This work has been supported by Elizabeth E. Kennedy Research Award.

.1 הכנת הזנים מפיק <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Streak זן דלקת ריאות להיבדק לייצור bacteriocin על 5% צלחת אגר סויה tryptic דם כבשים ממניות מקפיא -80 מעלות צלזיוס. הערה: השימוש בצלחות דם לצמיחה ראשונית של הזן להיבדק לעיכוב מגדיל מאוד את שחזור של assay. בנוסף, זנים שאינן דלקת ריאות אחרות המייצרים bacteriocins ניתן להשתמש למרות שתנאי גידול, ייתכן שיצטרכו להיות שונה לאורגניזם ספציפי. יש לנו להשתמש mitis Streptococcus וקטיס Lactococcus בassay הכיסוי בהצלחה. </li><li style=";text-align:right;direction:rtl"> דגירה את הצלחת למשך הלילה (O / N) על 37 מעלות צלזיוס עם 5% CO 2. </li><li style=";text-align:right;direction:rtl"> לאחר הדגירה של זן המפיק, להכין הצלחת אגר סויה tryptic (TSA) על ידי pipetting 3,000 4,000 יחידות של קטלאז על צלחות המכילות 25 מ&quot;ל של TSA ולהפיץ את פתרון קטלאז באמצעות חרוזים זכוכית סטרילית. בואו יבש הצלחת במשך כ10 דקות תחתארון בטיחות ביולוגי. </li><li style=";text-align:right;direction:rtl"> לאסוף כמות נראית לעין של חיידקים על קצה פיפטה. לדקור את קצה פיפטה לתוך צלחת TSA המיובש. הערה: יותר מ מתח מפיק אחד יכולה להיות נדקרת לתוך צלחת TSA יחידה. חשוב חלל את הדקירות, כך שהילות וכתוצאה מכך אינן חופפות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> במידת האפשר, כולל ידוע bacteriocin מפיק כביקורת חיובית ולהתאמץ כדי לשמש בשכבה כביקורת שלילית. הערה: בקרות חיוביות ושליליות הן כבר פורסמו בעבר, והם זמינים לפי בקשה 10. </li></ol></li><li style=";text-align:right;direction:rtl"> דגירה צלחת TSA מכיל דקירות מרובות על 37 מעלות צלזיוס עם 5% CO 2 למשך 6 שעות. </li></ol> 2 מניות גליצרול ביצוע של הזנים הכיסוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן מניות גליצרול (תרבויות גדלו שלב אמצע מעריכי ואז מאוחסנות ב-80 מעלות צלזיוס לאחר התוספת של גליצרול) לפני היום של assay הכיסוי. לחסן 5-10 מושבות של str דלקת הריאותעין להיבדק לרגישות או הפרשת פרומון במדיום טוד יואיט עם שמרי 0.5% תמצית (THY). הערה: בנייה של כתבים מושרה BlpC כבר תוארה בעבר 2 וזנים זמינים לפי בקשה. </li><li style=";text-align:right;direction:rtl"> דגירה 5-10 מושבות בצינורות זכוכית עם 5 מ&quot;ל של THY עם מכסים סגורים היטב באמבט מים 37 מעלות צלזיוס בלי לרעוד. </li><li style=";text-align:right;direction:rtl"> דגירה תרבויות על 37 מעלות צלזיוס עד שהם מגיעים OD 620 של 0.3-0.5. הערה: זה ייקח כ 4-5 שעות בהתאם לזן המשמש. