Name: using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins
Uploaded: 2014-09-30T15:00:00.000+00:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

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

1 Preparazione del Produttore Strain <ol><li> Streak un ceppo pneumococcica per essere testato per la produzione batteriocina su un sangue di pecora di soia trittico piastra di agar 5% da -80 ° C scorte congelatore. NOTA: L&#39;uso di piastre di sangue per la crescita iniziale del ceppo da testare per l&#39;inibizione aumenta notevolmente la riproducibilità del dosaggio. Inoltre, altri ceppi non-pneumococco che producono batteriocine possono essere utilizzati anche se le condizioni di crescita può avere bisogno di essere modificato per un organismo specifico. Abbiamo usato Streptococcus mitis e Lactococcus lactis nel saggio overlay con successo. </li><li> Incubare la piastra overnight (O / N) a 37 ° C con 5% di CO 2. </li><li> Dopo l&#39;incubazione del ceppo produttore, preparare il piatto trittico di agar soia (TSA) pipettando 3.000 a 4.000 unità di catalasi su piastre contenenti 25 ml di TSA e diffondere la soluzione catalasi utilizzando microsfere di vetro sterili. Lasciate asciugare la piastra per circa 10 minuti sottouna cappa di sicurezza biologica. </li><li> Raccogliere una quantità visibile di batteri su una punta della pipetta. Pugnalare la punta della pipetta nella piastra TSA secca. NOTA: Più di un ceppo produttore può essere accoltellato in un unico piatto TSA. È importante distanziare i colpi in modo che aloni risultanti non si sovrappongano. <ol><li> Se possibile, includere noto batteriocine produttore come controllo positivo e il ceppo da utilizzare nella sovrapposizione come controllo negativo. NOTA: I controlli positivi e negativi sono stati precedentemente pubblicati e sono disponibili su richiesta 10. </li></ol></li><li> Incubare la piastra TSA contenente più pugnalate a 37 ° C con il 5% di CO 2 per 6 ore. </li></ol> 2. Fare scorte glicerolo del ceppo Overlay <ol><li> Preparare le scorte glicerolo (colture cresciute a fase mid-esponenziale e poi conservati a -80 ° C previa aggiunta di glicerolo) prima del giorno del test di sovrapposizione. Inoculare 5-10 colonie di str pneumococcoain per essere testato per la sensibilità o la secrezione di feromoni in Todd-Hewitt media con estratto di 0,5% di lievito (THY). NOTA: Costruzione di giornalisti inducibili BlpC è stato precedentemente descritto e 2 ceppi sono disponibili su richiesta. </li><li> Incubare 5-10 colonie in tubi di vetro con 5 ml di THY con coperchi ben chiusi in un bagno d&#39;acqua a 37 ° senza agitare. </li><li> Incubare colture a 37 ° C fino a raggiungere un diametro esterno 620 di 0,3 a 0,5. NOTA: Questo richiederà circa 4-5 ore a seconda del ceppo utilizzato. <ol><li> Capovolgere delicatamente la provetta un paio di volte prima di misurare il diametro esterno. </li></ol></li><li> Se l&#39;esperimento non può essere effettuata il giorno stesso o il ceppo sarà utilizzato più volte in futuro, fare scorte di glicerolo e di riprendere l&#39;esperimento in una data successiva. Per gli stock di glicerolo, aggiungere glicerolo alle culture ad una concentrazione finale del 20%. Culture aliquote in provette da 1,5 ml microcentrifuga e conservare a -80 ° C. NOTA: I campioni possono essere ssbranati da mesi a -80 ° C. Se non fare scorte di glicerolo, utilizzare direttamente i campioni per l&#39;analisi di sovrapposizione. </li></ol> 3. Overlay saggio <ol><li> Poco prima di sovrapporre l&#39;applicazione, miscelare i componenti di sovrapposizione. In un tubo da 15 ml, aggiungere 5 ml di THY, 3000-4000 U di catalasi, e 200 ml di brodo di coltura ceppo di sovrapposizione che è stato sia cresciuto quel giorno o uno stock di glicerolo della cultura che è stato scongelato a temperatura ambiente. Se si utilizza il ceppo giornalista, aggiungere 50 ml di X-gal (40 mg / ml) alla miscela di sovrapposizione. Utilizzare un tubo conico per piastra. </li><li> Equilibrare miscela sovrapposizione a temperatura ambiente prima aggiunta di liquido TSA. </li><li> Rimuovere la piastra TSA con ceppo accoltellato da incubatrice. Sciogliere solido TSA in forno a microonde e mantenerlo a 55 ° C bagnomaria fino al momento dell&#39;uso. Aggiungere 3 ml di liquido TSA con il 1,5% agar raffreddata a 55 ° C per sovrapporre miscela. <ol><li> Preparare immediatamente la miscela di sovrapposizione prima pipettaggio, il composto inizierà a solidificarsi moltorapidamente. NOTA: Se la sovrapposizione inizia a solidificarsi prima di placcatura, i risultati saranno interpretabile. </li></ol></li><li> Mescolare il composto overlay pipettando su e giù parecchie volte con una pipetta 5 ml facendo attenzione a non introdurre bolle nella miscela. Lentamente pipettare miscela overlay direttamente alla piastra TSA accoltellato. Conservare la piastra sul banco per qualche minuto per consentire l&#39;agar per indurire. <ol><li> Non ruotare la piastra per distribuire la miscela overlay, questo tirerà crescita della cultura accoltellato nella sovrapposizione. </li></ol></li><li> Posizionare con cura piastra TSA con sovrapposizione in 37 ° C incubatore contenente il 5% di CO 2 O / N (circa 16-18 ore). </li><li> Osservare le piastre dopo la crescita durante la notte. Le piastre devono apparire opaca dalla crescita del ceppo sovrapposizione, tranne nelle zone in cui si è verificato l&#39;inibizione o feromone segnalazione. Questi appariranno come zone sia chiaro o blu attorno al ceppo accoltellato, rispettivamente. NOTA: Anche se measurements del diametro di compensazione blu o possono essere determinati per la valutazione quantitativa, differenze di diametro possono anche variare a seconda della quantità di batteri che è stato accoltellato nella piastra. Il saggio overlay è un test qualitativo, ogni valutazione quantitativa devono essere interpretati con cautela. Qualsiasi compensazione visibile rispetto al controllo negativo è considerata attività. </li></ol>

