Menenjit Vakfı'na ve MRC'ye bu çalışmaya finansman sağladıkları için teşekkür ederiz.

1. SOE-PCR23 ile mutasyona uğrayan amplikonun üretilmesi ve spektinomisin kasetinin amplifikasyonu
<ol><li>519/43 suşundan kat geninin yan bölgelerinin homoloji kollarının (sırasıyla kat 5' (488 bp) ve ply3' (715 bp) amplifikasyonu için PCR uygulayarak başlayın. plyFw1_NOTI (TTT GCGGCCGCCAGTAAATGACTTTATACTAGCTATG), ply5'R1_BamHI (CGAAATATAGACCAAAGGACGC GGATCC AGAACCAAACTTGACCTTGA), ply3'F1_BamHI (TCAAGGTCAAGTTTGGTTCTCTGGATCC GCGTCCTTTGGTCTATATTTCG) ve plyRv2_NotI (TTTGCGGCCGCCATTTTCTCTACCTCTCTCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTCTCCTCT) astarlarını kullanın.</li>
	<li>Ply5' için aşağıdaki PCR koşullarını kullanın: 60 sn için 94 °C'de denatür, adım 2: 30 s için 94 °C'de denatür, adım 3: 30 s için 58 °C'de tavlama, adım 4: 30 s için 72 °C'de uzatma, adım 5: adım 2'ye geri dönün ve 35 döngü için tekrarlayın, adım 6: 30 s için 72 °C'de son uzatma.<ol><li>60 s olması gereken adım 4 ve adım 6'daki uzatma süresi hariç, kat3' için aynı PCR koşullarını kullanın.</li>
	</ol></li>
	<li>PCR ürünlerini jel elektroforezi ile analiz edin ve amplikonu jelden çıkarın.</li>
	<li>PCR amplikonlarını, üreticinin talimatlarında (Malzeme Tablosu) açıklanan protokolü izleyerek saflaştırın.</li>
	<li>Her iki homoloji kolunun equimolar miktarlarını SOE-PCR'de şablon olarak kullanın. İki amplikonu primerleri kullanarak birleştirin plyFw1_ NOTI (TTT GCGGCCGCCAGTAAATGACTTTATACTAGCTATGTTATACTTATGTTATG) ve plyRv2_NotI (TTTGCGGCCGCCATTTTCTACCTCTCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTCTCCTCTCTCTCTCTCTCTCTCTTCTCCTCTCTCTCTCTCTCTCTCTTCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTCCC) iki amplikonu iki amplikonu birleştirin.</li>
	<li>Aşağıdaki SOE-PCR koşullarını kullanın (adım 1: 2 dakika boyunca 94 °C'de denatür, adım 2: 30 sn için 94 °C'de denatür, adım 3: 30 s için 58 °C'de tavlama, Adım 4: 60 s için 68 °C'de uzatma, adım 5: adım 2'ye geri dönün ve 25 döngü için tekrarlayın, adım 6: 90 s için 68 °C'de son uzatma).</li>
	<li>SOE-PCR ürününü jel elektroforezi ile analiz edin. Bir jel ekstraksiyon kiti kullanarak jelden tüketin ve verilen talimatları izleyin.</li>
	<li>Aşağıdaki PCR koşullarını kullanarak spektinomisin kasetini plazmid pR412'den yükseltin: adım 1: 60 s için 94 °C'de denatür, adım 2: 30 s için 94 °C'de denatüre etme, adım 3: 30 s için 55 °C'de tavlama, adım 4: 60 s için 68 °C'de uzatma, adım 5: adım 2'ye geri dönün ve 25 döngü boyunca tekrarlayın, Adım 6: 60 s için 68 ° C'de son uzatma.<ol><li>Astarları BamHI_SP2F2 (GGATCC CTA GAA CTA GTG GAT CCC CC) ve BamHI_SP2R2 (GGATCC AAT TCT GCA GAT TTT AC ATG ATC) kullanın. Plazmid pR412, Dr. Marc PrudHomme'dan (CNRS-Universite Paul Sabatier Toulouse Fransa) satın alındı.</li>
	</ol></li>
	<li>PCR amplikonlarını jel elektroforezi ile analiz edin. Elde edilen PCR amplikonunu yukarıda açıklandığı gibi tüketin ve saflaştırın.</li>
</ol>2. Plazmid pSD1 üretimi ve E. coli Dh5α'nın kimyasal dönüşümü
<ol><li>pGEMT-easy system I üretici talimatlarını (Malzeme Tablosu) izleyerek bir ligasyon gerçekleştirin. Bir mikrosantrifüj tüpünde, 20 μL'lik toplam hacme 5 μL 2x ligasyon tamponu, 1 μL pGEMTeasy, 2 μL ply_SOE ürünü, 1 μL T4 DNA ligaz ve su ekleyin. Gece boyunca 4 ° C'de inkübe edin. Bu, plazmid pSD1 üretir.</li>
	<li>Kimyasal olarak yetkin E. coli Dh5α'yı pSD1 ile dönüştürün. 50 μL kimyasal olarak yetkin E. coli Dh5α'yı buz üzerinde 15 dakika boyunca 3 μL pSD1 ligasyon reaksiyonu ile inkübe ederek başlayın. Daha sonra hücreleri termik şoka maruz bırakarak devam edin (42 ° C, 30 s). Hücreleri 2 dakika boyunca buzun üzerine yerleştirin.</li>
	<li>Hücreleri buzdan çıkarın ve 350 μL S.O.C ortamı ekleyin. Kültürü 37 °C, 120 rpm'de 2 saat boyunca inkübe edin.</li>
