Name: applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing
Uploaded: 2016-07-06T15:00:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

This work was supported by National Health and Medical Research Council (NHMRC) grants (Grant ID 1062383 and 1063646) awarded to HJN. EAL is supported by an Australian Postgraduate Award.

The authors have nothing to disclose.

Sekret-Systeme sowie der bakteriellen Effektor-Proteine ​​diese Systeme Transport in das Zytoplasma der Wirtszellen, sind eine wichtige Komponente, die Virulenz viele pathogene Bakterien verwenden, um eine Infektion innerhalb von einzigartigen replikativen Nischen zu etablieren. Im Mittelpunkt vieler Forschungsgruppen wurde das Zusammenspiel zwischen bakteriellen Effektoren und Wirtsproteinen und dem Einfluss diese Effektoren auf Host zellulären Wege haben zu untersuchen. Sehr begrenzte Forschung, wenn überhaupt, ...

Coxiella burnetii ist ein einzigartiges intrazelluläre Erreger der zoonotischen Infektionen beim Menschen Q - Fieber verursacht. Diese Krankheit wird mit einem breiten Spektrum von klinischen Präsentationen im Zusammenhang von asymptomatischen Serokonversion zu lebensbedrohlichen chronischen Infektion erstreckt , die als Endokarditis Jahre nach der Exposition 1 oft manifestiert. Die Infektion des Menschen erfolgt in erster Linie durch das Einatmen von kontaminierten Aerosolen mit Wiederkäuern der großen Reservoir für Infektionen, insbesondere Milchkühe, Schafe und Ziegen. Obwohl Coxiella Infektion bei diesen Tieren der Regel subklinischen ist, kann die Infektion Abtreibung auslösen und die erhebliche bakterielle Belastung innerhalb der Entbindungs ​​Flüssigkeit und Plazenta kann die lokale Umgebung 1 verunreinigen. Ein Beispiel für die enorme Belastung eine solche Kontamination kann sowohl auf die öffentliche Gesundheit haben und die Agrarindustrie vor kurzem in der Q - Fieber - Ausbruch beobachtet wurde , die in den Niederlanden 2 aufgetreten ist . Zwischen 2007 und2010 mehr als 4.000 menschliche Fälle von Q - Fieber diagnostiziert wurden und dieser Ausbruch zu erheblichen Kontamination von Ziegenbetriebe 3 wurde verknüpft. Darüber hinaus ist Coxiella eine potenzielle biologische Waffe, wie für Disease Control von den US Centers klassifiziert und Prävention, durch die Umweltstabilität der Bakterien und geringe Infektionsdosis erforderlich schwere Morbidität und Mortalität 4 zu verursachen. Coxiella existiert in zwei Phasen: Phase I Organismen, isoliert aus natürlichen Quellen, sind extrem virulent und Phase - II - Organismen in vivo stark gedämpft. Beispielsweise nach mehreren in vitro Passagen Coxiella burnetii Nine Mile Phase I Organismen, Phase II Bakterien produziert wurden , die eine irreversible chromosomale Deletion in abgeschnittene Lipopolysaccharid (LPS) enthalten 5. Dieser Stamm, C. burnetii NMII ist phänotypisch ähnlich ich in Gewebekulturmodellen zu Phase und bietet einen sichereren Modusl für Forscher Coxiella Pathogenese in Laboratorien zu untersuchen 5. In den letzten Jahren haben mehrere Durchbrüche auf dem Gebiet der Genetik Coxiella rasch vorangetrieben . Vor allem die Entwicklung von axenic Medien (Citrat Cystein Medium angesäuert - ACCM-2) hat die zellfreie Wachstum von Coxiella sowohl in flüssiger und auf festen Medien 6,7 erlaubt. Dies führte zu direkten Verbesserungen der genetischen Werkzeuge zur Verfügung , für Coxiella einschließlich eines induzierbaren Genexpressionssystem, Shuttle - Vektoren und zufällige Transposon - Systeme 11.08. In jüngster Zeit werden zwei Verfahren zur gezielten Geninaktivierung sind ebenfalls entwickelt worden, die den Weg für 12 spezifische Virulenzgen Kandidaten untersucht. Folgende Internalisierung durch Alveolarmakrophagen, repliziert Coxiella zu hohen Zahlen innerhalb eines membrangebundenen Kompartiment bezeichnet die Coxiella- enthält Vakuole (CCV). Der CCV erfordert Host endocytic Handel thrau frühen und späten Endosomen , bis es reift in ein Lysosom abgeleiteten Organell 13. Im Laufe dieses Prozesses erwirbt der CCV Wirtsfaktoren , die entweder vorübergehend erscheinen oder bleiben mit der Vakuole verbunden sind , einschließlich, aber nicht beschränkt auf, Rab5, Rab7, CD63 und LAMP-1 . 