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The authors have nothing to disclose.

Questo saggio overlay è un modo rapido per determinare la gamma di attività per i produttori batteriocina e dimostrare interazioni competitive che possono verificarsi tra i ceppi di pneumococco. Il dosaggio può essere adattato overlay da utilizzare per lo screening più ceppi per la produzione batteriocine una singola piastra. Abbiamo proiettato con successo grandi collezioni di deformazione con questo dosaggio utilizzando un replicatore 48-pin per inoculare le piastre SBA da una metà di una piastra a 96 pozzetti. D...

Streptococcus pneumoniae, un colonizzatore comune della comunità polimicrobica del rinofaringe, è in grado di competere con altri pneumococchi e specie strettamente collegate attraverso la produzione di peptidi antimicrobici batteri prodotte (batteriocine). Ogni ceppo pneumococcica completamente sequenziato esaminato fino ad oggi contiene una versione del b acteriocin- l ike p eptide locus, BLP. Produzione batteriocina da pneumococco ha dimostrato di essere importante nel risultato sia in vitro che in vivo la concorrenza 1-4. Dinamiche competitive sono influenzati dalla espressione di diverse batteriocine insieme a proteine ​​immunità affini in risposta ad uno specifico peptide feromone. Induzione del locus BLP è controllato da un sistema quorum sensing in cui un peptide feromone, BlpC, si lega alla chinasi sensore, BlpH, e inizia una cascata di segnalazione conseguentetrascrizione del locus 5,6 BLP. Ci sono quattro varianti alleliche di BlpC che ogni legano alla sua BlpH cognate 6. Per la cellula di sopravvivere agli effetti della propria batteriocina, i geni che codificano le proteine ​​immunitarie affini sono trascritti sullo stesso operone come ciascuno dei geni batteriocina. Uccidere verifica se un ceppo concorrente non è in grado di up-regolare il locus BLP in risposta a feromoni esogeno o, se il ceppo manca il gene immunità specifico richiesto per la protezione. Il complesso trasportatore, BlpAB è necessario per la secrezione e la lavorazione del feromone peptidico e peptidi batteriocina 2,4. Recentemente, è stato dimostrato che una percentuale significativa di ceppi di pneumococco sono &quot;imbroglioni&quot;, nel senso che hanno una mutazione nel gene conservato blpA che li rende incapaci di secernere feromoni e batteriocine 1,2. Questi ceppi sono in grado di rispondere alle BlpC esogeno 2 secreta da nitritoceppi noiosi consentono la produzione di proteine ​​di immunità. Anche se, preparazioni crude di batteriocine da surnatanti possono essere preparati da ceppi produttori utilizzando metodi biochimici passi per raggiungere la purezza, questo approccio non è utile per lo screening di grandi collezioni di isolati e se il livello di espressione batteriocina è bassa in colture in brodo cresciuta 2,7- 9. Il saggio di sovrapposizione viene usato come un modo rapido per studiare le dinamiche competitive tra i ceppi che possono esistere in vitro. Per fare ciò, un ceppo pneumococco è pre-inoculato su una piastra di agar e ha permesso di crescere fino a raggiungere concentrazioni batteriche locali sufficienti per l&#39;induzione del locus BLP. Un secondo ceppo viene quindi inoculato in una soluzione soft agar fuso che viene poi applicato sopra il ceppo pre-inoculato per verificare la sensibilità. Per valutare l&#39;attività del locus BLP (indipendente da attività inibitoria), i ceppi possono essere sovrapposti con una serie di three precedentemente descritti ceppi giornalista BlpC. Questi ceppi sono stati costruiti per contenere uno dei tre diversi alleli blpH che rispondono ai tre feromoni più comuni BlpC secreti dal pneumococchi. I ceppi giornalista hanno un gene lacZ integrato che è sotto il controllo di un promotore dipendente BlpH e portare una delezione nel gene blpC che impedisce auto-stimolazione 10,11.