	<li>Luria Bertani Agar (LBA) üzerindeki transformasyon, plakada büyüyen tüm kolonilerin plazmid omurgasına sahip olmasını sağlamak için mavi / beyaz seleksiyon için 0.4 mM IPTG, 0.24 mg / mL X-Gal ve 100 μg / mL ampisilin ile desteklenir. Beyaz koloniler pSD1 içerir.</li>
	<li>Üç beyaz koloni seçin ve 100 μg / mL ampisilin ile desteklenmiş 10 mL LB'de gece boyunca büyüme sağlayın. Kültürleri gece boyunca 37 ° C'de sallayarak inkübe edin.</li>
	<li>Ertesi gün kültürleri 3.082 x g'de santrifüj edin ve plazmid ekstraksiyonu için peleti kullanın.</li>
</ol>3. Plazmid DNA ekstraksiyonu, pSD1 ve spektinomisin geninin kısıtlanması sindirimi ve pSD2'nin montajı
<ol><li>Plazmid DNA'sını, ticari kit (Malzeme Tablosu) ile birlikte verilen talimatları izleyerek çıkarın.</li>
	<li>Hem pSD1 plazmid hem de daha önce güçlendirilmiş ve saflaştırılmış spektinomisin kaseti için bir BamHI kısıtlama sindirimi ayarlayın. Tablo 1'de açıklanan aşağıdaki koşulları ve miktarları kullanın.</li>
	<li>Kısıtlama sindirim reaksiyonlarını ve kontrollerini 3 saat boyunca 37 °C'de inkübe edin.</li>
	<li>Kısıtlama sindirimini elektroforez ile analiz edin, bandı tüketin ve ticari kit (Malzeme Tablosu) ile birlikte verilen talimatları izleyerek saflaştırın.</li>
	<li>Daha sonra, BamHI sindirilmiş pSD1 ve spektinomisin (adım 3.2'den itibaren) kullanarak üretici talimatlarını (Malzeme Tablosu) izleyerek bir ligasyon reaksiyonu hazırlayın. Bir mikrosantrifüj tüpüne, aşağıdaki reaksiyon bileşenlerini ekleyin: 5 μL 2x ligasyon tamponu, 2 μL pSD1, 2 μL spektinomisin kaseti, 1 μL T4 DNA ligaz ve 4 ° C'de gece boyunca inkübe edin. Bu, plazmid pSD2 üretir.</li>
	<li>Plazmid pSD2'yi, adım 2.2'de açıklandığı gibi kimyasal olarak yetkin E. coli Dh5α'ya dönüştürün.</li>
	<li>Plazmid pSD2 taşıyan transformantları, 100 μg / mL spektinomisin ve ampisilin ile desteklenmiş LBA'da büyüme yeteneklerine göre seçin.</li>
	<li>Yukarıda açıklandığı gibi ve üreticinin talimatlarını (μL) izleyerek bir plazmid DNA ekstraksiyonu (pSD2) gerçekleştirin.</li>
</ol>4. S. pneumoniae suşunun transformasyonu 519/43
<ol><li>BHI'da bir gecede S. pneumoniae 519/43 kültürü hazırlayın ve 37 ° C'de,% 5 CO2'de statik olarak büyümesine izin verin.</li>
	<li>Ertesi gün kültürleri 1:50 ve 1:100 olarak 10 mL taze BHI suyunda seyreltin. OD595nm 0.05 ile 0.1 arasında olana kadar kültürleri statik olarak 37 ° C'de inkübe edin (0.1 OD'ye daha yakın DNA'nın optimal kazanımı).</li>
	<li>0.1'lik bir OD'ye ulaşıldığında, 860 μL alın ve bir mikrosantrifüj tüpüne aktarın. Aynı mikrosantrifüj tüpünde şunları ekleyin: 100 μL 100 mM NaOH, 10 μL% 20 (w / v) BSA, 10 μL 100 mM CaCl 2,2 μL 50 ng / mL CSP124 ve 500 ng pSD2.</li>
	<li>Reaksiyonu 3 saat boyunca 37 °C'de statik olarak inkübe edin.</li>
	<li>Plaka 330 μL, 3 inkübasyon saati boyunca her saat başı 100 μg / mL spektinomisin ile desteklenen% 5 kan agar plakaları (BA) üzerine.</li>
	<li>Plakaları gece boyunca 37 ° C'de,% 5 CO2'de inkübe edin. Yama spektinomisin'e dirençli koloniler, 100 μg / mL spektinomisin ile desteklenmiş başka bir BA plakasına ve ayrıca 100 μg / mL ampisilin ile desteklenmiş BA plakalarına yerleştirin. Her iki plaka setini de yukarıda belirtilen koşullar altında gece boyunca inkübe edin. Ampisilin plakaları, plazmid omurgasının varlığını test etmek içindir.</li>
	<li>NOTI (TTT GCGGCCGCAGTAAATGACTTTATACTAGCTATG) ve plyFw1_ (TAATTCCTCTAAGTCATAATTTCCG) primerlerini kullanarak spektinomisin kasetinin varlığını PCR SPEC_REV ile onaylayın. Mutasyona uğramış bölgenin dışına bağlanan primerler plySCN1 (CCAATGGAAATCGCTAGGCAAGAGATAA) ve plySCN2 (ATTACTTAGTCCAACCACGGCTGAT) kullanarak PCR ile mutasyonu onaylayın.</li>
	<li>Primerler plySCN1 (CCAATGGAAATCGCTAGGCAAGAGATAA), plySCN2 (ATTACTTAGTCCAACCACGGCTGAT) ve ayrıca spektinomisin kaset sqr1 (CCTGATCCAAACATGTAAGTACC) sqf2 (CGTAGTTATCTTGGAGAGAATA) spec_sqf1 (GGTACTTACATGTTTGGATCAGGCAGG) ve spec_sqr2 TATTCTCTCCAAGATAACTACG içindeki bağlanma bölgelerine sahip primerleri kullanarak genomun doğru konumundaki entegrasyonu onaylayın.</li>
</ol>

