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. ח פילורי צמיחה בתנאים שונים של זמינות ברזל ושיתוף תרבות עם תאים אנושי הקיבה אפיתל <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר ה &#39; PMSS1 פילורי מתח למחקרים אלה כי יש לו אי פתוגניות CAG שלם ומבטא את מערכת הפרשה מסוג IV תפקוד. גם לנצל מוטציה כלוב isogenic (PMSSI Δ קייג &#39;) כביקורת שלילית. לגדול חיידקים על צלחות TSA בתוספת 5% דם כבשים (צלחות אגר דם) למשך 24 שעות על 37 מעלות צלזיוס בנוכחות של 5% CO 2. הערה: הכן חומרים כימיים ולהרכיב חומרים כפי שמתואר בטבלת חומרים וציוד. ריכוזים סופיים לכל מגיב רשום בסוגריים. </li><li style=";text-align:right;direction:rtl"> לגרד חיידקים מהצלחת באמצעות המוליך כותנה שקצה סטרילי ולחסן לתוך מרק ברוצלה שונה שהוכן ממרכיבים (ראה חומרים וציוד בלוח) והשלים עם כולסטרול. דגירה תרבויות הלילה בשעה 37° C באוויר חדר בתוספת 5% CO 2 עם רועד ב200 סיבובים לדקה (סל&quot;ד). הערה: כולסטרול משמש במקום של סרום שור עוברי בשל העובדה שהסרום הוא מורכב; המכיל מספר רב של מקורות ברזל תזונתי כגון heme הגורם לשונות בתוצאות. </li><li style=";text-align:right;direction:rtl"> ביום culturing חיידקים, זרע תאי האפיתל של הקיבה אנושי AGS (ATCC CRL-1739) על coverslips טופל poly-D- ליזין ב12 גם צלחות (כ 1 x 10 5 תאים לכל טוב). </li><li style=";text-align:right;direction:rtl"> למחרת, לדלל תרבויות חיידקים לOD600 של 0.3 במרק ברוצלה שונה לבד או בתוספת 100 מיקרומטר FeCl 3, dipyridyl 200 מיקרומטר (chelator ברזל סינטטי), או 200 dipyridyl מיקרומטר בתוספת 250 מיקרומטר FeCl 3. דגירה תרבויות אלה במשך 4 שעות על 37 מעלות צלזיוס באוויר חדר בתוספת 5% CO 2 עם הרועד בסל&quot;ד 200. </li><li style=";text-align:right;direction:rtl"> חיידקי צנטריפוגה XG ב 1000 לאיסוף תאים ולהסיר supernatants לפני resuspending אניn נפח שווה של מרק ברוצלה שונה טרי בתוספת כולסטרול. מדד OD600 של התרבות (OD600 = 1.0 = 5.5 x 10 8 תאים / מיליליטר) ולהוסיף חיידקים לתאי האפיתל בריבוי של זיהום (משרד הפנים) של 20: 1. לבצע דילולים סידוריים ותאי חיידקיים צלחת על גבי צלחות אגר דם כדי להעריך את כדאיות תא חיידק. </li><li style=";text-align:right;direction:rtl"> דגירה H. שיתוף תרבויות תאי האפיתל של הקיבה אנושי פילורי וAGS במשך 4 שעות על 37 מעלות צלזיוס בנוכחות של 5% CO 2 בתנאים סטטיים לפני הביצוע במיקרוסקופ אלקטרונים סורק הכנת מדגם (SEM). </li></ol> 2. SEM לדוגמא הכנה לדמיין H. פילורי CAG-T4SS פילי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר H. פילורי ושיתוף תרבויות AGS מsupernatant תרבות חממה ולמזוג (למעט IL-8 ELISA). לשטוף בעדינות שלוש פעמים עם 0.05 חיץ M נתרן cacodylate (pH 7.4). תקן את הדגימות ל2-4 שעות בטמפרטורת חדר בתמיסה של paraformaldehyde 2.0%, 2.5gluteraldehyde%, וחיץ cacodylate 0.05 M נתרן. לאחר קיבוע ראשוני, לשטוף דגימות שלוש פעמים עם חיץ cacodylate 0.05 M נתרן. </li><li style=";text-align:right;direction:rtl"> בצע קיבעון המשני באמצעות 0.1% tetroxide אוסמיום ב0.05 חיץ cacodylate M נתרן בשני 10 ברצף צעדי קיבעון דקות. לאחר קיבוע המשני, לשטוף דגימות שלוש פעמים עם חיץ cacodylate 0.05 M נתרן. </li><li style=";text-align:right;direction:rtl"> לאחר קיבוע ראשוני והמשני, ליבש דגימות על ידי שטיפה רציפה עם ריכוזים גוברים של אתנול לפי טבלת 1. </li><li style=";text-align:right;direction:rtl"> לאחר התייבשות אתנול, לבצע שלושה שוטף רציף של נוזל פחמן דו חמצני. דגימות ואז יבשות בנקודה קריטית באמצעות מכונה מייבש נקודה קריטית