &#39;ye eşdeğerdir) tahmin ediyoruz. </li><li> Yeterli L. hazırlanması % 1 FBS ile takviye edilmiş DMEM içinde seyreltme monocytogenes: EGDe.PrfA * gibi, yüksek oranda istilacı türleri kullanılarak, biz oyuk başına 30 ul ortam içinde 5E4 bakterilerin kullanımını (enfeksiyon çokluğu 2.000 hücreleri için 25 olarak tahmin edilir) göstermektedir. </li><li> Her çukurdaki hücre kültür ortamı (80 ul) çıkarın ve L. 30 ul eklenmesi ile değiştirin orta monocytogenes-ihtiva etmektedir. </li><li> Enfeksiyon süreci senkronize etmek için oda sıcaklığında 5 dakika boyunca 200 RCF plaka santrifüj. </li><li> Önceden ısıtılmış alüminyum blok üzerinde, 37 ° C&#39;de nemlendirilmiş% 5 CO2 içeren bir atmosfer altında 1 saat boyunca inkübe edin. </li><li> L. çıkarın de her bir ikinci orta monocytogenes-ihtiva eden ve% 10 FBS ile takviye edilmiş DMEM içinde önceden ısıtılmış 30 ul ekle ve40 ug / ml gentamisin dışı L. öldürmek için monocytogenes. </li><li> Önceden ısıtılmış metal blok üzerinde, 37 ° C&#39;de% 5 CO2 içeren bir atmosferde 4 saat boyunca inkübe edilir. </li><li> % 8 formaldehit (yerine paraformaldehid polimerlerden daha formaldehit monomerler kullanılır, ki bu çözelti, taze olarak hazırlanmalıdır) ile takviye edilmiş PBS içinde bir çözeltisi hazırlandı. </li><li> Hücre kültür ortamı atarak olmadan,% 8 formaldehit (nihai konsantrasyon:% 4) ile takviye edilmiş 30 ul PBS ekleyin ve oda sıcaklığında 15 dakika boyunca inkübe edin. </li><li> Fiksatif çıkarın ve (oyuk başına 80 ul PBS nihai hacmi içinde hücreleri tutun) oyuk başına 80 ul PBS ile hücreler üç kez yıkayın. </li><li> Hazırlama bir 1:250 PBS içinde tavşan anti-serumu inlc seyreltme% 0.2 saponin ile takviye edilmiştir. </li><li> De PBS çıkarılmasından sonra her bir birincil antikor çözeltisinin 10 ul ilave edin ve oda sıcaklığında 30 dakika boyunca inkübe edin. </li><li> Birincil atınAntikor çözeltisi ve oyuk başına 40 ul PBS ile dört kez yıkayın. </li><li> PBS içinde ikincil Alexa Fluor 546-bağlı anti-tavşan antikoru (1:250), DAPI çözeltisi (1:1,500) ve phalloidin-Dy647 (1:150) ile seyreltilir,% 0.2 saponin ile takviye edilmiştir. </li><li> Her çukuruna Bu ikinci boyama çözeltisi 10 ul ilave edin ve oda sıcaklığında 30 dakika boyunca inkübe edin. </li><li> İkinci boyama solüsyonu atın ve (her bir kuyucuğa 40 ul bir son hacim bırakın ve plaka mühür) oyuk başına 40 ul PBS ile dört kez yıkayın. </li><li> Plaka hemen görüntülü olabilir ya da bir sonraki analiz için ışıksız bir 4 ° C (alüminyum folyo ile kapak) saklanabilir. </li></ol> 4. Görüntü Toplama ve Analizi <ol><li> Otomatik bir mikroskop üzerine monte edilmiş bir 10X objektif (kuyu başına tercihen 9 görüntüleri elde) kullanarak üç farklı kanal (350 nm, 546 nm ve 647 nm) görüntü elde. </li><li> Inlc bilgi sinyali görüntü kullanılarak ölçülebilirsırasıyla DAPI ve phalloidin lekeleme kullanarak çekirdekleri ve hücre gövdeleri otomatik segmentasyon sağlar CellProfiler gibi analiz yazılımı. </li></ol>

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Biz ifşa hiçbir şey yok.