<ol>
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W., Yesilkaya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumococcal+6-phospho-&#946;-glucosidase+(BglA3)+is+involved+in+virulence+and+nutrient+metabolism.">Pneumococcal 6-phospho-&#946;-glucosidase (BglA3) is involved in virulence and nutrient metabolism.</a> Infection and Immunity. 84 (1), (2015).</li><li>Harvey, R. M., Ogunniyi, A. D., Chen, A. Y., Paton, 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=Pneumolysin+with+Low+Hemolytic+Activity+Confers+an+Early+Growth+Advantage+to+Streptococcus+pneumoniae+in+the+Blood.">Pneumolysin with Low Hemolytic Activity Confers an Early Growth Advantage to Streptococcus pneumoniae in the Blood.</a> Infection and Immunity. 79 (10), 4122 (2011).</li><li>Terra, V. S., Plumptre, C. D., Wall, E. C., Brown, J. S., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+pneumolysin+deficient+mutant+in+Streptococcus+pneumoniae+serotype+1+strain+519/43+and+phenotypic+characterisation.">Construction of a pneumolysin deficient mutant in Streptococcus pneumoniae serotype 1 strain 519/43 and phenotypic characterisation.</a> Microbial Pathogenesis. 141, 103999 (2020).</li></ol>

Yazarların açıklayacak hiçbir şeyleri yoktur.

