Bu çalışma için finansal destek Avrupa Topluluğu (ERC-STG INTRACELLTB Grant n° 260901, MM4TB Grant n° 260872), Agence Nationale de Recherche, Feder (12001407 (D-AL) Equipex Imaginex BioMed) ve Nord Pas de Calais Bölgesi tarafından sağlanmıştır. BICeL platformundan Gaspard Deloison, Elizabeth Werkmeister, Antonino Bongiovanni ve Frank Lafont'un teknik yardımını minnetle kabul ediyoruz.

1. Yüksek verimli Genom çapında siRNA Taraması
Yeşil Floresan Proteini (GFP) ifade eden Mtb H37Rv ile enfeksiyon üzerine insan Tip-II pnömosit modeli A549 hücre hattında tarama yapıldı. Bu yordam Şekil 1A'daözetlenmiştir.
<ol><li>4 μM konsantrasyona ulaşmak için 1x siRNA tamponu ile ana plakalarda (96 kuyu plakaları) depolanan kurutulmuş siRNA kütüphanesini yeniden kullanın, ardından karışımın 10 μl'lik kısmını 384 kuyulu bir kız plakasına (kız plakası 1) aktarın.</li>
	<li>SiRNA'yı 2 kat seyreltmek için 16 kız plakası 1'e 10 μl siRNA tamponu ekleyin. siRNA resüspensiyondan sonra, plakalar soyulabilir alüminyum conta ile kapatılır ve -20 °C'de en az 6 ay ve 2-3 yıla kadar saklanabilir, ancak depolama süresi siRNA kütüphane üreticisinin önerilerine bağlı olarak değişebilir.</li>
	<li>500 nM konsantrasyona ulaşmak için kız plakası 1'deki siRNA'ları kız plakası 2'ye seyreltin. siRNA resüspensiyondan sonra, plakalar soyulabilir alüminyum conta ile kapatılır ve -20 °C'de en az 6 ay ve 2-3 yıla kadar saklanabilir, ancak depolama süresi siRNA kütüphane üreticisinin önerilerine bağlı olarak değişebilir.</li>
	<li>Kullanmadan önce, kız plakası 2'yi oda sıcaklığında çözün.</li>
	<li>Kız plakası 2'den 2,5 μl siRNA alın ve 384 kuyulu bir test plakasına yerleştirin.</li>
	<li>Adım 1.5'te aynı 384 kuyu test plakasında, ilgili kuyularına 2.5 μl negatif ve pozitif kontrol siRNA ekleyin.</li>
	<li>Transfeksiyon reaktifini 1x D-PBS'de seyrelterek her kuyuda 0,1 μl transfeksiyon reaktifi sağlayacak kadar çözelti elde edin ve seyreltilmiş transfeksiyon çözeltisini oda sıcaklığında 5 dakika önceden kesin.</li>
	<li>Test plakasındaki her kuyuya 7,5 μl transfeksiyon reaktifi/PBS çözelti karışımı ekleyin ve oda sıcaklığında 30 dakika kuluçkaya yatırın.</li>
	<li>%10 fetal sığır serumu (FBS) ile desteklenmiş RPMI 1640 ortamında askıya alınan 40 μl A549 hücresi (1.500 hücre/kuyu) ekleyin. %5 CO2içeren bir atmosferde 37 °C'de 3 günlük kuluçka süresi boyunca hücreleri koruyun. Bu hücreler her 24 saatte bir bölünür, böylece transfeksiyondan üç gün sonra yaklaşık 12.000 hücre kuyulardadır.</li>
	<li>İki haftalık GFP ifade eden Mtb H37Rv kültürünü D-PBS (MgCl 2 ve CaCl2'den ücretsiz) ile5dakika boyunca 4.000 x g'da santrifüjleme ile yıkayın ve yıkamaları atın. Bu adımı 3x tekrarlayın. (GFP-Mtb H37Rv kültür koşulları için bkz. protokol 3).</li>
	<li>Bakteriyel peleti% 10 FBS ile desteklenmiş 10 ml RPMI 1640 ortamında askıya alın ve bakteriyel agregaların tortulanmasına izin vermek için oda sıcaklığında 1 saat dekant.</li>
