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. pylori Wachstum bei unterschiedlichen Bedingungen von Iron Verfügbarkeit und Co-Kultur mit Menschen Magenepithelzellen <ol><li> Wählen H. pylori-Stamm PMSS1 für diese Untersuchungen, weil es ein intaktes cag Pathogenität Insel und drückt eine IV-Sekretionssystem Funktionieren Typ. Auch verwenden einen isogenen Cage Mutante (PMSSI Δ Cage) als Negativkontrolle. Wachsen Bakterien auf TSA-Platten mit 5% Schafblut (Blut-Agar-Platten) für 24 h ergänzt war, bei 37 ° C in Gegenwart von 5% CO 2. HINWEIS: Bereiten Reagenzien und montieren Materialien wie in der Materialien und Geräte Tabelle skizziert. Endkonzentrationen für jedes Reagenz ist in Klammern aufgeführt. </li><li> Kratzen Bakterien von der Platte mit einem sterilen Wattestäbchen und impfen in modifizierte Brucellabrühe aus den Komponenten (siehe Materialien und Geräte Tabelle) und mit Cholesterin ergänzt. Kulturen über Nacht bei 37 inkubieren° C in Raumluft mit 5% CO 2, ergänzt mit 200 Umdrehungen pro Minute (rpm) schüttelt. HINWEIS: Cholesterin anstelle von fötalem Rinderserum auf die Tatsache zurückzuführen, dass Serum-Komplex verwendet; mit zahlreichen Quellen von Nährstoffen Eisen wie Häm, die Variabilität der Ergebnisse verursacht. </li><li> Am Tag der Kultivierung bakterieller, Samen AGS menschlichen Magen-Epithelzellen (ATCC CRL-1739) auf Poly-D-Lysin-behandelten Deckgläschen in 12-Well-Platten (ca. 1 x 10 5 Zellen pro Vertiefung). </li><li> Am folgenden Tag, verdünnte Bakterienkulturen bis zu einer OD600 von 0,3 in modifizierten Brucellabrühe allein oder mit 100 &amp; mgr; M FeCl 3, 200 &amp; mgr; M Dipyridyl (ein synthetisches Eisen-Chelator) oder 200 uM Dipyridyl plus 250 &amp; mgr; M FeCl 3 ergänzt. Diese Kulturen für 4 Stunden bei 37 ° C inkubieren in Raumluft mit 5% CO 2, ergänzt mit Schütteln bei 200 Upm. </li><li> Zentrifuge Bakterien bei 1.000 xg, um Zellen zu sammeln und die Überstände zu entfernen, bevor Resuspendieren in ein gleiches Volumen frischer modifiziert Brucella-Brühe, ergänzt mit Cholesterin. Maßnahme OD600 der Kultur (OD 600 = 1,0 = 5,5 x 10 8 Zellen / ml) und Bakterien in den Epithelzellen in einer Multiplizität der Infektion (MOI) von 20: 1. Führen serielle Verdünnungen und Platte Bakterienzellen auf Blutagarplatten, um bakterielle Zelllebensfähigkeit zu beurteilen. </li><li> Inkubieren H. pylori und AGS menschlichen Magen-Epithelzellen Co-Kulturen für 4 Stunden bei 37 ° C in Gegenwart von 5% CO 2 in statischen Bedingungen vor dem Durchführen der Rasterelektronenmikroskopie (SEM) Probenvorbereitung. </li></ol> 2. SEM Probenvorbereitung zur H. Visualisieren pylori Cag-T4SS Pili <ol><li> Entfernen H. pylori und AGS Co-Kulturen aus dem Inkubator und dekantieren Kulturüberstand (außer für IL-8 ELISA). Vorsichtig mit 0,05 M Natriumcacodylat-Puffer (pH 7,4), drei Mal waschen. Fix Proben für 2-4 Stunden bei Raumtemperatur in einer Lösung von 2,0% Paraformaldehyd, 2,5% Glutaraldehyd und 0,05 M Natriumcacodylatpuffer. Nach der primären Fixierung, waschen Proben dreimal mit 0,05 M Natriumcacodylatpuffer. </li><li> Zuführen Sekundärfixation mit 0,1% Osmiumtetroxid in 0,05 M Natriumcacodylatpuffer in zwei aufeinanderfolgenden 10 min Fixierungsschritte. Nach sekundäre Fixierung, waschen Proben dreimal mit 0,05 M Natriumcacodylatpuffer. </li><li> Nach der primären und sekundären Fixierung, Dehydratisierung Proben durch aufeinanderfolgende Waschen mit steigenden Konzentrationen von Ethanol gemäß Tabelle 1. </li><li> Nach Ethanol-Dehydratisierung, führen