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעדינות להפוך את הצינור כמה פעמים לפני מדידת OD. </li></ol></li><li style=";text-align:right;direction:rtl"> אם הניסוי לא ניתן לבצע באותו היום או הזן ישמש פעמים רבות בעתיד, להפוך את מניות גליצרול ולחדש את הניסוי במועד מאוחר יותר. עבור מניות גליצרול, להוסיף גליצרול לתרבויות לריכוז סופי של 20%. תרבויות Aliquot לתוך צינורות 1.5 מ&quot;ל microcentrifuge ולאחסן ב -80 מעלות צלזיוס. הערה: דוגמאות יכולות להיות שלהנמצאת בבמשך חודשים ב-80 מעלות צלזיוס. אם לא עושה מניות גליצרול, להשתמש דגימות ישירות לassay כיסוי. </li></ol> 3 הכיסוי Assay <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> רק לפני כיסוי יישום, לערבב את מרכיבי הכיסוי. בצינור חרוטי 15 מ&quot;ל, להוסיף 5 מ&quot;ל שלך, 3,000-4,000 U של קטלאז, ו200 μl של תרבות מרק מתח כיסוי שגם גדלה באותו יום או מניית גליצרול של התרבות שהיה מופשר בטמפרטורת חדר. אם אתם משתמשים בזן הכתב, להוסיף 50 μl של X-gal (40 מ&quot;ג / מ&quot;ל) לתערובת הכיסוי. השתמש בצינור חרוטי אחד לכל צלחת. </li><li style=";text-align:right;direction:rtl"> לאזן תערובת כיסוי בטמפרטורת חדר לפני תוספת של TSA המותך. </li><li style=";text-align:right;direction:rtl"> הסר את צלחת TSA עם מתח נדקר מחממה. ממסים TSA המוצק במיקרוגל ולשמור על 55 ° C אמבט מים עד מוכן לשימוש. הוסף 3 מ&quot;ל של TSA המותך עם אגר 1.5% מקורר 55 ° C כדי כיסוי תערובת. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מכין את תערובת הכיסוי מייד לפני pipetting, התערובת תתחיל לחזק מאודבמהירות. הערה: אם השכבה מתחילה לבסס לפני הציפוי, התוצאות תהיה uninterpretable. </li></ol></li><li style=";text-align:right;direction:rtl"> מערבבים את תערובת הכיסוי על ידי pipetting מעלה ומטה מספר פעמים עם 5 מ&quot;ל pipet נזהר שלא להכניס בועות לתוך התערובת. לאט לאט פיפטה תערובת כיסוי ישירות על צלחת TSA נדקרה. שמור את הצלחת על המעבדתיים למאפשרות אגר להקשיח כמה דקות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אל תסובב את הצלחת כדי לפזר את תערובת הכיסוי, זה ימשוך צמיחה מהתרבות נדקרה לכיסוי. </li></ol></li><li style=";text-align:right;direction:rtl"> בזהירות להניח צלחת TSA עם כיסוי ל37 חממת ° C המכילה 5% CO 2 O / N (כ 16-18 לשעה). </li><li style=";text-align:right;direction:rtl"> שים לב ללוחות לאחר צמיחת הלילה. הצלחות צריכה להראות אטום מצמיחה של זן הכיסוי למעט באזורים שבם איתות עיכוב או פרומון התרחשה. אלה יופיעו כאזורי או ברורים או כחולים סביב המתח נדקר, בהתאמה. שים לב: למרות measurements של הקוטר של קרחת יער כחול או יכול להיות שנקבע להערכה כמותית, הבדלים בקוטר יכולים גם משתנים בהתאם לכמות חיידקים שנדקרה לתוך הצלחת. Assay הכיסוי הוא assay איכותי, כל הערכה כמותית יש לפרש בזהירות. כל סליקה גלויה בהשוואה לביקורת השלילית נחשבת כפעילות. </li></ol>

