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 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 Förster 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.
Highlights
One of the current threats humanity faces is the rapid increase of antibiotic resistance seen amongst pathogenic microorganisms [1]
While an optical density reduction assay evaluates the ability of the lysin to lyse specific bacterial strains at a certain growth phase, it does not evaluate the cleavage activity of the lysin on the peptidoglycan. To overcome this problem and establish a more reproducible method to study lysin activity, we developed two methods based on fluorophore-quencher pair labeled peptides, adapted for analysis on either liquid chromatography mass spectrometry (LC-MS) or Forster resonance energy transfer (FRET) assays
As a proof of concept, we studied three distinct peptidoglycan hydrolases: the Staphylococcus simulans bacteriocin lysostaphin [16], the chimeric Staphylococcus aureus phage lysin ClyS [17], and the Streptococcus suis phage lysin PlySs2 [7], all with demonstrated activity on the Gram-positive pathogen S. aureus
Summary
One of the current threats humanity faces is the rapid increase of antibiotic resistance seen amongst pathogenic microorganisms [1]. A common method to evaluate the lysin’s activity is to test its capacity to degrade the peptidoglycan, by lysing and killing the bacterium This is usually studied by measuring ability of the lysin to reduce the optical density of a bacterial suspension, followed by plating the surviving bacteria and calculating the killing efficiency. While an optical density reduction assay evaluates the ability of the lysin to lyse specific bacterial strains at a certain growth phase, it does not evaluate the cleavage activity of the lysin on the peptidoglycan To overcome this problem and establish a more reproducible method to study lysin activity, we developed two methods based on fluorophore-quencher pair labeled peptides, adapted for analysis on either liquid chromatography mass spectrometry (LC-MS) or Forster resonance energy transfer (FRET) assays. We demonstrate the efficacy and ease of use of a high-throughput method to study the cleavage site(s) of peptidoglycan hydrolases using LC-MS on cleavage products from peptidoglycan-like synthetic peptides digested with the four different peptidoglycan hydrolases, as well as FRET assays for the study of lysin kinetics and enzymatic characteristics
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