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>

<ol>
	<li>Cover, T. L., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+in+health+and+disease.">Helicobacter pylori in health and disease.</a> Gastroenterology. (6), 1863-1873 (2009).</li><li>Sycuro, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+crosslinking+relaxation+promotes+Helicobacter+pylori's+helical+shape+and+stomach+colonization.">Peptidoglycan crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach colonization.</a> Cell. 141 (5), 822-833 (2010).</li><li>Kodaman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+Helicobacter+pylori+coevolution+shapes+the+risk+of+gastric+disease.">Human and Helicobacter pylori coevolution shapes the risk of gastric disease.</a> Proc. Natl. Acad. Sci. U.S.A. 111 (4), 1455-1460 (2014).</li><li>Tegtmeyer, N., Wessler, S., Backert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+cag-pathogenicity+island+encoded+type+IV+secretion+system+in+Helicobacter+pylori+pathogenesis.">Role of the cag-pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis.</a> FEBS J. 278 (8), 1190-1202 (2011).</li><li>Fischer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+molecular+mode+of+action+of+the+Helicobacter+pylori+Cag+type+IV+secretion+apparatus.">Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus.</a> FEBS J. 278 (8), 1203-1212 (2011).</li><li>Odenbreit, S., Puls, J., Sedlmaier, B., Gerland, E., Fischer, W., Haas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+Helicobacter+pylori+CagA+into+gastric+epithelial+cells+by+type+IV+secretion.">Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion.</a> Science. 287 (5457), 1497-1500 (2000).</li><li>Bourzac, K., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori-host+cell+interactions+mediated+by+type+IV+secretion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+cytoxin-associated+gene+A+activates+the+signal+transducer+and+activator+of+transcription+3+pathway+in+vitro+and+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+Helicobacter+pylori+triggered+by+contact+with+gastric+epithelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+sheathed+surface+organelle+of+the+Helicobacter+pylori+cag+type+IV+secretion+system.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet,+microbial+virulence+and+Helicobacter+pylori+induced+gastric+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+gastric+IL-1&#946;+concentration+and+iron+deficiency+parameters+in+Helicobacter+pylori+infected+children.">Increased gastric IL-1&#946; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori-associated+hypochlorhydria+and+development+of+iron+deficiency.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+deficiency+accelerates+Helicobacter+pylori-induced+carcinogenesis+in+rodents+and+humans.">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., Amieva, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+perturbs+iron+trafficking+in+the+epithelium+to+grow+on+the+cell+surface.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dissection+of+the+Helicobacter+pylori+Fur+regulatory+circuit+by+genome-wide+location+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+analysis+of+Helicobacter+pylori+Fur-regulated+promoters+reveals+a+Fur+box+core+sequence+and+novel+Fur-regulated+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+in+infection+and+immunity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+in+urinary+tract+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+iron+starvation+response+in+Bordatella+pertussis+provides+new+insights+into+siderophore+utilization+and+virulence+gene+expression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+factors+associated+with+disease+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+requirements+and+antibiotic+resistance+patterns+of+Helicobacter+species+in+chemically+defined.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+host+iron+utilization+mechanisms+of+Helicobacter+pylori+revealed+with+iron-deficient+chemically+defined+media.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+exploits+a+unique+repertoire+of+type+IV+secretion+system+components+for+pilus+assembly+at+the+bacteria-host+cell+interface.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+plasticity+in+the+type+IV+secretion+system+of+Helicobacter+pylori.">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.

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

15.0K 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

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultura do Sistema Nervoso Central Neonatal e Adulto

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Imunologia e Infecção

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Análise do dano epitelial Produzido pela<em&gt; Entamoeba histolytica</em&gt; Infecção

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Desenvolvimento de um<em&gt; In vitro</em&gt; Sistema modelo para estudar a interacção de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE ao seu receptor de elevada afinidade FceRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Métodos para aumentar a sensibilidade de alta resolução de derretimento Single Nucleotide Polymorphism Genotipagem em Malária

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organóides como modelo para Doenças Infecciosas: Cultura dos Direitos Humanos e Organóides e estômago murino Microinjection do Helicobacter pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Um passo de purificação cromatográfica de Negativo<em&gt; Helicobacter pylori</em&gt; Neutrófilos-proteína de activação sobre-expresso em<em&gt; Escherichia coli</em&gt; Em Modo Batch

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologias para o estudo<em&gt; B. subtilis</em&gt; Os biofilmes como um modelo para a caracterização de pequena molécula biofilme Inibidores

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Purificação de preparações de vesículas extracelulares de alto rendimento longe do vírus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Isolamento e caracterização de vesículas extracelulares produzidas por micobactérias limitadas por ferro

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

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.

Aplicando imagens de células vivas e tomografia crio-elétron para resolver características espaciales do Sistema de Secreção Legionella pneumophila Dot/ICM

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