This work has been supported by Elizabeth E. Kennedy Research Award.

Yapımcı Genleme 1. Hazırlık <ol><li> Şerit, bir pnömokok kökenli soyu -80 ° C dondurucu stoktan% 5 koyun kanı triptik soya agar plakası üzerine bakteriyosin üretimi için test edilecek. Not: inhibisyonu için test edilecek olan soyun ilk büyüme kan plakaların kullanımı büyük ölçüde tahlilin tekrarlanabilirliğini arttırır. Büyüme koşulları, belirli bir organizmanın değiştirilmesi gerekebilir, ancak ek olarak, bakteriyosin üreten diğer non-pnömokok suşları kullanılabilir. Biz başarıyla yayma deneyi Streptococcus mitis ve Lactococcus lactis kullandık. </li><li> % 5 CO2 ile 37 ° C &#39;de gece boyunca plaka (O / N) inkübe edin. </li><li> Üretici soyunun inkübasyondan sonra, TSA 25 ml ihtiva eden plakalar üzerine katalaz 3000 ila 4000 birimleri pipetleme triptik soya agar (TSA) plaka hazırlanması yapılmış ve steril cam boncuklar kullanılarak katalaz çözeltisi yayıldı. Yaklaşık 10 dakika altında plaka kurumasını bekleyinbiyolojik güvenlik kabini. </li><li> Bir pipet üzerine bakteri görünür bir miktar toplayın. Kurutulmuş TSA plakasına pipet bıçakla. NOT: Bir üretici suş daha fazlası tek bir TSA plakaya bıçakladı edilebilir. Oluşan haleler örtüşmeyen böylece bıçaklandığı uzayda önemlidir. <ol><li> Mümkünse, bir pozitif kontrol ve bir negatif kontrol olarak, kaplamayla kullanılacak soyu gibi bir üretici bakteriyosin bilinen maddeleri içerir. NOT: Pozitif ve negatif kontroller daha önce yayınlanmış ve istek 10 üzerine mevcuttur. </li></ol></li><li> 6 saat boyunca% 5 CO2 ile 37 ° C &#39;de birden fazla saplamanız içeren TSA plaka inkübe edin. </li></ol> Bindirme Genleme 2. Yapımı Gliserol Stokları <ol><li> Yayma deneyi bir gün önce (gliserol ilave edildikten sonra orta üslü faza kadar yetiştirilir ve daha sonra -80 ° C&#39;de saklandı kültürler), gliserol stokları hazırlayın. Pnömokok str 5-10 koloni aşılamakain% 0.5 maya ekstraktı (THY) Todd-Hewitt ortamda hassasiyet veya feromon salgılama açısından test edilmelidir. NOT: BlpC uyarılabilir gazetecilere İnşaatı daha önce 2 tarif edilmiştir ve suşları istek üzerine mevcuttur. </li><li> Çalkalamadan 37 ° C su banyosu içinde sıkıca kapalı kapak THY 5 ml cam tüpler içinde 5-10 koloniler inkübe edin. </li><li> Bunlar, bir 0.5, 0.3 OD 620 gelene kadar, 37 ° C kültürleri inkübe edin. NOT: Bu, kullanılan gerginlik bağlı olarak yaklaşık 4-5 saat sürer. <ol><li> Yavaşça tüpü OD ölçme önce bir kaç kez ters çevirin. </li></ol></li><li> Deneme aynı gün yapılamaz veya zorlanma gliserol stoğu yapmak ve daha sonraki bir tarihte deney devam, gelecekte birden çok kez kullanılabilir edilecektir. Gliserol stokları için,% 20 bir son konsantrasyona kadar kültürler gliserol ekleyin. -80 ° C&#39;de 1.5 mL mikrosantrifüj tüpleri ve mağaza içine kısım kültürleri. NOT: Örnekler s olabilir-80 ° C&#39;de ay boyunca yırtılmaktadır. Gliserol stokları yapmıyor ise, yayma deneyi doğrudan örnekleri kullanabilirsiniz. </li></ol> 3. Kaplama Testi <ol><li> Hemen önce, uygulamayı bindirme bindirme bileşenleri karıştırmak için. 15 ml konik bir tüp içinde 5 ml THY, katalaz 3,000-4,000 U ve ya hemen aynı günde ya da oda sıcaklığında eritildi, kültür, bir gliserol stokları yetiştirildi katlamalı soy çorba kültürü 200 ul ilave edin. Raportör suşu kullanılarak, kaplama karışımına, X-gal ve 50 ul (40 mg / ml) ilave edilmektedir. Plaka başına bir konik bir tüp içinde kullanın. </li><li> Önce erimiş TSA ilave edilmeden önce oda sıcaklığında katlamalı karışımı dengelenmesi. </li><li> Inkübatör bıçaklanarak zorlanma ile TSA plakasını sökün. Mikrodalga katı TSA eritin ve kullanmaya hazır olana kadar 55 ° C su banyosunda tutmak. % 1.5 agar erimiş TSA 3 ml ekleyin karışımı katlamalı 55 ° C&#39;ye kadar soğutuldu. <ol><li> Pipetlemeden önce hemen bindirme karışım hazırlayın, karışım çok katılaşmaya başlayacaktırhızlı. NOT: bindirme kaplama önce katılaşmaya başlar, sonuçlar uninterpretable olacaktır. </li></ol></li><li> Pipetleme ve 5 ml&#39;lik pipet karışımın içine kabarcıkları tanıtmak için dikkatli olmak ile birkaç kez aşağı bindirme karışımı karıştırın. Yavaşça bıçaklayarak TSA plakasına doğrudan bindirme karışımı pipetle. Agar sertleşmesine izin birkaç dakika için masa üstü plaka tutun. <ol><li> Bindirme karışımı dağıtmak için plakasını döndürmek etmeyin, bu bindirmenin içine bıçakladı kültüründen büyüme çeker. </li></ol></li><li> Dikkatle% 5 CO 2 O / N (yaklaşık 16-18 saat) içeren 37 ° C inkübatör içine kalıbı ile TSA plakasını yerleştirin. </li><li> Gecede büyümeden sonra plakaları gözlemleyin. Plakalar inhibisyon veya feromon sinyal oluştu alanlar haricinde bindirme suşu büyüme opak görünmelidir. Bunlar sırasıyla, bıçaklanarak zorlanma çevresinde net ya da mavi bölgeler olarak görünecektir. NOT: Her ne kadar kuvmavi veya açıklığın çapının enlemeleri, niceliksel değerlendirilmesi için belirlenebilir çapı farklılıklar da plakasına býçaklanmýţ bakteri miktarına bağlı olarak değişebilir. Bindirme tahlil herhangi bir nicel değerlendirme dikkatle yorumlanmalıdır gereken, kalitatif bir testtir. Negatif kontrol ile karşılaştırıldığında, herhangi bir görünür temizleme aktivite olarak kabul edilir. </li></ol>

