Yazarlar verdikleri destek için Ellison Tıp Vakfı ederim. Biz wormsorter kullanarak yardım için Abby Dernburg laboratuvar üyelerine teşekkür etmek de isterdim.

1.. Genç Yetişkin Hayvanlar bir Senkronize Kültürü Edinme <ol><li> OP50-1 E. ile ekildi birkaç NGM plakalar üzerinde solucanlar büyütün coli bakterisinin (10x doymuş bir kültürden konsantre) birçok solucan gebe aşamasına kadar. </li><li> Senkronize bir kültürü (yumurta) elde etmek için yumurta hazırlık solüsyonu ile gebe hayvanları tedavi. </li><li> Çok sayıda 60 mm plakalar NGM levhalar yumurta kabaca 150-200 yumurta / levha yoğunluğunda 10x konsantre OP50-1 ile tohumlandı. </li><li> (- Küçük yetişkin aşamada solucanlar L4 ulaşana kadar) 2 gün boyunca 25 ° C&#39;de inkübe edin. </li></ol> 2. Pseudomonas aeruginosa Plates hazırlanması ve Worms bulaşıyor <ol><li> Yumurta-prep (adım 1.1) de günde 100 ug / ml &#39;lik bir nihai konsantrasyonda King B içeren bir ortamda rifampisin, 2 ml bir PA14 :: GFP kültürü inoküle. Ajitasyon O / N 37 ° C &#39;de kültür inkübe </li><li> PA14 :: GFP cul PlateDeney için gerekli olduğu kadar çok yavaş öldürme Plakalar (SKP) üzerine Ture. Her 150 mm SKP Petri pipet üzerinde 75-100 doymuş kültür ul ve steril cam bir dağıtıcı ile yayılır. 20-24 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> Inkübatör plakaları kaldırmak ve onları RT (~ 20 dk) soğumaya bırakın. M9 tampon kullanarak, PA14 üzerine adım 1.4 solucanlar yıkama :: GFP plakaları bir sıvı minimal hacimde. 150 mm tabak kullanırken, birkaç yüz solucanlar, tek bir plaka üzerine yerleştirilebilir. </li><li> 18-21 saat boyunca 25 ° C&#39;de inkübe edin. P. için aeruginosa maruz kalan genç erişkin solucanlar, bu noncolonized hayvanların vs kolonize optimal dağılımını görüntüleyen zaman penceresi olduğunu. </li></ol> 3. Wormsorter ayarlama Örnek sıralama yapmadan önce, makinenin düzgün çalıştığından emin olun. Detaylar http://www.unionbio.com/support/documents.aspx?id=43 elde, ve inci olabilire gerekli adımlar aşağıda özetlenmiştir. <ol><li> Yaklaşık 5 psi (4.5-5.5 aralığı) için kılıf valf basıncını ayarlayın. </li><li> Bu kılıf sıvı akış hızı uygun doğrulamak dağıtıcının altında bir 15 ml konik tüp yerleştirmek, kılıf vana açmak ve sıralayıcı vanasını kapatın. 60 saniye için dağıtıcı tüpü altında tutun. Tüp içinde hacmi 9-10 ml olmalıdır. Bu aralığın dışında ise, buna kılıf valf basıncını ayarlamak. </li><li> (Adım 3.2 olarak ayarlanmış) bu kılıf sıvısı akış hızı 40 um kontrol parçacıkları kullanarak sıralama oranı test, doğru sıralamayı sağlayacak sağlamak için: </li><li> Kılıf vanasını kapatın. Hazneye kontrol parçacık çözeltisi yaklaşık 25 ml ilave edilir. </li><li> Kılıf vana ve numune vana