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Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage...

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Autores principales: Farquharson, Emma L., Lightbown, Ashlyn, Pulkkinen, Elsi, Russell, Téa, Werner, Brenda, Nugen, Sam R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8719110/
https://www.ncbi.nlm.nih.gov/pubmed/34975779
http://dx.doi.org/10.3389/fmicb.2021.741304
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author Farquharson, Emma L.
Lightbown, Ashlyn
Pulkkinen, Elsi
Russell, Téa
Werner, Brenda
Nugen, Sam R.
author_facet Farquharson, Emma L.
Lightbown, Ashlyn
Pulkkinen, Elsi
Russell, Téa
Werner, Brenda
Nugen, Sam R.
author_sort Farquharson, Emma L.
collection PubMed
description Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage’s ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage’s “adsorptive host range.” The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage’s adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein’s targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage’s adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4’s long tail fiber binding tip (gp37) to evaluate and quantify gp37’s relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay’s ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.
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spelling pubmed-87191102022-01-01 Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay Farquharson, Emma L. Lightbown, Ashlyn Pulkkinen, Elsi Russell, Téa Werner, Brenda Nugen, Sam R. Front Microbiol Microbiology Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage’s ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage’s “adsorptive host range.” The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage’s adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein’s targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage’s adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4’s long tail fiber binding tip (gp37) to evaluate and quantify gp37’s relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay’s ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins. Frontiers Media S.A. 2021-12-16 /pmc/articles/PMC8719110/ /pubmed/34975779 http://dx.doi.org/10.3389/fmicb.2021.741304 Text en Copyright © 2021 Farquharson, Lightbown, Pulkkinen, Russell, Werner and Nugen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Farquharson, Emma L.
Lightbown, Ashlyn
Pulkkinen, Elsi
Russell, Téa
Werner, Brenda
Nugen, Sam R.
Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay
title Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay
title_full Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay
title_fullStr Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay
title_full_unstemmed Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay
title_short Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay
title_sort evaluating phage tail fiber receptor-binding proteins using a luminescent flow-through 96-well plate assay
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8719110/
https://www.ncbi.nlm.nih.gov/pubmed/34975779
http://dx.doi.org/10.3389/fmicb.2021.741304
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