בטמפרטורה של 31.1 ºC ולחץ של 1073 PSI. </li><li style=";text-align:right;direction:rtl"> הר coverslips על ספחי מדגם האלומיניום SEM ולצייר עם קו דק של מי כסף בקצה המדגם כדי להשיג הארקה מתאימה ולהימנע מלחייב במהלך ההדמיה SEM. </li><li style=";text-align:right;direction:rtl"> דגימות מעיל עם 5 ננומטר שלזהב-פלדיום באמצעות מכונה מעיל גמגום להגדיל מדגם אותות משני אלקטרונים ואפקט קצה. </li></ol> 3. פרמטרי הדמיה לניתוחים ברזולוציה הגבוה SEM <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צפה בדוגמאות עם אקדח מיקרוסקופ סורק אלקטרוני פליטת שדה (FEG-SEM). </li><li style=";text-align:right;direction:rtl"> תמונה במצב ואקום גבוה במרחק עבודה של 5-10 מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> הגדר את המתח המאיץ עד 5 קילו וולט. </li><li style=";text-align:right;direction:rtl"> הגדר את גודל מקום ל2-2.5. </li><li style=";text-align:right;direction:rtl"> הטה את המדגם בין 15-25 מעלות כדי להשיג ראייה טובה יותר של ממשק המאכסן לפתוגן. </li><li style=";text-align:right;direction:rtl"> לתעדף הדמיה חיידקים חסיד לקצוות של תאי האפיתל לצפייה הגדלה גבוהה, כתחומים של אינטראקציה המאכסן לפתוגן אלה מועשרים לחיידקים יוצרי פילים. </li></ol> 4. ניתוח סטטיסטי כדי להעריך את הפילים Quantifications <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנתח H. פילי CAG-T4SS פילורי באמצעות תוכנת ImageJ לכמת מספר פילים / תא, כמו גם אחוז של תאים המציגים piliated Phenotype בהתאם להנחיות למטה. </li><li style=";text-align:right;direction:rtl"> קבצי מיקרוסקופ פתוחים בתוכנת ImageJ. לזהות פילים כמבנים שנוצרו בין תא החיידק והתא מארח, עם רוחב אחיד (10-13 ננומטר) ואורך (60-150 ננומטר). הערה: מבנה pilus קיים בזני חיידקי WT, אבל נעדרו בזני isogenic כגון זנים מוטנטים כלוב Δ, שנמל איון בגן המקודד את ATPase המרכזי שהרכבת pilus הפרשת IV סוג סמכויות. </li><li style=";text-align:right;direction:rtl"> כייל את כלי המדידה על ידי ציור קו עם כלי הקו הישר ולאחר מכן לחיצה על הכרטיסייה &quot;נתח&quot;. בחר &quot;סולם הגדר&quot; מהתפריט הנפתח והזן את הערך בר הגדלה לתוך התיבה &quot;מרחק ידוע&quot; שכותרתו ולהגדיר את יחידת האורך להגדרה המתאימה (ננומטר). לחץ על &quot;אישור&quot;. </li><li style=";text-align:right;direction:rtl"> למדוד פילי CAG-T4SS שימוש באפשרות הקו הישר לצייר על האורך או הרוחב של pilus ולאחר מכן לחץ על &quot;Ctrl-M&quot; למדוד כל pilus. שים לב תיבה דו-שיח עם הערכים שנמדדו. רשום את הערכים הללו או להעתיק ולהדביק לתוך תכנית גיליון אלקטרוני. </li><li style=";text-align:right;direction:rtl"> השתמש באורך ורוחב פרמטרים שנקבעו בעבר 24 תכונות התעלמות, שאינו מתאימות לפרופיל של CAG-T4SS. ואז לכמת את מספר הפילים המבוססים על ערכים הנמצאים בהפסקות 10-13 ננומטר רוחב ו60-150 ננומטר אורך. </li><li style=";text-align:right;direction:rtl"> נתח את הכימות של פילים סטטיסטיים על ידי מבחן t של סטודנט שני זנב באמצעות תוכנת GraphPad פריזמה. </li></ol> 5. אופציונלית: הערכה של IL-8 ההפרשה במיתאם עם Pilus הפקה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות supernatants משלב 2.1, לבצע ELISA IL-8 לפי הוראות יצרן עם הערכה (ראה חומרים וציוד בלוח). </li><li style=";text-align:right;direction:rtl"> נתח את IL-8 מופרש על ידי תאי מארח ולחשב את האינדוקציה IL-8 אחוזים ביחס לWT PMSS1 גדלו במדיום לבד. ביצוע ניתוחים סטטיסטיים באמצעות מבחן t של סטודנט שני זנב באמצעות GraphPad פריזמה כךftware. </li></ol>