	<li>Bakteriyel süpernatantı toplayın ve bakteri konsantrasyonu belirlemek için bir mikro plaka okuyucu kullanarakOD 600 (OD 600 0.6-0.8 arasında olmalıdır) ve GFP-floresan (RFU değeri) ölçün. Aynı koşullarda hazırlanmış başka bir kültür üzerinde denemeden önce oluşturulan RFU değeri = f (CFU değeri) gösteren bir referans regresyon çizgisi kullanarak süspansiyonun titresini hesaplayın. 5'lik çok miktarda enfeksiyona (MOI) karşılık gelen2,4 x 10 6 bakteri/ml içeren bakteri süspansiyonu hazırlayın.</li>
	<li>384 kuyulu test plakasındaki ortamı çıkarın ve 25 μl taze hazırlanmış bakteri süspansiyonu ekleyin.</li>
	<li>384 kuyulu test plakasını% 5 CO2içeren bir atmosferde 5 saat boyunca 37 ° C'de kuluçkaya yatırın.</li>
	<li>Ortamı çıkarın ve hücreleri% 10 FBS 3x ile desteklenmiş RPMI ortamı ile hafifçe yıkayın.</li>
	<li>Kalan hücre dışı bakterileri öldürmek için, %5 CO2içeren bir atmosferde 1 saat boyunca 37 °C'de 50 μg/ml amikasin içeren 50 μl taze RPMI-FBS ortamı ile hücreleri tedavi edin.</li>
	<li>Orta içeren amikacin'i çıkarın ve% 10 FBS ile desteklenmiş 50 μl taze RPMI ortamı ekleyin. 384 kuyulu test plakasını% 5 CO2içeren bir atmosferde 5 gün boyunca 37 ° C'de kuluçkaya yatırın.</li>
	<li>Görüntü alımından önce, PBS'de 10 μl taze hazırlanmış 30 μg/ml DAPI ekleyin (son konsantrasyon 5 μg/ml) ve 37 °C'de 10 dakika kuluçkaya yatırın.</li>
	<li>Plakayı otomatik konfokal mikroskopa yükleyin. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Pozlama parametrelerini ayarlayın. Emisyon filtresi 450 nm ile excitation lazer 405 nm ve emisyon filtresi 520 nm ile excitation lazer 488 nm kullanarak GFP floresan kullanarak DAPI floresan kaydedin. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Elde edilecek her kuyudaki kuyuları ve alanları seçin, daha sonra düzen ve alt katman parametreleri olarak adlandırılır. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>1.20 ve 1.21 adımlarındaki parametreleri kullanarak deneme dosyasını oluşturun ve otomatik alma işlemini çalıştırın. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Görüntüleri uzak sunucuya aktarın. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Görüntü analiz yazılımını kullanarak görüntüleri değerlendirin. Çekirdek algılama algoritmasını kullanarak DAPI kanalından hücre çekirdeklerini ve piksel yoğunluğu özellikleri algoritmasını kullanarak GFP kanalından bakteri alanını tespit edin (Şekil 4A).</li>
</ol>Not: Bu protokol, gen susturmasının hücre içi Mtb büyümesi üzerindeki etkisini incelemek için optimize edilmiştir. Mtb, her 20 saatte bir optimum koşullarda bölünen yavaş büyümeli bir bakteridir. Enfeksiyondan 5 gün sonra hücre dışı Mtb miktarı hücre lizizinin yokluğunda hala düşüktür ve analizin kalitesini etkilememiştir. Bu protokol, mycobacteria smegmatis ve Escherichia coli gibi yoğun olarak salınan ve yeni hücreleri enfekte edebilen hızlı büyüme bakterileri kullanılarak siRNA ekranları için uyarlanacak antibiyotik tedavisinin uzunluğu ve kuluçka süresi açısından optimize edilmelidir.
2. Yüksek verimli Bileşik Tarama
Tarama Mtb H37Rv enfekte konak hücrelerinde gerçekleştirildi. Bu yordam Şekil 1B'de özetlenmiştir.
<ol><li>DMSO'da çözünen bileşik kütüphaneyi içeren 384 kuyulu ana plakaları %100 çözün. %10 FBS ile desteklenmiş 10 μl RPMI 1640 ortamı içeren 384 kuyulu kız plakalarındaki bileşiklerin 0,5 μl'sini aktarın.</li>
