This research was supported by the Department of Veterans Affairs Career Development Award 1IK2BX001701 and the CTSA award UL1TR000445 from the National Center for Advancing Translational Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health. Scanning electron microscopy experiments were performed in part through the use of the VUMC Cell Imaging Shared Resource, supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126.

1. H. İnsan Gastrik Hücrelerine ile Demir Durumu ve Co-kültür Çeşitli Koşullar pylori Büyüme <ol><li> H. seçin Bu çalışmalar için pylori soyu PMSS1 bir sağlam cağ patojenite adası ve işleyen bir tip IV sekresyon sistemi ifade çünkü. Aynı zamanda, bir negatif kontrol olarak izogenik bir kafes mutant (PMSSI Δ kafesi) kullanmaktadır. % 5 CO2 varlığında 37 ° C&#39;de 24 saat boyunca% 5 koyun kanı (kan agar plakaları) ile takviye edilmiş TSA plakaları üzerine bakteri büyütün. NOT: reaktifleri hazırlayın ve Malzemeleri ve Ekipmanları Tablo belirtildiği gibi malzemeleri bir araya getirin. Her bir reaktifin son konsantrasyonları parantez içinde belirtilmiştir. </li><li> Steril pamuk uçlu bir aplikatör kullanılarak plaka bakteri kazıyın ve bileşenlerden hazırlanan modifiye brusella et suyu içine aşılamak (Malzeme ve Ekipmanları Tabloya bakınız) ve kolesterol ile desteklenmiş. 37 ° C&#39;de gece boyunca kültürler inkübeDakika (rpm), 200 dönüş çalkalayarak,% 5 CO2 ilave edilmiş, havada bulunan ° C. Not: Kolesterol bağlı olarak serum karmaşık olmasından fetal sığır serumu yerine kullanılır; Bu tür sonuçlar değişkenlik neden heme gibi besin demir sayısız kaynaklar içeren. </li><li> Bakteriyel kültür gününde, AGS insan gastrik epitel hücreleri, tohum (ATCC CRL-1739), 12-gözlü levhalar (oyuk başına yaklaşık 1 x 10 5 hücre) poli-D-lizin muamele edilmiş, kapak slipleri üzerine. </li><li> Ertesi gün, tadil edilmiş, Brucella et suyu, tek başına veya 100 uM FeCl3, 200 uM dipiridil (sentetik demir kenetleme maddesi) ya da 200 uM dipiridil artı 250 uM FeCl3 ile desteklenmiş 0.3 bir OD600&#39;e bakteri kültürleri seyreltin. 200 rpm&#39;de çalkalanarak,% 5 CO2 ilave edilmiş, havada bulunan 37 ° C de 4 saat süre ile, bu kültürleri inkübe edin. </li><li> 1000 xg&#39;de santrifüj bakteri hücreleri toplamak ve i resuspending önce süpernatanlan kaldırmak içinn, taze değiştirilmiş Brucella Et suyu eşit hacimde kolesterol ile takviye edilmiştir. Kültür (OD600 = 1.0 = 5.5 x 10 8 hücre / ml) ölçümü OD600 ve enfeksiyon 20 (İB) &#39;in bir çokluğu epitel hücrelerine bakteri ekleyin: 1 arasındadır. Bakteriyel hücre canlılığını değerlendirmek için kan agar plakaları üzerine seri dilüsyonlarını ve plaka bakteri hücreleri gerçekleştirin. </li><li> H. inkübe taramalı elektron mikroskobu (SEM) örnek hazırlama gerçekleştirmeden önce statik koşullarda% 5 CO2 varlığında 37 ° C&#39;de 4 saat boyunca pilori AGS insan mide epitel hücrelerinin ko-kültürler. </li></ol> 2. SEM H. Visualize&#39;a Numune Hazırlama H. pylori Çağ-T4SS Pili <ol><li> H. Kaldır H. pylori ve inkübatör ve dökme sıvı kültür süpernatanından AGS ko-kültürler (IL-8 ELISA ile) kaydedin. 0.05 M sodyum kakodilat buferi (pH 7.4) hafifçe üç kez yıkayın. ,% 2.0 paraformaldehit çözeltisi içinde 2,5, oda sıcaklığında 2-4 saat boyunca örnekleri saptamak% Gluteraldehit, ve 0.05 M sodyum kakodilat tamponu. Birincil tespit sonra örnekler 0.05 M sodyum kakodilat tamponu ile üç kez yıkayın. </li><li> İki ardışık 10 dakika sabitleştirme adımlarında 0.05 M sodyum kakodilat tampon maddesi içinde% 0.1 osmiyum tetroksit ile ikinci tespit gerçekleştirin. İkinci tespit sonra örnekler 0.05 M sodyum kakodilat tamponu ile üç kez yıkayın. </li><li> Birincil ve ikincil tespit edildikten sonra, Tablo 1 &#39;deki gibi etanol artan konsantrasyonları ile art arda yıkama ile örnekleri dihidrat. </li><li> Etanol dehidratasyon sonra sıvı karbon dioksit, sıralı üç yıkama yapar. 