Bu araştırma, Alerji ve Bulaşıcı Hastalıklar Ulusal Enstitüsü ABD Kamu Sağlığı Servisi hibe AI69233 ve AI073843 tarafından desteklenmiştir. EPS Bulaşıcı Hastalıklar Patogenez bir Burroughs Wellcome Üyesidir. KPH Hücresel ve Moleküler Mikrobiyoloji Eğitimi Hibe Programı 5 T32 A107611-10 tarafından finanse edildi.

1. Taze Kan gelen Hemoglobin saflaştırılması <ol><li> Bir antikoagülan ile desteklenmiş taze insan kanı edinin. Saflaştırma boyunca buz üzerinde veya 4 de kan ° C tutun. </li><li> 1,500 x g&#39;de 20 dakika boyunca kan santrifüjleyin. Kırmızı kan hücrelerinin (eritrositler), tüpün altında olacaktır. Dikkatlice süpernatan aspire ve yavaşça buz gibi soğuk% 0.9 (ağırlık / hacim) NaCl solüsyonu içinde pelet tekrar süspansiyon. Santrifüj tekrarlayın ve 3 kez yıkayın. </li><li> Buz gibi soğuk 10 mM Tris-HCl (pH 8.0) ve 1 hacim içinde pellet yeniden süspanse edin. Bu ozmotik basınç nedeniyle eritrositlerde parçalanma neden olacaktır. ~% 20 son hacim toluen ekleyin. </li><li> Gecede bir et lokantası 4 ° C&#39;de inkübe edin. </li><li> 1 saat boyunca 20.000 xg&#39;de lizat santrifüjleyin. Toluen (üstünde yüzen) ve pelet dokunulmazken ortasında hemolizat fraksiyonu toplayın. Orta kısmı toplamak için bir uzun saplı pipet kullanın. </li><li> 0.44 mikron şırınga filtresi Pass through. Çözelti partikül içeriyorsamadde ve adım 1.5 tekrarlayın, filtreden geçtikten edilemez. </li><li> Yüksek performanslı sıvı kromatografisi ile hemoglobin (Hb) Purify (HPLC) anyon değişim kolonuna (Varian, PL-SAX 1.000 Å 8 mikron, 150 mm × 4,6 mm). A, mobil faz mM Tris-HCl (pH 8.0) ve mobil faz B, 10 mM Tris-HCl (pH 8.0) + 0.5 M NaCl, 10. Çözücü B A% 0 -100% eğim 2.0 ml / dk akış oranında 2 dakika boyunca çalışır. (: 410 ve 280 nm λ) elüsyon emilimi esas izlenir. Parlak kırmızı renkli ve önemli bir emme zirve (Şekil 1) ile karakterize sadece fraksiyonu toplayın. </li><li> Karşı elüsyon Dialyze fosfat-tamponlu tekrar salin (PBS) bir karışımı, ve daha sonra birkaç saat karıştırıldı. 0.22 um bir şırınga filtresinden geçirerek sterilize edilir. </li><li> Hb konsantrasyonu ölçmek için, PBS içinde konsantrasyonu bilinen standart Hb çözümler hazırlamak. (Tr reaktifleri ile tabloya bakınız standart çözeltilerin karışımı ile örnek olarak Hb konsantrasyonunun tespit edilmesied) veya bir 1:1 oranında 2x Drabkin reaktifi (tozundan hazırlanan) ile numune çözeltisi. Örneğin, 96 oyuklu plakalar 100 ul 2x Drabkin reaktifi ile 100 ul Hb çözelti ilave edilir. 