Name: high resolution electron microscopy of the helicobacter pylori cag type iv secretion system pili produced in varying conditions of iron availability
Uploaded: 2014-11-21T14:00:00
Description: 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

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 carne...

<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>

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 ...

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

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Determinação de estruturas moleculares de glicoproteínas do envelope HIV usando Cryo-Electron Tomografia e Média Sub-tomogram Automated

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Imunologia e Infecção

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Microondas Assistida Diagnóstico Rápido de Doenças vírus de planta por Microscopia Eletrônica de Transmissão

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

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

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

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

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

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

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

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

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

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

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) ...