This work was funded by the Creative Inquiry Program for Undergraduate Research and the Calhoun Honors College at Clemson University.

1. Genel Trypanosome Yetiştiriciliği <ol><li> SDM79 ortamlarının hazırlanması (Tablo 1), katı madde tartılır. </li><li> 4 50 ml konik ° C veya bir plastik torba saklayınız. NOT: Reaktifler, en az 6 ay stabildir. </li><li> Çözülme fetal inek, bir 37 ° C su banyosunda serumu (FBS) ve tersine periyodik olarak karıştırın. NOT: FBS steril bir çözelti olarak tedarikçiden alınır. FBS sterilize parazit büyümeyi desteklemek kabiliyetini azaltır. </li><li> Tüm şişe çözülmüş sonra, ısı serum bileşenlerinin çökelmesini azaltmak için periyodik karıştırma, 56 ° C su banyosu içinde 30 dakika boyunca serum etkisiz hale getirirler. </li><li> Çözülme penisilin / streptomisin solüsyonu (pen / strep), Hemin (50 mM NaOH içinde 2 mg / ml), ve oda sıcaklığında bazal ortam kartal vitamin çözeltisi. </li><li> 1.000 ml&#39;lik dereceli bir silindir için, sıvı bileşenler (Tablo 2) SDM79 katı (Tablo 1) 20.2 g &#39;ını ve bir karıştırma levhası üzerinde iyice karıştırın. </li><li> 7,35 pH ayarlayın ve su ile 850 ml hacim getirmek. </li><li> Filtre bir filtre şişe altına bir vakum hortumu takarak ortamı sterilize. Çözelti filtre kadar vakum uygulayın. </li><li> Biyogüvenlik kabini filtre üst çıkarın ve FBS ısı 150 ml ekleyin. Steril kap ve mühür medya şişe plastik kaplamayı çıkarın. </li><li> Aşağıdaki istisnalar dışında SDM79 gibi aynı şekilde SDM80 ortamını hazırlayın; glikoz veya glukozamin ekleyebilir ve glutamin olmadan MEM kullanmayın. FBS, ısı 150 ml yerine, 135 diyalizlenmiş FBS, ısı ml ve FBS, ısı 15 ml ilave ediniz. </li><li> 27 ° C de, 10 ml bir uygun ortam ile 25 cm2 şişeye ve% 5 CO2 Kültür PCF parazitleri. Not: Hücreler bir hemositometre sitometresi veya bir akışı kullanılarak günlük olarak kabul edilmelidir (bölüm 6) ve kültürler 1 x 10 5 ve 5 x 10 6 hücre / ml arası bir yoğunlukta muhafaza edilmesi gereklidir. </li></ol> 2. TrFloresan Raportörü Yapısının ile PCF Parazitlerin ansfection Not: geliştirilmiş sarı floresan proteinine aldolaz peroksisomal hedefleme sekansı (PTS2) ihtiva eden bir raportör proteini parazitler olarak ifade edilir, glycosome dinamikleri izleyin. Füzyon proteini kodlayan sekansı procyclin promotörünü ve tübülin intergenik bölgeleri, genom içerisine doğrudan homolog rekombinasyon ve seçilmesi için blasticidin direnç geni içeren pXS2bla 12 içine klonlanır. T7 polimeraz ve tetrasiklin bastırıcı (PF29-13) kodlayan genleri barındıran Procyclic hücre çizgileri hedefleme konstruktu, pXS2 ile transforme edilmiştir: PTS2eYFP. <ol><li> Tablo 3&#39;te sıralanan unsurların karıştırılması ile cytomix hazırlayın. Oda sıcaklığında sterilize etmek ve saklamak Filtre. </li><li> DNA (1 ug / ml), 5 ul sınırlayıcı enzim tamponu, 33 ul su, 10 ul pipetleme Mlul ile linearize plazmid DNA ve 2 μ, Bir mikrosantrifüj tüpüne Mlul enzim l. 1 saat boyunca 37 ° C&#39;de inkübe edilir. </li><li> Kısıtlama sınırlama enzimi sindirimi ve bağlama tamponu içinde 1 hacim ekleyerek sindirmek saflaştınlır. Vorteks yoluyla bağlayıcı tampon ve sindirilmiş DNA karıştırın. Kısaca tüpün altındaki örnek toplamak için santrifüj. </li><li> 2 ml&#39;lik bir toplama tüpü içine sütunu, DNA bağlayıcı bir ekleme, ve transfer