Brug Overlay Assay til Kvalitativt foranstaltning Bakteriel Produktion af og følsomhed til Pneumokok Bakteriociner
Summary
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.
Abstract
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.
Introduction
Streptococcus pneumoniae, en fælles kolonisator af polymikrobiale samfund af nasopharynx, er i stand til at konkurrere med andre pneumokokker og nært beslægtede arter gennem produktion af bakterielt produceret antimikrobielle peptider (bakteriociner). Hvert fuldt sekventeret pneumokok-stamme undersøgt til dato indeholder en version af b acteriocin- l ike p eptide locus BLP. Bakteriocinproduktion af pneumokokker har vist sig at være vigtig i resultatet af både in vitro og in vivo konkurrence 1-4. Konkurrencedynamik påvirkes af ekspressionen af diverse bakteriociner sammen med beslægtede om immunitet proteiner som svar på et specifikt peptid feromon. Induktion af BLP locus styres af et quorum sensing system, i hvilket et peptid feromon, BlpC binder til sensoren kinase, BlpH og initierer en signaleringskaskade resulterer itransskription af BLP locus 5,6. Der er fire allelle variationer af BlpC at hver binder til dets beslægtede BlpH 6. For cellen at overleve virkningerne af sin egen bakteriocin, er de gener, der koder beslægtede immunitet proteiner transkriberet på samme operon som hver af bakteriocin gener. Killing opstår, hvis en konkurrent stamme er ikke i stand til at opregulere BLP locus reaktion på exogent feromon eller, hvis stammen mangler specifik immunitet gen nødvendig for beskyttelsen. Transportøren kompleks er BlpAB kræves til sekretion og behandling af peptidet feromon og bakteriocin peptiderne 2,4. For nylig er det blevet vist, at en betydelig del af pneumokokstammer er "snydere", som betyder at de har en bevaret mutation i blpA gen, som gør dem ude af stand til at udskille feromoner og bakteriociner 1,2. Disse stammer er i stand til at reagere på eksogen BlpC 2 udskilt af vrinskekedelige stammer muliggør produktion med immunitet proteiner.
Selv rå præparater af bakteriociner fra supernatanter kan fremstilles ud fra producentpriserne stammer hjælp biokemiske metoder skridt for at opnå renhed, denne tilgang er ikke nyttigt til screening af store samlinger af isolater, og hvis niveauet af bakteriocinet udtryk er lav i bouillon dyrket kulturer 2,7- 9. Overlapningskonturen analysen bruges som en hurtig måde til at undersøge de konkurrencemæssige dynamik mellem stammer, der kan findes in vitro. For at gøre dette, en pneumokok-stamme er pre-podet på en agarplade og lov til at vokse til at opnå lokale bakterielle koncentrationer er tilstrækkelige til induktion af BLP locus. En anden stamme podes derefter i en smeltet blød agar opløsning, som påføres derefter over præ-inokuleret stamme til at teste for sensitivitet. For at vurdere om aktiviteten af BLP locus (uafhængigt af inhiberende aktivitet), kan stammer overlejres med en række three tidligere beskrevne BlpC reporter stammer. Disse stammer blev konstrueret til at indeholde en af tre forskellige blpH alleler, der reagerer på de tre mest almindelige BlpC feromoner udskilles af pneumokokker. De reporter stammer har en integreret lacZ-gen, der er under kontrol af en BlpH promotor og bære en deletion i blpC gen, der forhindrer selv-stimulering 10,11.
Protocol
Representative Results
Discussion
Denne overlay-analysen er en hurtig måde at bestemme området for aktivitet for bakteriocin producenter og demonstrere konkurrencedygtige interaktioner, der kan opstå blandt pneumokokstammer. Overlay-assay kan tilpasses til brug for screening af flere stammer til bakteriocinproduktion på en enkelt plade. Vi har med succes screenet store strain samlinger med dette assay ved anvendelse af en 48-polet replikator at pode SBA plader fra den ene halvdel af en 96-brønds plade. Efter inkubation natten over væksten overfør…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work has been supported by Elizabeth E. Kennedy Research Award.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Trypticase Soy Agar with 5% Sheep Blood | Fisher | B21261X | For growth from freezer cultures |
| Catalase | Sigma | C100-500mg | 4,741 Units per plate |
| Todd Hewitt Broth + Yeast Extract (THY) | Fisher | DF0492-17-6 | 30 g of THB, 5 g yeast extract, 1 L ddH20 autoclave |
| Trypticase Soy Broth + Agar plates (TSA) | Fisher | B11768 | 30 g of TS, 15 g agar, 1 L ddH20 autoclave |
| 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) | Sigma | B4252-50MG | Dissolve in dimethylformamide |
| Petri dishes | Fisher | FB0875712 | |
| Spectrophometer | Measures the OD620 of cultures | ||
| 37 °C water bath | |||
| 37 °C incubator with 5% CO2 | |||
| 15 ml conical tube |
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