Yazarlar Goodrich-Blair laboratuar geçmiş üyelerine teşekkür etmek istiyorum: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard ve Youngjin Park bu protokolün gelişmesine katkılarından dolayı. Bu çalışma, Ulusal Bilim Vakfı Hibe IOS-0950873 ve Sağlık NRSA dostluk FAI084441Z Ulusal Sağlık Enstitüleri tarafından finanse edildi.

1. Böcek Yumurta Sterilizasyon ve Yetiştirilmesi <ol><li> 900-1,000 ml H 2 O içinde sağlanan agar ilk otoklavlanarak 15 g diyet hazırlayın Hemen sonra iyi bir laboratuvar blender 166 gr buğday tohumu diyet ve harmanı ile otoklav, karıştırın. 4 de alüminyum folyo için diyet aktarmak, ardından serinlemek için bir tabak (veya yemekleri) dökün, sıkıca sarın ve mağaza ° C </li><li> Girişte, M. sterilize ara sıra karıştırarak, 90 mm ​​filtre kağıdı ile bir cam filtre tutucu ve termos aparat, 2-3 dakika süreyle% 0.6 çamaşır suyu çözeltisi 250 ml Sexta yumurta. </li><li> Yıkama başına 250 ml steril distile H 2 O ile çamaşır suyu çözeltisi ve yıkama yumurta 3-4 kez tahliye için vakum açın. </li><li> Bir Petri plaka kapağı yerleştirilmiş taze 90 mm filtre kağıdı yumurta aktarın ve onları kurutun kadar birlikte artık sopa (20-30 dk.) </li><li> Her kabı yaklaşık 40 yumurta koyarak, böcek diyet ile plastik kaplar altına yumurta aktarın(Şekil 1A). Yumurta nem kaynaklı fungal kontaminasyonu önlemek için diyet ayrılır. 8 saat karanlık fotoperiyodun: 16 saat ışık ile 26 yumurta ° C koruyun. Enkübasyon öncesinde sarı-beyaz renk hafifletir. </li><li> Kuluçka tamamlandıktan sonra, dikkatli bir şekilde alt (Şekil 1B) üzerinde bir diyet ile yeni konteyner yaklaşık 25 larvası her bir aktarın. Bu onların kırılgan erken gelişim aşamalarında onlara zarar vermemek için forseps kullanarak uzun siyah boynuz tarafından her larva almak için önemlidir. Yukarıdaki gibi 2 gün için inkübe edin. Larva ölüm bu aşamada alışılmadık olsa da, bazı ölü ya da daha büyük olasılıkla, gelişimsel bodur larva görülebilir. Bu tür böcekler fazla çalışma dışı ve donma sonucu feda edilmelidir. </li><li> Yamyamlık davranışları, yemek küçük parçalar (Şekil 1C) ile bireysel kaplara transferi larva önlemek ve yukarıdaki gibi inkübe etmek. Böcek gelişimini izlemek, yemek yerine ve temizlik dışkı ouher gün konteyner t onlar üçüncü larva dökücü (genellikle 6-9 gün yumurtadan sonra) geçmesi kadar. Bu böcek gövdesi (Şekil 1D) boyunca çizgili her proleg ve artan önem üzerinde siyah kanca benzeri Kroşe ve görünümü ile karakterize, 4. evre larva aşamasında olduğunu. Larva boyutu önemli ölçüde değişebilir, ancak genellikle 4. evre evre girdikten 0.15-0.4 gr ağırlığındadır. Benzer boyuttaki larvaları enjeksiyon için seçilmelidir. </li></ol> 2. Enjeksiyon için Bakteri hazırlanması <ol><li> Eğer test etmek isteyen büyüme fazı için standart prosedürlere uygun olarak, test edilecek olan bakteri suşu (ler) büyütün. </li><li> Her bir bakteri suşunun Pelet 500 ul, oda sıcaklığında 13,000 rpm&#39;de bir mikrosantrifüj içinde 2 dakika için eğirme ile test edilmelidir. </li><li> 1 ml steril 1x fosfat süspanse hücreleri yukarıdaki gibi tekrar tamponlu salin (PBS) ve pelet. </li><li> 0.5 ml steril 1x PBS ve mea içinde süspanse edin hücrelerisüspansiyon emin optik yoğunluk (OD). Gerekirse, aynı OD her süspansiyonlar seyreltilir. İstenirse, steril büyüme ortamı aynı zamanda, 1 x PBS yerine hücre yeniden süspansiyon ve seyreltme için kullanılabilir. </li></ol> 3. 