Streptococcus pneumoniae, özellikle serotip 1, invaziv pnömokok hastalığına ve menenjite neden olan küresel bir tehdit olmaya devam etmektedir. Serotip 1'e karşı koruyucu olması gereken çeşitli aşıların uygulanmasına rağmen, Afrika'da bu serotip hala yüksek morbidite ve mortaliteye yol açan salgınlara neden olabilir13. Bu serotipi genetik olarak manipüle etme yeteneği, klinik alaka düzeyi nedeniyle kritik öneme sahiptir. Bu çalışmada açıklanan yöntem, bu serot...

Streptococcus pneumoniae (S. pneumoniae, pnömokok) tüm dünyada morbidite ve mortalitenin başlıca nedenlerinden biridir. Yakın zamana kadar, 100'e yakın S. pneumoniae serotipi 1,2,3,4,5,6,7 keşfedilmiştir. Yıllık, invaziv pnömokok hastalığı (IPD), 5 yaşından küçük çocukların yaklaşık 700.000 ölümünü iddiaetmektedir 8. S. pneumoniae, tüm dünyada bakteriyel pnömoni, otitis media, menenjit ve septiseminin başlıca nedenidir9.
Afrika menenjit kuşağında, serotip 1, son derece virülan bir sekans tipi olan sekans tipi (ST) ST217'nin baskın olduğu menenjit salgınlarından sorumludur10,11,12,13,14,15. Menenjit patolojisindeki önemi, Afrika menenjit kuşağı16'daki Neisseria meningitidis'inkine benzetilmiştir. Serotip 1 genellikle IPD'nin ana nedenidir; ancak, taşımacılıkta çok nadiren bulunur. Aslında, Gambiya'da, bu serotip tüm invaziv hastalıkların% 20'sinden sorumludur, ancak sağlıklı taşıyıcıların sadece% 0.5'inde bulunmuştur14,17,18,19. Yetkin pnömokoklarda genetik değişim ve rekombinasyon genellikle invaziv hastalıktan ziyade taşıyıcılıkta meydana gelir20. Ayrıca, serotip 1'in pnömokoklar arasında tarif edilen en kısa taşıma oranlarından birine sahip olduğu gösterilmiştir (sadece 9 gün). Bu nedenle, bu serotipin diğerlerinden çok daha düşük bir rekombinasyon oranına sahip olabileceği öne sürülmüştür21.
Serotip 1 suşlarının düşük taşıma hızının arkasındaki nedeni ve Sahra altı Afrika'daki invaziv hastalıklardaki önemini anlamak için derinlemesine çalışmalar gereklidir.
Burada, belirli bir serotip 1 suşunun genom çapında mutagenezine izin veren bir protokol sunuyoruz, 519/43. Bu suş, yeni DNA'yı kolayca elde edebilir ve genomuna yeniden birleştirebilir. Bu yöntem henüz gerginlikler arasında değildir, ancak 519/43 arka planında yapıldığında çok etkilidir (diğer hedefler mutasyona uğramıştır, el yazmaları hazırlanmaktadır). Sadece 519/43 suşunu kullanarak ve doğal yeterliliğinden yararlanarak ve ekzojen DNA'nın sağlanma şeklini değiştirerek, bu serotip 1 suşunda pnömolizin genini (kat) mutasyona uğratabildik. Bu yöntem, Harvey ve ark.22 tarafından sunulana göre bir gelişmeyi temsil eder, çünkü DNA'yı farklı bir serotipten geçirmeye gerek kalmadan tek adımda yapılır. Bununla birlikte, gerinimler arası değişkenlik nedeniyle, hiçbir yöntem tüm suşlar için standartlaştırılmamıştır. Belirli genleri mutasyona uğratma ve etkilerini gözlemleme yeteneği, serotip 1 S. pneumoniae suşlarının derinlemesine anlaşılmasını sağlayacak ve bu suşların Sahra altı Afrika'daki menenjitteki rolüne cevaplar sağlayacaktır.