	<li>İki haftalık GFP ifade eden Mtb H37Rv kültürünü D-PBS (MgCl 2 ve CaCl2'den ücretsiz) ile5dakika boyunca 4.000 x g'da santrifüjleme ile yıkayın ve yıkamaları atın. Bu adımı 3x yineleyin (GFP-Mtb H37Rv kültür koşulları için bkz. protokol 3).</li>
	<li>Bakteriyel peleti% 10 FBS ile desteklenmiş 10 ml RPMI 1640 ortamında askıya alın ve bakteriyel agregaların tortulanmasına izin vermek için oda sıcaklığında 1 saat dekant.</li>
	<li>Bakteriyel süpernatantı toplayın ve mikro plaka okuyucu kullanarak OD600 (OD600 0.6-0.8 arasında olmalıdır) ve GFP floresanını (RFU değeri) ölçün. Denemeden önce oluşturulan RFU değeri = f (CFU değeri) görüntüleyen bir referans regresyon çizgisi kullanarak süspansiyonun titresini hesaplayın. Tipik konsantrasyon 1 x 108 bakteri/ml'dir.</li>
	<li>%10 fetal sığır serumu (FBS) ve50 ng/ml rekombinant insan M-CSF ile desteklenmiş RPMI 1640 ortamında 4 x 10 5 hücre/ml'de 6 günlük birincil insan makrofajlarını hasat edin (İnsan Periferik Kan Monosit Hücreleri saflaştırma ve makrofajların farklılaşması için bkz. Protokol 4).</li>
	<li>Seyreltilmiş birincil hücreleri, 1-5 arasında değişen farklı MOI'de basil ile kuluçkaya yaslanın, süspansiyonda 90 rpm'de 37 °C'de 2 saat hafif sallanarak.</li>
	<li>Hücre dışı bakterileri temizlemek için enfekte hücreleri 350 x g'da santrifüjleme ile yıkayın. Her santrifüj adımından sonra peletin %10 FBS ile desteklenmiş RPMI 1640 ortamında yeniden kullanılabilir. Bu adımı 2x tekrarlayın.</li>
	<li>RPMI 1640 ortamındaki enfekte hücreleri % 10 FBS ve amikacin ile 50 μg / ml'de askıya alın ve süspansiyonu 37 ° C'de 1 saat hafif sallama ile kuluçkaya yatırın.</li>
	<li>Amikacin içeren hücre kültürü ortamını 350 x g'da santrifüjleme ile çıkarın ve enfekte olmuş hücreleri% 10 FBS ve 50 ng / ml rekombinant insan M-CSF ile desteklenmiş eksiksiz RPMI 1640 ortamı ile yıkayın. Bir kez tekrarlayın.</li>
	<li>Zaten bileşik seyreltmelerin 10 μl'sini içeren 2.1 adımında aynı test plakasına 40 μl enfekte makrofaj süspansiyonu ekleyin. DMSO'nun her kuyudaki son konsantrasyonu artık % 1'e ulaştı.</li>
	<li>%5 CO2içeren bir atmosferde 37 °C'de 5 gün boyunca test plakalarını kuluçkaya yatırın.</li>
	<li>Canlı hücreleri hücre geçirgen uzak kırmızı floresan boya ile lekelenin.</li>
	<li>Plakayı otomatik konfokal mikroskopa yükleyin. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Pozlama parametrelerini ayarlayın. Emisyon filtresi 690 nm ile excitation lazer 640 nm ve emisyon filtresi 520 nm ile excitation lazer 488 nm kullanarak GFP floresan kullanarak uzak kırmızı floresan kaydedin. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Elde edilecek her kuyudaki kuyuları ve alanları seçin, daha sonra düzen ve alt katman parametreleri olarak adlandırılır. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>2.14 ve 2.15 adımlarındaki parametreleri kullanarak deneme dosyasını oluşturun ve otomatik alma işlemini çalıştırın. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Görüntüleri uzak sunucuya aktarın. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">Daha büyük resmi görüntülemek için burayı tıklatın.</a></li>
	<li>Görüntü analiz yazılımını kullanarak görüntüleri değerlendirin. Piksel yoğunluğu özellikleri algoritması kullanarak uzak kırmızı kanaldan hücre alanını ve piksel yoğunluğu özellikleri algoritması(Şekil 4B)kullanarak GFP kanalından bakteri alanını algılayın.</li>