4. evre larvalar Enjeksiyon <ol><li> Her bir seyreltme için yeni bir pipet ucu kullanılarak steril PBS içerisinde 1 x 96-kuyu mikrotiter plaka 6 kuyu içinde ilk olarak bakteri suşunun seri 10-kat dilüsyonu, (Şekil 2A) hazırlayın. Her bir kuyudaki hücre süspansiyonu nihai hacimli enjeksiyon (böcek başına 10 ul) artı plaka için 20 ul inokulum (adım 3.2 ve 3.7 &#39;ye bakınız) için gerekli hacim aşmalıdır. </li><li> Uygun bir büyüme ortamı içeren bir Petri plaka üst kısmındaki 5 dilüsyonları (10 -6 ile 10 -2) için spot 10 ul her. Noktalar merkezi (Şekil 2B) yayılmış böylece kompaktörü yatırın. </li><li> Buz üzerinde kaplarda böcek diyet ve dışkı ve yerde böcekler kaldıryaklaşık 5 dk. </li><li> 3 kez% 70-100 etanol, 2 tüplerin her biri, steril H2O (Şekil 2C) bir tüp ardından durulama yoluyla şırınga iğnesi sterilize edin. Sıvı hacmi batırmak iğne için yeterli olmalıdır. </li><li> Hemasitometre kullanarak bir mikroskop altında seyreltik hücre süspansiyonları quantitate. İstenen inokulum bağlı olarak, iyi bir uygun mikro-titer (Şekil 2B) 10 ul çizin. </li><li> Svab böcek% 95 etanol ile yüzey ve karın prolegs biri arkasından (45 ° &#39;den daha az), bir açıyla böcek içine 10 ml hücre süspansiyonu enjekte edilir. Ponksiyon gut vermemeye özen ve sadece epidermal tabakasında (Şekil 2E) altında içeriğini enjekte. Hemolimf bazı kaybı (berrak, yeşil-mavi sıvı) normal olsa da, enjeksiyon yerinde çıkan partikül madde ve sarı sıvı bağırsak delinmesi göstergesidir. Bu durum oluşursa, böcek kurban ve yeniden yeni bir larva eldeyerleştirin. </li><li> İlk enjeksiyon grubunda kalan her böcek için adım 3.6 tekrarlayın. İğne sterilizasyon kirlenme meydana gelmedikçe bakteri suşu ile aynı seyreltme ile enjekte edilen her bir böcek arasında gerekli değildir. Alternatif olarak, tekrar eden dağıtıcı örnekleri arasında enjeksiyon hacmi daha fazla tutarlılık için kullanılabilir. </li><li> Bakteri suşu ile birinci her böcek enjekte edildikten sonra, bu süre plakasının ortasında, hücre süspansiyonları lekelenme ve alt kısmından başlayarak akış (Şekil 2F) sağlayarak, aşama 3.2 &#39;tekrarlayın. Uygun sıcaklıkta inkübe plakaları ve inokulum Numaralandırılacak kolonileri saymayın. Bu ikinci adım, bu kaplama, birinci ve ikinci kaplamalar arasında farklı koloni sayısı neden olacaktır gibi örnekleri, enjeksiyon işlemi sırasında kontamine olmadığını doğrulamak için kullanılır. </li><li> Tekrar denemeye bakteri her bir suş için 3.8 ile 3.1 adım. Son olarak, bir ster kullanarak steril 1x PBS ile 3-5 böcekler enjekteBir negatif kontrol grubu olarak Ile iğne. 8 saat karanlık fotoperiyodun: 16 saat ışık ile 26 ° C&#39;de inkübe böcekler. </li><li> Enjeksiyonundan sonra zamanla böcek hayatta izleyin. Böcek ölümü stimülasyonu üzerine istemli hareket eksikliği olarak karakterize edilir. Böcekler genellikle Ölmeden önce enfeksiyon sırasında iştah kaybı, ishal (sulu, sarı dışkı), ve / veya su kaybı (&quot;terleme&quot;) sergilerler; bu özelliklerin yanı sıra kayda değer olabilir. </li></ol>

