Yazarlar balık bakımı için Şekil 4, Ulrike Nehrdich ve Davy de Witt görüntüleri ve yararlı tartışmalar için diğer laboratuvar üyeleri için, Chao Cui teşekkür Kat de Kort, Esther Stoop ve Ralph Carvalho istiyorum. Bu çalışma Ekonomik İlişkiler Hollanda Bakanlığı Smart Mix Programı ve Eğitim, Kültür ve Bilim, Avrupa Komisyonu 7. Çerçeve projesi ZF-SAĞLIK (SAĞLIK-F4-2010-242048), Avrupa Marie-Curie Bakanlığı tarafından desteklenmektedir İlk Eğitim Ağı FishForPharma (PITN-GA-2011-289209) ve Avustralya NHMRC (637.394) tarafından. Avustralya Rejeneratif Tıp Enstitüsü Victoria ve Avustralya Hükümeti Eyalet Hükümeti hibe ile desteklenmektedir.

<ol>
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Yazarlar ifşa hiçbir şey yok.

Bu yazıda tarif enfeksiyonu yöntemler genellikle bilgisayar doğuştan gelen bağışıklık genlerinin ya da bakteriyel virülans genler 1 işlevini incelemek için kullanılır. Intravenöz mikro-enjeksiyon yöntemleri (protokolleri 5 ve 6.1) bu tür çalışmalar için en sık kullanılan. Posterior kan adada kaudal ven 1-günlük embriyoların intravenöz enjeksiyon için en uygun yerdir. Kaudal ven sonraki aşamalarında nüfuz için daha zor hale geldikçe, biz 2-3 dpf&#39;e az embriyo için intravenöz enjeksiyon bölgesi olarak Cuvier ve Kanal tercih. Her durumda kob sayımı için kaplama enjeksiyon inokulum tarafından kontrol edilmelidir bakteri tutarlı numaraları ile embriyo enjekte etmek önemlidir. Aşağıdaki hususların enjeksiyonlar tekrarlanabilir ulaşmak için dikkate alınmalıdır. İlk olarak, yüksek kaliteli cam microcapillary enjeksiyon iğneleri olması şarttır. Iğne ucu kadar büyükse, enjeksiyon meydana kanama için yeterince büyük bir deliği oluşturur veenjekte edilen bakterilerin kan dolaşımını dışarı akacaktır. İkinci olarak, bakteri kan dolaşımı içine doğrudan enjekte edilmesi gerekir. Iğne ucu yolk sac uzatma enjeksiyon sıvısı yayılır damar içine veya eğer değilse, embriyo deneyden atılmalıdır. Üçüncü olarak, M. enjekte etmek için bir taşıyıcı olarak PVP40 kullanılarak marinum cam iğne batıyor gelen bakteri önlemede yardımcı olacaktır. PVP40 enjeksiyon süresi boyunca daha tekrarlanabilir inokulum sonuçlanan, süspansiyon homojen olmasını sağlar. PVP40 da S. için kullanılabilir büyük boyut ve bakterilerin parlak floresan deneyler sırasında enjeksiyon inokulum üzerinde görsel kontrolü sağlar çünkü typhimurium enjeksiyonları, ama burada daha az önemlidir. Enjeksiyonlar uygulama için biz fenol kırmızı boya (% 1 v / v) veya 1 mikron farklı renkler (Invitrogen) mevcut floresan kürelerin kullanılması tavsiye edilir. Teknik hakim edildikten sonra, bu yaklaşık olarak 30 minut alıres agaroz plakalar üzerinde embriyo hizalayarak ve bakteriyel enjeksiyon ses ayarlamak için gereken zaman dahil, 50-100 embriyo enjekte etmek. Kan ada (protokol 5) veya Cuvier (protokol 6.1) kanal içine intravenöz enjeksiyon en sık kullanılan edilirken, bu video makalede açıklanan enfeksiyon alternatif güzergahlar (protokolleri 6.2, 6.3 makrofaj ve nötrofil kemotaksisini çalışmak için yararlı olan ve 6.4), zayıflatılmış bakteri suşları (protokol 6.5) büyüme incelemek ve yüksek hacimli uygulamalar (protokol 6.6) 4-8 zebrabalıkları enfeksiyon modelinde uyarlamak. Açıklandığı mikro-enjeksiyon yöntemleri de virüsler 18, 19, mantar sporları 14, 