drei sequentielle Wasch von flüssigem Kohlendioxid. Dann trockenen Proben an einem kritischen Punkt mit einem kritischen Punkt Trockner bei einer Temperatur von 31,1 ° C und einem Druck von 1073 PSI. </li><li> Berg Deckgläser auf Aluminium SEM Proben Stubs und malen Sie mit einer dünnen Linie von kolloidalem Silber an der Probenkante geeignete Erdung zu erreichen und zu vermeiden Lade während SEM-Bildgebung. </li><li> Coat Proben mit 5 nmGold-Palladium mit einem Sputter-Mantel Maschine Probe Sekundärelektronensignal und Kanteneffekt zu erhöhen. </li></ol> 3. Imaging Parameter für die hochauflösende SEM Analysen <ol><li> Ansicht Proben mit einem Feldemissions-Gun Rasterelektronenmikroskop (FEG-SEM). </li><li> Bild in einem Hochvakuumbetrieb bei einem Arbeitsabstand von 5-10 mm. </li><li> Stellen Sie die Beschleunigungsspannung bis 5 kV. </li><li> Stellen Punktgröße auf 2-2,5. </li><li> Kippen Sie die Probe zwischen 15-25 Grad, um eine bessere Sicht auf die Wirt-Pathogen-Schnittstelle zu erreichen. </li><li> Priorisieren Abbilden Bakterien haft zu den Kanten der Epithelzellen bei hoher Vergrößerung Betrachtung, da diese Bereiche der Wirt-Pathogen-Wechselwirkungen sind für die Pili-bildenden Bakterien angereichert. </li></ol> 4. Statistische Analysen zu Pili Quantifizierungen auswerten <ol><li> Analysieren H. pylori CAG T4SS Pili mit ImageJ Software Anzahl von Pili / Zelle sowie Prozent der Zellen zu quantifizieren zeigt die piliated pheNOTYPE pro Erklärung unten. </li><li> Offene mikroskopische Aufnahme Dateien in ImageJ Software. Identifizierung Pili als Strukturen zwischen der Bakterienzelle und Wirtszelle mit einheitlicher Breite (10-13 nm) und Länge (60-150 nm) gebildet. HINWEIS: Die Pilus Struktur auf WT Bakterienstämme vorhanden, aber abwesend isogenen Stämme wie Δ Cage Mutantenstämme, die eine Inaktivierung des Gens die Haupt ATPase, die Befugnisse Typ IV Sekretion Pilus-Zusammenbau kodiert Hafen ist. </li><li> Kalibrieren Sie das Messwerkzeug durch Ziehen einer Linie mit der Geraden Werkzeug und dann auf der Registerkarte &quot;Analysieren&quot;. Wählen Sie &quot;Scale&quot; aus dem Dropdown-Menü und geben Sie den Vergrößerungs bar Wert in das Feld &quot;bekannte Strecke&quot; und stellen Sie die Einheit der Länge in der entsprechenden Einstellung (nm). Klicken Sie auf &quot;OK&quot;. </li><li> Messen Sie Cag-T4SS Pili mit der geraden Linie Werkzeug, um über die Länge oder Breite des Pilus ziehen und drücken Sie &quot;Strg-M&quot;, um jeden Pilus messen. Beachten Sie eine Dialogbox mit den gemessenen Werten. Notieren Sie diese Werte oder Kopieren und Einfügen in ein Tabellenkalkulationsprogramm. </li><li> Verwenden Sie die Länge und Breite Parameter vorher festgelegten 24, Missachtung Features, die passen nicht in das Profil der Cag-T4SS. Dann quantifizieren die Anzahl der Pili basierend auf Werten, die in den 10 bis 13 nm Breite und 60 bis 150 nm Länge Cutoffs sind. </li><li> Analysieren Sie den Quantifizierung von Pili statistisch durch zweiseitigen Student t-Test mit GraphPad Prism Software. </li></ol> 5. Optional: Auswertung der IL-8-Sekretion mit Pilus Produktion Korrelieren <ol><li> Mit Überstände aus Schritt 2.1, führen Sie eine IL-8 ELISA gemäß den Herstellerangaben mit dem Kit (siehe Materialien und Ausstattungstabelle). </li><li> Analysieren Sie den IL-8 durch Wirtszellen sezerniert und berechnen die prozentuale IL-8 Induktion nach WT PMSS1 allein im Medium gezüchtet. Statistische Analysen mit zweiseitigen Student t-Test mit GraphPad Prism software. </li></ol>

