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 Pfx 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 S. pneumoniae transformants</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine</td>
			<td>sigma</td>
			<td>55470</td>
			<td>used for S. pneumoniae Transformation</td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td>used to grow S. pneumoniae cells</td>
		</tr>
		<tr>
			<td>Calcium Chloride Cacl2</td>
			<td>Sigma</td>
			<td>449709</td>
			<td>used for S. pneumoniae Transformation</td>
		</tr>
		<tr>
			<td>Competence stimulating peptide 1</td>
			<td>AnaSpec</td>
			<td>AS-63779</td>
			<td>used for S. pneumoniae 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&#39;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 S.pneumoniae 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&#945; 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

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Canlı Hücre Görüntüleme Bacillus subtilis Ve Streptococcus pneumoniae Otomatik Zaman atlamalı Mikroskopi

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

İmmünoloji ve Enfeksiyon

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

Isolate A Genetik Ekran Toxoplasma gondii Host-hücre çıkış Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

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 ...

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

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

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

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

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

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

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 ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Kapsüler Serotipleme<em&gt; Streptococcus pneumoniae</em&gt; Lateks Aglutinasyon tarafından

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Immünkompetan Kemirgenler bir Sağlam Pnömoni Modeli karşı antibakteriyel etkinliğini değerlendirmektir<i&gt; S. pneumoniae</i&gt;<i&gt; H. influenza</i&gt;<i&gt; K. pneumoniae</i&gt;<em&gt; P. aeruginosa</em&gt; ya da<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

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 ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomyces gelişimsel mutantlar görsel ve mikroskobik değerlendirilmesi

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...