açın. </li><li> Bilgisayar yazılımı, &quot;kontrol parçacık modu&quot; gidin ve Al&#39;ı tıklatın. </li><li> Parçacık akış hızı genişliğine yakın bir TOF (uçuş süresi) ile, ~ 5-8 parçacıklar / sn olduğundan emin olunParçacığın, 40 um. Böyle bir TOF tek bir 40 um amacı, bir zamanda geçerek olduğunu gösterir. Değerleri bu aralığın dışında ise, buna göre (yukarıdaki yönergelerle) ayarlarını yapın. </li></ol> 4. Noncolonized Worms vs kolonileştirildiğini, sıralama <ol><li> M9 kullanarak, 15 ml&#39;lik konik tüplere solucanlar yıkayın. Yetişkin solucanlar yerçekimiyle tüpün dibine çöker ve süpernatant kaldırmak için izin verin. Taze M9 ile tüp doldurun. Bu işlemi 3-4x tekrarlayın. Bu, larva büyük bir kısmı hem de aşırı bakteri yok eder ve yaklaşık 10 dakika sürer. </li><li> Bir yavaşça karıştırma küçük karıştırma çubuğu ile wormsorter rezervuar solucanlar ekleyin. </li><li> 200 600 Yeşil PMT ayarlama ve Kırmızı ve Sarı PMT tarafından ilk sinyal kazancı ayarlayın. </li><li> Ayarlarını yapmak için veri elde başlayın. </li><li> Saniyede 25-30 olaylar (solucanlar) bir çeşit oranı hedefliyoruz. Sayısı hızı çok yüksek ise, buna göre M9 ile rezervuar içinde solucanlar seyreltin. Oranı iseo düşük, M9 küçük bir hacimde daha solucanlar ekleyin. </li><li> Deney noncolonized solucanlar vs kolonize çok sıkı ayrılığı gerektiriyorsa, &quot;tesadüf kontrol&quot; için &quot;saf&quot; olarak ayarlayın. Bu olay iki nesne birbirine çok yakın olması veya aynı açılan, makine yerine toplamaktan daha düşüş reddeder sağlar. </li><li> , Ilgi nüfusu sıralamak boyutunu (erişkin solucanlar elde etmek için) ve (noncolonized düşük kolonize için yani yüksek) floresan yoğunluğunu gösteren eksenleri etrafında kapıları çizmek için. Floresan yolluk için değerler deneysel olarak tespit edilmelidir. </li><li> Solucanlar başlamak üzere sıralama sıralama vanasını açın solucanları toplamak için bir çok plaka veya dağıtıcı altında bir Petri kabı yerleştirin. </li><li> Kriteri nüfus ilgi nüfusu mikroskop altında doğrulamak için hayvanların küçük bir nüfusa toplayın. Eğer değilse, buna göre yolluk parametrelerini ayarlamak ve örnek toplama ile devam ediyor. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Biz tarif yöntemi solucanı ve çevre arasındaki etkileşimleri takip etmek, solucanın dışında kişilerin floresan etiketleme yararlanır. Sunduğumuz durumda, ayırma, bir patojenin etiketleme dayanmaktadır ve hiçbir (veya ışık) yük olan ağır patojen yük ayrı solucanlar için istihdam edilmiştir. Sonraki gen ekspresyon analizleri onlar patojen yük bağımsız olduğunu düşündüren iki grup arasında bağışıklık yanıtlarında fark bulunamadı. Yanıtı başlatan sinyal fiziksel olarak, bu sinya...

