Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays
(2017) In PLoS ONE 12(3).- Abstract
The necessity of identifying novel methods to combat infections caused by antibiotic resistant bacteria is increasing each year. Recent advancements in the development of peptidoglycan hydrolases (e.g. lysins) from bacterial viruses (bacteriophages) have revealed the efficiency of this class of enzymes in treating serious bacterial infections. Though promising results have been obtained regarding the lethal action of lysin on bacterial pathogens both in vitro and in vivo, an often-overlooked factor in these studies is precisely identifying their peptidoglycan cleavage site. This knowledge would be useful for following the activity of the enzyme during development, without the need for whole-organism lytic assays. However, more... (More)
The necessity of identifying novel methods to combat infections caused by antibiotic resistant bacteria is increasing each year. Recent advancements in the development of peptidoglycan hydrolases (e.g. lysins) from bacterial viruses (bacteriophages) have revealed the efficiency of this class of enzymes in treating serious bacterial infections. Though promising results have been obtained regarding the lethal action of lysin on bacterial pathogens both in vitro and in vivo, an often-overlooked factor in these studies is precisely identifying their peptidoglycan cleavage site. This knowledge would be useful for following the activity of the enzyme during development, without the need for whole-organism lytic assays. However, more importantly, it would enable the selection of lysins with different cleavage activities that would act synergistically for enhanced efficacy. Here, we have developed two new methods to accurately identify the cleavage site of lysins using liquid chromatography mass spectrometry (LC-MS) on peptidoglycan-like fluorophore-quencher modified synthetic peptides, as well as determining the enzymatic action and kinetics of the enzymes on modified peptides in a FoÈrster resonance energy transfer (FRET) assay. These methods should facilitate progress within the lysin field, accelerating the development of therapeutic lysins to combat antibiotic resistant bacterial infections.
(Less)
- author
- Lood, Rolf LU ; Molina, Henrik and Fischetti, Vincent A
- organization
- publishing date
- 2017-03-01
- type
- Contribution to journal
- publication status
- published
- subject
- in
- PLoS ONE
- volume
- 12
- issue
- 3
- article number
- e0173919
- publisher
- Public Library of Science (PLoS)
- external identifiers
-
- pmid:28296948
- wos:000396311700078
- scopus:85015328605
- ISSN
- 1932-6203
- DOI
- 10.1371/journal.pone.0173919
- language
- English
- LU publication?
- yes
- id
- 7b9e4f20-209c-4c58-b466-42d34bbf3880
- date added to LUP
- 2017-04-11 15:48:11
- date last changed
- 2024-06-23 15:12:30
@article{7b9e4f20-209c-4c58-b466-42d34bbf3880,
abstract = {{<p>The necessity of identifying novel methods to combat infections caused by antibiotic resistant bacteria is increasing each year. Recent advancements in the development of peptidoglycan hydrolases (e.g. lysins) from bacterial viruses (bacteriophages) have revealed the efficiency of this class of enzymes in treating serious bacterial infections. Though promising results have been obtained regarding the lethal action of lysin on bacterial pathogens both in vitro and in vivo, an often-overlooked factor in these studies is precisely identifying their peptidoglycan cleavage site. This knowledge would be useful for following the activity of the enzyme during development, without the need for whole-organism lytic assays. However, more importantly, it would enable the selection of lysins with different cleavage activities that would act synergistically for enhanced efficacy. Here, we have developed two new methods to accurately identify the cleavage site of lysins using liquid chromatography mass spectrometry (LC-MS) on peptidoglycan-like fluorophore-quencher modified synthetic peptides, as well as determining the enzymatic action and kinetics of the enzymes on modified peptides in a FoÈrster resonance energy transfer (FRET) assay. These methods should facilitate progress within the lysin field, accelerating the development of therapeutic lysins to combat antibiotic resistant bacterial infections.</p>}},
author = {{Lood, Rolf and Molina, Henrik and Fischetti, Vincent A}},
issn = {{1932-6203}},
language = {{eng}},
month = {{03}},
number = {{3}},
publisher = {{Public Library of Science (PLoS)}},
series = {{PLoS ONE}},
title = {{Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays}},
url = {{http://dx.doi.org/10.1371/journal.pone.0173919}},
doi = {{10.1371/journal.pone.0173919}},
volume = {{12}},
year = {{2017}},
}