</ol>Not: Bu protokol, floresan proteini (Bir kuyu/Bir mutant) ifade eden mutantlar tarafından bileşiklerin değiştirilmesiyle Mtb mutant kütüphanesi taraması için uyarlanabilir (Şekil 1C, ayrıca bkz. 4). Floresan mutantlar ilk olarak kuyularda tohumlanır (kuyu başına 20 μl bakteri süspansiyonu). Bakteriler daha sonra 30 μl hücre süspansiyonu ile geri kazanilir. 1 dakika boyunca 350 x g'da santrifüjlemeden sonra, plaka% 5 CO2içeren bir atmosferde 37 ° C'de inkübe edilir. Kuluçka süresi ve MOI teste bağlıdır. Örnek olarak, fagozom asitleştirme gibi erken hücresel olayların görselleştirilmesi için, hücreler 1-20 arasında değişen MOI ile 2 saat boyunca enfekte edilebilir. Lizozomlar% 5 CO 2 içeren bir atmosferde 37 ° C'de 1.5 saat boyunca2 μM'de Lysotracker boyası kullanılarak lekelenir ve daha sonra% 10 formalin veya% 4 paraformaldehit (PFA) ile sabitlenir. Konfokal görüntüler, lizozom tespiti ve hücre altı yerelleştirme için uygun algoritmalara sahip görüntü analizi komut dosyaları kullanılarak elde edilir ve son olarak analiz edilir4.
3. Mikobakteri tüberkülozu ifade eden Yeşil Floresan Protein H37Rv (GFP-H37Rv) Kültür Koşulları
Uzun süreli depolama için, GFP-H37Rv D-PBS'de donduruldu (şişe başına yaklaşık 1 x10 8 mikobakteri).
<ol><li>Middlebrook OADC zenginleştirmesi %10, Gliserol %0,5, Ara-80%0,05 ve Hygromycin B (50 μg/ml) ile desteklenmiş 50 ml 7H9 et suyu ortamı içeren bir Erlenmeyer şişesinde bir adet donmuş stok GFP-H37Rv şişesini yeniden depola.</li>
	<li>37 °C'de 8 gün kuluçkaya yaslanın.</li>
	<li>GFP-H37Rv kültürünün OD600'lerini ölçün.</li>
	<li>GFP-H37Rv kültürünü seyrelterek OD600 = 0.1'i Middlebrook OADC zenginleştirmesi ile desteklenmiş taze 7H9 et suyu ortamında %10, Gliserol%0.5, Ara-80%0.05 ve Hygromycin B (50 μg/ml) ile destekleyin.</li>
	<li>Test için kullanmadan önce GFP-H37Rv'u 8 gün daha 37 °C'de kuluçkaya yatırın.</li>
</ol>4. İnsan Periferik Kan Monosit Hücreleri Tam Kan veya Buffy-coat Preparatından Arındırma
<ol><li>Kan kesesini % 1 FBS içeren 1x D-PBS'de (MgCl2 ve CaCl 2'den ücretsiz)2xseyreltin.</li>
	<li>Monositleri 20 dakika boyunca 400 x g'da Ficoll yoğunluk gradyan santrifüjleme ile izole edin.</li>
	<li>Yalıtılmış monositleri toplayın.</li>
	<li>Monositleri 3x'i oda sıcaklığında 10 dakika boyunca 400 x g'da santrifüjleme ile%1 FBS içeren 1x D-PBS (MgCl 2 ve CaCl2içermez) ile yıkayın.</li>
	<li>Hücreleri 1 x 107 hücre/ml'ye kadar konsantre edin.</li>
	<li>Cd14 manyetik boncukları kullanarak monositleri üreticinin protokolüne göre arındırın (bkz. Malzemeler).</li>
	<li>CD14-monositlerin saflaştırılmasından sonra, RPMI 1640'ta 1,5 x 106 hücre/ml'deki hücrelerin tohumlamaları% 10 FBS ve 40 ng / ml insan Makrofaj koloni uyarıcı faktörü (hM-CSF) ile tamamlanmış ve% 5 CO2içeren bir atmosferde 37 ° C'de 4 gün boyunca inkübe edilir.</li>
	<li>4 gün sonra ortamı% 10 FBS ve 40 ng / ml hM-CSF ile tamamlanan ve% 5 CO 2 içeren bir atmosferde 37 ° C'de 2 gün boyunca inkübe edilen taze RPMI1640ile değiştirin.</li>
	<li>6 gün sonra, hücreler test için kullanılabilir.</li>
</ol>