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<li>Bourzac, K., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+pylori-host+cell+interactions+mediated+by+type+IV+secretion%5BTitle%5D)+AND+%22Cell+Microbiol%22%5BJournal%5D)">Helicobacter pylori-host cell interactions mediated by type IV secretion.</a> Cell Microbiol. 7 (7), 911-919 (2005).</li>
<li>Bronte-Tinkew, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+pylori+cytoxin-associated+gene+A+activates+the+signal+transducer+and+activator+of+transcription+3+pathway+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Helicobacter pylori cytoxin-associated gene A activates the signal transducer and activator of transcription 3 pathway in vitro and in vivo.</a> Cancer Res. 69 (2), 632-639 (2009).</li>
<li>Johnson, E. M., Gaddy, J. A., Cover, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alterations+in+Helicobacter+pylori+triggered+by+contact+with+gastric+epithelial+cells%5BTitle%5D)+AND+%22Front.+Cell.+Infect.+Microbiol%22%5BJournal%5D)">Alterations in Helicobacter pylori triggered by contact with gastric epithelial cells.</a> Front. Cell. Infect. Microbiol. (2), 17(2012).</li>
<li>Rohde, M., Puls, J., Buhrdorf, R., Fischer, W., Haas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+sheathed+surface+organelle+of+the+Helicobacter+pylori+cag+type+IV+secretion+system%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system.</a> Mol. Microbiol. 49 (1), 219-234 (2003).</li>
<li>Cover, T. L., Peek, R. M. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diet%2C+microbial+virulence+and+Helicobacter+pylori+induced+gastric+cancer%5BTitle%5D)+AND+%22Gut+Microbes%22%5BJournal%5D)">Diet, microbial virulence and Helicobacter pylori induced gastric cancer.</a> Gut Microbes. 4 (6), 482-493 (2013).</li>
<li>Queiroz, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+gastric+IL-1%CE%B2+concentration+and+iron+deficiency+parameters+in+Helicobacter+pylori+infected+children%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Increased gastric IL-1β concentration and iron deficiency parameters in Helicobacter pylori infected children.</a> PLoS One. 8 (2), 57420(2013).</li>
<li>Harris, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+pylori-associated+hypochlorhydria+and+development+of+iron+deficiency%5BTitle%5D)+AND+%22J.+Clin.+Pathol%22%5BJournal%5D)">Helicobacter pylori-associated hypochlorhydria and development of iron deficiency.</a> J. Clin. Pathol. 66 (4), 343-347 (2013).</li>
<li>Noto, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron+deficiency+accelerates+Helicobacter+pylori-induced+carcinogenesis+in+rodents+and+humans%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans.</a> J. Clin. Invest. 123 (1), 479-492 (2013).</li>
<li>Tan, S., Noto, J. M., Romero-Gallo, J., Peek, R. M. Jr, Amieva, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+pylori+perturbs+iron+trafficking+in+the+epithelium+to+grow+on+the+cell+surface%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface.</a> PLoS Pathog. 7 (5), (2011).</li>
<li>Danielli, A., Roncarati, D., Delany, I., Chiarini, V., Rappouli, R., Scarlato, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+dissection+of+the+Helicobacter+pylori+Fur+regulatory+circuit+by+genome-wide+location+analysis%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis.</a> J. Bacteriol. 188 (13), 4654-4662 (2006).</li>
