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. Crecimiento pylori en distintas situaciones de disponibilidad de hierro y co-cultivo con células gástricas epiteliales humanas <ol><li> Seleccione H. PMSS1 pylori cepa para estos estudios debido a que tiene una isla de patogenicidad cag intacta y expresa un sistema de secreción de tipo IV funcionamiento. También utilizar un mutante cagE isogénica (PMSSI Δ jaula) como un control negativo. Crecer las bacterias en placas de TSA suplementadas con 5% de sangre de oveja (placas de agar sangre) durante 24 horas a 37 ° C en presencia de 5% de CO 2. NOTA: Preparar reactivos y ensamblar materiales como se indica en la Tabla de Materiales y Equipos. Las concentraciones finales de cada reactivo se enumeran entre paréntesis. </li><li> Raspe las bacterias de la placa utilizando un aplicador con punta de algodón estéril e inocular en caldo brucella modificado preparado a partir de los componentes (ver Materiales y Equipos tabla) y se completará con el colesterol. Incubar durante una noche a 37 culturas° C en aire ambiente, complementado con 5% de CO 2 con agitación a 200 revoluciones por minuto (rpm). NOTA: El colesterol se utiliza en lugar de suero bovino fetal debido al hecho de que el suero es complejo; que contiene numerosas fuentes de hierro de nutrientes tales como heme que provoca variabilidad en los resultados. </li><li> En el día de cultivo bacteriano, semilla de células epiteliales gástricas humanas AGS (ATCC CRL-1739) sobre cubreobjetos tratados con poli-D-lisina en placas de 12 pocillos (alrededor de 1 x 10 5 células por pocillo). </li><li> El día siguiente, diluir cultivos bacterianos a una DO600 de 0,3 en caldo Brucella modificado solo o suplementado con 100 mM FeCl 3, 200 dipiridilo mu M (un quelante de hierro sintético), o 200 mM dipiridilo más 250 M FeCl 3. Incubar estos cultivos durante 4 horas a 37 ° C en aire ambiente, complementado con 5% de CO 2 con agitación a 200 rpm. </li><li> Bacterias centrifugar a 1000 xg para recoger las células y eliminar los sobrenadantes antes de resuspender in un volumen igual de caldo de Brucella modificado fresco suplementado con colesterol. Medida DO600 del cultivo (OD600 = 1,0 = 5,5 x 10 8 células / ml) y añadir las bacterias a las células epiteliales en una multiplicidad de infección (MOI) de 20: 1. Realizar diluciones en serie y las células bacterianas de la placa sobre placas de agar de sangre para evaluar la viabilidad celular bacteriana. </li><li> Incubar H. células epiteliales gástricas humanas pylori y AGS co-cultivos de 4 horas a 37 ° C en presencia de 5% de CO 2 en condiciones estáticas antes de realizar la microscopía electrónica de barrido (SEM) de preparación de muestras. </li></ol> 2. Preparación de muestras para SEM Visualice H. pylori CagA-T4SS Pili <ol><li> Retire H. pylori y co-cultivos AGS de la incubadora y decantar el sobrenadante de cultivo (a ahorrar para la IL-8 ELISA). Lavar suavemente tres veces con 0,05 M tampón de cacodilato de sodio (pH 7,4). Fijar las muestras durante 2-4 horas a temperatura ambiente en una solución de 2,0% de paraformaldehído, 2.5% De glutaraldehído, y tampón de cacodilato de sodio 0,05 M. Después de la fijación primaria, lavar las muestras tres veces con tampón de cacodilato de sodio 0,05 M. </li><li> Realizar fijación secundaria usando 0,1% de tetróxido de osmio en tampón de cacodilato de sodio 0,05 M secuencial en dos pasos 10 min de fijación. Después de la fijación secundaria, lavar las muestras tres veces con tampón de cacodilato de sodio 0,05 M. </li><li> Después de la fijación primaria y secundaria, deshidratar muestras por lavado secuencial con concentraciones crecientes de etanol según la Tabla 1. </li><li> Después de la deshidratación de etanol, realizar tres lavados secuenciales de dióxido de carbono líquido. A continuación, las muestras secas en un punto crítico utilizando una máquina secadora de punto crítico a una temperatura de 31,1 ° C y la presión de 1,073 psi. </li><li> Monte cubreobjetos en aluminio SEM talones de la muestra y la pintura con una línea delgada de plata coloidal en el borde de la muestra para lograr la conexión a tierra apropiada y evitar la carga durante la proyección de imagen SEM. </li><li> Muestras Coat con 5 nm deoro-paladio usando una máquina de capa por pulverización catódica para aumentar la señal de electrones secundarios de la muestra y efecto de borde. </li></ol> 3. Los parámetros de imagen para análisis de alta resolución de SEM <ol><li> Ver muestras con una emisión de campo Pistola microscopio electrónico de