20 veya protozoon paraziti (Maria Forlenza, kişisel iletişim) enjeksiyonu için uygulanabilir. Bu video makalesinde tarif enjeksiyon prosedürler yararlı bir ek zebr bakterilerin subkutan enjeksiyon için kısa bir süre önce anlatılan prosedürüafish embriyoların 21. Bu prosedür, yüzeyler üzerinde etkili bir doku engulf bakteri olduğu tespit edilmiştir, ancak nötrofiller, bakterilerin fagositozunu incelemek için uygulanmış, makrofajlar aksine, kan içinde ya da sıvı dolu bir kavite içine enjeksiyon gövdesi üzerine bakteriler fagosite neredeyse olamamıştır. Subkutan enjeksiyonlar embriyolar için bu video makalede açıklanan kuyruk kas enjeksiyon için benzer yerleştirilmiş, ancak iğne bir hücre gruplarının üzerinde bakteri enjekte derinin hemen altında eklenir. Bu mikro-enjeksiyon esas enfeksiyon yapay bir yol olduğunu dikkate almak önemlidir. Ancak, erken embriyo bakteriyel patojenler dış maruz karşı oldukça dirençlidir. Aslında, embriyolar bakteriyel süspansiyon içinde kalmış olan batırma testleri, bizim elimizde 22 tekrarlanabilir değildir. Bazı embriyolar daldırma üzerine bulaşabilir ederken, ölüm oranları ve ekspres büyük bir değişim gözlemledikBu tür testler bireysel embriyoları arasında inflamatuvar belirteç genlerin iyon. Bu makalede açıklanan mikro-enjeksiyon yöntemleri tekrarlanabilir enfeksiyonları ulaşmak ve host doğuştan gelen bağışıklık hücreleri ile bakteriyel etkileşimleri çalışmak için özellikle faydalıdır. Bireysel embriyolarında bakteriyel enfeksiyon ölçmek için etkin bir yaklaşım ısmarlama, özel piksel ölçümü yazılımı 17, 23 ile enfekte embriyoların floresan görüntüleri analiz etmektir. Bu yaklaşım enfekte embriyoların sonra kaplama kob sayımı sonuçları ile iyi bir korelasyon olduğu gösterilmiştir. Benzer şekilde, embriyo başına lökosit sayısında göreceli değişiklikler transgenik lökosit fluorofor muhabiri hatlarında dijital görüntü analizi ile ölçülebilir edilmiştir; bu yaklaşım enfeksiyon 24 konak leukopoietic yanıt miktarının için geçerli olacaktır. Konak doğuştan gelen bağışıklık genlerin işlevine tarif enfeksiyonu modelleri birleştirerek in vivo olarak etkili bir şekilde araştırılmıştır edilebilirmorfolino devirme ile. Morpholinos belirli bir hedef RNA ve bu Journal 10, 25 in diğer video makaleler gösterildiği gibi, 1-hücre aşaması zebrafish embriyosu içine enjekte edildiğinde gen ürünleri ifadesi azaltmak sentetik antisens oligonükleotidler şunlardır. Morfolino Knockdown çalışmalarda, karşılaştırılmak üzere tüm embriyo gruplar bakteriyel enjeksiyonları öncesinde doğru sahnelenir büyük önem taşımaktadır. Embriyo zaman içinde giderek yetkin hale gelişmekte olan bir bağışıklık sistemine sahip, çünkü bu özellikle önemlidir. Zebra balığı embriyolarının en önemli doğal immün alt, makrofaj ve nötrofiller, ayırt etmek için şu anda birçok transgenik muhabiri hatları vardır. Mpx ve lyz arttırıcılar nötrofil raportör çizgileri 16, 26, 27 üretmek için kullanılmış olan ve csf1r (FMS) ve mpeg1 arttırıcılar makrofaj gazetecilere 14, 28 üretmek için kullanıldı. Iken csf1r (FMS) ayrıca xanthophores ifade edilir, MPEG1 ifade makrofajlarda münhasıran. Bu video madde de gösterildiği üzere, yeni geliştirilen mpeg1 raportör hatları makrofajların görüntüleme bakteriyel fagositozunu için son derece yararlıdır.