540 nm&#39;de 15 dk ve önlem absorbans inkübe. Bir standart eğrisi çizilir ve örnek olarak Hb konsantrasyonu belirlenir. Beş ila on beş mg / ml verimleri tipiktir. </li><li> Çoğaltmak% 15 SDS-PAGE üzerine hemoglobin saflaştırılmış 15-20 mikrogram çalıştırın. Jellerin bir Leke; hemoglobin için bir nitroselüloz zar ve immunoblot (Şekil 2) üzerine başka bir jel proteinleri aktarın. </li><li> Freeze ve sıvı azot içinde hemoglobin 1 ml alikotları saklayın. </li></ol> 2. Hazırlanması Demir deplete Büyüme Medya <ol><li> Üreticiler ve% 1 cassamino asit (CA) (w / v) tarafından tavsiye edildiği gibi sodyum bikarbonat eklemek, su içinde eritilerek toz RPMI Roswell Park Memorial Institute (RPMI) et suyu hazırlayın. 0.2 um bir filtreden ve mağaza refrig geçirilerek sterilizeçalıştırılamaz. </li><li> % 7 (ağırlık / hacim) Chelex-100 ekleme ve bir karıştırma plakası üzerinde gece boyunca karıştırma ile metal-azalmış RPMI (NRPMI) hazırlayın. 0.2 um bir filtreden ve buzdolabında depo üzerinden geçerek Chelex-100 çıkarın. 25 uM ZnCl 2, 25 uM MnCl 2, 100 mM CaCl2 ve 1 mM steril 1.000 x çözüm olarak önceden hazırlanmış MgCl2: esansiyel demir dışı metaller ile medya tamamlayın. Yeniden kullanılabilir malzemeler demir kontaminasyonu önlemek için bu adımı için tek kullanımlık plastik kaplar kullanın. </li></ol> Bir Sole Demir Kaynağı Olarak Hemoglobin kullanarak 3. Staphylococcus aureus Büyüme <ol><li> Streak S. Donmuş bir stoktan triptik soya agar (TSA) üzerinde yalıtım aureus. 20-24 saat süre ile 37 ° C&#39;de inkübe edin. </li><li> Yeniden süspanse etilendiamin-N, 100 mM e kadar susuz etanol içinde N&#39;-bis (2-hidroksifenilasetik asit) (EDDHA). EDDHA çözüm içine gitmez ama etanol ile steril edilmiştir. </li><li> 0.5 mM nihai konsantrasyon RPMI için EDDHA ekleyin. Izin vermekEDDHA sonraki adıma geçmeden önce, en az 30 dakika eritin. Partiden partiye varyasyon nedeniyle, nihai konsantrasyon EDDHA bakteri oluşumuna izin vermek için 0.25 mM e indirilmesi gerekir. </li><li> S. tek koloniler inoküle RPMI 15 ml&#39;lik vida kapaklı konik bir tüp içinde EDDHA ihtiva eden 5 ml içine aureus. 16-20 saat boyunca dakika (rpm) başına 180 devir az sallama ile 37 ° C&#39;de inkübe edin. </li><li> 7.500 x g&#39;de 5 dakika boyunca gecede kültürleri santrifüjleyin ve NRPMI 0.5 mM EDDHA içeren yılında pelet tekrar süspansiyon haline getirin. ~ 3 OD 600 Normalize. </li><li> NRPMI ml Hb başına 2.5 mikrogram ve 0.1-1.0 mM EDDHA içeren hazırlayın. Gruplar arası varyasyon nedeniyle, NRPMI serbest demir şelatların döküntülerini büyütmek için gerekli olan konsantrasyon EDDHA değişebilir. </li><li> 15 ml vidalı kapaklı konik tüp içinde 1 ml NRPMI + EDDHA + Hb içine adım 3,5 bakteriyel süspansiyonun Altkültür 10 ul. </li><li> 180 rpm&#39;de veya haddeleme davul sallayarak ile 48 saat için 37 ° C&#39;de kültür inkübe edin. </li><li&gt; Her 6-12 saat 96-kuyucuğu içinde kültür 50 ul kaldırma ve PBS 150 ul ile karıştırılarak OD 600 okuma alabilir. </li></ol>