<table><tbody><tr><td>AccuPrime &lt;em&gt;Pfx&lt;/em&gt; DNA polymerase</td><td>Invitrogen</td><td>12344024</td><td>Used for amplification of the fragments</td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A9518</td><td>Used for bacterial selection on stage 1(pSD1)</td></tr><tr><td>Blood Agar Base</td><td>Oxoid</td><td>CM0055</td><td>Used to plate &lt;em&gt;S. pneumoniae&lt;/em&gt; transformants</td></tr><tr><td>Bovine Serum Albumine</td><td>sigma</td><td>55470</td><td>used for &lt;em&gt;S. pneumoniae&lt;/em&gt; Transformation</td></tr><tr><td>Brain Heart Infusion</td><td>Oxoid</td><td>CM1135</td><td>used to grow &lt;em&gt;S. pneumoniae&lt;/em&gt; cells</td></tr><tr><td>Calcium Chloride Cacl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>449709</td><td>used for &lt;em&gt;S. pneumoniae&lt;/em&gt; Transformation</td></tr><tr><td>Competence stimulating peptide 1</td><td>AnaSpec</td><td>AS-63779</td><td>used for &lt;em&gt;S. pneumoniae&lt;/em&gt; Transformation</td></tr><tr><td>Luria Broth Agar</td><td>Gibco</td><td>22700025</td><td>used for plating and selection of pSD1 and pSD2</td></tr><tr><td>Luria Broth Base (Miller&#039;s formulation)</td><td>Gibco</td><td>12795027</td><td>used for plating and selection of pSD1 and pSD2</td></tr><tr><td>Monarch Gel Extraction Kit</td><td>NEB</td><td>T1020S</td><td>Used to extract the bands from the DNA gel</td></tr><tr><td>Monarch Plasmid Miniprep Kit</td><td>NEB</td><td>T1010S</td><td>Used to extract plasmid from the cells</td></tr><tr><td>pGEM T-easy</td><td>Promega</td><td>A1360</td><td>used as suicide plasmid</td></tr><tr><td>S.O.C.</td><td>Invitrogen</td><td>15544034</td><td>used for recovery of cells after transformation</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma</td><td>S0899</td><td>used for &lt;em&gt;S.pneumoniae&lt;/em&gt; Transformation</td></tr><tr><td>Spectinomycin Hydrochloride</td><td>SigmaAldrich</td><td>PHR1426</td><td>Used for bacterial selection</td></tr><tr><td>Subcloning Efficiency DH5&amp;alpha; Competent Cells</td><td>Invitrogen</td><td>18265017</td><td>used for the creation of pSD1 and pSD2</td></tr></tbody></table>

constructing mutants in serotype 1 streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotip 1, düşük ve orta gelirli ülkelerde, özellikle Sahra altı Afrika'da büyük bir sorun olmaya devam etmektedir. Önemine rağmen, bu serotipteki çalışmalar, onu değiştirmek için genetik araçların eksikliği nedeniyle engellenmiştir. Bu çalışmada, bir serotip 1 klinik izolatı genetik olarak değiştirmek için bir yöntem tarif edilmiştir (suş 519/43). İlginç bir şekilde, bu, Pnömokokların doğal olarak DNA elde etme yeteneğinden yararlanarak elde edildi. Bununla birlikte, çoğu pnömokoktan farklı olarak, lineer DNA kullanımı başarılı değildi; Bu önemli suşu mutasyona uğratmak için bir intihar plazmidinin kullanılması gerekiyordu. Bu metodoloji, hem biyolojisi hem de patojenitesi açısından bu zor serotipin daha derin bir şekilde anlaşılması için araçlar sağlamıştır. Yöntemi doğrulamak için, bilinen başlıca pnömokok toksini olan pnömolizin, iyi bilinen ve takip edilmesi kolay bir fenotipe sahip olduğu için mutasyona uğradı. Mutantın, beklendiği gibi, kırmızı kan hücrelerini lize etme yeteneğini kaybettiğini gösterdik. İlgilenilen serotipte önemli bir geni mutasyona uğratarak, intraperitoneal ve intranazal enfeksiyonlar üzerine fonksiyon kaybı mutantları için diğer serotipler için gözlenenlerden farklı fenotipler gözlemleyebildik. Özetle, bu çalışma 519/43 suşunun (serotip 1) genetik olarak değiştirilebileceğini kanıtlamaktadır.