1073 PSI 31.1 ºC sıcaklık ve basınçta bir kritik nokta kurutma makinesi kullanılarak kritik bir noktada sonra kuru örnekleri. </li><li> Dağı alüminyum SEM örnek taslakları üzerine lamelleri ve uygun topraklama sağlamak ve SEM görüntüleme sırasında şarj önlemek için numune kenarında kolloidal gümüş ince bir çizgi ile boyayın. </li><li> Coat örnekleri 5 nm ileörnek ikincil elektron sinyal ve kenar etkisini artırmak için bir püskürtme ceket makinesi kullanarak altın-paladyum. </li></ol> Yüksek Çözünürlüklü SEM Analizleri 3. Görüntüleme Parametreleri <ol><li> Saha-Emisyon Gun Taramalı Elektron Mikroskobu (FEG-SEM) ile View örnekler. </li><li> 5-10 mm çalışma mesafesi yüksek bir vakum modunda görüntü. </li><li> 5 kV hızlandırma voltajı ayarlayın. </li><li> 2-2,5 spot boyutunu ayarlayın. </li><li> Konak-patojen arayüzü daha iyi bir görünüm elde etmek için 15-25 derece arasında örnek eğin. </li><li> Konak-patojen etkileşimlerinin bu alanlarda Pili oluşturan bakteriler için zenginleştirilmiş olarak, yüksek büyütme görüntüleme için epitel hücrelerinin kenarlarına yapışmış bakteri görüntüleme öncelik. </li></ol> 4. İstatistiksel Analizler Pili quantifications değerlendirin <ol><li> H. analiz ImageJ yazılımı kullanılarak H. pylori Çağ-T4SS piluslar piliated Phe arzeden hücre zarları / hücre sayısının yanı sıra yüzde ölçmek içinAşağıdaki talimatlara göre notype. </li><li> ImageJ yazılım açık mikrografiğidir dosyaları. Düzgün genişlikte (10-13 nm) ve uzunluğu (60-150 nm) bakteriyel hücre ve konakçı hücre arasında oluşturulan yapılar olarak Pili belirlenmesi. Not: pilus yapısı büyük ATPaz güçler tip IV sekresyon pilus düzeneği kodlayan gende bir etkisiz hale liman bu Δ kafesini mutant soylar olarak izogenik suşları ilgilidir WT bakteriyel suşlar üzerinde mevcut olan, ancak mevcut değildir. </li><li> &quot;Analiz&quot; sekmesine tıklayarak ardından düz çizgi aracıyla bir çizgi çizerek tarafından ölçme aracı kalibre. Açılan menüden &quot;Set Ölçeği&quot; seçin ve kutuya etiketli &quot;Bilinen uzaklık&quot; içine büyütme bar değerini girin ve uygun ayarı (nm) uzunluğu birimini ayarlayın. &quot;Tamam&quot; ı tıklayın. </li><li> Pilus uzunluğu veya genişliği boyunca çizmek için düz çizgi aracını kullanarak Çağ-T4SS Pili ölçün ve sonra her Pilus ölçmek için &quot;Ctrl-M&quot; tuşuna basın. Ölçülen değerler ile bir diyalog kutusu gözlemlemek. Bu değerleri kaydedin veya kopyalayın ve bir elektronik tablo programı yapıştırın. </li><li> Uzunluğu kullanın ve genişlik parametreleri önceden Çağ-T4SS profilini uymayan 24, göz ardı özellikleri kurdu. Sonra 10-13 nm genişlik ve 60-150 nm uzunluk eşik dahilinde değerlere dayalı pili sayısını ölçmek. </li><li> GraphPad Prism yazılımı kullanılarak iki kuyruklu Student t testi ile istatistiksel pili ve miktarının analiz. </li></ol> 5. İsteğe bağlı: IL-8 salgısını değerlendirilmesi Pilus üretimi ile ilişkilendirir için <ol><li> Adım 2.1 süpernatanlarm kullanarak, kit ile üreticinin talimatlarına (Malzeme ve Ekipmanları Tablo bakınız) başına bir IL-8 ELISA gerçekleştirin. </li><li> IL-8 konakçı hücreler tarafından salgılanan analiz eder ve tek başına ortam içinde yetiştirildiğinde, WT PMSS1 göre yüzde IL-8, indüksiyon hesaplar. Böylece GraphPad Prism kullanılarak iki kuyruklu Student t testi kullanılarak istatistiksel analizftware. </li></ol>