<table border="1"><tbody><tr><td> Reaktif </td><td> Satıcı </td><td> Katalog numarası </td></tr><tr><td> Fenol Red </td><td> Sigma </td><td> P0290 </td></tr><tr><td> Gliserin </td><td> Sigma-Aldrich </td><td> 228210 </td></tr><tr><td> Difco Middlebrook 7H10 agar </td><td> Becton, Dickenson ve Şirket </td><td> 262710 </td></tr><tr><td> BBL Middlebrook OADC Zenginleştirme </td><td> Becton, Dickenson ve Şirket </td><td> 211886 </td></tr><tr><td> Difco Middlebrook 7H9 Broth </td><td> Becton, Dickenson ve Şirket </td><td> 271310 </td></tr><tr><td> BBL Middlebrook ADC Zenginleştirme </td><td> Becton, Dickenson ve Şirket </td><td> 211887 </td></tr><tr><td> Tween 80 </td><td> Sigma-Aldrich </td><td> P1754 </td></tr><tr><td> Polivinilpirrolidon (PVP40) </td><td> Calbiochem, Merck KGaA </td><td> 529504 </td></tr><tr><td> N &lt;/ Em&gt;-Phenylthiourea (PTU) </td><td> Sigma-Aldrich </td><td> P7629 </td></tr><tr><td> Etil 3-aminobenzoat metansülfonat tuzu (Tricaine) </td><td> Sigma-Aldrich </td><td> A5040 </td></tr><tr><td> Metil selüloz </td><td> Sigma-Aldrich </td><td> M0387 </td></tr><tr><td> SeaPlaque Agaroz (düşük erime noktası agaroz) </td><td> Lonza A.Ş. </td><td> 50100 </td></tr><tr><td> FluoSpheres (1.0 mikron, kırmızı floresan (580/605)) </td><td> Invitrogen </td><td> F8821 </td></tr><tr><td> FluoSpheres (1.0 mikron, (505/515) sarı-yeşil flüoresan) </td><td> Invitrogen </td><td> F8823 </td></tr></tbody></table>

infection of zebrafish embryos with intracellular bacterial pathogens

Zebra balığı (Danio rerio) embriyo giderek-konak ilişkilerinin 1 omurgalı doğuştan gelen bağışıklık sisteminin fonksiyonunu incelemek için bir model olarak kullanılmaktadır. Doğuştan gelen bağışıklık sisteminin önemli hücre tipleri, makrofaj ve nötrofiller, önce adaptif immün yanıtları için gerekli olan lenfositlerin olgunlaşması için embriyogenezisin ilk günlerinde gelişir. Embriyoların sayıda elde kolaylığı, dış gelişme, embriyonik ve larva aşamalarını optik şeffaflık nedeniyle erişilebilirlik, genetik araçları, transgenik muhabiri hatları geniş mutant kaynakları ve koleksiyonları, geniş bir yelpazede tüm zebrabalıkları çok yönlülüğünü eklemek modeli. Salmonella enterica serovar Typhimurium (S. typhimurium) ve Mycobacterium marinum makrofaj hücre içinde bulunabilir ve sık Zebra balığı embriyolarının-konak ilişkilerinin incelemek için kullanılır. Bu iki enfeksiyonu süreçlerbakteriyel patojenler karşılaştırmak ilginç çünkü S. typhimurium enfeksiyonu M. ise, akut ve bir gün içinde ölümcüldür marinum enfeksiyonu kronik ve larva dönemi 2, 3 kadar görüntülenebilir. Embriyo (Şekil 1) bakterilerin mikro-enjeksiyon sitesi enfeksiyon hızla sistemik olacak veya başlangıçta lokalize kalır belirler. Hızlı bir sistemik enfeksiyon ile kurulabilir mikro-enjeksiyon doğrudan posterior kan adada kaudal ven yoluyla kan dolaşımı içine veya Cuvier, gövde damar kalbe bağlayan yolk kesesi üzerinde geniş bir sirkülasyon kanalı Kanal yoluyla bakteri. 1 dpf&#39;e anda, bu aşamada embriyo phagocytically aktif makrofajlar var ama nötrofiller henüz kan adaya enjekte, olgun olmadığı zaman tercih edilir. Embriyolar da fonksiyonel (miyeloperoksidaz üretmeyen) nötrofil, geliştirdik 2-3 dpf&#39;e, az enjeksiyon için Cuvier ve Kanal t olarak tercih edilmektedirdiye enjeksiyon yerinde. Lokal enfeksiyonlar doğru miyeloid hücrelerin yönettiği göç çalışmak için, bakteri kuyruk kas, otik vezikül veya arka beyin ventrikül 4-6 enjekte edilebilir. Buna ek olarak, notokord miyeloid hücrelere normal erişilmez gibi görünen bir yapı, lokal enfeksiyon 7 son derece duyarlı. Yüksek hacimli uygulamalar için kullanışlı bir alternatif gübreleme 8 sonraki ilk saat içinde embriyo sarısı içine bakteri enjekte edilmesidir. Floresan makrofaj veya nötrofil ile floresan bakteri ve transgenik zebrabalıkları çizgileri birleştirerek-konak ilişkilerinin çok-renkli görüntüleme için ideal şartlar oluşturur. Bu video makalede S. ile Zebra balığı embriyolarının damardan ve lokal enfeksiyon için ayrıntılı protokoller açıklayacağız typhimurium veya M. marinum bakteri ve doğuştan gelen bağışıklık sistemi hücreleri ile etkileşim sonraki floresans görüntüleme için.