Burada açıklanan protokol, sol ve sağ homoloji kollarını yükseltmek için PCR kullanarak başlar ve aynı zamanda kat geninin orta bölgesinden 191 bp'yi siler. PCR yapılırken sol homoloji kolunun 3' ucunda ve sağ homoloji kolunun 5' ucunda bir BamHI bölgesi tanıtılır (Şekil 1A). Bunu, sol ve sağ homoloji kollarının bir amplikon halinde kaynaştırıldığı PCR-SOE izler (Şekil 1B). Bu SOE-PCR amplikonu daha sonra plazmid pSD1 üretme...

Watch this Scientific Journal Video about Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43 at JoVE.com

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Burada, doğal olarak DNA ve intihar plazmidini elde etme kabiliyeti kullanılarak genetik olarak değiştirilebilen bir S. pneumoniae serotip 1 suşu 519/43'ü tanımlıyoruz. İlke kanıtı olarak, pnömolysin (kat) geninde izojenik bir mutant yapıldı.

Serotip 1 Streptococcus pneumoniae suşunda Mutantların İnşası 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

constructing-mutants-serotype-1-streptococcus-pneumoniae-strain

Research

JoVE Journal

Immunology and Infection

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

1.6K Görüntüleme.  London School of Hygiene and Tropical Medicine. Burada, doğal olarak DNA ve intihar plazmidini elde etme kabiliyeti kullanılarak genetik olarak değiştirilebilen bir S. pneumoniae serotip 1 suşu 519/43'ü tanımlıyoruz. İlke kanıtı olarak, pnömolysin (kat) geninde izojenik bir mutant yapıldı.

Video: Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Nesil bir gen kesintiye Streptococcus mutans çoğunlukla zorlanma olmadan Gen Klonlama

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Genetik

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Streptococcus pneumoniae ile İntranazal Kolonizasyon Sırasında İnflamatuar Yanıtların Karakterizasyonu

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

İmmünoloji ve Enfeksiyon

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Biyoluminesen Bakteriler Kullanılarak Gerçek Zamanda Bir Akut Fare Pnömoni Modelinde Pnömokok virulans faktörlerinin etkisi aşağıdaki

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

Of kapsül Serotipleme<em&gt; Streptococcus pneumoniae</em&gt; Kapsül şişme deneyi kullanarak Reaksiyon

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

Görselleştirme<em&gt; Streptococcus pneumoniae</em&gt; Kalp Microlesions ve sonraki Kardiyak Tadilat içinde

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Tıp

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Sipariş edilmiş, Pseudomonas aeruginosa baskı PA14 Transposon ekleme mutantlar Nonredundant Kütüphane çoğaltılması

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new protein functions or to have access to protein conjugates. Stop codon suppression, in particular amber codon suppression, has emerged as the most popular method to genetically introduce UAAs at defined positions. This methodology is herein applied to the preparation of a carrier protein containing an UAA harboring a bioorthogonal functional group. This reactive handle can next be used to specifically and efficiently graft a synthetic oligosaccharide hapten to provide a homogeneous glycoconjugate vaccine. The protocol is limited to the synthesis of glycoconjugates in a 1:1 carbohydrate hapten/carrier protein ratio but amenable to numerous pairs of biorthogonal functional groups. Glycococonjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization, thereby, satisfying more and more demanding drug regulatory agency recommendations, a criterion which is unmet by classical conjugation strategies. Moreover, this protocol makes it possible to finely tune the structure of the actual conjugate vaccine, giving rise to tools to address structure-immunogenicity relationships.

Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

Homojen Glikokonkonjuggate Kombine Doğal Olmayan Amino Asit Birleşme ve Tıklama-Kimya Aşı Amaçlı Tarafından Üretilen

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...

Biyomühendislik

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Streptococcus mutans'larda Yüksek Moleküler Kütle Proteininin Saflaştırılması

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

İntrasisternal Doğum yoluyla Farelerde Meningokok Menenjit Serogroup C Indükleyen

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Pseudomonas aeruginosa In-Frame Gen Silme Mutantlar Üretimi ve Enfeksiyon Basit Bir Fare Modelinde Virülans Zayıflama sıiçin Test

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Serotip 1 Streptococcus pneumoniae suşunda Mutantların İnşası 519/43

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