<li>Pich, O. Q., Carpenter, B. M., Gilbreath, J. J., Merrell, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detailed+analysis+of+Helicobacter+pylori+Fur-regulated+promoters+reveals+a+Fur+box+core+sequence+and+novel+Fur-regulated+genes%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">Detailed analysis of Helicobacter pylori Fur-regulated promoters reveals a Fur box core sequence and novel Fur-regulated genes.</a> Mol. Microbiol. 84 (5), 921-941 (2012).</li>
<li>Cassat, J. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron+in+infection+and+immunity%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">Iron in infection and immunity.</a> Cell Host Microbe. 13 (5), 509-519 (2013).</li>
<li>Nielubowicz, G. R., Mobley, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host-pathogen+interactions+in+urinary+tract+infection%5BTitle%5D)+AND+%22Nat.+Rev.+Urol%22%5BJournal%5D)">Host-pathogen interactions in urinary tract infection.</a> Nat. Rev. Urol. 7 (8), 430-441 (2010).</li>
<li>Brickman, T. J., Cummings, C. A., Liew, S. Y., Relman, D. A., Armstrong, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+profiling+of+the+iron+starvation+response+in+Bordatella+pertussis+provides+new+insights+into+siderophore+utilization+and+virulence+gene+expression%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Transcriptional profiling of the iron starvation response in Bordatella pertussis provides new insights into siderophore utilization and virulence gene expression.</a> J. Bacteriol. 193 (18), 4798-4812 (2011).</li>
<li>Mobley, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+pylori+factors+associated+with+disease+development%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Helicobacter pylori factors associated with disease development.</a> Gastroenterology. 113 (6), 21-28 (1997).</li>
<li>Testerman, T. L., Conn, P. B., Mobley, H. L., McGee, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nutritional+requirements+and+antibiotic+resistance+patterns+of+Helicobacter+species+in+chemically+defined%5BTitle%5D)+AND+%22J.+Clin.+Microbiol%22%5BJournal%5D)">Nutritional requirements and antibiotic resistance patterns of Helicobacter species in chemically defined.</a> J. Clin. Microbiol. 44 (5), 1650-1658 (2006).</li>
<li>Senkovich, O., Ceaser, S., McGee, D. J., Testerman, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unique+host+iron+utilization+mechanisms+of+Helicobacter+pylori+revealed+with+iron-deficient+chemically+defined+media%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Unique host iron utilization mechanisms of Helicobacter pylori revealed with iron-deficient chemically defined media.</a> Infect. Immun. 78 (5), 1841-1849 (2010).</li>
<li>Shaffer, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Helicobacter+pylori+exploits+a+unique+repertoire+of+type+IV+secretion+system+components+for+pilus+assembly+at+the+bacteria-host+cell+interface%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.</a> PLoS Pathog. 7 (9), (2011).</li>
<li>Barrozo, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+plasticity+in+the+type+IV+secretion+system+of+Helicobacter+pylori%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Functional plasticity in the type IV secretion system of Helicobacter pylori.</a> PLoS Pathog. 9 (2), (2013).</li>
</ol>