<ol>
	<li>Dawid, S., Roche, A. M., Weiser, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blp+bacteriocins+of+Streptococcus+pneumoniae+mediate+intraspecies+competition+both+in+vitro+and+in+vivo.">The blp bacteriocins of Streptococcus pneumoniae mediate intraspecies competition both in vitro and in vivo.</a> Infect Immun. 75 (1), 443-451 (2007).</li><li>Sawa, N., 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=Isolation+and+characterization+of+enterocin+W,+a+novel+two-peptide+lantibiotic+produced+by+Enterococcus+faecalis+NKR-4-1.">Isolation and characterization of enterocin W, a novel two-peptide lantibiotic produced by Enterococcus faecalis NKR-4-1.</a> Appl Environ Microbiol. 78 (3), 900-903 (2012).</li><li>Kochan, T. J., Dawid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HtrA+protease+of+Streptococcus+pneumoniae+controls+density-dependent+stimulation+of+the+bacteriocin+blp+locus+via+disruption+of+pheromone+secretion.">The HtrA protease of Streptococcus pneumoniae controls density-dependent stimulation of the bacteriocin blp locus via disruption of pheromone secretion.</a> J Bacteriol. 195 (7), 1561-1572 (2013).</li><li>Lux, T., Nuhn, M., Hakenbeck, R., Reichmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+bacteriocins+and+activity+spectrum+in+Streptococcus+pneumoniae.">Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae.</a> J Bacteriol. 189 (21), 7741-7751 (2007).</li><li>Reichmann, P., Hakenbeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+a+peptide-inducible+two-component+system+of+Streptococcus+pneumoniae.">Allelic variation in a peptide-inducible two-component system of Streptococcus pneumoniae.</a> FEMS Microbiol Lett. 190 (2), 231-236 (2000).</li><li>Saizieu, 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=Microarray-based+identification+of+a+novel+Streptococcus+pneumoniae+regulon+controlled+by+an+autoinduced+peptide.">Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide.</a> J Bacteriol. 182 (17), 4696-4703 (2000).</li><li>Barbour, A., Philip, K., Muniandy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+production,+purification,+characterization+and+mechanism+of+action+of+salivaricin+9+lantibiotic+produced+by+Streptococcus+salivarius+NU10.">Enhanced production, purification, characterization and mechanism of action of salivaricin 9 lantibiotic produced by Streptococcus salivarius NU10.</a> PLoS One. 8 (10), 77751 (2013).</li><li>Wladyka, B., 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=Isolation,+biochemical+characterization,+and+cloning+of+a+bacteriocin+from+the+poultry-associated+Staphylococcus+aureus+strain+CH-91.">Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91.</a> Appl Microbiol Biotechnol. 97 (16), 7229-7239 (2013).</li><li>Shea, E. F., 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=Bactofencin+a,+a+new+type+of+cationic+bacteriocin+with+unusual+immunity.">Bactofencin a, a new type of cationic bacteriocin with unusual immunity.</a> 4 (6), 00498-00413 (2013).</li><li>Son, M. 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=Conserved+mutations+in+the+pneumococcal+bacteriocin+transporter+gene,+blpA,+result+in+a+complex+population+consisting+of+producers+and+cheaters.">Conserved mutations in the pneumococcal bacteriocin transporter gene, blpA, result in a complex population consisting of producers and cheaters.</a> MBio. 2 (5), (2011).</li><li>Kochan, T. J., Dawid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HtrA+protease+of+Streptococcus+pneumoniae+controls+density-dependent+stimulation+of+the+bacteriocin+blp+locus+via+disruption+of+pheromone+secretion.">The HtrA protease of Streptococcus pneumoniae controls density-dependent stimulation of the bacteriocin blp locus via disruption of pheromone secretion.</a> J Bacteriol. 195, 1561-1572 (2013).</li><li>Widdick, D. 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=Cloning+and+engineering+of+the+cinnamycin+biosynthetic+gene+cluster+from+Streptomyces+cinnamoneus+cinnamoneus+DSM+40005.">Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005.</a> Proc Natl Acad Sci U S A. 100 (7), 4316-4321 (2003).</li><li>Begley, M., Cotter, P. D., Hill, C., Ross, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+two-peptide+lantibiotic,+lichenicidin,+following+rational+genome+mining+for+LanM+proteins.">Identification of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining for LanM proteins.</a> Appl Environ Microbiol. 75 (17), 5451-5460 (2009).</li></ol>

