Name: a high-throughput, high-content, liquid-based c. elegans pathosystem
Uploaded: 2018-07-01T15:00:00
Description: Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

Diese Studie wurde von der Krebsprävention und Research Institute of Texas (CPRIT) Award RR150044, Welch Foundation Research Grant C-1930, und von den nationalen Instituten der Gesundheit K22 AI110552 verliehen an NVK unterstützt. Die Geldgeber hatten keine Rolle beim Studiendesign, Datenerhebung und Analyse, Entscheidung, zu veröffentlichen oder der Manuskripterstellung.

Achtung: P. Aeruginosa und E. Faecalis sind Biosafety Level 2 Krankheitserreger und angemessene Sicherheitsvorkehrungen getroffen, um versehentliche Infektion zu verhindern und Verschmutzung von Oberflächen zu minimieren. Alle Medien und Materialien, die in Kontakt mit Krankheitserregern kommen müssen sterilisiert bzw. verworfen werden. Weitere Richtlinien sind bei der CDC Publikation Biosicherheit in Mikrobiologie und biomedizinischen Labors (BMBL), 5. Auflage erhältlich.
1...

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Dieser Assay (oder ähnliche Tests wo andere Krankheitserreger für p. Aeruginosa oder E. Faecalisersetzt werden) eignet sich für eine Vielzahl von Zwecken, einschließlich der Arzneimittelforschung. Es ist auch nützlich für grundlegende biologische Fragestellungen, z. B. Virulenzfaktoren, die Aufklärung der Host Verteidigung Bahnen, Identifizierung und Bestimmung der regulatorischen Maschinen der Wirt-Erreger-Interaktion beteiligt.
Obwohl p. Aeruginosa flüssige...

