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. Crescimento pylori em diferentes condições de Ferro Disponibilidade e Co-cultura com gástrico humano células epiteliais <ol><li> Selecione H. pylori PMSS1 tensão para estes estudos porque tem uma ilha cag patogenicidade intacta e expressa um sistema de secreção do tipo IV em funcionamento. Também utilizam uma gaiola mutante isogênico (PMSSI Δ Cage) como controle negativo. Cresça as bactérias em placas de TSA suplementadas com 5% de sangue de carneiro (placas de agar de sangue) durante 24 horas a 37 ° C na presença de 5% de CO 2. NOTA: Preparar reagentes e montar materiais, conforme descrito na Tabela de Materiais e Equipamentos. Concentrações finais para cada reagente é listado entre parênteses. </li><li> Raspar as bactérias da placa, utilizando um aplicador com ponta de algodão esterilizado e inocular em caldo Brucella modificado preparado a partir de componentes (ver Materiais e Equipamento Tabela) e suplementado com colesterol. Incubar durante a noite a 37 culturas° C em ar ambiente enriquecido com 5% de CO 2 com agitação a 200 rotações por minuto (rpm). NOTA: O colesterol é utilizado em vez de soro fetal de bovino devido ao fato de que o soro é complexo; contendo numerosas fontes de nutrientes tais como ferro heme que faz com que a variabilidade nos resultados. </li><li> No dia da cultura bacteriana, semear as células epiteliais gástricas humanas AGS (ATCC CRL-1739) para poli-D-lisina lamelas tratadas, em placas de 12 poços (cerca de 1 x 10 5 células por poço). </li><li> No dia seguinte, dilui-se culturas bacterianas para um OD600 de 0,3 em caldo Brucella modificado sozinho ou suplementado com 100 | iM de FeCl3, 200 uM dipiridilo (um quelante de ferro sintético), ou 200 uM dipiridilo mais 250? M FeCl3. Incubar estas culturas durante 4 horas a 37 ° C em ar ambiente enriquecido com 5% de CO 2 com agitação a 200 rpm. </li><li> Bactérias centrifugar a 1000 xg para coletar células e remover o sobrenadante antes de ressuspender in um volume igual de caldo de Brucella modificado fresco suplementado com colesterol. Medida OD600 da cultura (DO600 = 1.0 = 5.5 x 10 8 células / ml) e adicionar bactérias às células epiteliais de uma multiplicidade de infecção (MOI) de 20: 1. Efectuar diluições em série e as células bacterianas placa em placas de agar de sangue para avaliar a viabilidade celular bacteriana. </li><li> Incubar H. células epiteliais gástricas humanas pylori e AGS co-culturas durante 4 horas a 37 ° C na presença de 5% de CO 2 em condições estáticas, antes de realizar a microscopia electrónica de varrimento (MEV), a preparação da amostra. </li></ol> 2. SEM Preparação de Amostras para Visualize H. pylori Cag-T4SS Pili <ol><li> Remover H. pylori e AGS co-culturas a partir do sobrenadante incubadora e decantar cultura (com exceção de IL-8 ELISA). Lavar cuidadosamente três vezes com tampão de 0,05 M de cacodilato de sódio (pH 7,4). Fix amostras para 2-4 horas à temperatura ambiente numa solução de 2,0% de paraformaldeído, 2,5% De glutaraldeido, e tampão de cacodilato de sódio 0,05 M. Após a fixação primária, lavar as amostras três vezes com tampão de cacodilato de sódio 0,05 M. </li><li> Realizar fixação secundária usando 0,1% de tetróxido de ósmio em tampão de cacodilato de sódio 0,05 M em dois passos sequenciais de 10 min de fixação. Após a fixação secundária, lavar as amostras três vezes com tampão de cacodilato de sódio 0,05 M. </li><li> Após a fixação primária e secundária, desidratar amostras por lavagem sequencial com concentrações crescentes de etanol conforme a Tabela 1. </li><li> Após a desidratação de etanol, realizar três lavagens seqüenciais de dióxido de carbono líquido. Em seguida, as amostras secas em um ponto crítico, utilizando uma máquina de secar ponto crítico a uma temperatura de 31,1 ºC e pressão de 1073 psi. </li><li> Monte lamelas em topos de amostra de alumínio SEM e pintar com uma linha fina de prata coloidal na borda da amostra para alcançar o aterramento adequado e evitar a cobrança durante SEM imagem. </li><li> Amostras de revestimento com 5 nm deouro-paládio usando uma máquina de revestimento por pulverização catódica para aumentar sinal de elétrons secundários da amostra e efeito de borda. </li></ol> 3. Parâmetros de imagem para análises alta resolução MEV <ol><li> Ver amostras com um campo-Emission Gun Microscópio Eletrônico de Varredura (FEG-SEM). </li><li> Imagem