sütuna bağlanma tampon çözeltisi / DNA ile sindirilmiş. </li><li> 1 dakika 10.000 xg için santrifüj. Santrifüj işleminden sonra toplama tüpünden süzüntü atın ve toplama tüpüne sütun dönmek. </li><li> 1 dakika boyunca 10,000 x g&#39;de Dikenlikurt&#39;un yıkama tamponu ve santrifüj 700 ul ilave edin. </li><li> Toplama tüpünden atın süzüntü, toplama tüpüne sütun dönmek ve Dikenlikurt&#39;un yıkama adımı ve tekrar santrifüj. </li><li> 10,000 x g&#39;de 3 dakika boyunca atın Filtratın, toplama tüpüne geri sütun ve santrifüj boş kolon kalan etanolü atmak üzere. </li><li> Bir biyogüvenlik kabini içinde, col atmaksteril mikrosantrifüj tüpü içinde lection boru ve yer kolon. </li><li> , DNA elüt sütunu için steril su içinde 25 ul ilave edin ve 2 dakika boyunca oda sıcaklığında inkübe etmek. 1 dakika 10.000 x g&#39;de santrifüj. </li><li> Sütun biyo-güvenlik başlığı içinde steril su içinde bir 25 ul ilave edin ve 2 dakika boyunca oda sıcaklığında inkübe edin. </li><li> 1 dakika 10.000 xg hızda santrifüj. </li><li> Bir biyogüvenlik kabini içinde yeni bir steril mikrosantrifüj tüp DNA&#39;nın tüm hacim transferi. NOT: Bu adım santrifüj sırasında açık kapaktan kirlenmesini önler. </li><li> Filtre ve sterilize edilen cytomix 400 ul steril, saflaştırılmış, linearize DNA (10 ug) ilave edin. </li><li> Oda sıcaklığında 15 dakika boyunca 800 x g&#39;de santrifüj ile 5 x 10 7 -10 8 hücrelerin bölüm 6&#39;da tarif edilen ve hasat gibi hücre sayımı. </li><li> Süpernatantı dökün ve 1 ml&#39;lik bir serolojik bir pipet kullanarak DNA + cytomix 450 ul hücre pelletini. </li><li> Ce içeren Transfer çözüm<html> rf, elektroporasyon odasında steril 4 mm boşluk küvet ve yer küvete DNA ve cytomix. </li><li> &quot;Üstel çürüme&quot; seçin ve elle aşağıdaki ayarları girin: Gerilim: 1.5 kV, Kapasitans: 25 mF, Resistance: Ω, ve Küvet: 4 mm. Basın darbe. Darbe tamamlandıktan sonra, elektroporasyon odasından küvet kaldırmak ve biyo-güvenlik kabini geri döner. </li><li> Yeni bir steril bir şişeye, pipet SDM79, 10 ml. SDM79 10 ml şişeye üzere dönüşmüş hücreleri aktarın. </li><li> 27 ° C,% 5 CO2 seviyesinde 24 saat süre ile ilaç seçim olmadan inkübe edin. 24 saat sonra, G418 (15 ug / ml), higromisin (50 ug / ml) ve blastisidin (ug / ml 10 ug) ilave edilir. Ilaç ile SDM79 9 ml ilaç ile dönüştürülmüş hücrelerin 1 ml geçirin. </li><li> Bölüm 6 ve 7 hücreleri, floresan içinde tanımlanan, ve x, 10 7/1 mi bir hücre yoğunluğuna ulaşıldığında gibi uzun süreli depolama (3.1-3.3) hale stabilates için, günlük hücreleri analiz edin. </li></ol>ve_title &quot;&gt; 3. Stabilates Yapımı <ol><li> Dondurma medya yapmak için sterilize cytomix ve filtre% 100 gliserol eşit hacimde ekleyin. </li><li> Kriyotüpe hücre 800 ul (1 x 10 7) benzer şekilde dondurma ortamı 200 ul, iki raf arasında köpük inversiyon ve yerine göre, yavaşça karıştırın ve -80 ° C&#39;de dondurun. </li><li> (~ 24 saat) sonra, donmuş sıvı nitrojen transfer hücreleri. NOT: C hücre yaşamı sürdürme kabiliyetinde bir azalmaya yol açmaktadır ° -80 Bu daha uzun depolama gibi, 24 saat sonra 2 LN hücreleri içine taşımak için önemlidir. </li></ol> 4. Çözülme Dondurulmuş Stoklar <ol><li> Yaklaşık 10 dakika boyunca oda sıcaklığında C ve -80 ° &#39;den çözülme dondurulmuş şişesinden çıkarın. </li><li> Yeni bir steril şişe