Die Identifikation und Entwicklung von wirksamen, Breitspektrum-Antibiotika, nun vor fast einem Jahrhundert führte zu einem Wendepunkt im Gesundheitswesen gab es eine weit verbreitete Überzeugung, dass infektiöse Krankheit wäre eine Geißel der Vergangenheit. Innerhalb von wenigen Jahrzehnten begann dieser Optimismus zu schwinden als Erreger nach Erreger Resistenzmechanismen entwickelt, die diese einmal wunderbaren Behandlungen begrenzt. Eine Zeitlang schien das Wettrüsten zwischen Drug Discovery Bemühungen und die Erreger ausgeglichen. Der Missbrauch von Antibiotika hat jedoch vor kurzem bei der Entstehung von Pan-resistente Stämme von Klebsiella Pneumoniae, Acinetobacter Baumanii, Serratia Marcescensund p. Aeruginosa1gipfelte, 2,3,4.
P. Aeruginosa ist ein opportunistisch, Gram negative, Multi-Host Erreger, eine schwere Bedrohung für Patienten mit schweren Verbrennungen, diejenigen, die sind immungeschwächte oder Mukoviszidose. Es wird auch zunehmend als ein Erreger in schweren Nosokomialinfektionen, vor allem durch die laufende Übernahme von Antibiotikaresistenzen identifiziert. Um zu beginnen, um dieser Bedrohung zu begegnen, haben wir den gut dokumentierten C. Elegans-p. Aeruginosa Infektion System5verwendet. Unser Labor hat genutzt, dieses System zu entwickeln eine Liquid-basierter, Hochdurchsatz-, High-Content Screening-Plattform um neue Verbindungen zu identifizieren, die die Fähigkeit des Erregers, die Host-6töten zu begrenzen. Faszinierend, scheinen diese Verbindungen mindestens drei allgemeine Kategorien, einschließlich Antibiotika7 und Virulenz Inhibitoren8anzugehören. Andere High-Content-Droge-Entdeckung-Assays in C. Elegans für Mycobacterium Tuberculosum, Chlamydia Trachomatis, Yersinia Pestis, Listeria Monocytogenes, Francisella Tularensis, Staphylococcus Aureus, Candida Albicans, gemeldet wurden und Enterococcus Faecalis, unter anderem9,10,11,12,13,14,15,16. Diese Arten von Tests haben mehrere allgemein anerkannten Vorteile, wie die Begrenzung falsche positive Treffer, die möglicherweise giftig für beide der Host und der Erreger, erhöhte Wahrscheinlichkeit der Bioverfügbarkeit im Vergleich zu einem chemischen Bildschirm und Hits jenseits zu identifizieren einfach die Begrenzung mikrobiellen Wachstums, wie Anti-Virulents, immun stimulierende Moleküle oder Verbindungen, die sonst das Gleichgewicht der Wirt-Pathogen Interaktionen zu Gunsten der ehemaligen kippen. Darüber hinaus sind die Verbindungen entdeckt in diesen Bildschirmen oft wirksam bei Säugetieren Gastgeber.
Es ist erwähnenswert, dass mindestens zwei anderen Assays17,18 sind zur Durchführung von Hochdurchsatz-Bildschirme in C. Elegans in Flüssigkeit. Jeder dieser Tests ist jedoch eine Änderung, die die prototypische intestinale Kolonisation Assay, bekannt als langsam-Tötung, in Flüssigkeit, durchgeführt werden kann Erhöhung des Durchsatzes und ermöglicht Verbindungen leichter untersucht werden. Achten Sie darauf, dass Charakterisierung schlüssig nachgewiesen, dass die Mechanismen der bakteriellen Virulenz dieser Assays und unsere Liquid-basierter Bildschirm7unterschiedlich sind. Da beide Arten von Virulenz bei Säugetieren Systemen beobachtet werden, ist es wichtig zu überlegen, welche Virulenz Determinante für den Experimentator Interessen vor Assay Auswahl am relevantesten ist.
Hier zeigen wir eine optimierte Version der Liquid-basierter C. Elegans-P. Aeruginosa Assay. Wir berichten auch die Anpassung der unsere Liquid-basierter Assay-Methode, um den grampositiven bakteriellen Erreger beherbergen Enterococcus Faecalis. Wie p. Aeruginosaist E. Faecalis zunehmend als schwere nosokomiale Bedrohung mit einer wachsenden Bewaffnung der Antibiotikaresistenz Wege1erkannt. Obwohl eine frühere Methode für Hochdurchsatz-Screening von E. Faecalis 14vorhanden ist, erfordert es Preinfection mit dem Erreger, die das Verfahren kompliziert und erhöht die Wahrscheinlichkeit einer Kontaminierung Ausrüstung wie die COPAS-FlowSort. Unser Protokoll überflüssig vor Infektion, das Sicherheitsprofil zu verbessern. Zu guter Letzt berichten wir über ein Mittel, mit denen entweder diese Assays kombiniert werden können, mit der Fütterung RNAi, so dass der Benutzer suchen für Host-Faktoren, die eine bei der Schaffung Rolle oder Widerstandskraft gegen Infektionen.