Çeşitli parametre inlc sinyalinin kesin bir algılanmasına izin vermek için yeteri kadar büyük bir sitoplazma gösteren sağlıklı hücre çizgilerinin kullanımı da dahil olmak üzere inlc bilgi algılama protokolün başarısına için kritik öneme sahiptir. Deneyde biz nedeniyle sitosolik boşluğun genişletilmesi için tahlil için özellikle uygun olan HeLa CCL2 hücrelerin kullanımı önermek bu madde içinde mevcut bu tür Kyoto HeLa hücreleri gibi diğer HeLa klonlar daha küçük bir ekran ancak sit...

A correction was made to <a href="http://www.jove.com/video/51043">Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes</a>. Three authors were omitted from the article at the time of publication. The acknowledgments section was also updated. The author list has been updated from:
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020
to
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Jason Mercer4, Mario Emmenlauer5, Christoph Dehio5, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute,2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Z&#252;rich 5Focal Area Infection Biology, Biozentrum, University of Basel
The acknowledgments where update from:
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We thank Jason Mercer for optimizing the cellular transfection protocol.
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Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We acknowledge support by grant 51RT 0_126008 for the Research and Technology Development (RTD) project InfectX in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology (to C.D.).

A correction to author list has been made for the article Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes. Three authors were omitted from the article at the time of publication.

