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 crescita in condizioni particolari di ferro Disponibilità e co-coltura con cellule umane epiteliali gastriche <ol><li> Seleziona H. pylori ceppo PMSS1 per questi studi perché ha un intatto patogenicità cag isola ed esprime un tipo di funzionamento del sistema di secrezione IV. Utilizzare anche una gabbia mutante isogenico (PMSSI Δ Cage) come controllo negativo. Grow batteri su piastre di TSA addizionato con 5% di sangue di pecora (piastre di agar sangue) per 24 ore a 37 ° C in presenza del 5% di CO 2. NOTA: Preparare i reagenti e assemblare materiali come indicato in materiali e attrezzature della tabella. Le concentrazioni finali per ciascun reagente è elencato tra parentesi. </li><li> Raschiare i batteri dalla piastra utilizzando un applicatore con punta di cotone sterili e inoculare in brodo di Brucella modificato preparata dai componenti (vedi Materiali e attrezzature Table) e integrato con il colesterolo. Incubare le culture per una notte a 37° C in aria ambiente integrato con il 5% di CO 2 con agitazione a 200 giri al minuto (rpm). NOTA: colesterolo viene utilizzato al posto di siero fetale bovino a causa del fatto che il siero è complesso; contenente numerose fonti di ferro nutrienti come eme che provoca variabilità nei risultati. </li><li> Il giorno della coltura batterica, seminare le cellule epiteliali gastriche umane AGS (ATCC CRL-1739) su poli-D-lisina coprioggetto trattati in piastre da 12 pozzetti (circa 1 x 10 5 cellule per pozzetto). </li><li> Il giorno seguente, diluire colture batteriche ad una OD600 di 0,3 in solo o integrato con 100 mM FeCl 3, 200 mM dipiridile (un chelante del ferro sintetico), o 200 micron dipiridile più 250 micron FeCl 3 brodo brucella modificato. Incubare le colture per 4 ore a 37 ° C in aria ambiente supplementato con 5% di CO 2 con agitazione a 200 rpm. </li><li> Batteri centrifugare a 1000 xg per raccogliere le cellule e rimuovere surnatanti prima di risospendere in un volume uguale di fresco Brucella modificato brodo integrata con colesterolo. Misura OD600 della cultura (OD600 = 1,0 = 5,5 x 10 8 cellule / ml) e aggiungere batteri alle cellule epiteliali in una molteplicità di infezione (MOI) di 20: 1. Effettuare diluizioni seriali e le cellule batteriche piastra su piastre di agar sangue per valutare la vitalità cellulare batterica. </li><li> Incubare H. cellule epiteliali gastriche umane pylori e AGS co-coltura per 4 ore a 37 ° C in presenza del 5% di CO 2 in condizioni statiche prima di eseguire la microscopia elettronica a scansione (SEM) preparazione del campione. </li></ol> 2. SEM Preparazione del campione per visualizzare H. pylori Cag-T4SS Pili <ol><li> Rimuovere H. pylori e co-colture AGS dal surnatante incubatore e decantare la cultura (salvo per IL-8 ELISA). Lavare delicatamente tre volte con sodio 0,05 M tampone cacodilato (pH 7,4). Fix campioni per 2-4 ore a temperatura ambiente in una soluzione di 2,0% paraformaldeide, 2.5% Glutaraldeide, e tampone cacodilato di sodio 0,05 M. Dopo fissazione primaria, lavare campioni tre volte con tampone cacodilato di sodio 0,05 M. </li><li> Eseguire ancoraggio secondario utilizzando 0,1% tetrossido di osmio in 0,05 M di sodio tampone cacodilato in due fasi sequenziali 10 min di fissaggio. Dopo la fissazione secondaria, lavare campioni tre volte con tampone cacodilato di sodio 0,05 M. </li><li> Dopo fissazione primaria e secondaria, disidratano campioni di lavaggio sequenziale con concentrazioni crescenti di etanolo come da Tabella 1. </li><li> Dopo etanolo disidratazione, effettuare tre lavaggi sequenziali di anidride carbonica liquida. Poi i campioni a secco in un punto critico con una macchina asciugatrice punto critico ad una temperatura di 31,1 ° C e pressione di 1073 PSI. </li><li> Monte coprioggetto sul stub campione alluminio SEM e dipingere con una linea sottile di argento colloidale sul bordo del campione per ottenere adeguata messa a terra ed evitare di ricarica durante l&#39;imaging SEM. </li><li> Campioni cappotto con 5 nm dioro-palladio utilizzando una macchina cappotto polverizzazione di aumentare campione di segnale degli elettroni secondari e l&#39;effetto bordo. </li></ol> 3. I parametri di imaging per analisi ad alta risoluzione SEM <ol><li> Vedi i campioni con un campo-Emission Gun microscopio elettronico a scansione (SEM-FEG). </li><li> Immagine in una modalità di aspirazione ad alta ad una distanza di lavoro di 5-10 mm. </li><li> Impostare la tensione di accelerazione di 5 kV. </li><li> Impostare dimensione del punto di 2-2,5. </li><li> Inclinare il campione tra i 15-25 ° per ottenere una migliore visualizzazione dell&#39;interfaccia ospite-patogeno. </li><li> Priorità di imaging batteri aderenti ai bordi delle cellule epiteliali per la visualizzazione ad alto ingrandimento, in quanto queste aree di interazione ospite-patogeno sono arricchiti per i batteri-Pili formando. </li></ol> 4. Analisi statistica per valutare Pili Quantificazione <ol><li> Analizzare H. pylori Cag-T4SS pili utilizzando il software ImageJ per quantificare il numero di pili / cellule e per cento delle cellule esibendo il piliated phenotype secondo le istruzioni riportate di seguito. </li><li> Aprire i file microfotografia nel software ImageJ. Identificare pili come strutture formate tra la cellula batterica e cellula ospite, con larghezza uniforme (10-13 nm) e la lunghezza (60-150 nm). NOTA: La struttura pilus è presente su ceppi batterici WT, ma assente in ceppi isogeni quali Δ Cage ceppi mutanti, che ospitano l&#39;inattivazione del gene che codifica per il principale ATPasi che l&#39;assemblaggio tipo poteri secrezione IV pilus. </li><li> Calibrare lo strumento di misurazione tracciando una linea con lo strumento linea retta e quindi facendo clic sulla scheda &quot;Analizza&quot;. Selezionare &quot;Imposta Scala&quot; dal menu a discesa e immettere il valore bar di ingrandimento nella casella &quot;Noto Distanza&quot; e impostare l&#39;unità di misura per l&#39;impostazione appropriata (nm). Fare clic su &quot;OK&quot;. </li><li> Misurare Cag-T4SS pili utilizzando lo strumento linea retta per disegnare su tutta la lunghezza o la larghezza del pilus e poi premere &quot;Ctrl-M&quot; per misurare ogni pilus. Osservare una finestra di dialogo con i valori misurati. Registrare questi valori o copiare e incollare in un programma di foglio di calcolo. </li><li> Utilizzare la lunghezza e la larghezza dei parametri stabiliti in precedenza 24, caratteristiche non tengono conto che non si adattano al profilo di Cag-T4SS. Poi quantificare il numero di pili in base ai valori che rientrano nelle cut-off 10-13 nm di larghezza e 60-150 nm di lunghezza. </li><li> Analizzare la quantificazione dei pili statisticamente con il test t di Student a due code utilizzando il software GraphPad Prism. </li></ol> 5. Opzionale: Valutazione della secrezione di IL-8 in correlazione con Pilus produzione <ol><li> Utilizzando surnatanti dal punto 2.1, eseguire un IL-8 ELISA secondo le istruzioni del produttore con il kit (vedi Materiali e attrezzature Tabella). </li><li> Analizzare l&#39;IL-8 secreto dalle cellule ospiti e calcolare la percentuale di IL-8 induzione rispetto al WT PMSS1 coltivate in terreno da solo. Eseguire l&#39;analisi statistica con due code il test t di Student con GraphPad Prism cosìftware. </li></ol>