<ol>
	<li>Bintrim, S. B., Ensign, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+inactivation+of+genes+encoding+the+crystalline+inclusion+proteins+of+Photorhabdus+luminescens+results+in+mutants+with+pleiotropic+phenotypes.">Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes.</a> J. Bacteriol. 180, 1261-1269 (1998).</li><li>Schesser, J. H., Kramer, K. J., Bulla, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioassay+for+homogeneous+parasporal+crystal+of+Bacillus+thuringiensis+using+the+tobacco+hornworm,+Manduca+sexta.">Bioassay for homogeneous parasporal crystal of Bacillus thuringiensis using the tobacco hornworm, Manduca sexta.</a> Appl. Environ. Microbiol. 33, 878-880 (1977).</li><li>P&#233;chy-Tarr, M., Bruck, D. J., Maurhofer, M., Fischer, E., Vogne, C., Henkels, M. D., Donahue, K. M., Grunder, J., Loper, J. E., Keel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+a+novel+gene+cluster+encoding+an+insect+toxin+in+plant-associated+strains+of+Pseudomonas+fluorescens.">Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens.</a> Environ. Microbiol. 10, 2368-2386 (2008).</li><li>Nu&#241;ez-Valdez, M. E., Calder&#243;n, M. A., Aranda, E., Hern&#225;ndez, L., Ram&#237;rez-Gama, R. M., Lina, L., Rodr&#237;guez-Segura, Z., Guti&#233;rrez Mdel, C., Villalobos, F. J. <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+putative+Mexican+strain+of+Serratia+entomophila+pathogenic+against+root-damaging+larvae+of+Scarabaeidae+(Coleoptera).">Identification of a putative Mexican strain of Serratia entomophila pathogenic against root-damaging larvae of Scarabaeidae (Coleoptera).</a> Appl. Environ. Microbiol. 74, 802-810 (2008).</li><li>Forst, S. A., Tabatabai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+histidine+kinase,+EnvZ,+in+the+production+of+outer+membrane+proteins+in+the+symbiotic-pathogenic+bacterium+Xenorhabdus+nematophilus.">Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.</a> Appl. Environ. Microbiol. 63, 962-968 (1997).</li><li>Kanost, M. R., Jiang, H., Yu, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+a+lepidopteran+insect,+Manduca+sexta.">Innate immune responses of a lepidopteran insect, Manduca sexta.</a> Immunol. Rev. 198, 97-105 (2004).</li><li>Yu, X. Q., Zhu, Y. F., Ma, C., Fabrick, J. A., Kanost, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+proteins+in+Manduca+sexta+plasma.">Pattern recognition proteins in Manduca sexta plasma.</a> Insect Biochem. Mol. Biol. 32, 1287-1293 (2002).</li><li>Eleftherianos, I., ffrench-Constant, R. H., Clarke, D. J., Dowling, A. J., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+immune+response+to+the+entomopathogen+Photorhabdus.">Dissecting the immune response to the entomopathogen Photorhabdus.</a> Trends Microbiol. 18, 552-560 (2010).</li><li>Herbert, E. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friend+and+foe:+the+two+faces+of+Xenorhabdus+nematophila.">Friend and foe: the two faces of Xenorhabdus nematophila.</a> Nat. Rev. Microbiol. 5, 634-646 (2007).</li><li>D'Argenio, D. A., Gallagher, L. A., Berg, C. A., Manoil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+host+for+Pseudomonas+aeruginosa+Infection.">Drosophila as a model host for Pseudomonas aeruginosa Infection.</a> J. Bacteriol. 183, 1466-1471 (2001).</li><li>Waterfield, N., Dowling, A., Sharma, S., Daborn, P. J., Potter, U., Ffrench-Constant, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+toxicity+of+Photorhabdus+luminescens+W14+toxin+complexes+in+Escherichia+coli.">Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.</a> Appl. Environ. Microbiol. 67, 5017-5024 (2001).</li><li>Park, Y., Herbert, E. E., Cowles, C. E., Cowles, K. N., Menard, M. L., Orchard, S. S., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+variation+in+Xenorhabdus+nematophila+virulence+and+suppression+of+Manduca+sexta+immunity.">Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity.</a> Cell. Microbiol. 9, 645-656 (2007).</li><li>Park, Y., Kim, Y., Putnam, S. M., Stanley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterium+Xenorhabdus+nematophilus+depresses+nodulation+reactions+to+infection+by+inhibiting+eicosanoid+biosynthesis+in+tobacco+hornworms,+Manduca+sexta.">The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms, Manduca sexta.</a> Arch. Insect Biochem. Physiol. 52, 71-80 (2003).</li><li>Cowles, K. N., Cowles, C. E., Richards, G. R., Martens, E. C., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+regulator+Lrp+contributes+to+mutualism,+pathogenesis+and+phenotypic+variation+in+the+bacterium+Xenorhabdus+nematophila.">The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila.</a> Cell. Microbiol. 9, 1311-1323 (2007).</li><li>Cowles, K. N., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+activity+of+a+Xenorhabdus+nematophila+haemolysin+required+for+full+virulence+towards+Manduca+sexta+insects.">Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects.</a> Cell. Microbiol. 7, 209-219 (2005).</li><li>Goodrich-Blair, H., Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutualism+and+pathogenesis+in+Xenorhabdus+and+Photorhabdus:+two+roads+to+the+same+destination.">Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination.</a> Mol. Microbiol. 64, 260-268 (2007).</li><li>Eleftherianos, I., Baldwin, H., ffrench-Constant, R. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+modulation+of+immunity:+changes+within+the+feeding+period+of+the+fifth+larval+stage+in+the+defence+reactions+of+Manduca+sexta+to+infection+by+Photorhabdus.">Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.</a> J. Insect Physiol. 54, 309-318 (2008).</li><li>Kavanagh, K., Reeves, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+potential+of+insects+for+in+vivo+pathogenicity+testing+of+microbial+pathogens.">Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens.</a> FEMS Microbiol. Rev. 28, 101-112 (2004).</li></ol>