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The authors declare that they have no competing financial interests.

Demir, bakteriyel patojenler dahil olmak üzere yaşamın birçok formları için önemli bir mikrobesleyicidir. Mikroorganizmaları istila canlılığı kısıtlamak için bir çaba, omurgalı ana &quot;beslenme dokunulmazlığı&quot; 18 olarak bilinen bir süreç içinde besin demir müsadere. Buna karşılık olarak, bakteriyel patojenler çevrelerini algılamak ve demir toplama sistemleri, toksinler ve toksin salgı makine 19,20 olarak virülans özelliklerinin detaylandırılması düzenleyen...

H. pylori infection is a significant risk factor for gastric cancer1. However, disease outcomes vary and depend on numerous factors such as host genetics, genetic diversity of H. pylori strains, and environmental elements such as host diet11. Previous reports have established that a correlation exists between H. pylori infection, iron deficiency (as measured by decreased blood ferritin and hemoglobin concentrations), and increased proinflammatory cytokine production, including IL-8 secretion, which ultimately leads to increased gastric disease progression12. Acute H. pylori infection is also associated with hypochlorhydria which impairs the host&#8217;s ability to absorb nutrient iron, and ultimately leads to changes in iron homeostasis13. These clinical findings suggest that iron availability within the gastic niche could be an important factor in disease outcome. In fact, animal models of H. pylori infection have demonstrated that low dietary iron consumption exacerbates gastric disease14. The reduced iron levels in these animals necessitate that H. pylori induce an iron-acquisition response in order to obtain the iron needed for bacterial replication. H. pylori has the capacity to perturb iron trafficking within host cells to facilitate bacterial replication in a CagA-dependent fashion15. Interestingly, the cag-pathogenicity island has been shown to be regulated by the iron-responsive transcription factor Fur16,17. Furthermore, Cag+ strains are associated with increased inflammation and gastric diseases such as cancer1. These findings support a model whereby H. pylori alters Cag-T4SS expression in an effort to obtain iron from host cells that reside in an iron deplete environment resulting in exacerbated disease outcomes. 
Two factors that increase inflammation and morbidity are Cag expression and low dietary iron intake. These facts support the hypothesis that reduced iron availability increases the production of Cag-T4SS pili at the host pathogen interface resulting in worse gastric disease11-14. The goal of the method provided in this manuscript is to establish the role of the micronutrient iron in the regulation of the Cag-T4SS pilus biogenesis. In previous work, we utilized two approaches to observe an iron-dependent increase in Cag-T4SS expression. First, output strains from animals maintained on high and low iron diets were analyzed and revealed that low-iron diet output strains produced more Cag-T4SS pili than high-iron diet strains14. Second, growing the H. pylori 7.13 strain in vitro in iron replete conditions resulted in reduced pili formation while cells grown in the presence of an iron chelator produced significantly more pili. 
We have continued to investigate the iron-dependent regulation of Cag-T4SS pili phenotype and offer the following optimized protocol and representative results performed with an additional Helicobacter pylori strain, PMSS1. The rationale behind the development of this technique was to correlate increased Cag-T4SS activity in conditions of iron-limitation with increased Cag-T4SS pilus formation. The broader implication and use of this technique will provide optimized culture conditions that result in elevated production of the Cag-T4SS pili. This assay will be useful to researchers seeking to determine the composition and architecture of the Cag-T4SS by enriching for this important bacterial surface feature. The sample preparation and visualization by field-emission gun electron microscopy has numerous advantages over alternative techniques such as light-microscopy methods to visualize the Cag-T4SS and will be appropriate to investigators interested in studying the regulation of this organelle10.