<table>
	<tbody>
		<tr>
			<td>COPAS FP BioSorter</td>
			<td>Union Biometrica</td>
			<td></td>
			<td>Large object flow cytometer/worm sorter</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EL406 Washer Dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron Pro</td>
			<td>Infors HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 Deep-Well RB Block</td>
			<td>Thermo Fisher Scientific</td>
			<td>CS15124</td>
			<td></td>
		</tr>
		<tr>
			<td>384-Well plate</td>
			<td>Greiner Bio-One</td>
			<td>MPG-781091</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Media (NGM)</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) plates</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) media</td>
			<td></td>
			<td></td>
			<td>Amount per liter:&#160; 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>USBiological Life Sciences</td>
			<td>L1520</td>
			<td></td>
		</tr>
		<tr>
			<td>Brian Heart Infusion broth (BHI)</td>
			<td>Research Products International Corp</td>
			<td>50-488-526</td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Bleach Solution</td>
			<td></td>
			<td></td>
			<td>Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water</td>
		</tr>
		<tr>
			<td>S Basal</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 5.85 grams NaCl, 6 grams KH2PO4, 1 gram K2HPO4, and 1 Liter of milli-Q water</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>USBiological Life Sciences</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>USBiological Life Sciences</td>
			<td>S5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>USBiological Life Sciences</td>
			<td>P3300</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>USBiological Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffer</td>
			<td></td>
			<td></td>
			<td>amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Acros Organics</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>USBiological Life Sciences</td>
			<td>P5100</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Sodium Hypochlorite Solution</td>
			<td>BICCA</td>
			<td>7495.5-32</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution</td>
			<td>Fisher Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-easy</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Nucleic Acid Stain</td>
			<td>Fisher Scientific</td>
			<td>S11368</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P. aeruginosa (PA14)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. faecalis(OG1RF)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli superfood (OP50)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAi expressing bacteria (HT115)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput, high-content, liquid-based c. elegans pathosystem

Die Zahl der neuen Medikamente identifiziert durch traditionelle, in-vitro- Bildschirme hat nachgelassen, den Erfolg dieses Ansatzes bei der Suche nach neuen Waffen gegen mehrere Resistenzen reduzieren. Dies führte zu dem Schluss, dass Forscher nicht nur Notwendigkeit, neue Medikamente zu finden, sondern auch neue Wege zu finden, sie zu entwickeln müssen. Methoden gehören des vielversprechendsten Kandidat ganzen Organismus, in Vivo Tests dieser Verwendung Hochdurchsatz-, phänotypische auslesen und beherbergt, die von Caenorhabditis Elegans , Danio Rerioreichen. Diese Hosts haben mehrere leistungsstarke Vorteile, einschließlich dramatische Kürzungen in falsche positive Treffer, wie Verbindungen, die giftig für die Host- und/oder Biounavailable sind in der Regel bis in das Einstiegsbild vor kostspieligen folgen gelöscht werden.
Hier zeigen wir, wie unser Test verwendet wurde, um Host-Variation in den gut dokumentierten C. Eleganszu verhören –Pseudomonas Aeruginosa flüssige Tötung Pathosystem. Wir zeigen auch mehrere Erweiterungen dieser gut ausgearbeitete Technik. Wir sind beispielsweise Hochdurchsatz-genetische Bildschirme mit RNAi in 24 oder 96-Well Plattenformate Abfrage Host Faktoren in diesem Wirt-Pathogen Interaktionen durchführen zu können. Mit Hilfe dieser Assay, können gesamte Genom-Bildschirme in nur wenigen Monaten abgeschlossen werden die drastisch vereinfachen kann die Aufgabe Drogeziele potentiell ohne mühsame biochemische Reinigung Ansätze zu ermitteln.
Außerdem berichten wir hier eine Variation unserer Methode, die der gram-positiven Bakterium Enterococcus Faecalis für gramnegative Erreger p. Aeruginosaersetzt. Ähnlich wie bei p. Aeruginosader Fall ist, ist die Tötung von E. Faecalis zeitabhängig. Im Gegensatz zu früheren C. Elegans—E. Faecalis -Assays, unsere Assay für E. Faecalis ohne Preinfection auskommt, Verbesserung seiner Sicherheitsprofil und reduzieren die Wahrscheinlichkeit von kontaminierenden Flüssigkeiten Handhabungsgeräte. Der Test ist sehr robust, ~ 95 % Sterbeziffern 96 h Post Infektion zeigen.

Wichtige Parameter für die Testleistung
Ein richtiges Verständnis der zugrunde liegenden dieser Assay Biologie ist notwendig für die Problembehandlung und Optimierung der Assay. Zu diesem Zweck verweisen wir zunächst auf einige wichtige Papiere, die Aufklärung der Mechanismen der Pathogenese von p. Aeruginosa-vermittelte Tötung in liquid7,20. Unte...

Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Hier beschreiben wir eine Protokoll, die ist ein anpassungsfähiges, ganze Host, High-Content-Screening-Tool, das genutzt werden kann, um Wirt-Pathogen Interaktionen zu untersuchen und für die Wirkstoffforschung verwendet werden.

Ein High-Throughput, High-Content, Liquid-basierter C. Elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

This method can answer questions in host pathogen interaction and drug discovery, such as the the importance of various virulence factors and the ability of small molecules to ameliorate with the genesis. The main advantage of this technique is that it takes place in a liquid format with small volumes. To begin, while working inside a BSL 2 biosafety cabinet, streak P.aeruginosa from a frozen stock onto an LB Agar plate.Incubate the plate at 37 degrees C for 16 to 24 hours, then store the plate at four degrees Celsius for up to one week. Two days prior to setting up assay plates, use a single colony from the plate to inoculate three to five milliliters of sterile LB broth. Incubate the culture at 37 degrees Celsius for 12 to 16 hours.After preparing the slow kill or SK media according to the text protocol, with 350 milliliters of p. Aeruginosa from the fresh overnight LB culture, seed each 10 centimeter SK plate. Use a sterile bacterial spreader to evenly spread the bacteria, and allow the plates to dry in the biosafety cabinet.Then, incubate the plates at 37 degrees Celsius for 24 hours. To test multiple RNAi bacterial strains in parallel in a single well of a 24 well deep well plate, inoculate a single colony from each clone of previously prepared RNAi containing bacteria into four milliliters of carbenicillin supplemented LB.Place the plates into a shaking incubator optimized for multi-well plates at 37 degrees Celsius and 950 rpm for 16 hours, then collect the bacteria by centrifugation at 2, 000 g for 5 minutes. Decant the supernatant by inverting the plate and vigorously shaking it.Using 100 microliters of S Basal resuspend the RNAi bacteria. Pipette the resuspended bacteria into an appropriate number of wells of a multi-well NGM plate supplemented with IPTG and carbenicillin and allow the plate to dry. After preparing L1 worms according to the text protocol, prepare plates for basic experiments by pipetting approximately 5, 000 worms per 10 centimeter plate, seeded with RNAi or OP50 superfood.To set up for an RNAi screen, pipette approximately 300 worms per well in a 24 well plate seeded with RNAi. If the strain being used is temperature-sterile, incubate the worms at 15 degrees Celsius for approximately 16 hours and then transfer them to 25 degrees Celsius for 44 hours to complete development and prevent embryogenesis. To carry out a liquid killing assay use a cell scraper to remove the P.aeruginosa from an SK plateand resuspend the bacteria in approximately 5 milliliters of S.Basal.Using a spectrophotometer meaure the OD 600 of the bacterial suspension. Prepare 24 milliliters of diluted stock of P.Aeruginosa in S.Basal at an OD 600 equal to 09, then add 21 milliliters of liquid SK media, and, using a multichannel pipette, transfer 45 microliters of bacteria and media to each well of a 384 well plate. Wash the worms from their source into a 50 milliliter conical tube, and allow them to settle under gravitational force.Aspirate the supernatant to 5 milliliters, then use a total of 50 milliliters of S.Basal to resuspend the worms, and repeat the wash twice more. Using a worm sorter, sort approximately 22 worms into each well of a 384 well plate, according to the text protocol, then use a gas-permeable film to seal the plate and incubate it at 25 degrees Celsius for 24 to 48 hours. At the desired time, use a microplate washer and S.Basal to wash the 384 well plate a total of 5 times.One of the easiest mistakes to make is to aspirate the 384 well plate before worms have completely settled. It takes practice to accurately see whether the worms have settled completely. After the final wash, aspirate the supernatant down to 20 microliters.Then, add 50 microliters of 98 micromolar nucleic acid stain per well, for a final concentration of 0.7 micromolar. Incubate the plate at room temperature for 12 to 16 hours. After the desired incubation period, use the microplate washer to wash the plates a minimum of three times.For a data acquisition use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence. Add one milliliter of S Basal per well of a 24 well plate containing 300 worms per well. Gently agitate the worms by shaking the plate, then transfer worms to an empty, sterile 24 deep well plate.Allow the worms to settle gravitationally, about five minutes. Aspirate the supernatant, leaving approximately one milliliter per well, Then add 7 milliliters of S Basal to each well. Repeat the wash two more times, and then after the final wash aspirate all but approximately 400 microliters of supernatant.After pipetting the bacteria into 384 well plates to avoid starvation, using the resampler function of the worm sorter sort 22 worms into each well of a 384 well plate. Finally, following sorting, add small molecules or other experiment specific materials. As illustrated in this graph, when the steps outlined in this video are followed, a time dependent killing of C.elegans will be observed only in the presence of P.aeruginosa.In contrast, in the absence of key bacterial nutritional supplements, little to no killing will be observed. As shown here, the two step incubation of P.aeruginosa at 37 degrees Celsius and then 25 degrees Celsius, which is critical for conventional slow killing assays, and was originally implemented in liquid killing, is dispensable in this assay. The protocol tolerates a wide range of initial bacterial concentrations, from as low as an OD 600 equal to 0025, and still exhibits time and concentration dependent killing, although the timing does shift.In addition, as few as four wells is frequently sufficient to obtain statistically significant data. An example of the utility of processing is shown here when the signal-to-noise ratio is high, as in this example, analysis is simple and discriminating between positive and negative conditions is trivial. In these cases, even weak hits can be readily identified.Once mastered, this technique can be performed in about 60 to 75 minutes of hands on time per 384 well plate, not including sorting. While attempting this procedure, it's critical to remember to add all the components to your media or virulence will be compromised. This assay simplifies the application of whole organism phenotype based screening to drug discovery in host-pathogen research.After watching this video, you should have a good understanding of how to carry out liquid-based C elegans pathogenesis assays. This procedure can be modified by substituting with other pathogens, such as E.Faecalis for P.Aeruginosa, facilitating the search for treatments for other bacterial infections. Don't forget that working with infectious bacteria like P.Aeruginosa or E.Faecalis can be dangerous.Appropriate precautions, such as proper training, good personal protective equipment, and proper technique, should always be taken while performing this procedure.