The authors have nothing to disclose.

Bu bindirme tahlil bakteriyosin üreticileri için faaliyet alanını belirlemek ve pnömokok suşları arasında oluşabilecek rekabet etkileşimleri göstermek için hızlı bir yoldur. Bindirme deney, tek bir plaka üzerinde birden fazla bakteriyosin üretimi suşları taranması için kullanmak üzere adapte edilebilir. Başarıyla, 96 oyuklu bir plakanın yarısını SBA plakalarının aşılanması için bir 48 pimli çoğaltıcı kullanarak, bu deneyde büyük gerilme koleksiyon gösterildi. Gece boyunca inkübas...

Streptococcus pneumoniae, nazofarenks polimikrobiyaldir eden ortak bir şekilde koloni, bakteriyel olarak üretilmiş antimikrobiyal peptitler (bakteriosinler) üretimi aracılığıyla diğer pneumococci ve yakından ilişkili türleri ile rekabet edebilmektedir. Bugüne kadar incelenen her tam sıralandı pnömokok suşu b acteriocin- l ike p peptit odağı blp bir sürümünü içerir. Pnömokok bakteriyosin üretimi, hem in vitro ve in vivo sonuç 1-4 rekabet önemli olduğu gösterilmiştir. Rekabetçi dinamikleri, spesifik peptit feromon tepki olarak aynı kökenli bağışıklık proteinlerle birlikte çeşitli bakteriyosinlerin ekspresyonu ile etkilenmektedir. Blp lokusunun indüksiyonu bir peptit feromon, BlpC, sensör kinaz BlpH bağlanan olan bir çekirdek algılama sistemi ile kontrol ve sonuçlanan bir sinyal akışını başlatır edilirblp lokus 5,6 transkripsiyonu. BlpC dört alelik varyasyonlar vardır ki, kendi doğasındaki BlpH 6 her bağlanmaktadır. Hücre, kendi bakteriyosin etkilerini hayatta kalabilmesi için ortak kökenli bağışıklık proteinleri kodlayan genler bakteriyosin genlerin her biri ile aynı operonunun transkribe edilmektedir. Soy korunması için gerekli olan özel gen bağışıklık yetersiz kalması durumunda, bir rakip soyu dışsal feromon tepki olarak blp lokusu yukarı doğru düzenleme mümkün olup olmadığını ya Öldürme oluşur. Taşıyıcı kompleksi, BlpAB salgısı ile işleme peptit feromon ve bakteriyosin peptidler 2,4 için gereklidir. Son zamanlarda, bu pnömokok suşları önemli bir bölümünün de bir durumdur bu feromonu ve bakteriyosin 1,2 salgılamasına hale blpA geninde bir mutasyon korunmuş olması, yani &quot;dolandırıcı&quot; olduğu gösterilmiştir. Bu suşlar kişnemek tarafından salgılanan eksojen BlpC 2 yanıt edebiliyoruzbağışıklık proteinlerin üretimi için izin sıkıcı suşları. Süpernatanlarından bakteriyosinlerin, ham preparatlar saflık elde etmek için biyokimyasal yöntemler kullanılarak adımları Üretici türlerde hazırlanabilir, ancak, bu yaklaşım, büyük izolat koleksiyonlarının taranmasında yararlı değildir ve bakteriyosin ekspresyon seviyesinin suyu büyüyen kültürlerden düşükse 2,7- 9. Bindirme deney in vitro olarak mevcut olabilir suşları arasındaki rekabet dinamiğini incelemek için hızlı bir yöntem olarak kullanılmaktadır. Bunu yapmak için, bir pnömokok soyu önceden aşılanmış agar plaka üzerine ve blp lokusunun uyarılması için yeterli lokal konsantrasyonları elde etmek için bakteriyel büyümeye izin verilir. Ikinci bir soy daha sonra hassasiyeti test etmek için önceden aşılanmış soyu üzerine uygulandığında, erimiş bir yumuşak agar çözeltisi içine aşılanır. (Inhibe edici aktivitesi bağımsız olarak) blp lokusunun değerlendirmek aktivitesi için, suşlar th bir dizi ile kaplanabilirDaha önce tarif BlpC haberci soylarını ree. Bu suşlar, pnömokok tarafından salgılanan en yaygın üç BlpC feromonlar yanıt üç farklı blpH allellerinden biri içeren yapılmıştır. Haberci soylarının bir BlpH bağımlı promotörün kontrolü altındadır ve kendini uyarma 10,11 engeller blpC geninde bir delesyon yerine entegre bir lacZ genini sahiptir.