Erratum: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Gram pozitif bakteri Listeria monocytogenes, konakçı hücreleri işgal onun içselleştirme vakuol bozar ve konakçı hücreler 1 sitoplazması içinde replike olan bir gıda kaynaklı patojendir. L. hücre ve hayvan modellerinde görülen bakteriyel hastalık özelliklerin kalıcılığı ile ilişkili laboratuar bağlamında (hızlı büyüme, sağlıklı bireyler için düşük toksisite) manipülasyon kolaylığı monocytogenes (hemolitik aktivite, lökositoz) için önemli bir model olarak 1960 yılında ilk kullanım izin hücre içi asalaklık ve enfeksiyondan 2 karşı hücresel bağışıklığın teorik temellerinin kurulması için çalışma. 1980&#39;lerin sonu ve 1990&#39;ların başlarında, bakteri hücre içi döngüsü 3 yanı sıra en önemli bakteriyel virulans moleküler karakterizasyonu diseksiyonu 4-7 L. kullanımını tercih faktörleri manipülasyon ve damızlık için önemli bir moleküler araç olarak monocytogeneskonakçı hücre fonksiyonlarının y. Listeria cinsinin Avirulent varlığı (L. innocua) ve öldürücü (L. monocytogenes) türleri karşılaştırmalı genomik çalışmalar 8 önünü açtığını, birlikte tam L. son kurulması ile monocytogenes L. evrim anlayışımızı artış, 9 transcriptome bir insan patojeni olarak ve enfeksiyonu için bir model sistem olarak monocytogenes 10 çalışmalar. L. monocytogenes ev sahibi hücre reseptörleri, sırasıyla E-kadherin ve Met, 11-12 ile bakteriyel yüzey proteinleri ve InlA ınlB etkileşimi üzerine konakçı hücreler içine içselleşmesini uyarır. Başlangıç ​​aday bazlı çalışmaları ınlB-kritik bir effektör olarak InlA istilası yolunun 13 ve fosfoinositid 3-kinaz (PI 3-K) önemli bir bileşeni olarak α / kateninler β-aktin bağlantı tanımlanmasına yol açtı bağımlı işgali Cascade 14-15. Proteomik ve fonksiyonel bazlı deneyler sonradan izin roman sitoskeletal elemanları 16 ve konakçı hücre işgali için gerekli lipid ikinci haberciler 17 tanımlama. Transkripsiyonal çalışmalar 18 ve kütle spektrometresi tabanlı nicel proteomiks 19 Yakın zamanlarda sinyal şelaleden aktivasyonu ve L. sırasında bağışıklık tepkilerinin baskılara ilişkin yeni bir ışık tutmuştur monocytogenesin. Sistem biyolojisi küçük müdahale RNA genlerin (kinomes, tam genomları) büyük setleri inaktive dayalı yaklaşımlar (siRNA) susturulması son zamanlarda fagositozundan dahil, spesifik hücresel fonksiyonları bağlamında sinyal basamaklarının küresel ana analizi için yeni yollar açtı ve var patojen içselleştirilmesi 20. Genom siRNA&#39;nın ekranlar önceden L. enfeksiyonu için gerekli hücresel kaskadlar araştırmak için yapılmıştır fagositik Drosophila monocytogenes </em&gt; S2 hücreleri 21-22 ancak bu tip analiz fagositik olmayan hücrelerde gerçekleştirilen edilmemiştir, in vivo enfeksiyonu için önemli hedefleri temsil eder. Biz, L. tarafından enfeksiyonun geç aşamalarında mikroskobik tespiti için bir protokol optimize epitel hücre içinde bakteri giriş yüksek verimli siRNA çalışmaları için uygundur monocytogenes. Bizim deney oldukça invazif L. yararlanır PrfA, L. majör kopyalama düzenleyicisi olarak, bir nokta mutasyon sunulur gerginlik monocytogenes 6, hastalık oluşturma faktörleri monocytogenes (PrfA * olarak adlandırılır) bu mutasyon 23 PrfA kurucu bir şekilde etkin hale getirir ve bu yüzden başka türlü kötü enfekte fagositik olmayan hücrelere bakteriyel girişi lehine, istila protein InlA ve ınlB bir artış çıkmasına yol açar. Enfeksiyon Bizim okuma salgılanan bakteriyel protein inlc sitozolik birikiminin saptanması üzerine dayanmaktadır: Bu molekülintra-sitoplazmik L. tarafından tercihen eksprese edilen bir pleiotropik efektör monocytogenes 9 ve katılır bakteri hücreden hücreye yayılan 24 değil, aynı zamanda konak immün yanıtları modüle 25, bunlar sadece. Hücre içi bakterilerin inlc salgılanmasının floresan etiketleme açıkça enfekte olmayan hücrelerin enfekte ayırt etmesine olanak veren, aynı zamanda, bir uç-nokta, daha sonra, farklı adımlarda enfeksiyon incelemek için kullanılabilir okuma temsil eder, sadece: girişi, vakuolar kaçış, sitosolik bakteriyel çoğalması ve hücre-hücre yayıldı. Bu mikroskopi tabanlı protokol Bu nedenle L. tarafından konak hücrelerin enfeksiyonu ile ilgili hücresel yolları okumak için siRNA ekranlara bağlanabilir monocytogenes.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td>For liquid BHI preparation</td>
		 </tr>
		 <tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>Supplement to liquid BHI for BHI agar plates </td>
		 </tr>
		 <tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51830-500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamicin</td>
			<td>Sigma </td>
			<td>G1397-10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Formaldehyde (16%)</td>
			<td>EMS</td>
			<td>15710</td>
			<td>Prepare fresh before each experiment</td>
		 </tr>
		 <tr>
			<td>Anti-Rabbit Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A-11035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D-1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phalloidin Dy647</td>
			<td>Dyomics</td>
			<td>647-33</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Scramble</td>
			<td>Dharmacon</td>
			<td>D-001810-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Met</td>
			<td>Dharmacon</td>
			<td>L-003156-00-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 384-well microscopy cell culture plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AxioObserver Z1 microscope</td>
			<td>Zeiss</td>
			<td>431007 9901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Neo</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Metamorph analysis software</td>
			<td>Molecular Devices</td>
			<td>4000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CellProfiler analysis software</td>
			<td>Broad Institute</td>
			<td>Public software available at <a href="http://www.cellprofiler.org/" target="_blank">http://www.cellprofiler.org/</a></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes

Bakteriyel hücre içi patojenler zarif işlemek ve ana hedef dokuların enfeksiyonu için gerekli olan hücre fonksiyonlarını yıkmak için kendi kapasitesi nedeniyle hücresel sinyal kaskadlar incelemek için moleküler araçlar olarak tasavvur edilebilir. Bu bakteriyel patojenler arasında, Listeria monocytogenes hücresel bağışıklık tepkilerinin karakterizasyonunda hücre içi parazitlenme için bir paradigma olarak kullanılan bir Gram pozitif mikroorganizma ve hücre iskeleti ve membran ticareti dinamiklerini kontrol moleküler yollarının bulunması etkili rol oynamıştır. Bu yazıda, L. geç hücre enfeksiyonu aşamalarının saptanması için bir tahlili tarif sağlam mikroskobik monocytogenes inlc, enfekte olmuş hücrelerin sitoplazması içinde biriken bir salgılanan protein bakteriyel floresan etiketleme göre, bu deney, Char için otomatik yüksek verimli, küçük müdahale edici RNA ekranlara bağlanabilirenfeksiyon yukarı-veya aşağı-regülasyonunda rol oynayan hücresel sinyal yolları acterize.

Sitoplazmik inlc floresan etiketleme L. tarafından hücre enfeksiyon için sağlam okunmasını sağlar monocytogenes, Şekil 1 &#39;de gösterildiği gibi: mikrografta merkez hücre yüksek P14.PrfA * 23 suşu ile enfekte edilir bu faz kontrast çekimi (ok uçları, Şekil 1A) gözlenebilir ve bu sinyali DAPI ile doğrulanır gibi olduğu Bireysel bakteri açık (Şekil 1B) ayırt edilebilir. (Şekil 1 C &#39;de DAPI lekelemesi ile üst ü...

Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes sıklıkla hücre içi asalaklığın çalışma için büyük bir model olarak kullanılan bir Gram pozitif bakteri patojendir. Geç L. Görüntüleme küçük müdahale edici RNA ekranlar bağlamında enfeksiyon aşamaları monocytogenes, hedef konakçı hücrelerin bakteriyel bir enfeksiyon için gerekli hücresel yolların küresel çalışma sağlar.

Bakteriyel Patojen Hücresel Enfeksiyon Soruşturma inlc salgılanmasına Görüntüleme<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

imaging-inlc-secretion-to-investigate-cellular-infection-bacterial

Research

JoVE Journal

Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

9.8K Görüntüleme.  Pasteur Institute.  Listeria monocytogenes sıklıkla hücre içi asalaklığın çalışma için büyük bir model olarak kullanılan bir Gram pozitif bakteri patojendir. Geç L. Görüntüleme küçük müdahale edici RNA ekranlar bağlamında enfeksiyon aşamaları monocytogenes, hedef konakçı hücrelerin bakteriyel bir enfeksiyon için gerekli hücresel yolların küresel çalışma sağlar. 

Video: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Yılları arasında sinerjistik Araştırma Bioluminescent Görüntüleme kullanma Streptococcus pneumoniae Bebek Fare ve İnfluenza A Virüsü

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

İmmünoloji ve Enfeksiyon

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.

Bakteriyel Patojen İnsan Hücreleri Invasion

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

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Ile Enfekte Fare Bakteriyel Yük ve Bağışıklık Yanıtları Ölçüm Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

Böcek<em&gt; Galleria mellonella</em&gt; Bakteriyel Patogenez İnceleyecek Güçlü Enfeksiyon Model olarak

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Sözlü İletim<em&gt; Listeria monocytogenes</em&gt; Kirlenmiş Gıda Yutma ile Farelerde

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Otomatik Ayırma<em&gt; C. elegans</em&gt; Değişken derecelerde Bakteriyel Patojen tarafından kolonileştirildiğini

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes Kullanarak Enfekte Farelerde Kültür ve Kalp Kolonizasyonunda Kardiyak Hücrelerin Bakteriyel İstilasının Değerlendirilmesi

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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.

RNA Arıtma Hücre içindeki dan Yetiştirilen<em&gt; Listeria monocytogenes</em&gt; Makrofaj

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

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Deneysel Enfeksiyon<em&gt; Listeria monocytogenes</em&gt; Ana İnterferon-γ Yanıtları incelenmesi için Bir Model Olarak

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.