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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.

ברזל הוא יסודות קורט חיוני עבור רוב צורות חיים, כוללים חיידקים פתוגנים. במאמץ להגביל את יכולת הקיום של פלישת מיקרואורגניזמים, מארחי חוליות לעקל ברזל מזין בתהליך המכונה &quot;חסינות תזונתית&quot; 18. בתגובה לכך, חיידקים פתוגנים התפתחו להשתמש בברזל כמולקולת איתות גלוב...

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.

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			<td colspan="2" height="21" style="height:21px;width:475px;">Modified brucella broth</td>
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			<td style="width:167px;"></td>
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		<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>
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		<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>
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			<td height="20" style="height:20px;width:216px;">Yeast extract (2g/L)</td>
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			<td style="width:196px;">92144</td>
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			<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>
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		<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:216px;"></td>
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			<td colspan="2" height="20" style="height:20px;width:475px;">Modified RPMI</td>
			<td style="width:196px;"></td>
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			<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>
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		<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>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:259px;"></td>
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			<td colspan="2" height="20" style="height:20px;width:475px;">Electron Microscopy Preparation</td>
			<td style="width:196px;"></td>
			<td style="width:167px;"></td>
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		<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>
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			<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>
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			<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.

בדוח זה, אנחנו הוכחנו כי יש להם תנאים משתנים זמינות ברזל היכולת לווסת את ה biogenesis pilus פילורי CAG-T4SS בממשק שהפתוגן המארח. כאשר בתרבית בינונית בלבד, ח &#39; פילורי מהווה ממוצע של 3 פילים / תא. כאשר ה &#39; פילורי הוא גדל בברזל לרוקן את תנאים (באמצעות dipyridyl chelator ה?...

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.

רזולוציה גבוהה מיקרוסקופית אלקטרונים של<em&gt; הליקובקטר פילורי</em&gt; CAG Pili סוג IV הפרשת המערכת הופק במשתנים תנאים של זמינות הברזל

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

14.9K Views.  Vanderbilt University School of Medicine. Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

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

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

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

קביעת מבנים מולקולריים של גליקופרוטאינים HIV מעטפה באמצעות אלקטרונים cryo-טומוגרפיה ו ממוצעים אוטומטי תת tomogram

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 ...

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

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

מיקרוגל Assisted אבחון מהיר של מחל וירוס צמח על ידי מיקרוסקופי אלקטרוני הילוכים

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

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.

ניתוח של נזק אפיתל הופק על ידי<em&gt; Histolytica Entamoeba</em&gt; זיהום

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.

פיתוח<em&gt; במבחנה</em&gt; מערכת מודל לחקר האינטראקציה של<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE עם קולטן גבוה הזיקה שלה FcεRI

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.

שיטות כדי להגדיל את הרגישות של רזולוציה גבוהה התכה יחידה נוקלאוטיד פולימורפיזם Genotyping במלריה

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.

Organoids כמודל למחלות זיהומיות: התרבות של האדם וOrganoids הקיבה Murine וMicroinjection של הליקובקטר פילורי

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.

צעד אחד טיהור זיהוי תכונות חומרים שלילית של<em&gt; הליקובקטר פילורי</em&gt; חלבון נויטרופילים-הפעלת ביתר<em&gt; Coli Escherichia</em&gt; במצב אצווה

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.

מתודולוגיות לימוד<em&gt; B. subtilis</em&gt; Biofilms כמודל לאפיון מולקולה קטנה Biofilm מעכבי

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.

טיהור של תשואה גבוהה לחילוץ ההכנות הרחק וירוס

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.

בידוד ואפיון מסחטות ושלפוחיות מתוצרת ברזל-בקטריה מוגבלת

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