içine ilaç ile SDM79 Pipet 9 mi. Bu şişeye, çözülmesi hücreleri ekleyin. (1 x 10 5 ug / ml nihai konsantrasyon), yeni bir şişe içinde SDM79 9 ml bu kültürün 1 ml ilave etmek suretiyle hücreleri 01:10 geçirin. </li><li> Hücreler, 10 7 / ml &#39;lik bir yoğunluğa ulaşmadan önce, sitometresi set başlaryukarı ve seyreltme testleri. </li></ol> 5. Cytometer Kur <ol><li> Sitometrede akış açın ve cflow Artı programını açın. NOT: Herhangi bir örnek çalıştırmadan önce, fluidik adımlar 5,2-5,7 göre akıtmaya ve durulanmalıdır. </li><li> SIP boş bir tüp ile depolandığı Nanopure suyun tüp değiştirin. </li><li> &quot;Backflush&quot; düğmesini seçin. </li><li> Backflush tamamlandıktan sonra, yudum ve ıskarta gelen mikrosantrifüj tüpü çıkarın. </li><li> Yudum yeni Nanopure su 1 ml ihtiva eden yeni bir mikrosantrifüj tüpü yerleştirin. </li><li> Cflow programda yeni bir kuyu seçin. &quot;Çalıştır Sınırları&quot; altında 2 dakika için zaman ayarlayın. &quot;Fluidics&#39;in&quot; altında &quot;hızlı&quot; ve &quot;Çalıştır&quot; seçeneğini seçin. </li><li> 2 dakikalık bir zaman sınırı tamamlandıktan sonra, &quot;Örnek Verileri Sil&quot; düğmesine tıklayın. </li></ol> Akış Sitometreyi kullanarak 6. Hücre Sayım <ol><li> Numune su yerine sayılacak. &quot;SetRun Sınırları &quot;30 sn,&quot; hızlı &quot;için&quot; Fluidics Çalıştır &quot;&quot; ve sonra seçin &quot;. </li><li> 30 sn zaman sınırı cflow, tam artı ul &quot;Son Çalıştır.&quot; Her kültür için tekrarlayın sayımı altında olayları / sağlayacaktır sonra. NOT: Hücreler, 1 x 10 7 hücre / ml ulaşmadan önce, seyreltme testi ile devam edin. Bir seyreltme deney tamamlandıktan sonra hücreler kesilmelidir. Uzun süreler boyunca kültüre hücreler çevre koşullarına yanıt yeteneğini kaybeder. </li></ol> Akış Sitometreyi kullanarak 7. Ölçme Floresan <ol><li> Floresan değerlendirmek için, 10.000 olaylar &quot;Run Limitleri&quot; ve &quot;yavaş&quot; için &quot;Fluidics&quot; olarak ayarlayın. &quot;Set eşiği&quot; seçeneğini seçin. &quot;Birincil Eşik&quot; altında (kutuda aşağı açılan), &quot;Sürekli olayları ortadan kaldırmak&quot; &quot;FSC-H&quot; seçin ve daha az 30,000 girin. Ardından &quot;kapatmak&quot;, &quot;uygulamak&quot; tıklayın. </li><li> Seçin220; Histogram &quot;yeni arsa pencerelerden birinde düğmesine basıp&quot; FSC-A FL1-A &quot;&quot; Açılır listeden itibaren, seçin &quot;. NOT: 530 nm dalga boyunda FL1-A önlemler floresan. </li><li> &quot;Çalıştır&quot; ı seçin ve tüm kültürler için tekrarlayın. </li><li> 2 dakika 2 dakika ve su için fluidik hattı ile çözüme temizleme çalıştırın. </li></ol> 8. Seyreltme Tahliller <ol><li> 25 cm 2 kültürü şişesinde SDM79 3 ml içinde 1 x 10 5 hücre / ml &#39;lik bir yoğunluğa hücrelerin Pass ve hemen geçtikten sonra ve daha sonra 3 saat ve 24 saat sonra floresans ölçer. </li></ol> 9. Veri Analizi <ol><li> Seçin cflow Plus sekmesi &quot;Analiz&quot;. </li><li> Click &quot;yeni bir komplo yapın.&quot; Seç &quot;FSC-A&quot; ve bir damla listesi altında &quot;Histogram&quot; düğmesi belirir. Kanal seç &quot;FL1-A.&quot; </li><li> Analiz etmek için bir kuyu vurgulayın. &quot;Kapısı&quot; düğmesini seçin, ve elle bir kapı çizmekilgi nüfus için. </li><li> Akış Artı hücre bu kapı içinde &quot;Sayısı&quot; ve &quot;Bu Plot%&quot; sağlayacaktır. </li></ol>