barrido (FEG-SEM). </li><li> Image en un modo de alto vacío a una distancia de trabajo de 5-10 mm. </li><li> Ajuste el voltaje de aceleración a 5 kV. </li><li> Establecer tamaño de punto a 2-2,5. </li><li> Incline la muestra entre 15-25 grados para conseguir una mejor vista de la interfaz de huésped-patógeno. </li><li> Dar prioridad a la formación de imágenes bacterias adheridas a los bordes de las células epiteliales para la visualización de gran aumento, ya que estas áreas de interacción huésped-patógeno se enriquecen para las bacterias de formación de pili. </li></ol> 4. Los análisis estadísticos para Evaluar Pili cuantificaciones <ol><li> Analizar H. pili pylori Cag-T4SS utilizando el software ImageJ para cuantificar número de pili / célula, así como por ciento de células que presentan el piliated phenotype según las instrucciones de abajo. </li><li> Micrografía archivos abiertos en el software ImageJ. Identificar pili como estructuras formadas entre la célula bacteriana y la célula huésped, con anchura uniforme (10-13 nm) y la longitud (60-150 nm). NOTA: La estructura pilus está presente en cepas bacterianas WT, pero ausente en las cepas isogénicas tales como cepas mutantes Δ jaula, que albergan una inactivación en el gen que codifica la ATPasa importante que el conjunto de la secreción de tipo IV poderes pilus. </li><li> Calibrar la herramienta de medición dibujando una línea con la herramienta de línea recta y luego hacer clic en la pestaña &quot;Analizar&quot;. Seleccione &quot;Escala de Ajuste&quot; en el menú desplegable e introduzca el valor de la barra de ampliación en el cuadro denominado &quot;distancia conocida&quot; y establecer la unidad de longitud en el valor adecuado (nm). Haga clic en &quot;Aceptar&quot;. </li><li> Mida pili Cag-T4SS utilizando la herramienta de línea recta para dibujar sobre la longitud o anchura del pilus y luego presione &quot;Ctrl-M&quot; para medir cada pilus. Observar un cuadro de diálogo con los valores medidos. Anote estos valores o copiar y pegar en un programa de hoja de cálculo. </li><li> Utilice la longitud y el ancho de los parámetros previamente establecidos 24, hacen caso omiso de las características que no se ajustan al perfil de Cag-T4SS. Entonces cuantificar el número de pili sobre la base de los valores que están dentro de los puntos de corte 10-13 nm de anchura y 60-150 nm de longitud. </li><li> Analizar la cuantificación de pili estadísticamente por la prueba t de Student de dos colas utilizando el software GraphPad Prism. </li></ol> 5. Opcional: Evaluación de la IL-8 secreción que se correlaciona con Pilus Producción <ol><li> El uso de sobrenadantes de la Etapa 2.1, realizar una IL-8 ELISA según las instrucciones del fabricante con el kit (véase Materiales y Equipo Tabla). </li><li> Analizar la IL-8 secretada por las células huésped y calcular el porcentaje de inducción de IL-8 con respecto a WT PMSS1 crecido en medio solo. Realizar el análisis estadístico utilizando dos colas la prueba t de Student utilizando GraphPad Prisma asíftware. </li></ol>

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The authors declare that they have no competing financial interests.

El hierro es un micronutriente esencial para la mayoría de las formas de vida, incluyendo los patógenos bacterianos. En un esfuerzo para restringir la viabilidad de los microorganismos invasores, los huéspedes vertebrados secuestran hierro de nutrientes en un proceso conocido como &quot;inmunidad nutricional&quot; 18. En respuesta a esto, los patógenos bacterianos han evolucionado para utilizar el hierro como una molécula de señalización global para percibir su entorno y regular la elaboración de cara...

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.

En este informe, hemos demostrado que las condiciones de variación de la disponibilidad de hierro tienen la capacidad de modular H. pylori CagA-T4SS biogénesis pilus en la interfaz de patógenos de acogida. Cuando se cultivan en medio solo, H. pylori forma un promedio de 3 pili / célula. Cuando H. pylori se cultiva en condiciones de agotar hierro (utilizando el dipiridilo quelante sintético) que son sub-inhibidor al crecimiento bacteriano (Figura 1), las bacterias producen...

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.

Microscopía Electrónica de Alta Resolución de la<em&gt; Helicobacter pylori</em&gt; Cag secreción tipo IV Sistema Pili Producido en condiciones variables de Hierro disponibilidad

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 vistas.  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