Otomatik solucan sıralama, bir tüp içinde düzleştirilmiş geçtikçe (genellikle raportör proteinlerin transgenik ekspresyonu tarafından sağlanan) bir solucan bir flüoresan sinyal ölçümü ile çalışan yönlendirme ya da bir toplama tüpüne / oyuk ya da bir atık kabına göre sağlayan, çok FACS gibidir araştırmacı 1 tarafından belirlenen parametreleri yolluk için. Wormsorter birçok yönden araştırma kolaylaştırabilir; analitik bir araç olarak kullanan bir örnek neredeyse 1.000 genler 2 için yükseltici faaliyet zamanmekansal modellerini takip eden bir çalışmadır. Bununla birlikte, wormsorter en önemli kullanım hedef gen ekspresyon düzeyleri veya solucanın 3-5 ekseni boyunca floresan protein lokalizasyonu sonra, genetik ekranlar yer almaktadır. Burada, bir floresan etiketli patojenin solucanın kolonizasyonu Aşağıdaki, wormsorter için yeni bir uygulama açıklanmaktadır. Bu bir araç olarak biz Pathog arasındaki ilişkiye odaklanmıştırkolonizasyon / yük ve immün yanıt tr, solucan bağışıklık tepkilerinin başlamasından sorumlu mekanizmalarına yeni anlayışlar kazanmak için. Bugüne kadar incelenen hemen hemen tüm organizmalarda, mikrobiyal patojenlere karşı doğal bağışıklık tepkilerinin başlaması patojen ilişkili moleküler desenleri (PAMPs) tanınması bağlıdır, ve / veya tehlike / zarar-ilişkili moleküler desenleri (sönümlendirir) 6,7. Mikrobiyal hücre duvarı olarak, flagellum&#39;un, ya da lipid çift katlı 6 bileşenlerini içerir ilk korunmuş mikrobiyal yapılar, ikinci, hem de serbest molekülleri (örneğin ATP 8), değiştirilmiş proteinler veya değiştirilmiş hücresel süreçlerin 9,10 diğer işaretleri içerir. Sinyallerin iki tip spesifik üzerine bir desen molekülün bağlanma koruyucu bir tepki yol açan olaylar zincirini aktive örüntü tanıma reseptörleri (PRR) olarak belirlenen proteinler tarafından tanınır. C&#39;yi elegans bir TRACTA olarak son derece yararlı olmuşturble modeli konak-patojen etkileşimleri çeşitli yönlerini incelemek, ama iyi anlaşılmış değildir bir şey bağışıklık tepkileri solucan başlatılan nasıl da. Diğer organizmalarda eden örüntü tanıma reseptöleri (PRR) hiç ortolog olan farazi reseptör hiçbiri PAMPs bağlandığı gösterilmiştir ve diğer organizmalarda bağışıklık yanıtları için önemli olan PRRS en ortologlar birçok solucan patojen tepkilerinin şaşırtıcı bir şekilde sınırlı bir katkı göstermektedir ve direnç. Örneğin Gram pozitif patojenler direnç göstermek için gerekli olan Drosophila Toll reseptör, C temsil edilir belirten verilerle kombine ancak test edilen diğer Gram-negatif ya da-pozitif patojenlerin 11,12 dan, Gram negatif bir patojen Salmonella Typhimurium 11 korunmasına katkıda bulunur. Bu gözlemler, bir tek homolog, tol-1, ile elegans bu bağışıklık tepkileri Hücresel protein için h bozarak neden olabilirled gibi bazı önermek için o C. elegans öncelikle sönümlendirir 9,13,14 algılar. Bununla birlikte, bağışıklık karşılıklarının uyarılması için ölü patojenlerin yeteneğini tarif raporları PAMP bağlayıcı C&#39;de patojen tanınmasında önemli bir role sahip olabileceğini göstermektedir elegans 15,16. Yaş-senkronize genetik özdeş C bağışıklık yanıtları odaklanarak Önceki iş elegans nüfus, bakteri Gram-negatif patojen Pseudomonas aeruginosa tarafından intestinal kolonizasyonu büyük bireysel değişkenlik gösterdi. Ancak, transkripsiyonel profilleme çalışmaları bir varlık 17,18 olarak bu değişken-kolonize popülasyonları tedavi. Bu değişkenlik yararlanan, GFP-ifade P. maruz Caenorhabditis elegans farklı şekilde kolonize popülasyonları ayırmak için otomatik bir wormsorter odaklanan bir protokol geliştirdi aeruginosa. Farklı sömürgeleştirilen toplumlarda gen ifadesini li incelenmesipatojen yük (ve ilişkili hasar) ve immün yanıtları ve C patojen tanıma konusunda sağlanan yeni anlayışlar arasındaki ilişkinin değerlendirilmesi litated elegans 19. Biz herhangi bir floresan etiketli patojen ile enfekte solucanlar sıralamak için uygulanan olabilir protokolü, tanımlayacağız. Potansiyel kullanıcılar için dizildi gereken kurtların sayısı sayılır kullanımında müteakip analizler ve protokoller niteliğine bağlı olduğu unutulmamalıdır. Örneğin, mikro-dizi gen ekspresyon analizi durumunda,&gt; 1000 solucanlar standart protokoller kullanıldığında, yeterli RNA elde etmek için gerekli, ancak amplifikasyon kullanılması halinde ~ 100 solucanlar yeterli olacaktır, malzemenin hızlı bir koleksiyon sağlar ve bu nedenle stres en aza indirmek solucanlar.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">M9 Buffer</td>
			<td style="width:139px;">Prepared in house</td>
			<td style="width:527px;">Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin&#160;</td>
			<td>Sigma</td>
			<td>R3501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg prep solution&#160;</td>
			<td>Prepared in house</td>
			<td>50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGM plates&#160;</td>
			<td>Prepared in house</td>
			<td>Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKP plates</td>
			<td>Prepared in house</td>
			<td>Recipe same as NGM only 0.35% peptone instead of 0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control test particles&#160;</td>
			<td>Union Biometrica&#160;</td>
			<td>310-5071-001</td>
		</tr>
	</tbody>
</table>