13-15 Die Replikation von Coxiella innerhalb von Wirtszellen ist völlig abhängig von einem voll funktionsfähigen Dot / Icm Typ IVB - Sekretionssystem (T4SS) 8,16,17. Dieses Sekretionssystem ist ein Multi-Proteinstruktur ancestrally Konjugation Systemen und erstreckt sich über sowohl bakterielle als auch vacuolar Membranen bakterielle Proteine ​​zu liefern, bezeichnet als Effektoren in das Wirts Zytoplasma 18. Die Coxiella T4SS funktionell sehr ähnlich dem gut charakterisierten Typ IVB Dot / Icm Sekretionssystem von Legionellen pneumophila 19,20. Interessanterweise Aktivierung des T4SS und nachfolgende Translokation Effektor nicht auftritt , bis die saure Coxiella Lysosom-derived erreichtOrganell, etwa 8 Stunden nach der Infektion 17,21. Bis heute sind über 130 Dot / Icm Effektoren wurden 9,17,22-24 identifiziert. Viele dieser Effektoren spielen wahrscheinlich eine wichtige Rolle bei der Replikation von Coxiella in Wirtszellen; jedoch nur wenige Effektoren wurden funktionell 25-29 charakterisiert. In dieser Studie nutzen wir eine fluoreszenzbasierten Translokation Assay, der auf der Spaltung des (bezeichnet im Folgenden als BlaM Substrat) CCF2-AM FRET Substrat stützt über β-Lactamase - Aktivität in der Wirtszelle Zytoplasma (Abbildung 1). Das Gen von Interesse fusioniert an TEM-1-β-Lactamase (BlaM) auf einem Reporterplasmid, das eine konstitutive Expression sorgt. Das BlaM Substrat besteht aus zwei Fluorophore (Cumarin und Fluorescein), die ein FRET-Paar bilden. Anregung der Cumarin Ergebnisse in FRET des Fluorescein und grüne Fluoreszenzemission in Abwesenheit von Effektorzellen Translokation; Wenn jedoch die BlaM-Effektor-Fusionsprotein wird in das Wirts Zytoplasma transloziert, die sich ergebende β-Lactamase-Aktivität spaltet die β-Lactam-Ring des BlaM Substrat, wobei das FRET-Paar produzieren blaue Fluoreszenzemission nach Anregung zu trennen. Diese Translokation Assay wurde als ein Ansatz zur Identifizierung Effektor - Proteine ​​aus einer Reihe von verschiedenen intra- und extrazellulären Bakterien gut bewährt, einschließlich C. burnetii, L. pneumophila, L. longbeachae, Chlamydia trachomatis, darmpathogene E. coli, Salmonella und Brucella 17,30-35. Um die Rolle der spezifischen Wirtsfaktoren auf Coxiella Effektor Translokation bestimmen verwenden wir ein gut etabliertes Verfahren zur Gen - Silencing als RNA - Interferenz bekannt, insbesondere small interfering RNA (siRNA). Ursprünglich in Caenorhabditis elegans identifiziert, ist die RNA - Interferenz ein konserviertes endogenen zellulären Prozess für angeborene defe verwendetnse gegen Viren sowie der Genregulation 36,37. Von sequenzspezifischen siRNA, Abbau von mRNA erfolgt durch RISC (RNA-induced silencing complex) , was zu spezifischen Gen - Silencing oder Zuschlags 38 nach der Bindung. In dieser Studie wurde siRNA zwei Wirtsproteine ​​zu zielen, RAB5A und Rab7A, die wichtige Regulatoren des endocytischen sind. Die Auswirkungen der RAB5A und Rab7A auf Effektor - Translokation zum Schweigen zu bringen wurde mit C ermittelt burnetii pBlaM-CBU0077. CBU0077 ausgewählt wurde , wie es zuvor durch die Dot / Icm Sekretionssystem von Coxiella 17 transloziert werden , wurde gezeigt. Unter Verwendung sowohl siRNA Gen - Silencing und die fluorescencebased Translokation Assay hier beschrieben, beginnen wir eine Rolle für die Wirtsfaktoren bei der Translokation von Effektor - Proteine ​​, die durch Coxiella zu etablieren. Dieser Ansatz kann auf eine breite Palette von sowohl intra- und extrazellulären Bakterien angewendet werden, die ähnliche secretio besitzenn-Systeme, die für die Translokation von Effektor-Proteine.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C0759</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S4641</td><td>ACCM-2 medium component</td></tr><tr><td>Potassium phosphate</td><td>Sigma</td><td>60218</td><td>ACCM-2 medium component</td></tr><tr><td>Magnesium chloride</td><td>Calbiochem</td><td>442611</td><td>ACCM-2 medium component</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080</td><td>ACCM-2 medium component</td></tr><tr><td>Iron sulfate</td><td>Fisher</td><td>S93248</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625</td><td>ACCM-2 medium component</td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>C6852</td><td>ACCM-2 medium component</td></tr><tr><td>Bacto-neopeptone</td><td>BD</td><td>211681</td><td>ACCM-2 medium component</td></tr><tr><td>Casamino acids</td><td>Fisher</td><td>BP1424</td><td>ACCM-2 medium component</td></tr><tr><td>Methyl beta cyclodextrin</td><td>Sigma</td><td>C4555</td><td>ACCM-2 medium component</td></tr><tr><td>RPMI + Glutamax</td><td>ThermoFisher Scientific</td><td>61870-036</td><td>ACCM-2 medium component</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td>For bacterial culture</td></tr><tr><td>ON-TARGETplus Non-targeting Control Pool (OTP)</td><td>Dharmacon</td><td>D-001810-10-05</td><td>Non-targeting control</td></tr><tr><td>siGENOME Human PLK1 (5347) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-003290-01-005</td><td>Causes cell death; measure of transfection efficiency</td></tr><tr><td>siGENOME Human RAB5A (5968) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-004009-00-0005</td><td></td></tr><tr><td>siGENOME Human RAB7A (7879) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-010388-00-0005</td><td></td></tr><tr><td>5x siRNA buffer</td><td>Dharmacon</td><td>B-002000-UB-100</td><td>Use sterile RNase-free water to dilute 5x siRNA buffer to 1x siRNA buffer</td></tr><tr><td>DharmaFECT-1 Transfection Reagent</td><td>Dharmacon</td><td>T-2001-01</td><td>For transfection</td></tr><tr><td>Opti-MEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>51985-034</td><td>Reduced-serum medium used for transfection</td></tr><tr><td>DMEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>10567-014</td><td>For cell culture and infection</td></tr><tr><td>Heat-inactivated fetal calf serum (FCS)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Can use alternate equivalent product</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>For storage of Coxiella strain</td></tr><tr><td>PBS</td><td></td><td></td><td>For cell culture</td></tr><tr><td>0.05% Trypsin + EDTA</td><td>ThermoFisher Scientific</td><td>25300-054</td><td>For cell culture</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>For dilution of samples and standards for qPCR</td></tr><tr><td>Quick-gDNA Mini Prep</td><td>ZYMO Research</td><td>D3007</td><td>Can use alternate equivalent product to extract gDNA</td></tr><tr><td>SensiFAST SYBR No-ROX Kit</td><td>Bioline</td><td>BIO-98020</td><td>Can use alternate equivalent qPCR master mix product for qPCR reaction</td></tr><tr><td>LiveBLAzer FRET-B/G Loading kit with CCF2-AM</td><td>Invitrogen</td><td>K1032</td><td>For measurement of translocation using fluorescence and generation of the 6X loading solution (contains Solution A, B, C and DMSO)</td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106469</td><td>Probenicid solution component</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>71505</td><td>Probenicid solution component</td></tr><tr><td>di-Sodium hydrogen orthophosphate</td><td>Merck</td><td>106586</td><td>Probenicid solution component</td></tr><tr><td>Probenicid</td><td>Sigma</td><td>P8761</td><td>Probenicid solution component</td></tr><tr><td>DRAQ5 Fluorescent Probe Solution (5 mM)</td><td>ThermoFisher Scientific</td><td>62251</td><td>Nuclei stain to determine cell viablity. Use 1:4000 diluted in PBS.&amp;nbsp;</td></tr><tr><td>4% PFA (paraformaldehyde) solution in PBS</td><td>Sigma</td><td>P6148</td><td>Fixing solution for HeLa 229 cells</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430639</td><td>For growth of bacterial strain</td></tr><tr><td>96 Well Flat Clear bottom Black Polystyrene TC-Treated Microplates, Individually wrapped, with Lid, Sterile</td><td>Corning</td><td>3603</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430641</td><td>For growth of HeLa 229 cells</td></tr><tr><td>175 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>431080</td><td>For growth of HeLa 229 cells</td></tr><tr><td>Haemocytometer</td><td></td><td></td><td>For quantification of HeLa 229 cells</td></tr><tr><td>Tear-A-Way 96 Well PCR plates</td><td>4titude</td><td>4ti-0750/TA</td><td>For qPCR reaction</td></tr><tr><td>8-Lid chain, flat</td><td>Sarstedt</td><td>65.989.002</td><td>For qPCR reaction</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bench top microfuge</td><td></td><td></td><td></td></tr><tr><td>Bench top vortex</td><td></td><td></td><td></td></tr><tr><td>Orbital mixer</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td>For pelleting bacterial culture</td></tr><tr><td>Nanodrop</td><td></td><td></td><td>For gDNA quantification</td></tr><tr><td>Mx3005P QPCR machine</td><td>Aligent Technologies</td><td>401456</td><td>For quantification of Coxiella genomes in 7 day culture</td></tr><tr><td>ClarioSTAR microplate reader</td><td>BMG LabTech</td><td></td><td>For measurement of fluorescence</td></tr></tbody></table>

applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing

Coxiella burnetii, der Erreger des Q - Fiebers, ist ein intrazelluläres Pathogen , das IV Dot / Icm Sekretionssystem auf einer Art setzt eine replikative Nische zu etablieren. Eine Kohorte von Effektoren werden durch dieses System in die Wirtszelle Host-Prozesse transloziert und ermöglichen die Schaffung eines einzigartigen Lysosom abgeleiteten Vakuole für die Replikation zu manipulieren. Die hier vorgestellte Methode beinhaltet die Kombination von zwei gut etablierte Techniken: spezifische Silencing Gen siRNA und Messung der Effektor-Translokation mit einem FRET-basierten Substrat verwenden, die auf β-Lactamase-Aktivität beruht. Die Anwendung dieser beiden Ansätze können wir damit beginnen, die Rolle von Wirtsfaktoren in bakteriellen Sekretionssystem Funktion und Effektor-Translokation zu verstehen. In dieser Studie untersuchten wir die Rolle der RAB5A und Rab7A, die beide wichtige Regulatoren des endocytic Handel Weg. Wir zeigen, dass eine Abnahme der Effektor translocatio die Expression von Protein Ergebnisse Silencingn Effizienz. Diese Verfahren können leicht modifiziert werden, um andere intra- und extrazellulären Krankheitserregern zu untersuchen, die auch die Sekretion Systeme nutzen. Auf diese Weise kann aufgedeckt werden ein globales Bild Faktoren Wirt in bakteriellen Effektor-Translokation beteiligt.