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C., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetric+behavior+of+hemoglobin+alpha-+and+beta-+subunits+during+acid-induced+denaturation+observed+by+electrospray+mass+spectrometry.">Symmetric behavior of hemoglobin alpha- and beta- subunits during acid-induced denaturation observed by electrospray mass spectrometry.</a> Biochemistry. 46, 10675-10684 (2007).</li><li>Williams, R. C., Tsay, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+chromatographic+method+for+the+preparation+of+human+hemoglobin.">A convenient chromatographic method for the preparation of human hemoglobin.</a> Analytical Biochemistry. 54, 137-145 (1973).</li><li>Shen, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+unmodified+human+adult+hemoglobin+in+Escherichia+coli.">Production of unmodified human adult hemoglobin in Escherichia coli.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8108-8112 (1993).</li><li>Manjula, B. N., Acharya, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+molecular+analysis+of+hemoglobin+by+high-performance+liquid+chromatography.">Purification and molecular analysis of hemoglobin by high-performance liquid chromatography.</a> Methods Mol. Med. 82, 31-47 (2003).</li><li>Neilands, J. 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Biz ifşa etmek başka bir şey var.

Demir yaşamın 15 tüm krallıklardan organizmalar tarafından gerekli olan temel bir besindir. Omurgalılar, demir bu öğe tarafından kaynaklanan zehirlenme önlemek için tecrit edilir. Bu haciz de beslenme bağışıklık 16 olarak bilinen bir süreçte mikroplar istila demir gizler. Buna karşılık, patojenler beslenme bağışıklık engelleyecek stratejiler geliştirmişlerdir. Böyle bir mekanizma hemoglobin dayanır, hangi ana 17 içindeki demirin en bol kaynağıdır. Hemogl...

<table border="1"><tbody><tr><td> Reaktif / Malzeme Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> HPLC anyon değişim kolonuna </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Drabkin reaktifi </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> Hemoglobin standart </td><td> Pointe Scientific </td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> HyClone </td><td> SH30011.02 </td><td></td></tr><tr><td> Chelex 100 sodyum formu </td><td> Sigma </td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC Standartları GmbH </td><td> ANC 001 </td><td></td></tr><tr><td> Hemoglobin bir antikor </td><td> Santa Cruz Biotechnology, Inc </td><td> SC-21005 </td><td> &lt;/ Td&gt; </tr><tr><td> Triptik Soy Agar </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. aureus hayati metabolik fonksiyonları ve hastalık yapan yürütmek için demir gerektiren bir patojen bakteridir. Insan konak içindeki demirin en bol rezervuar hemoglobin kofaktörüdür heme vardır. Hemoglobin demir elde etmek için, S. aureus demir-regüle yüzey belirleyicisi (Isd) sistemi 1 olarak bilinen karmaşık bir sistem kullanır. Isd sistem ilk bind konak hemoglobin Bileşenleri, daha sonra ayıklamak ve heme ithalat ve nihayet bakteriyel sitoplazma 2,3 heme demir kurtarmak. Bu yol vitro çalışmalar 4-9 sayısız aracılığıyla diseke edilmiştir. Ayrıca, enfeksiyon için Isd sistemin katkısı art arda 8,10-14 fare modellerinde ortaya konmuştur. Hemoglobin kaynaklı demir edinimi Isd sistemin katkısı kurulması ve büyüme daha zorlu olduğu kanıtlanmıştır. Bir tek demir kaynağı olarak hemoglobin kullanarak Büyüme deneyleri b karmaşıktıry büyüme ortamında serbest demir kirletici ticari olarak temin hemoglobin kararsızlık, ve demir şelatlar ile ilişkili toksisite. İşte biz bu sınırlamalarının üstesinden bir yöntem sunuyoruz. Yüksek kaliteli hemoglobin taze kandan hazırlanmıştır ve sıvı azot içinde saklanır. Arıtılmış hemoglobin içine ilave edilir, orta omurgalı konukçu içerisindeki patojenlerin karşılaştığı demir-fakir ortamda taklit demir tüketmek. S. açlıktan tarafından serbest demir aureus ve biz, hemoglobin bağlamak heme ayıklamak, bakteri hücre zarfı ile heme geçmek ve sitoplazmada heme aşağılamak yeteneğini tamamen bağımlı bir şekilde büyümesine neden hemoglobin minimal manipüle formu ile ilave. Bu tahlil S. hemoglobin-/heme-derived demir iktisap mekanizmaları aydınlatmak isteyen araştırmacılar için yararlı olacaktır aureus ve muhtemelen diğer bakteriyel patojenler.

Biz HPLC (Protokol adım 1.7) ile hemolizat insan hemoglobin Temizlendikleri. Şekil 1 gösterir 280 ve 410 nm dalga boylarında eluat absorbans kaydedildi. Fraksiyon 5 toplandı ve diğer fraksiyonlar atıldı. Eluat mililitrede hemoglobin beş ile on beş miligram Verimleri genellikle elde edilir. Saflaştırılmış hemoglobin kopya halinde SDS-PAGE ile analiz edildi ve jeller ya da proteinler için boyandı ya da nitroselüloz üzerine transfer edildi ve (Protokol adım 1.10, Şekil 2)</stro...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Burada bir büyüme tahlil tarif<em&gt; Staphylococcus aureus</em&gt; Mevcut besin demir tek kaynak olarak hemoglobin kullanarak. Bu testte elde edilen hemoglobin demir elde etme ile ilgili bakteri faktörlerin rolü kurar.