num modo alto vácuo a uma distância de trabalho de 5-10 mm. </li><li> Defina a tensão de aceleração de 5 kV. </li><li> Defina o tamanho do ponto de 2-2,5. </li><li> Incline a amostra entre 15-25 graus para alcançar uma melhor visão da interface do patógeno-hospedeiro. </li><li> Priorize imagiologia bactérias aderentes às bordas das células epiteliais para visualização de alta ampliação, uma vez que estas áreas de interacção hospedeiro-patogénio são enriquecidas para as bactérias formadoras de pili. </li></ol> 4. As análises estatísticas para avaliar as Pili quantificações <ol><li> Analisar H. pylori pili-Cag T4SS utilizando o software ImageJ para quantificar o número de pili / célula, bem como a percentagem de células que manifestam o piliated phenotype acordo com as instruções abaixo. </li><li> Arquivos micrografia abertas em software ImageJ. Identificar pili como estruturas formadas entre a célula e célula hospedeira bacteriana, com largura uniforme (10-13 nm) e comprimento (60-150 nm). NOTA: A estrutura pilus está presente em cepas bacterianas WT, mas ausente em linhagens isogênicas como Δ gaiola cepas mutantes, que abrigam uma inativação no gene que codifica o principal ATPase que a montagem tipo poderes secreção IV pilus. </li><li> Calibrar a ferramenta de medição por desenhar uma linha com a ferramenta de linha reta e em seguida, clicando na guia &quot;Analisar&quot;. Selecione &quot;Set Scale&quot; no menu suspenso e digite o valor bar ampliação na caixa chamada &quot;Distância Known&quot; e defina a unidade de comprimento para a definição apropriada (nm). Clique em &quot;OK&quot;. </li><li> Meça Cag-T4SS pili usando a ferramenta de linha reta para desenhar sobre o comprimento ou a largura do pilus e pressione &quot;Ctrl-M&quot; para medir cada pilus. Observar uma caixa de diálogo com os valores medidos. Anote esses valores ou copiar e colar em um programa de planilha. </li><li> Use o comprimento ea largura de parâmetros previamente estabelecidos 24, características da negligência que não se encaixam no perfil de CAG-T4SS. Em seguida, quantificar o número de pili com base em valores que estão dentro dos pontos de corte 10-13 nm de largura e comprimento de 60-150 nm. </li><li> Analisar a quantificação dos pili estatisticamente pelo teste t bicaudal de Student utilizando o software GraphPad Prism. </li></ol> 5. Opcional: Avaliação da secreção de IL-8 para correlacionar com Pilus Produção <ol><li> Usando sobrenadantes do Passo 2.1, realizar um ELISA de IL-8 de acordo com as instruções do fabricante, com o kit (Ver Materiais e Equipamento Tabela). </li><li> Analisar a IL-8 segregada pelas células hospedeiras e calcular a percentagem de IL-8 com respeito à indução WT PMSS1 cultivadas em meio sozinho. Realizar a análise estatística usando bicaudal teste t de Student utilizando GraphPad Prism assimftware. </li></ol>

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The authors declare that they have no competing financial interests.

O ferro é um micronutriente essencial para a maioria das formas de vida, incluindo patógenos bacterianos. Num esforço para restringir a viabilidade dos microorganismos invasores, hospedeiros vertebrados sequestrar ferro nutriente num processo conhecido como &quot;imunidade nutricional&quot; 18. Em resposta a isso, patógenos bacterianos evoluíram para usar o ferro como uma molécula de sinalização global para sentir seu entorno e regular a elaboração de características de virulência, tais como siste...

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.

Neste relatório, nós demonstramos que as condições de disponibilidade diferenciada de ferro tem a capacidade de modular H. pylori Cag-T4SS biogénese pilus na interface patógeno hospedeiro. Quando cultivados em meio sozinho, H. pylori constitui uma média de 3 pili / célula. Quando H. pylori é cultivada em condições de ferro esgotar (utilizando o quelante de dipiridilo sintética) que são sub-inibitório para o crescimento bacteriano (Figura 1), as bactérias produze...

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.

Alta resolução de Microscopia Eletrônica da<em&gt; Helicobacter pylori</em&gt; Cag Tipo IV Secreção Sistema Pili Produzido em diferentes condições de Ferro Disponibilidade

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 visualizações.  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.

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