Für diese Studie wurden die C. burnetii pBlaM-CBU0077 Stamm wurde ausgewählt als CBU0077 wurde zuvor ein transloziert Effektor des Coxiella Dot / Icm Sekretionssystem 17 gezeigt ist. Vor der Infektion, die Gesamtzahl der Genome / ml in der 7 Tage C. burnetii pBlaM-CBU0077 Kultur aufgezählt wurde qPCR unter Verwendung dar. 4 zeigt ein Beispiel des Zyklus Schwellenwert (Ct) -Werte von beiden Standards erwartet und Proben nach qPCR. ...

Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Untersuchung der Wechselwirkungen zwischen bakteriellen Erregern und ihren Wirten ist ein wichtiger Bereich der biologischen Forschung. Hier beschreiben wir die notwendigen Techniken Effektor - Translokation durch Coxiella burnetii während siRNA Gen - Silencing mit BlaM Substrat zu messen.

Anwenden von Fluorescence Resonance Energy Transfer (FRET) zu untersuchen Effektor Translokation Effizienz durch<em&gt; Coxiella burnetii</em&gt; Während siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

applying-fluorescence-resonance-energy-transfer-fret-to-examine

Research

JoVE Journal

Immunology and Infection

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

10.6K Aufrufe.  University of Melbourne.  Untersuchung der Wechselwirkungen zwischen bakteriellen Erregern und ihren Wirten ist ein wichtiger Bereich der biologischen Forschung. Hier beschreiben wir die notwendigen Techniken Effektor - Translokation durch Coxiella burnetii während siRNA Gen - Silencing mit BlaM Substrat zu messen. 

Serie: Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

In-vitro-Fluoreszenz Resonanz Energietransfer verwenden, um die Dynamik der Proteinkomplexe auf einer Millisekunde Zeitskala studieren

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Forschung

Biochemie

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

Screening und Identifizierung von RNA-Silencing Suppressoren von sezernierten Effektoren von Pflanzenpathogenen

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Genetik

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Immunologie und Infektion

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analyse von<em&gt; Yersinia enterocolitica</em&gt; Effektor Translokation in Wirtszellen unter Verwendung Beta-Lactamase-Effektor-Fusionen

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

In Situ Detektion von Ribonucleoprotein Complex Assembly in der C. elegans Germline mit Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Entwicklungsbiologie

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Einzel-Molekül-Imaging der Genregulation<em&gt; In vivo</em&gt; Verwenden cotranslationalen Aktivierung durch Spaltung (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Biologie

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA-Katalysator als Reporter für Drogen-Screening gegen RNA-Editing in Trypanosomen

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

Workflow für Hoch Inhalt, einzelne Zell Quantifizierung der Fluoreszenzmarker von Universal Mikroskop Daten, Unterstützung von Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within &#60;10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

Untersuchung von Wechselwirkungen zwischen histonmodifizierenden Enzymen und Transkriptionsfaktoren in vivo durch Fluoreszenzresonanz-Energietransfer

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the DNA can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides.
This video describes the basics behind chemical mediated transfections and introduces some of the most commonly-used reagents, including charged lipids, polymers, and calcium phosphate. Each step is described from the preparation of cells for transfection through analysis of transfection efficiency. Additionally, the applications section of this video-article describes the use of electroporation and a biolistic transfection as alternative methods for introducing nucleic acid into mammalian cells. It also describes an advanced use of transfection where co-transfection of interfering RNA and DNA are introduced as a way to down-regulate a naturally occurring protein while at the same time producing a mutant variant of it within the same cell.

Transfection: Inserting Genetic Materials into Mammalian Cells

Eine Einführung in die Transfektion

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

Anwenden von Fluorescence Resonance Energy Transfer (FRET) zu untersuchen Effektor Translokation Effizienz durch

Zusammenfassung

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