<ol>
	<li>Gandhi, N. R., 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=Multidrug-resistant+and+extensively+drug-resistant+tuberculosis:+a+threat+to+global+control+of+tuberculosis.">Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis.</a> Lancet. 375, 1830-1843 (2010).</li><li>Barry, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+latent+tuberculosis:+rethinking+the+biology+and+intervention+strategies.">The spectrum of latent tuberculosis: rethinking the biology and intervention strategies.</a> Nat. Rev. Microbiol. 7, 845-855 (2009).</li><li>Christophe, T., Ewann, F., Jeon, H. K., Cechetto, J., Brodin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+imaging+of+Mycobacterium+tuberculosis-infected+macrophages:+an+in+vitro+model+for+tuberculosis+drug+discovery.">High-content imaging of Mycobacterium tuberculosis-infected macrophages: an in vitro model for tuberculosis drug discovery.</a> Future Med. Chem. 2, 1283-1293 (2010).</li><li>Brodin, P., 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=High+content+phenotypic+cell-based+visual+screen+identifies+Mycobacterium+tuberculosis+acyltrehalose-containing+glycolipids+involved+in+phagosome+remodeling.">High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.</a> PLoS Pathog. 6, (2010).</li><li>Bermudez, L. E., Goodman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+invades+and+replicates+within+type+II+alveolar+cells.">Mycobacterium tuberculosis invades and replicates within type II alveolar cells.</a> Infect. Immun. 64, 1400-1406 (1996).</li><li>Dobos, K. M., Spotts, E. A., Quinn, F. D., King, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrosis+of+lung+epithelial+cells+during+infection+with+Mycobacterium+tuberculosis+is+preceded+by+cell+permeation.">Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation.</a> Infect. Immun. 68, 6300-6310 (2000).</li><li>McDonough, K. A., Kress, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+for+lung+epithelial+cells+is+a+virulence-associated+phenotype+of+Mycobacterium+tuberculosis.">Cytotoxicity for lung epithelial cells is a virulence-associated phenotype of Mycobacterium tuberculosis.</a> Infect. Immun. 63, 4802-4811 (1995).</li><li>Lee, H. M., Yuk, J. M., Shin, D. M., Jo, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dectin-1+is+inducible+and+plays+an+essential+role+for+mycobacteria-induced+innate+immune+responses+in+airway+epithelial+cells.">Dectin-1 is inducible and plays an essential role for mycobacteria-induced innate immune responses in airway epithelial cells.</a> J. Clin. Immunol. 29, 795-805 (2009).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J. Biomol. Screen. 4, 67-73 (1999).</li><li>Birmingham, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+methods+for+analysis+of+high-throughput+RNA+interference+screens.">Statistical methods for analysis of high-throughput RNA interference screens.</a> Nat. Methods. 6, 569-575 (2009).</li><li>Carralot, J. P., 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=Automated+high-throughput+siRNA+transfection+in+raw+264.7+macrophages:+a+case+study+for+optimization+procedure.">Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.</a> J. Biomol. Screen. 14, 151-160 (2009).</li><li>Zhang, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illustration+of+SSMD,+z+score,+SSMD*,+z*+score,+and+t+statistic+for+hit+selection+in+RNAi+high-throughput+screens.">Illustration of SSMD, z score, SSMD*, z* score, and t statistic for hit selection in RNAi high-throughput screens.</a> J. Biomol. Screen. 16, 775-785 (2011).</li><li>Song, O. R., 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=Confocal-based+method+for+quantification+of+diffusion+kinetics+in+microwell+plates+and+its+application+for+identifying+a+rapid+mixing+method+for+high-content/throughput+screening.">Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for identifying a rapid mixing method for high-content/throughput screening.</a> J. Biomol. Screen. 15, 138-147 (2010).</li></ol>