<table><tbody><tr><td>Modified brucella broth</td><td></td><td></td><td></td></tr><tr><td>Peptone from casein (10 g/L)</td><td>Sigma</td><td>70172</td><td></td></tr><tr><td>Peptic digest of animal tissue (10 g/L)</td><td>Sigma</td><td>70174</td><td></td></tr><tr><td>Yeast extract (2 g/L)</td><td>Sigma</td><td>92144</td><td></td></tr><tr><td>Dextrose (1 g/L)</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Sodium chloride (5 g/L)</td><td>Thermo Fisher</td><td>S271-10</td><td></td></tr><tr><td>Cholesterol (250X) (4 ml/L)</td><td>Life Technologies</td><td>12531018</td><td></td></tr><tr><td>Ferric chloride (100 or 250 uM)</td><td>Sigma</td><td>157740-100G</td><td></td></tr><tr><td>Dipyridyl (200 uM)</td><td>Sigma</td><td>D216305-100G</td><td></td></tr><tr><td>Modified RPMI</td><td></td><td></td><td></td></tr><tr><td>RPMI+HEPES (1X)</td><td>Life Technologies</td><td>22400-121</td><td></td></tr><tr><td>Fetal bovine serum (100 ml/L)</td><td>Life Technologies</td><td>10438-026</td><td></td></tr><tr><td>Electron Microscopy Preparation</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde (2.0% aqueous)</td><td>Electron Microscopy Sciences</td><td>15713</td><td></td></tr><tr><td>Gluteraldehyde (2.5% aqueous)</td><td>Electron Microscopy Sciences</td><td>16220</td><td></td></tr><tr><td>Sodium cacodylate (0.05 M)</td><td>Electron Microscopy Sciences</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide (0.1% aqueous)</td><td>Electron Microscopy Sciences</td><td>19150</td><td></td></tr><tr><td>Ethanol (absolute)</td><td>Sigma</td><td>E7023</td><td></td></tr><tr><td>Colloidal silver paint</td><td>Electron Microscopy Sciences</td><td>12630</td><td></td></tr><tr><td>SEM sample stubs</td><td>Electron Microscopy Sciences</td><td>75220</td><td></td></tr><tr><td>Coverslips</td><td>Thermo Fisher</td><td>08-774-383</td><td></td></tr><tr><td>IL-8 Secretion Evaluation</td><td></td><td></td><td></td></tr><tr><td>Quantikine IL-8 ELISA kit</td><td>R&amp;amp;D Systems</td><td>D8000C</td><td></td></tr></tbody></table>

high resolution electron microscopy of the helicobacter pylori cag type iv secretion system pili produced in varying conditions of iron availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

Bu yazıda, demir kullanılabilirliği değişen koşulları H. modüle kapasitesine sahip olduğunu göstermiştir ana patojen arayüzünde pilori Çağ-T4SS pilus biyogenez. H. tek başına ortamda kültürlenen zaman H. pylori 3 Pili / hücre ortalama oluşturur. H. H. pylori demir yetiştirilir tüketen bakteri büyümesi (Şekil 1), alt inhibitördür (sentetik kenetleyici dipiridil kullanılarak) şartları altında, bakteriler üreten bir çok Çağ-T4...