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T., Schumann, U., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+maintenance+of+mammalian+peroxisomes+involves+a+de+novo+PEX16-dependent+pathway+from+the+ER.">The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER.</a> The Journal of cell biology. 173, 521-532 (2006).</li><li>Hoepfner, D., Schildknegt, D., Braakman, I., Philippsen, P., Tabak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+endoplasmic+reticulum+to+peroxisome+formation.">Contribution of the endoplasmic reticulum to peroxisome formation.</a> Cell. 122, 85-95 (2005).</li><li>Furuya, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+is+toxic+to+glycosome-deficient+trypanosomes.">Glucose is toxic to glycosome-deficient trypanosomes.</a> Proc Natl Acad Sci U S A. 99, 14177-14182 (2002).</li></ol>

The authors have nothing to disclose.

Glycosomes temel, dinamik, parazit özgü organellerdir. Bu organellerin biyogenezi, bakım, çoğalması ve biçimlenme düzenleyen süreçler olası terapötik amaçlar için istismar edilebilir ilaç hedefleri arasındadır. Bu tür ilaç hedefleri potansiyel olarak yüksek bolluğuna rağmen, daha glycosome biyogenez alan baskın nedeniyle, hızlı ve dinamik organel karşılıklarının izlenmesi için olan bir izlenebilir, yüksek hacimli sistem eksikliği, bir başka organizmalarda benzer süreçlerle ilgili çal...