Watch this Scientific Journal Video about Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens at JoVE.com

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Şeffaf zebrabalıkları embriyolar gibi görselleştirmek için faydalı model bilgisayarlar ve doğuştan gelen bağışıklık hücreleri ve hücre içi bakteriyel patojenler arasında işlevsel çalışma etkileşimleri, kanıtladık<em&gt; Salmonella typhimurium</em&gt; Ve<em&gt; Mycobacterium marinum</em&gt;. Bakteri ve çok-renkli floresans görüntüleme mikro-enjeksiyon zebrafish embriyo enfeksiyonu modellerinin uygulaması ile ilgili önemli tekniklerdir.

Hücre içi bakteriyel patojenler ile Zebra balığı Embryolarının Enfeksiyon

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

infection-of-zebrafish-embryos-with-intracellular-bacterial-pathogens

Research

JoVE Journal

Immunology and Infection

43.5K Görüntüleme.  Leiden University. Şeffaf zebrabalıkları embriyolar gibi görselleştirmek için faydalı model bilgisayarlar ve doğuştan gelen bağışıklık hücreleri ve hücre içi bakteriyel patojenler arasında işlevsel çalışma etkileşimleri, kanıtladık Salmonella typhimurium Ve Mycobacterium marinum. Bakteri ve çok-renkli floresans görüntüleme mikro-enjeksiyon zebrafish embriyo enfeksiyonu modellerinin uygulaması ile ilgili önemli tekniklerdir.

Video: Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Kategori Algılama Bir Bakteriyel Patojenler &#39;Bioluminescent&#39; Muhabir Faj

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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 ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Amoebal kokültürü ve Amoebal Zenginleştirme Yaklaşımlar New hücre içi patojenlerin keşif

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Bir uyarılabilir İfade Sistemi uygulamak Hücre içi Sinyali ile Bakteriyel Virulans Faktörler Girişim Eğitim için

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNA gen Sıralama ve MALDI-TOF MS tarafından Nadir Bakteriyel patojenlerin tanımlanması

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

İdrar yolu enfeksiyonu aşağı akım tek hücreli analiz için bir fare modelinden oluşturulan tek hücre içi bakteriyel toplulukların yalıtım

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic öldürme tahlil bakteriyel patojenlere karşı immünolojik yanıt değerlendirmek için

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

İntravital Mikroskopi ile Zebrabalığı Embriyolarında Mikobakteriyel Enfeksiyon Sırasında Makrofaj Litik Hücre Ölümünün Görüntülenmesi

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Luciferase Tabanlı Büyüme Testini Kullanarak Hücre İçi Toksoplazma Gondii Büyümesine Karşı Kimyasal Inhibitör Verimliliğin Belirlenmesi

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...