Çıkar çatışması ilan etti.

M. direkt enjeksiyon Burada açıklandığı gibi entomopatojen bakterilerle Sexta larva, bakteriyel virulans analiz için basit ve etkili bir araç olarak hizmet vermektedir. Yöntemi farklı deneysel konularda ve / veya koşullara göre aynı zamanda son derece uyarlanabilir. Bakteriler enjeksiyon öncesinde çeşitli şekillerde hazırlanabilir. X&#39;in durumda nematophila, orta-günlük faz besin açısından zengin Luria-Bertani (LB) ortamda yetişen yabani türü hücre enjeksi...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> 90 mm filtre kağıdı </td><td> Whatman </td><td> 1001 090 </td><td></td></tr><tr><td> Cam filtre tutucusu </td><td> Millipore </td><td> XX1004700 </td><td></td></tr><tr><td> Manduca Sexta yumurta </td><td> Carolina Biological Supply </td><td> 143880 </td><td></td></tr><tr><td> Gypsy Moth Diyet + agar </td><td> MP Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5.5 oz. Plastik kaplar ve kapakları </td><td> Yalnız Kupası Şirketi </td><td> URC55-0090 PL4-0090 </td><td></td></tr><tr><td> 1 oz. Plastik kaplar ve kapakları </td><td> DART Konteyner A.Ş. </td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1x PBS </td><td></td&gt; <td></td><td> 137 mM NaCl, 2,7 mM KCl, 8 mM 2 Na HPO 4, 1,46 mM KH 2 PO 4, pH 7.4 &#39; </td></tr><tr><td> Şırınga </td><td> Hamilton </td><td> 80208 </td><td> 30 gauge, 0.375 &quot;uzunluğunda, gelin tarzı 2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