Demir Kaynağı Olarak İnsan Hemoglobin kullanarak Staphylococcus aureus Büyüme

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

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Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.5K Görüntüleme.  Vanderbilt University Medical School. Burada bir büyüme tahlil tarif Staphylococcus aureus Mevcut besin demir tek kaynak olarak hemoglobin kullanarak. Bu testte elde edilen hemoglobin demir elde etme ile ilgili bakteri faktörlerin rolü kurar.

Video: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

metabolit analizi için bir Tandem Sıvı Kromatografi-Kütle Spektrometresi tabanlı yaklaşım<em&gt; Stafilokok aureus</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Biyokimya

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Yatay Gen Transferi Çalışmasında Metodolojisi<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

İmmünoloji ve Enfeksiyon

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

İnsan Polimorfonükleer Lökositlere Karşı Staphyloccus aureus Sitortoksitesinin Ölçülmesi

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

Neisseria gonorrhoeae'nin Metal-Sınırlı Büyümesi, Ev Sahibi Ligandlardan Metal Duyarlı Genlerin Karakterizasyonu ve Metal Edinimi için

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein ...

T3SS Activity in Yersinia pseudotuberculosis Under Iron Depletion & Anaerobic Stress

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 &#176;C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

Bacteria colonize host tissues that vary in oxygen and iron bioavailability, yet most approaches to studying bacteria use aerated, rich media. This protocol describes culturing the human pathogen Yersinia pseudotuberculosis under varying iron concentrations and oxygen tension, and quantifying activity of the Yersinia type III secretion system, which is an important virulence factor.

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or ...

<meta charset="utf8"></head><body>Hücre İçi S. aureus Enfeksiyon Çalışmaları için Optimize Edilmiş İn Vivo Modeller

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.

<meta charset="utf8"></head><body>Staphylococcus aureus için Hücre İçi Enfeksiyon Modellerinin Geliştirilmesi ve Değerlendirilmesi

Staphylococcus aureus (S. aureus) has the capability to disseminate throughout the body, causing persistent and recurrent infections. To better understand these processes, this study establishes an intracellular infection model for S. aureus. This model will provide a crucial foundation for investigating the mechanisms behind intracellular infections.

Staphylococcus aureus için Hücre İçi Enfeksiyon Modellerinin Geliştirilmesi ve Değerlendirilmesi

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Antimikrobiyal Bileşiklerin Staphylococcus aureus Internalization ve Hücre İçi Etkinliğini İncelemek için Geliştirilmiş Enzim Koruma Tahlilleri

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Biyoloji

Pseudomonas aeruginosa İzolatlarından Siderofor Üretiminin Değerlendirilmesi

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.
The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Yazar Spotlight: Enfeksiyon Kontrolü için Sideroforların ve Piyochelin'in Ölçülmesi 

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Pseudomonas aeruginosa'dan Siderofor Üretiminin Kalitatif ve Kantitatif Analizi

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

Demir Kaynağı Olarak İnsan Hemoglobin kullanarak Staphylococcus aureus Büyüme

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metabolit analizi için bir Tandem Sıvı Kromatografi-Kütle Spektrometresi tabanlı yaklaşım

Yatay Gen Transferi Çalışmasında Metodolojisi

İnsan Polimorfonükleer Lökositlere Karşı Staphyloccus aureus Sitortoksitesinin Ölçülmesi

Neisseria gonorrhoeae'nin Metal-Sınırlı Büyümesi, Ev Sahibi Ligandlardan Metal Duyarlı Genlerin Karakterizasyonu ve Metal Edinimi için

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

Staphylococcus aureus için Hücre İçi Enfeksiyon Modellerinin Geliştirilmesi ve Değerlendirilmesi

Staphylococcus aureus için Hücre İçi Enfeksiyon Modellerinin Geliştirilmesi ve Değerlendirilmesi

Staphylococcus aureus için Hücre İçi Enfeksiyon Modellerinin Geliştirilmesi ve Değerlendirilmesi

Antimikrobiyal Bileşiklerin Staphylococcus aureus Internalization ve Hücre İçi Etkinliğini İncelemek için Geliştirilmiş Enzim Koruma Tahlilleri

Pseudomonas aeruginosa'dan Siderofor Üretiminin Kalitatif ve Kantitatif Analizi