automated separation of c. elegans variably colonized by a bacterial pathogen

Wormsorter Caenorhabditis elegans çalışmalarda kullanılan bir FACS makinesine benzer bir alettir, tipik olarak bir flüoresan raportör ekspresyonu göre solucanlar sıralamak. Burada, bir GFP-ifade patojenin kolonizasyon derecelerine göre solucanlar sıralama için bu aletin alternatif bir kullanım gelmek. Bu yeni kullanım ki, bu solucan bağırsak ve bağışıklık sisteminin indüksiyonunun kolonizasyon arasındaki ilişkiyi ele bırakıldı. Ederken C. farklı patojenlere elegans bağışıklık yanıtları, bunları başlatır ne hala bilinmiyor, dokümante edilmiştir. (Birbirini dışlayan değil) iki ana olasılık patojen ilişkili moleküler desen tanıma ve enfeksiyona neden hasar tespiti vardır. Iki olasılık arasında ayrım için, patojene maruz sebep olduğu hasar ayrılmış olmalıdır. Gram-n tarafından yaygın-kolonize edildi solucanlar wormsorter etkin ayrılıkkolonize benzer maruz bırakıldı solucanlardan muhtemel patojen yükün yol açtığı hasar, ancak, ya da marjinal, ile egative patojen Pseudomonas aeruginosa,. Bu farklı popülasyonlar patojen yük ve transkripsiyonel bağışıklık tepkilerinin indüklenmesinde arasındaki ilişkiyi değerlendirmek için kullanıldı. Sonuçlar, iki patojen tanıma olasılığını destek, ayrışmış olduğunu göstermektedir.

Yaştaki zaman, genetik olarak özdeş C. elegans P. maruz aeruginosa, geniş bir dağıtım kolonizasyon (Şekil 1A) seviyeleri gözlenir. Kolonize solucanlardan noncolonized verimli ayırma (Şekil 1 B) elde edilebilir, burada açıklanan protokolü yardımıyla. Noncolonized solucanlar aksine, sömürgeleştirilmiş solucanlar gibi uyuşukluk ve azaltılmış dışkılama 19 olarak hasar belirtileri göstermektedir. İkincisi solucanlar P. taraf?...

Watch this Scientific Journal Video about Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen at JoVE.com

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

Wormsorter floresan gazetecilere ifadeye göre solucanlar sıralayarak Caenorhabditis elegans genetik ekranları kolaylaştırır. Burada, yeni bir kullanımını tanımlamak: GFP-ifade patojen tarafından kolonizasyon göre sıralama, ve biz bağışıklık tepkilerini başlatarak patojen tanıma anlaşılamamıştır rolünü incelemek için istihdam.

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

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

automated-separation-c-elegans-variably-colonized-bacterial

Research

JoVE Journal

Immunology and Infection

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

8.3K Görüntüleme.  University of California, Berkeley.  Wormsorter floresan gazetecilere ifadeye göre solucanlar sıralayarak Caenorhabditis elegans genetik ekranları kolaylaştırır. Burada, yeni bir kullanımını tanımlamak: GFP-ifade patojen tarafından kolonizasyon göre sıralama, ve biz bağışıklık tepkilerini başlatarak patojen tanıma anlaşılamamıştır rolünü incelemek için istihdam. 

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

Invasion of Human Cells by a Bacterial Pathogen

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

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

Bakteriyel Patojen İnsan Hücreleri Invasion

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

İmmünoloji ve Enfeksiyon

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

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

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

Kan örneklerinden Bakteriyel Patojen Algılama ve Antimikrobiyal Direnç Testi için bir günlük iş akışı şeması

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

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Canlı Hücre Floresan Mikroskopi kullanma Bakteriyel Toksin İsteyerek Yanıtların Görselleştirme

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

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

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

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

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

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

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

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 ...

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

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

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

Bir Maya İki hibrid Ekran kullanarak Olmayan ekilebilir Bitki Patojen bir Bakteriyel Efektör Fonksiyonu aydınlatmak

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

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Zenginleştirme bakteriyel lipoproteinler ve kütle spektrometresi tarafından yapısal kararlılık için N-terminal Lipopeptides hazırlanması

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

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 ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Otomatik davranış analizi büyük C. elegans nüfus kullanarak bir geniş alan bakış açısı izleme platformu

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...