Trypanosoma brucei sığır Afrika insanlarda uyku hastalığından ve israf hastalığı, nagana neden olur. Bu hastalıkların tedavisinde kullanılan ilaçlar, aşılar mevcut değildir, eskimiş ve son derece zehirlidir ve ilaç direnci gelişimi için potansiyel yeni ilaç hedefleri 1 için arama gerektirir. Ömrünün, T. sırasında brucei, bir böcek vektör ve memeli ev sahibi arasında dönüşümlü; parazit hayatta gerekir hangi çok farklı ortamlarda sergileyen iki ana. Parazit farklı çevre koşullarına maruz gibi metabolik ve morfolojik değişikliklerin bir dizi ortaya çıkar. En dramatik değişikliklerden bazıları tek-zar-sınırlı parazit özgü Mikrocisimler gözlenir, glycosomes 13 adlandırılır. Glukoz seviyeleri nispeten yüksek (~ 5 mM) kan ve kan parazitler (BSF) glikolisis wh ile sadece ATP üretmekIle mitokondriyal metabolizma 14 bastırılır. Ökaryotlarda farklı olarak, glikoliz, sitoplazma, T. oluşur brucei glycosomes 14,15 glikolitik enzimlerin çoğu compartmentalizes. Parazitler, bir bloodmeal sırasında çeçe sineği tarafından alınır ve sinek tarafından sindirildikten 15 dakika içinde tespit edilemez seviyelere düşer glikozunda bir düşmenin, yaşarlar. Böcek metabolizması, procyclic formu (PCF), parazit daha esnek ve glukoz gibi, prolin gibi amino asitler, ATP, 16-18 sentezinde kullanılabilir olduğunu. Karşılaştırmalı proteomik çalışmalar yaşam döngüsü glycosomal bağımlı değişiklikler ve kan parazitlerin artan glikolitik proteinler ile mitokondriyal proteinler ve TCA döngüsü ve solunum zincirinde 13,19 katılan mitokondriyal proteinleri ortaya. Birçok çalışma BSF ve PCF glycosomes arasındaki farklar odaklanmış olsa da, küçük env tepki olarak ortaya PCF glycosomes değişiklikler hakkında bilinmektedirironmental değişir. Sineğin hindgut, glukoz düzeyleri bir beslenme 20 sırasında geçici artışlar ile düşüktür. In vitro çalışmalar çoğunda, PCF parazitler glikoz içeren ortamda yetiştirilmektedir. Ancak son çalışmalar önemli ölçüde yanıt PCF metabolizma değişiklikleri durumunu 17 glikoza olduğunu göstermiştir. Glukoz, prolin alım ve prolin dehidrojenaz aktivitesi artışı 18 yokluğunda. Mitokondrial metabolizmanın bu değişiklik muhtemelen glycosome kompozisyon ve morfolojisinde bir değişime de eşlik eder, bununla birlikte, bu direkt olarak değerlendirilmemiştir. Elektron ve floresan mikroskobu yaygın teknikler T. glycosome dinamiklerini incelemek için kullanılır brucei 2,21-24. Bu protokoller, zaman ve yoğun, pahalı ve gerçek zamanlı çalışma ve yüksek verimli protokollere adapte zor doğum. Bir floresan-organel muhabiri sistemi u bu sınırlamayı aşmak içinmemeli hücreleri ve maya sistemlerinin organellere çalışma sed T. kullanılmak üzere modifiye edilmiştir brucei 12. Floresan-organel haberci sistemleri yaygın gibi maya, bitki ve memeli hücreleri de 25-27 gibi daha yüksek ökaryotlardaki kullanılmıştır. Bu gibi sistemlerde, bir flüoresan proteini spesifik organellere proteini hedefler bir amino asit sekansına kaynaştırılır. Hedef proteinlerin bozulması veya sentez floresan ölçülmektedir ve organel bileşim değişiklikler hücre flüoresanlıktaki değişikliklerle yansıtılmaktadır. Geliştirilmiş sarı floresan proteini (EYFP) &#39;nin açık okuma çerçevesi, bir tip II peroksisomal hedefleme sekansı (PTS2) ve 12 bağlı olduğunda, olgun bir protein PTS2eYFP, ithal yetkin glycosomes ve floresan içine alınır, akış sitometrisi vasıtasıyla izlenebilir. Glycosome bileşim içinde varyasyonlar hücresel flüoresanlıktaki değişikliklerle yansıtılmaktadır. Bu sistem, resol yardımcı olabilirglycosome bileşim içinde çevreden kaynaklanan değişiklikler belirleyen mekanizmaları VING. Bu yazıda canlı parazitlere gerçek zamanlı glycosome dinamiklerini izlemek için akış sitometrisi ile bağlantılı PCF parazitler bir glycosome raportör sistemin üretimini tarif ve farklı ortamlarda yanıt olarak glycosome bileşim değişimlerin izlenmesi için kullanılmıştır bir örnek sağlamaktadır. Taze ortam glycosome kompozisyonunda değişikliklere tetikler içine Özet olarak, bileşim, glycosome hücre dışı glükoz konsantrasyonu ve log-faz geçişi kültürlerin etkilenir. Bu sistem tripanozomlarına ve diğer parazitlerin diğer organellerin dinamik davranışlarını incelemek için modifiye edilebilir.