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The authors declare that they have no competing financial interests.

Il ferro è un micronutriente essenziale per la maggior parte delle forme di vita, compresi i batteri patogeni. Nel tentativo di limitare la validità di microrganismi invasori, ospiti vertebrati sequestrano il ferro di nutrienti in un processo noto come &quot;immunità nutrizionale&quot; 18. In risposta a questo, batteri patogeni sono evoluti per utilizzare il ferro come molecola di segnalazione globale di percepire l&#39;ambiente circostante e regolano l&#39;elaborazione delle caratteristiche di virulenza c...

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 border="0" cellpadding="0" cellspacing="0" width="837">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:259px;"></td>
			<td style="width:196px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:475px;">Modified brucella broth</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Peptone from casein (10g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">70172</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Peptic digest of animal tissue (10g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">70174</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Yeast extract (2g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">92144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dextrose (1g/L)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">D9434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium chloride (5g/L)</td>
			<td style="width:259px;">Thermo Fisher</td>
			<td style="width:196px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cholesterol (250X) (4mL/L)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:196px;">12531018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ferric chloride (100 or 250 uM)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">157740-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dipyridyl (200 uM)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">D216305-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:259px;"></td>
			<td style="width:196px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;width:475px;">Modified RPMI</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RPMI+HEPES (1X)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:196px;">22400-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Fetal bovine serum (100 mL/L)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:196px;">10438-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:259px;"></td>
			<td style="width:196px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;width:475px;">Electron Microscopy Preparation</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Paraformaldehyde (2.0% aqueous)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">15713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gluteraldehyde (2.5% aqueous)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">16220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium cacodylate (0.05 M)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">12300</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Osmium tetroxide (0.1% aqueous)</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">19150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ethanol (absolute)</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:196px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Colloidal silver paint</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">12630</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SEM sample stubs</td>
			<td style="width:259px;">Electron Microscopy Sciences</td>
			<td style="width:196px;">75220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:259px;">Thermo Fisher</td>
			<td style="width:196px;">08-774-383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:475px;">IL-8 Secretion Evaluation</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Quantikine IL-8 ELISA kit</td>
			<td style="width:259px;">R&#38;D Systems</td>
			<td style="width:196px;">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 questa relazione, abbiamo dimostrato che le condizioni di diversa disponibilità di ferro hanno la capacità di modulare H. pylori Cag-T4SS pilus biogenesi all&#39;interfaccia patogeno host. Quando coltivate in terreno da solo, H. pylori fa una media di 3 pili / cell. Quando H. pylori è coltivato in ferro esaurire condizioni (utilizzando il chelante dipiridile sintetico) che sono sub-inibitorio per la crescita batterica (Figura 1), i batteri producono numerosi Cag-T4SS pil...

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.

Ad alta risoluzione microscopia elettronica del<em&gt; Helicobacter pylori</em&gt; Cag secrezione di tipo IV Sistema Pili Prodotto in diverse condizioni di Ferro Disponibilità

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

14.8K Views.  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