<table><tbody><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>Fisher</td><td>B21261X</td><td>For growth from freezer cultures</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C100-500mg</td><td>4,741 Units per plate</td></tr><tr><td>Todd Hewitt Broth + Yeast Extract (THY)</td><td>Fisher</td><td>DF0492-17-6&amp;nbsp;</td><td>30 g of THB, 5 g yeast extract, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>Trypticase Soy Broth + Agar plates (TSA)</td><td>Fisher</td><td>B11768&amp;nbsp;</td><td>30 g of TS, 15 g agar, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside (X-gal)</td><td>Sigma</td><td>B4252-50MG</td><td>Dissolve in dimethylformamide</td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Spectrophometer</td><td></td><td></td><td>Measures the OD&lt;sub&gt;620&lt;/sub&gt; of cultures</td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO2</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td></td><td></td><td></td></tr></tbody></table>

using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Yayma deneyi iki olası sonuç vardır. Gece boyunca inkübasyondan sonra bıçakladı suşunun büyümesini görmek mümkün olmalıdır. Şekil 1A, üst tabaka gerilme Bakteriyosinler üretilen duyarlıdır. Atılımının bir bölge bıçakladı gerginlik çevredeki görülebilir. Buna ek olarak, kaplama karışımı içine bıçakladı soyun dönen mümkündür ve Şekil 1A &#39;da görülebilir. Şekil 1B, üst tabaka gerilme bıçak çevreleyen bir halo olmaması ...

Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Kaplama Testi kullanarak niteliksel Pnömokok Bakteriyosinlerin Bakteriyel Üretim ve Hassasiyet Tedbir

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

using-overlay-assay-to-qualitatively-measure-bacterial-production

Research

JoVE Journal

Immunology and Infection

13.4K Görüntüleme.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. 

Video: Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

Bakterilerde protein RNA bağlayıcı ölçümü için bir tahlil

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Genetik

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

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

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

T6SS aracılı Bakteriyel Rekabet Monitör A Görsel Assay

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

İmmünoloji ve Enfeksiyon

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Vakuolar Kopma Tek Hücre ölçümleri Hücre içi patojenlerin neden

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

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

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

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

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

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

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

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Bir kullanma Pnömokok Kolonizasyon üzerinde Probiyotiklerin Etkileri incelenmesi<em&gt; In Vitro</em&gt; Yapışma Deneyi

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bakteri-konak Etkileşimleri karakterize etmek Biyomimetik Malzemeler

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

Yumuşak agar üst tabaka tekniği kullanımı bakteriyel olarak üretilmiş önleyici bileşiklerin taranması için

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Bir yüksek-den geçerek Shigella-belirli bakterisidal tahlil

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Ertelenmiş Büyüme İnhibisyon Tahlili Rekabet Bakteri kaynaklı Antimikrobiyal Etkisi belirlememize

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

Kaplama Testi kullanarak niteliksel Pnömokok Bakteriyosinlerin Bakteriyel Üretim ve Hassasiyet Tedbir

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