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using fluorescent proteins to monitor glycosome dynamics in the african trypanosome

Trypanosoma brucei sığır 1, insan Afrika trypanosomiasis (HAT), ya da uyku hastalığı ve israf hastalığı, nagana neden olan bir kinetoplastida parazittir. Memeli konakçının kan ve çeçe sineği vektörü arasındaki parazit seçim yapar. Pek çok hücresel organellerin bileşimi bu farklı hücre dışı koşullara 2-5 cevaben değişir. Glycosomes glikoliz katılan enzimlerin çoğu bölmeli olan yüksek seviyede uzmanlaşmış peroksizomlar bulunmaktadır. Gelişimsel ve çevresel düzenlenmiş şekilde 4-11 Glycosome kompozisyon değişir. Şu anda, glycosome dinamiklerini incelemek için kullanılan en yaygın teknikler elektron ve floresan mikroskopisi; pahalı, zaman ve emek yoğun ve yüksek verimlilik analizleri kolayca adapte değildir teknikleri. Bu sınırlamaları, bir flüoresan raportör sistemi-glycosome üstesinden gelebilmek için,12 glycosomes için, füzyon proteini yönlendiren peroksizom hedefleme dizisi (PTS2) kaynaşır sarı floresan protein (EYFP) geliştirilmiş olan kurulmuştur. PTS2eYFP füzyon proteininin ithalinde, glycosomes floresan olur. Organel bozulması ve akış sitometrisi ile ölçülebilir floresans kaybı geri dönüşüm ile sonuçlanır. Hücrelerin büyük sayılar (5,000 hücre / sn) gibi tespit ve montaj gibi geniş numune hazırlama olmadan gerçek zamanlı olarak analiz edilebilir. Bu yöntem, çevre koşulları dalgalı yanıt olarak bir organel kompozisyonunda değişikliklere tespit hızlı bir yol sunar.

Bu sistemde, glycosome bileşim içinde bir glukoza bağımlı bir değişiklik gözlenmemiştir. Hücreleri glikoz içeren ortamda yetiştirildiği zaman, iki nüfus gözlenir; Bir parlak ve bir loş (Şekil 2A). Dim hücreler parlak hücreler olgun ve olgunlaşmamış glycosomes 12 bir karışımını barındıran süre PTS2eYFP ithal değil olgunlaşmamış glycosomes, liman. Glikoz ortamı içinde mevcut olduğunda, protein glycosome mislocalization 15,28 öldürücü ve glycos...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome at JoVE.com

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

using-fluorescent-proteins-to-monitor-glycosome-dynamics-african

Research

JoVE Journal

Immunology and Infection

8.9K Görüntüleme.  Clemson University Eukaryotic Pathogens Innovation Center. Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Video: Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS aracılı Bakteriyel Rekabet Monitör A Görsel Assay

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

İmmünoloji ve Enfeksiyon

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Kullanımı<em&gt; In vivo</em&gt; Görüntüleme Yenidoğanlar Deneysel Fare Sitomegalovirüs Enfeksiyon ilerleme Monitör

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membran Kutup, Membran Dürüstlük ve Hücre içi İyon Konsantrasyonlarında İzleme değişiklikler<em&gt; Streptococcus pneumoniae</em&gt; Floresan boyalar kullanarak

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Görselleştirme ve Miktarının Belirlenmesi için floresan proteinleri kullanarak<em&gt; Chlamydia</em&gt; Vakuol Büyüme Dinamikleri Yaşayan Hücrelerde

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

Mezenterik Lenf Kanal Kanüllü Rat Modeli: Bağırsak Lenfatik İlaç Ulaştırma Değerlendirilmesine Uygulaması

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Hepatit C Virüsü Karşı İnsan laktoferrinin Hücresel Alımı ve İlişkili Antiviral Aktivite Monitör İmmünofloresan

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Biyoparlaklık Görüntüleme Afrika Trypanosomiasis Geç Evre Enfeksiyon Algılama için

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagozomun Kapatma Deneyi&quot; kullanma Üç Boyutta fagozomun Formasyonu Visualize&#39;a Toplam İç Yansıma Floresan Mikroskobu (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Bir Hepatit B Virüsü Muhabir Sisteminin Geliştirilmesi Çoğaltma Döngüsü Erken Aşamaları Monitör

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...