Çıkar çatışması bildirilmedi.

Burada, yüksek içerikli / Yüksek verimli ekranlar için uygun hale getiren floresan etiketli konak hücreleri enfekte etmek için GFP ifade eden bir Mtb H37Rv suşu kullanarak fenotipik bir test için gerekli yöntemleri açıklıyoruz. Bu protokol çok çeşitli bileşiklere, floresan problara ve Mtb mutantlarına uygulanabilir. Yukarıda açıklanan her protokol için, görüntü alımından önce fiksasyon ve immünolabeling adımları gerçekleştirilebilir. Görüntü elde etmek için 20X (NA 0....

Son yıllarda bildirilen ortaya çıkan ve yeniden ortaya çıkan bulaşıcı patojenler arasında, Mycobacterium tuberculosis (Mtb),2011 yılında 1,4 milyon ölümden ve 8,7 milyon yeni enfeksiyondan sorumlu olarak önemli bir yer tutmaktadır (Küresel tüberküloz raporu 2012, www.who.int/topics/tuberculosis/en/). Multidrug tedavilerinin mevcudiyetine rağmen, enfekte insan sayısı hala artmaktadır ve çok halıya dayanıklı (MDR) ve yaygın olarak ilaca dirençli (XDR) Mtb hızla tüm dünyaya yayılmaktadır1. Ayrıca, Mtb antijenlerinin varlığı dikkate alındığında, küresel nüfusun üçte birinin Mtb tarafından gizlice enfekte olduğu düşünülmektedir. İstatistiksel olarak, on vakadan birinde, sonraki klinik semptomlarla hastalığın aktif formuna doğru evrim vardır2. Bu nedenle, Mtb ile savaşmak için acilen yeni araçlara ihtiyaç vardır. Bu kapsamda, otomatik konfokal floresan mikroskopisi3 . Tahlilin 384 kuyulu mikrotiter plakalarda otomatik görüntü alma ve analiz ile birlikte uyarlanması, orta ölçekli bileşik, siRNA ve bakteriyel mutant kütüphanelerinin Yüksek içerikli / Yüksek verimli Taramaya (HC / HTS) izin verdi. Bu fenotipik test üzerinde genom geniş bir RNAi kütüphanesinin taranması, böylece Mtb kaçakçılığı ve hücre içi replikasyonda yer alan temel konak faktörlerinin tanımlanmasını ve aynı zamanda tüberkül basil basili tarafından sömürülen konak yolların aydınlatılmasını sağladı. Bu özel fenotipik tahlilin bir diğer adaptasyonu da Mtb intra-fagozomal kalıcılık için gerekli bakteriyel faktörlerin tanımlanmasıydı. Örneğin, fagozom olgunlaşmasının tutuklanması, Mtb'nin makrofajda hayatta kalmasını ve replikasyonunu kolaylaştıran önemli mekanizmalardan biri olarak kabul edilir. Mtb nakavt mutantlarının floresan etiketli-asidik bölmelerde hücre altı lokalizasyonunun izlenmesi, sağkalım sürecinde yer alan bakteri genlerinin tanımlanmasına izin verdi4. Son olarak, Mtb'nin yüksek içerikli görüntülemesi, hücre içi bakteri üremesi gibi çeşitli fenomenleri inhibe etmek için ilaç verimliliğini ölçmek için mükemmel bir yöntem sunar3. Tamamen, bu tür yüksek verimli fenotipik test, TB'ye karşı ilaç keşfini hızlandırmaya izin verir ve bu farklı yaklaşımlarla toplanan veriler Mtbtarafından uygulanan konak manipülasyonunun daha iyi anlaşılmasına katkıda bulunur.