<table><tbody><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>Fisher</td><td>B21261X</td><td>For growth from freezer cultures</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C100-500mg</td><td>4,741 Units per plate</td></tr><tr><td>Todd Hewitt Broth + Yeast Extract (THY)</td><td>Fisher</td><td>DF0492-17-6&amp;nbsp;</td><td>30 g of THB, 5 g yeast extract, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>Trypticase Soy Broth + Agar plates (TSA)</td><td>Fisher</td><td>B11768&amp;nbsp;</td><td>30 g of TS, 15 g agar, 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O autoclave</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside (X-gal)</td><td>Sigma</td><td>B4252-50MG</td><td>Dissolve in dimethylformamide</td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Spectrophometer</td><td></td><td></td><td>Measures the OD&lt;sub&gt;620&lt;/sub&gt; of cultures</td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO2</td><td></td><td></td><td></td></tr><tr><td>15 ml conical tube</td><td></td><td></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.

Ci sono due possibili risultati nel test di sovrapposizione. Dovrebbe essere possibile vedere la crescita del ceppo accoltellato dopo incubazione durante la notte. Nella Figura 1A, il ceppo sovrapposizione è sensibile alle batteriocine prodotte. Una zona libera può essere visualizzata circonda il ceppo accoltellato. Inoltre, vorticoso del ceppo pugnalato nella miscela sovrapposizione è possibile e può essere visto in Figura 1A. Nella Figura 1B, il ceppo overlay è i...

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.

Utilizzando il Overlay saggio misurare Qualitativamente produzione batterica di sensibilità e di pneumococcico batteriocine

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

13.5K 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

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.

Un test per il monitoraggio visivo T6SS-mediata Concorso batterica

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

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Immunologia e infezioni

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.

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

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.

Trattabili infezioni cellula di mammifero con protozoo-innescate batteri

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

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

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.

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in<em&gt; Streptococcus pneumoniae</em&gt; Utilizzo di tinture fluorescenti

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.

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un<em&gt; In Vitro</em&gt; Aderenza Assay

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.

I materiali biomimetici per caratterizzare le interazioni batteri-ospite

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.

Saggi qualitativi e quantitativi per il rilevamento e la caratterizzazione di proteine ​​antimicrobici

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.

L&#39;utilizzo della tecnica di sovrapposizione Soft-agar per lo screening composti inibitori bacterially Prodotto

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

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Quantificazione della competizione interbatterica utilizzando l'imaging a fluorescenza a singola cellula

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Utilizzando il Overlay saggio misurare Qualitativamente produzione batterica di sensibilità e di pneumococcico batteriocine

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un test per il monitoraggio visivo T6SS-mediata Concorso batterica

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

Trattabili infezioni cellula di mammifero con protozoo-innescate batteri

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un

I materiali biomimetici per caratterizzare le interazioni batteri-ospite

Saggi qualitativi e quantitativi per il rilevamento e la caratterizzazione di proteine antimicrobici

L'utilizzo della tecnica di sovrapposizione Soft-agar per lo screening composti inibitori bacterially Prodotto

Quantificazione della competizione interbatterica utilizzando l'imaging a fluorescenza a singola cellula

Utilizzando il Overlay saggio misurare Qualitativamente produzione batterica di sensibilità e di pneumococcico batteriocine

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un test per il monitoraggio visivo T6SS-mediata Concorso batterica

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

Trattabili infezioni cellula di mammifero con protozoo-innescate batteri

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un

I materiali biomimetici per caratterizzare le interazioni batteri-ospite

Saggi qualitativi e quantitativi per il rilevamento e la caratterizzazione di proteine ​​antimicrobici

L'utilizzo della tecnica di sovrapposizione Soft-agar per lo screening composti inibitori bacterially Prodotto

Quantificazione della competizione interbatterica utilizzando l'imaging a fluorescenza a singola cellula

Saggi qualitativi e quantitativi per il rilevamento e la caratterizzazione di proteine antimicrobici