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The authors have nothing to disclose.

assay הכיסוי זוהי דרך מהירה כדי לקבוע את הטווח של פעילות עבור יצרני bacteriocin ולהפגין אינטראקציות תחרותיות שעלולות להתרחש בין זני דלקת ריאות. Assay הכיסוי יכול להיות מותאם לשימוש עבור הקרנת זנים מרובים לייצור bacteriocin על צלחת אחת. יש לנו הוקרנו בהצלחה אוספי מתח גדולים עם assay זה ...

Streptococcus pneumoniae, קולוניאלי משותפת של קהילת polymicrobial של לוע האף, הוא מסוגל להתחרות עם pneumococci אחר ומינים קרובים באמצעות הייצור של פפטידים מיקרוביאלית מיוצרים bacterially (bacteriocins). כל זן דלקת ריאות באופן מלא רצף שנבחן עד היום מכיל גרסה של מוקד acteriocin- ב אייק l eptide עמ &#39;, BLP. ייצור Bacteriocin ידי pneumococcus הודגם להיות חשוב בתוצאות הן במבחנה בתחרות vivo 1-4. דינמיקה תחרותית מושפעת מהביטוי של bacteriocins המגוון יחד עם חלבוני חסינותו מקור בתגובה לפרומון פפטיד ספציפי. אינדוקציה של מוקד BLP נשלטת על ידי מערכת חישת מניין שבו פרומון פפטיד, BlpC, נקשר לקינאז החיישן, BlpH, ויוזם מפל איתות וכתוצאה מכךשעתוק של 5,6 מוקד BLP. ישנם ארבע וריאציות allelic של BlpC שכל נקשרים לBlpH המקור שלה 6. לתא כדי לשרוד את ההשפעות של bacteriocin משלו, הגנים המקודדים את חלבוני חסינותו מקור מתועתקים באותו אופרון ככל אחד מהגנים bacteriocin. הרג מתרחש אם זן מתחרה הוא לא מסוגל upregulate מוקד BLP בתגובה לפרומון אקסוגניים או, אם הזן חסר את גן החסינות הספציפי הנדרש להגנה. המורכב טרנספורטר, BlpAB נדרש להפרשה והעיבוד של פרומון פפטיד ופפטידים bacteriocin 2,4. לאחרונה, הוכח שחלק משמעותי מזני דלקת ריאות הם &quot;רמאים&quot;, כלומר יש להם מוטציה שמורה בגן blpA אשר הופך אותם מסוגל להפריש פרומונים וbacteriocins 1,2. זנים אלה מסוגלים להגיב לBlpC אקסוגניים 2 מופרש על ידי צהלהזנים משעממים המאפשרים הייצור של חלבוני חסינות. אמנם, הכנות גולמי של bacteriocins מsupernatants ניתן להכין מזני מפיק באמצעות צעדי שיטות ביוכימיות להשגת טוהר, גישה זו אינה שימושית לסינון אוסף גדולה של בידודים ואם רמת ביטוי bacteriocin היא נמוכה בתרבויות מרק גדל 2,7- 9. Assay הכיסוי משמש כדרך מהירה כדי לחקור את הדינמיקה התחרותית בין זנים שעלולים להתקיים במבחנה. לשם כך, מתח דלקת ריאות הוא על צלחת אגר ואפשרו לגדול כדי להשיג ריכוזי חיידקים מקומיים מספיקים לאינדוקציה של מוקד BLP-מחוסן מראש. זן שני הוא מחוסן אז לפתרון אגר רך מותך אשר לאחר מכן מיושם על זן מחוסן מראש לבדיקת רגישות. כדי להעריך את הפעילות של מוקד BLP (עצמאית של פעילות מעכבת), יכולים להיות מעולף זנים בסדרה של הרי זני כתב BlpC שתוארו קודם לכן. זנים אלו נבנו כדי להכיל אחד משלושה אללים blpH שונים שמגיבים לשלושה פרומונים BlpC הנפוצים ביותר מופרשים על ידי pneumococci. יש לי זני כתב גן lacZ משולב שנמצא תחת השליטה של אמרגן תלוי BlpH ולבצע מחיקה בגן blpC המונע גירוי עצמי 10,11.

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:351px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Trypticase Soy Agar with 5% Sheep Blood</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B21261X&#160;</td>
			<td>For growth from freezer cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Catalase</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C100-500mg</td>
			<td>4,741 Units per plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Todd Hewitt Broth + Yeast Extract (THY)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">DF0492-17-6&#160;</td>
			<td>30 g of THB, 5 g yeast extract, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Trypticase Soy Broth + Agar plates (TSA)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B11768&#160;</td>
			<td>30 g of TS, 15 g agar, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">5-Bromo-4-chloro-3-indolyl &#946;-D-galactopyranoside (X-gal)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">B4252-50MG</td>
			<td>Dissolve in dimethylformamide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Petri dishes</td>
			<td style="width:71px;">Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Spectrophometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Measures the OD620 of cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C incubator with 5% CO2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">15 ml conical tube</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

ישנן שתי תוצאות אפשריות בassay הכיסוי. זה צריך להיות אפשרי לראות צמיחה של הזן נדקר לאחר דגירה הלילה. באיור 1 א, מתח הכיסוי רגיש לbacteriocins שיופקו. אזור של פינוי יכול להיות דמיין המקיף את המתח נדקר. בנוסף, מתערבל של המתח נדקר לתוך תערובת הכיסוי אפשרי ושניתן לראות <strong...

Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

באמצעות assay הכיסוי למדוד את הייצור בקטריאלי ורגישות לדלקת הריאות Bacteriocins איכותי

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

using-overlay-assay-to-qualitatively-measure-bacterial-production

Research

JoVE Journal

Immunology and Infection

13.4K Views.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. 

Video: Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

שיטת בדיקת מעטפת חלבון-RNA כריכת חיידקים

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Genetics

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Assay חזותי לפקח תחרות קטריאלי T6SS בתיווך

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

מידות תא יחיד של קרע vacuolar נגרמים על ידי פתוגנים תאיים

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

זיהומים של תאי יונקים צייתנים עם חיידקי protozoan דרוך-

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

ניטור שינויים בממברנה קוטביות, ממברנה יושרה, וריכוזי יון תאיים ב<em&gt; Streptococcus pneumoniae</em&gt; שימוש צבעי ניאון

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

חוקר את ההשפעות של פרוביוטיקה על דלקת ריאות התיישבות שימוש<em&gt; במבחנה</em&gt; Assay דבקות

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

חומרי Biomimetic לאפיין אינטראקציות חיידקים-מארח

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

מבחנים איכותיים וכמוני עבור זיהוי ואפיון של antimicrobials החלבון

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

השימוש בטכניקת הכיסוי הרך-אגר למסך תרכובות מעכבות הופקו bacterially

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

תפוקה גבוהה שיגלה-Assay אחרים ספציפיים

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...