Watch this Scientific Journal Video about High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability at JoVE.c

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Yüksek çözünürlüklü elektron mikroskobu<em&gt; Helikobakter pilori</emDemir Kullanılabilirlik Değişen Koşullarda Üretilen&gt; Çağ Tip IV Salgı Sistemi Pili

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

high-resolution-electron-microscopy-helicobacter-pylori-cag-type-iv

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

15.0K Görüntüleme.  Vanderbilt University School of Medicine. Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Video: High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE Kullanılarak Elektron Kriyo-mikroskopi Görüntülerinden Yüksek Çözünürlüklü Tek Parçacık Analizi

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Biyokimya

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12&#39;nin Ekspresyon, Saflaştırma ve Antimikrobiyal Aktivitesi

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

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 ...

İmmünoloji ve Enfeksiyon

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.
X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented.

Ayagin Gram-negatif bakterilerde temel Metal alımı: x-ışını Floresans, Radioisotopes ve hücre ayırma

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for ...

Kimya

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Ana Hücreleri ile Anaerobik Bakteriler Etkileşim değerlendirilmesi için bir model organizma olarak Porphyromonas gingivalis

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

Rolü seğirmesi Motilitesi görselleştirme ve Karakterizasyonu<em&gt; PilG</em&gt; &#39;de<em&gt; Xylella fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Fare modelleri Helicobacter enfeksiyonu ve gastrik patolojiler

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Legionella pnömophila Dot/Icm Salgı Sisteminin Spatiotemporal Özelliklerini Çözmek İçin Canlı Hücre Görüntüleme ve Kriyo-Elektron Tomografisi Uygulama

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

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 ...

Impact of Helicobacter pylori Infection on Retina and Choroid Using SS-OCT

Retinal and Choroidal Thickness Changes in Populations with Helicobacter pylori Infection by Swept-Source Optical Coherence Tomography

Around half of the world&#39;s population is infected with Helicobacter pylori (H. pylori), which is closely related to several ocular diseases. The study aims to evaluate the retinal and choroidal thickness changes in subjects with H. pylori infection by swept-source optical coherence tomography (SS-OCT). The ophthalmic examination and 13C-urea breath test (13C-UBT) were performed on all subjects participating in the cross-sectional study. The participants were divided into H. pylori (+) and H. pylori (&#8722;) groups depending on the 13C-UBT results. This study covered 2574 right eyes from 2574 subjects with H. pylori infection and 2574 right eyes from 2574 age- and sex-matched individuals without H. pylori infection. Out of the nine sectors of the early treatment diabetic retinopathy study (ETDRS) grid, the maximum retinal thickness was in the inner superior sector, while the minimum was in the center sector. The maximum choroidal thickness was in the inner superior sector, while the minimum was in the outer nasal sector. The choroid of each area of the ETDRS subfield in the H. pylori (+) group was significantly thicker than that in the H. pylori (&#8722;) group, but retinal thickness did not show any difference between the two groups. Increased choroidal thickness may be an early indicator of H. pylori-associated retinal or choroidal diseases.

Here, we compared macular retinal and choroidal thickness in populations with and without Helicobacter pylori infection using a swept-source optical coherence tomography (SS-OCT) device, which is one of the recent milestones in the development of retinal and choroidal visualization.

Around half of the world&#39;s population is infected with Helicobacter pylori (H. pylori), which is closely related to several ocular diseases. The study ...

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