Yaygın olarak bilinen bir tütün hornworm Manduca Sexta, domates ve tütün içeren solanaceous bitkilerle beslenen, önemli bir tarımsal zararlıların olarak kabul edilir. M. duyarlılık entomopatojenik bakteri türleri 1-5, hem de böcek bağışıklık sistemi 6-8 ile ilgili olarak mevcut olan bilgi birikimini çeşitli Sexta larva ve beklemede olan genom sekansı 9 bu konak-mikrop etkileşimleri araştırılmasında kullanım için iyi bir model organizma yapmak patogenezi sırasında. Buna ek olarak, M. Sexta larvaları diğer duyarlı böcek türüne laboratuar göreli olarak işlemek ve sürdürmek için nispeten büyük ve kolay. Onların büyük boyutu da enfeksiyona konak yanıtının analizi için etkili doku / hemolimf ekstraksiyon kolaylaştırır. Burada sunulan yöntem M. hemocoel içine bakterilerin direkt enjeksiyon (kan boşluğu) açıklar Sexta larva. Bu yaklaşımsadece enjeksiyonundan sonra böcek ölüme zaman izleyerek, çeşitli bakteri türleri, suşları, ya da mutantlarının virülans özelliklerini incelemek ve karşılaştırmak için kullanılabilir. Bu yöntem genellikle böcek girmesinden kazanmak için bir araç olarak nematod vektörleri ile ilişkilendirmek Xenorhabdus ve Photorhabdus türlerin patojenite incelemek için geliştirilmiştir. Entomopatojen nematodlar genellikle doğal sindirim veya solunum açıklıklar yoluyla larva enfekte ve böcek hemolimf (kan) kısa bir süre sonra 10 içine onların simbiyotik bakteri içeriğini yayınlayacak. Burada açıklanan yöntem ve böylece enjeksiyon böcek üzerinde bakteri ve nematod etkileri ayrılmasının bir nematod vektör için ihtiyaç atlar. Bu yöntem, nickleme 11 ve oral toksisite deneyleri 12 de dahil olmak üzere, entomopathogenesis analiz etmek için var olan diğer yöntemler kullanılarak mümkün değildir inokulum, içinde bulaşıcı malzemenin doğru sayımı (hücre ya da protein) için olanak sağlar <edirekt enjeksiyon yöntemi tam hücreli inokulum virülans adresleri ise m&gt;. Ayrıca, oral toksisite testleri, larvaların sindirim sistemine girmiş salgılanan toksinler virülans ele. Burada açıklandığı gibi direkt enjeksiyon yöntemi programı Böcek ölümleri takip ederek bakteriyel patogenez analiz etmektir. Bununla birlikte, bu metod M. üzerindeki enfeksiyon üzerindeki etkilerini araştırmak kullanılmak üzere genişletilebilir Sexta bağışıklık sistemi. Böcek humoral ve hücresel yanıtları hem yoluyla enfeksiyona yanıt verir. Beden irinterine ait tepki ile ilişkili bakteri desen ve tanıma farklı antimikrobiyal peptitler daha sonraki üretim 7 içerir; bu peptitlerin kodlayan genlerin ekspresyonu RNA ekstraksiyonu ve PCR kantitatif 13 aracılığıyla doğrudan enfeksiyonu takiben izlenebilir. Enfeksiyon hücresel yanıt nodülasyon, kapsülleme ve hemocytes 6 Enfeksiyöz ajanlar fagositozu içerir </sup&gt;. Bu tepkileri analiz etmek için, enjekte böcekler disseke ve mikroskopi 13, 14 tarafından görüntülenebilir.

Böcek ölümleri bir tahlil temsili bir örneği Şekil 3&#39;te gösterilmektedir. Bu deneyde, böcekler ya da vahşi tip (ATCC19061) veya orta-log fazı (zorlanma başına n = 6 böcekler) için yetiştirilen Xenorhabdus nematophila bir zayıflatılmış mutant suşu (LRP 13) yaklaşık 50 koloni oluşturan birim (CFU) enjekte edildi. Böcekler yaklaşık olarak 72 saat süre ile izlenir ve her bir timepoint hâlâ hayatta enjekte edilen böceklerin yüzde kaydedildi. Bu ...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Yöntemin hemocoel içine entomopatojenik bakteri doğrudan enjeksiyon kullanır burada açıklanan<em&gt; Manduca Sexta</em&gt; Böcek larvaları.<em&gt; M. Sexta</em&gt; Piyasada bulunan ve iyi çalışılmış böcek türüdür. Bu nedenle, bu yöntem, bir veya her iki ortağın perspektifinden konak-bakteri etkileşimleri analiz için basit bir yaklaşımı temsil etmektedir.

Ve Yetiştirme ve Enjeksiyon<em&gt; Manduca Sexta</emBakteriyel virülans Değerlendirememesi&gt; Larva

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

rearing-injection-manduca-sexta-larvae-to-assess-bacterial

Research

JoVE Journal

Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K Görüntüleme.  University of Wisconsin-Madison. Yöntemin hemocoel içine entomopatojenik bakteri doğrudan enjeksiyon kullanır burada açıklanan Manduca Sexta Böcek larvaları. M. Sexta Piyasada bulunan ve iyi çalışılmış böcek türüdür. Bu nedenle, bu yöntem, bir veya her iki ortağın perspektifinden konak-bakteri etkileşimleri analiz için basit bir yaklaşımı temsil etmektedir.

Video: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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 ...

İmmünoloji ve Enfeksiyon

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

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 ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Hamam böceği virülans, Patogenez ve uyuşturucu etkinliği araştırmak için alternatif bir Non-memeli hayvan Model tıslama Madagaskar

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

Mycobacterium abscessus virülans işaretleri tanımlaması fagositler hücre içi çoğaltma için

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

Virülans ve Aeromonas enfeksiyon Caenorhabditis elegans modelindeki patogenezinde değerlendirilmesi

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Bir fare modeli için değerlendirmek doğuştan gelen bağışıklık yanıtı Staphylococcus aureus enfeksiyon

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

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 ...

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 ...