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			<td height="21" style="height:21px;width:287px;">&#181;clear-plate black, 384 well</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781091</td>
			<td style="width:388px;">127.8/86/15 MM with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CellCarrier 384 well plate</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;">6007550</td>
			<td>Black, Clear Bottom, with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">V-bottom white , 384 well-plate</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">sealing tape, breathable, sterile</td>
			<td style="width:160px;">Corning</td>
			<td style="width:175px;">3345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMax</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">13778150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Dimethyl sulfoxide</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">34943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">RPMI 1640 + GlutaMAX-I</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">61870-010</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [-]MgCl2/[-]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-094</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [+]MgCl2/[+]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-091</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Fetal bovine serum</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">2610040-79</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">FICOLL PAQUE PLUS</td>
			<td style="width:160px;">DUTSCHER</td>
			<td style="width:175px;">17-1440-03</td>
			<td>Ficoll for Peripherical Blood Monocyte Cells purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CD14 MicroBeads, human</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-050-201</td>
			<td>Purification of CD14+ Monocytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Human M-CSF, premium gr. (1000 &#956;g)</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-096-493</td>
			<td>Macrophage Colony Stimulating Factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">LS Columns</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-042-401</td>
			<td>Columns for CD14+ Monocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tween 80</td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">2002-A</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glycerol high purity </td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">50405-EX</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;"> Middlebrook OADC enrichment</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">211886</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7H9</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">W1701P</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Versene 1X</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">15040033</td>
			<td>Non enzymatic cell dissociation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DAPI</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">D1306</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hoechst 33342</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">H3570</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syto60</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">S11342</td>
			<td>Nuclei/cytoplasm dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Formalin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">HT5014</td>
			<td>Cell fixation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siRNA targeting Dectin-1</td>
			<td style="width:160px;">Santa-Cruz</td>
			<td style="width:175px;">sc-63276</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">*siGenome* Non targeted siRNA pool </td>
			<td style="width:160px;">Dharmacon</td>
			<td style="width:175px;">D-001206-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Rifampicin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">R3501</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoniazid (INH)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">I3377-50G</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hygromycin B</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">10687-010</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Amikacin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">A1774</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Automated Confocal Microscope OPERA</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Columbus 2.3.1 Server Database</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Data transfert, storage and analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acapella 2.6 software</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image-based analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GraphPad Prism5 software</td>
			<td style="width:160px;">GraphPad </td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:287px;">Excel 2010</td>
			<td style="width:160px;">Microsoft</td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
	</tbody>
</table>

a microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening

Tedavi ve aşı mevcudiyetine rağmen, tüberküloz (TB) dünyadaki en ölümcül ve yaygın bakteriyel enfeksiyonlardan biri olmaya devam etmektedir. Birkaç on yıldan bu yana, çok ve yaygın olarak ilaca dirençli suşların ani patlaması tüberkülozun kontrolü için ciddi bir tehdittir. Bu nedenle, tüberkülozun etken maddesi olan Mycobacterium tuberculosis(Mtb)için kritik olan yeni hedeflerin ve yolların belirlenmesi ve TB ilaçları olabilecek yeni kimyasalların araştırılması esastır. Bir yaklaşım, samanlıkta iğne aranmasını sağlayan büyük ölçekli kütüphanelerin genetik ve kimyasal ekranlarına uygun yöntemler kurmaktır. Bu amaçla, otomatik konfokal mikroskopi kullanarak floresan etiketli konak hücreler içinde floresan etiketli Mtb'nin tespitine dayanan fenotipik bir test geliştirdik. Bu in vitro test, Mtb'nin kolonileşme sürecinin konakçıya görüntü bazlı nicelleştirilmesine izin verir ve siRNA, kimyasal bileşik veya Mtb mutant kütüphanelerinin ekranları için uygun olan 384 kuyu mikro plaka formatı için optimize edilmiştir. Görüntüler daha sonra konak hücreler içindeki Mtb patogenezinde okuma çıkarımını sağlayan çokparametrik analiz için işlenir.

Yüksek verimli genom çapında siRNA taraması
Mtb, immün hücreleri in vitro ve diğer birkaç akciğer epitel hücresini kolonize edebilir. Örneğin, Mtb,insan tipi II pnömosilatlar için model olarak yaygın olarak kullanılan A549 epitel hücrelerini enfekte edebilir ve zarar verebilir5-7. Dectin-1, A549 hücrelerinde hücre içi mikobakteriyel büyüme üzerinde Mtb alımı, proinflamatuar yanıt ve antibakteriyel etkide yer alan bir konak h...

Watch this Scientific Journal Video about A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening at JoVE.com

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Burada, küçük müdahale eden sentetik RNA (siRNA), kimyasal bileşik ve Mycobacterium tuberculosis mutant kütüphanelerinin Yüksek verimli / Yüksek içerikli ekranları için geçerli olan fenotipik bir test açıklıyoruz. Bu yöntem, otomatik konfokal mikroskopi kullanılarak floresan etiketli konak hücre içinde floresan etiketli Mycobacterium tuberculosis'in tespitine dayanır.

Yüksek Verimli/Yüksek İçerikli Taramaya Uyarlanmış Hücre İçi Mikobakterilerin Nicelleştirilmesi için Mikroskobik Fenotipik Bir Tahlil

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

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Research

JoVE Journal

Immunology and Infection

7.6K Görüntüleme.  Université de Lille. Burada, küçük müdahale eden sentetik RNA (siRNA), kimyasal bileşik ve Mycobacterium tuberculosis mutant kütüphanelerinin Yüksek verimli / Yüksek içerikli ekranları için geçerli olan fenotipik bir test açıklıyoruz. Bu yöntem, otomatik konfokal mikroskopi kullanılarak floresan etiketli konak hücre içinde floresan etiketli Mycobacterium tuberculosis'in tespitine dayanır.

Video: A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Mikobakteriler canlılık Tedbir Fonksiyonel Tam Kan Testi, Reporter Gen BCG veya M. Tb (BCG Takip edilen konu kullanarak Lüks / M. Tb Lüks)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

İmmünoloji ve Enfeksiyon

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Hücre içi Karşı Antileishmanial Tarama için Parasite Kurtarma ve Dönüşüm Testi<em&gt; Leishmania donovani</emTHP1 İnsan Akut Monositik Lösemi Hücre Hattı&gt; Amastigotes

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Termostabil Bacteriolytic Enzimlerin yönlendirilmiş evrimi için Yeni Tarama Yöntemi

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

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.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

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

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

Mikobakteriler Yarı kantitatif, Orta hacimli Sayımı İçin Kömür Agar Resazurin Testi görselleştirme

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Köpek Nötrofil Ekstrasellüler Trap Release Niceleme için Basit Floresan Testi

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

Küçük moleküllerin yüksek verimli tarama için uygun bir floresan bazlı lenfositi analizi

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Hücre içi Hedefleme İnhibitörleri Etkinliği ve Sitotoksisite Taraması için sistem<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Hızlı ve güvenilir yöntem LEISHMANIA parazit virülans değerlendirmek için hücre içi büyüme makrofajlar içinde miktar olduğunu

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Yüksek işlem hacmi, yüksek-içerik, sıvı tabanlı C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...