The authors declare that they have no competing financial interests.

Eisen ist ein wesentliches Spurenelement für die meisten Lebensformen, einschließlich bakterieller Pathogene. In dem Bemühen, die Lebensfähigkeit der eindringenden Mikroorganismen zu beschränken, Wirbeltierwirten maskieren Nährstoff Eisen in einem Prozess als &quot;Ernährungs Immunität&quot; 18 bekannt. In Reaktion darauf wurden bakterielle Pathogene entwickelt, um Eisen als globales Signal-Molekül zu verwenden, um die Umgebung zu erfassen und zu regeln für die Ausarbeitung von Virulenz-Eigenschafte...

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.

In diesem Bericht haben wir gezeigt, dass die Bedingungen der unterschiedlichen Eisenverfügbarkeit die Fähigkeit, H. modulieren pylori Cag-T4SS Pilus-Biogenese im Wirt-Pathogen-Schnittstelle. Wenn in Medium allein, H. kultiviert pylori bildet einen Durchschnitt von 3 Pili / Zelle. Wenn H. pylori ist in Eisen gewachsen erschöpfen Bedingungen (unter Verwendung des synthetischen Chelator Dipyridyl), die Unter hemmend auf das Bakterienwachstum (Abbildung 1) si...

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.

Hohe Auflösung Elektronenmikroskopie der<em&gt; Helicobacter pylori</em&gt; Cag Typ IV-Sekretionssystem Pili Produziert in variierenden Bedingungen von Iron Verfügbarkeit

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

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

15.1K Aufrufe.  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.

Serie: 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.

Hochauflösende Einzelteilchenanalyse aus Elektronen-Kryomikroskopie-Bildern mit SPHIRE

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

Forschung

Biochemie

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Engineering adhärenten Bakterien durch Schaffung eines einheitlichen Synthetic Curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Biotechnik

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.

Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

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

Immunologie und Infektion

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.

Wesentlichen Metall Aufnahme in Gram-negativen Bakterien: Röntgen-Fluoreszenz, Radioisotopen, und Zelle Fraktionierung

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

Chemie

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.

Porphyromonas gingivalis als Modellorganismus für die Beurteilung der Interaktion von anaeroben Bakterien mit Wirtszellen

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.

Visualisierung von Twitching Motilität und Charakterisierung der Rolle des<em&gt; Pilg</em&gt; in<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.

Maus-Modellen der Helicobacter -Infektion und Magen-Erkrankungen

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.

Anwendung von Live Cell Imaging und Cryo-Electron Tomography zur Auflösung der raumzeitlichen Merkmale des Legionella pneumophila Dot/Icm Sekretionssystems

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

<meta charset="utf8"></head><body>T3SS-Aktivität bei Yersinia pseudotuberculosis unter Eisenmangel und anaerobem 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.

<meta charset="utf8"></head><body>Quantifizierung der Aktivität des Yersinia pseudotuberculosis Typ III Sekretionssystems nach Eisenmangel und anaerobem Wachstum

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.

Quantifizierung der Aktivität des Yersinia pseudotuberculosis Typ III Sekretionssystems nach Eisenmangel und anaerobem Wachstum

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

Hohe Auflösung Elektronenmikroskopie der

Zusammenfassung

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Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

Wesentlichen Metall Aufnahme in Gram-negativen Bakterien: Röntgen-Fluoreszenz, Radioisotopen, und Zelle Fraktionierung

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Quantifizierung der Aktivität des Yersinia pseudotuberculosis Typ III Sekretionssystems nach Eisenmangel und anaerobem Wachstum

Quantifizierung der Aktivität des Yersinia pseudotuberculosis Typ III Sekretionssystems nach Eisenmangel und anaerobem Wachstum