Peptidoglycan Compositional Analysis of Enterococcus faecalis Biofilm by Stable Isotope Labeling by Amino Acids in a Bacterial Culture.

Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into the bacterium's cellular functions and responses to external stimuli, but this analysis remains challenging because of various chemical modifications to PG-repeat subunits. We characterized changes to the PG composition of E. faecalis grown as planktonic bacteria and biofilm by developing "stable isotope labeling by amino acids in bacterial culture" (SILAB), optimized for bacterial cultures with incomplete amino acid labeling. This comparative analysis by mass spectrometry was performed by labeling E. faecalis in biofilm with heavy Lys (l-[13C6,2D9,15N2]Lys) and planktonic bacteria with natural abundance l-Lys, then mixing equal amounts of bacteria from each condition, and performing cell wall isolation and mutanolysin digestion necessary for liquid chromatography and mass spectrometry. An analytical method was developed to determine muropeptide abundances using correction factors to compensate for incomplete heavy Lys isotopic enrichment (98.33 ± 0.05%) and incorporation (83.23 ± 1.16%). Forty-seven pairs of PG fragment ions from isolated cell walls of planktonic and biofilm samples were selected for SILAB analysis. We found that the PG in biofilm showed an increased level of PG cross-linking, an increased level of N-deacetylation of GlcNAc, a decreased level of O-acetylation of MurNAc, and an increased number of stem modifications by d,d- and l,d-carboxypeptidases.

Inhibition of Staphylococcus aureus Cell Wall Biosynthesis by Desleucyl-Oritavancin: a Quantitative Peptidoglycan Composition Analysis by Mass Spectrometry.

Oritavancin is a lipoglycopeptide antibiotic that exhibits potent activities against vancomycin-resistant Gram-positive pathogens. Oritavancin differs from vancomycin by a hydrophobic side chain attached to the drug disaccharide, which forms a secondary binding site to enable oritavancin binding to the cross-linked peptidoglycan in the cell wall. The mode of action of secondary binding site was investigated by measuring the changes in the peptidoglycan composition of Staphylococcus aureus grown in the presence of desleucyl-oritavancin at subinhibitory concentration using liquid chromatography-mass spectrometry (LC-MS). Desleucyl-oritavancin is an Edman degradation product of oritavancin that exhibits potent antibacterial activities despite the damaged d-Ala-d-Ala binding site due to its functional secondary binding site. Accurate quantitative peptidoglycan composition analysis based on 83 muropeptide ions determined that cell walls of S. aureus grown in the presence of desleucyl-oritavancin showed a reduction of peptidoglycan cross-linking, increased muropeptides with a tetrapeptide-stem structure, decreased O-acetylation of MurNAc, and increased N-deacetylation of GlcNAc. The changes in peptidoglycan composition suggest that desleucyl-oritavancin targets the peptidoglycan template to induce cell wall disorder and interferes with cell wall maturation.IMPORTANCE Oritavancin is a lipoglycopeptide antibiotic with a secondary binding site that targets the cross-linked peptidoglycan bridge structure in the cell wall. Even after the loss of its primary d-Ala-d-Ala binding site through Edman degradation, desleucyl-oritavancin exhibits potent antimicrobial activities through its still-functioning secondary binding site. In this study, we characterized the mode of action for desleucyl-oritavancin's secondary binding site using LC-MS. Peptidoglycan composition analysis of desleucyl-oritavancin-treated S. aureus was performed by determining the relative abundances of 83 muropeptide ions matched from a precalculated library through integrating extracted ion chromatograms. Our work highlights the use of quantitative peptidoglycan composition analysis by LC-MS to provide insights into the mode of action of glycopeptide antibiotics.

Peptidoglycan O-acetylation increases in response to vancomycin treatment in vancomycin-resistant Enterococcus faecalis.

Vancomycin resistance is conferred upon vancomycin-resistant enterococci (VRE) through the replacement of peptidoglycan (PG) stem terminal d-Ala-d-Ala with d-Ala-d-Lac. The d-Ala-d-Lac incorporation can affect both the fitness and virulence of VRE. Here we comprehensively investigate the changes to PG composition in vancomycin-resistant Enterococcus faecalis following the growth in presence of vancomycin using liquid chromatography-mass spectrometry. Using high-resolution mass spectrometry, 104 unique muropeptides fragments were identified and the relative abundance of each fragment was accurately quantified by integrating the ion current of a selected ion using extracted-ion chromatogram. The analysis indicates reduced PG cross-linking, increased carboxypeptidase activities, increased N-deacetylation, and increased O-acetylation in VRE when grown in the presence of vancomycin. We found that O-acetylation preferentially occurred on muropeptides fragments with reduced cross-linking with a pentapeptide stem that terminated in d-Ala-d-Lac. These findings show that O-acetylation preferentially occurred in regions of the cell wall with reduced PG cross-linking on PG units that have stems terminating in d-Ala-d-Lac, serving as markers to prevent both the PG-stem modification by carboxypeptidases and the cell wall degradation by autolysins. Accurate quantitative PG composition analysis provided compositional insights into altered cell wall biosynthesis and modification processes in VRE that contribute to lysozyme resistance and enhanced virulence for VRE grown in the presence of vancomycin.

Quantification of the d-Ala-d-Lac-Terminated Peptidoglycan Structure in Vancomycin-Resistant Enterococcus faecalis Using a Combined Solid-State Nuclear Magnetic Resonance and Mass Spectrometry Analysis.

Induction of vancomycin resistance in vancomycin-resistant enterococci (VRE) involves replacement of the d-Ala-d-Ala terminus of peptidoglycan (PG) stems with d-Ala-d-Lac, dramatically reducing the binding affinity of vancomycin for lipid II. Effects from vancomycin resistance induction in Enterococcus faecalis (ATCC 51299) were characterized using a combined solid-state nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. Solid-state NMR directly measured the total amounts of d-Lac and l,d-Ala metabolized from [2-13C]pyruvate, accumulated Park's nucleotide, and changes to the PG bridge-linking density during the early exponential growth phase (OD660 = 0.4) in intact whole cells of VRE. A high level of accumulation of depsipeptide-substituted Park's nucleotide consistent with the inhibition of the transglycosylation step of PG biosynthesis during the initial phase of vancomycin resistance was observed, while no changes to the PG bridge-linking density following the induction of vancomycin resistance were detected. This indicated that the attachment of the PG bridge to lipid II by the peptidyl transferases was not inhibited by the d-Ala-d-Lac-substituted PG stem structure in VRE. Compositions of mutanolysin-digested isolated cell walls of VRE grown with and without vancomycin resistance induction were determined by LC-MS. Muropeptides with PG stems terminating in d-Ala-d-Lac were found only in VRE grown in the presence of vancomycin. Percentages of muropeptides with a pentapeptide stem terminating in d-Ala-d-Lac for VRE grown in the presence of vancomycin were 26% for the midexponential phase (OD660 = 0.6) and 57% for the stationary growth phase (OD660 = 1.0). These high percentages indicate that d-Ala-d-Lac-substituted lipid II was efficiently utilized for PG biosynthesis in VRE.