a-high-throughput-high-content-liquid-based-c-elegans-pathosystem

Research

JoVE Journal

Immunology and Infection

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Die Erleichterung Drug Discovery: Eine automatisierte High-Content-Assay Entzündung im Zebrafisch

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Forschung

Immunologie und Infektion

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Automatische Trennung von<em&gt; C. elegans</em&gt; Variabel durch einen bakteriellen Erreger kolonisiert

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Ein mikroskopischer phänotypischer Assay zur Quantifizierung intrazellulärer Mykobakterien angepasst für High-Throughput/High-Content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Hochdurchsatz-Assay zum Phänotyp<em&gt; Salmonella enterica</em&gt; Typhimurium Association, Invasion und Replikation in Makrophagen

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Einrichtung eines Setup-Hochdurchsatz-Screening für kleine Moleküle, die Modulate c-di-GMP-Signalisierung in<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

Ein Fluoreszenz-basierte Lymphocyte Assay geeignet für Hochdurchsatz-Screening von kleinen Molekülen

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Zellfreie biochemische fluorometrisch enzymatische Assays für Hochdurchsatz-Messung der Lipidperoxidation in High-Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Automatisierte verhaltensbasierte Analyse der großen C. Elegans Populationen verwenden ein breites Sichtfeld Tracking-Plattform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

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.

Ein High-Throughput- Shigella-spezifischen bakterizide Assay

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